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~ T CELL ACTIVATION AND CYTOKINE
I PRODUCTION IN EXPERIMENTAL ALLERGIC
ENCEPHALOMYELITIS
for the Department of MICROBIOLOGY & IMMUNOLOGY,
McGILL UNIVERSITY, MONTREAL
July 1997
A thesis submitted to the Faculty of Graduate Studies and
Research in partial fulfilment of the requirements of the degree of
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TABLE OF CONTENTS
INDEX OF FIGURE LEGENDS .................................. 5
ACKNOWLEDGEMENTS ...................................... 7
PREFACE .................................................... 10
ABSTRACT .................................................. 11
L'ABSTRACT ................................................. 13
........................... ABBREVIATIONS & DESIGNATIONS 15
CLAlMSTOORlGlNALlTY ..................................... 17
.............................................. INTRODUCTION 20
CHAPTER 1
LITERATURE REVIEW ......................................... 23
Experirnental Allergic Encephalomyelitis :
A model for lmmunological Responses in the CNS .......... 23
Multiple Sclerosis ....................................... 24
Cellular and Molecular lmmunology of EAE
with cornparisons to MS ................................. 27
Validity of EAE as a model for MS ......................... 46
References ............................................. 48
PREFACE TO CHAPTER 2 ..................................... 75
CHAPTER 2
The central netvous system environment controls effector C04+ T cell
cytokine profile in experimental allergic encephalomyelitis ......... 76
CopyrigMwaiver ........................................ 85
PREFACE TO CHAPTER 3 ..................................... 86
CHAPTER 3
IFN-y Confers Resistance to Experimental Allergic Encephalomyelitis 87
........................................ Copyright waiver 94
PREFACE 10 CHAPTER 4 ..................................... 95
CHAPTER 4
CNS Specific IFN y Expression ................................. 96
Abstract ................................................ 97
Introduction ............................................ 98
Materials & Methods .................................... 100
Results ............................................... 103
Discussion ............................................ 107
Acknowledgments ..................................... 109
References ............................................ 110
Figures ............................................. 113
PREFACETOCHAPTER5 .................................... 125
CHAPTER 5
Bystander CD4+ T Cells do not contribute to
Experimental Allergic Encephalomyelitis .................. 126
Abstract ............................................... 127
Introduction ........................................... 129
Materials & M8th0ds .................................... 131
Results ............................................... 135
Discussion ............................................ 139
..................................... Acknowledgments 144
............................................ References 145
Figures ............................................... 150
CHAPTER 6
...................................... Conclusions 8 Summary 168
............................................ References 171
Figures .............................................. 172
Figure 4.1 : DNA PCR for the neomycin cassette and endogenous
IFNygene ............................................ 113
......................... Figure 4.2. Genotype analysis of IFN y 115
Figure 4.3. Genotype screening for G91 mice ................... 117
. . . . . Figure 4.4. RT-PCR analysis of IFN y mRNA levels in G9 mice 119
Figure 4.5. RT-PCR analysis of IFN y mRNA levels in G91 mice ... 122
Figure 5.1 : Short terni culture of LN selects for T cells reactive to
.................................................. OVA 150
Figure 5.2: Short term culture of LN selects for CD4+. CD45R8int T
cells .................................................. 152
Figure 5.3. Protocol for bystander OVA-specific T cell transfer ..... 154
Figure 5.4. OVA-reactive T cells trafic to the LN ................. 156
Figure 5.5. OVA-reactiveTcells trafficto the CNS ............... 158
Figure 5.6: Protocol for DO1 1.1 0 'bystander mouse" experiment . . 160
Figure 5.7. Characterization of the Anti-MBP T cell line ........... 162
Figure 5.8. Transgenic bystander T cells of the LN ............... 164
............. Figure 5.9. Transgenic bystander T cells of the CNS 166
Figure 6.1 Role of lFNq in EAE ................................ 171
Figure 6.2 T cell Encephalitogenicity vs IFN-y Actions ............ 173
Figure 6.3 Peripheral T cell Activation Leads to Inflammation
withintheCNS ........................................ 175
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my wouk LIT mncd s~eps . 1 wodb pa~ncufuKiy hke TO ~bunk ~ i chae l Juliw FOR yaahanntj and encounaçinç my m v e TO d ~ e Owens L<lr in 1991 and FOR aM bis sensikh advice ~ b e w u p ? ~ . Neil Gashman, J0sepbim ~ h t ~ l u , Pbdippe Poussie~, and W a p e Lapp also Gave peely oy r b e i ~ nme (and Reptence lerre~s
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Annie Johnson & Maya Anqelou
PREFACE
Candidates have the option of including, as part of the thesis, the
text of one or more papers submitted or to be submitted for publication, or
clearly-duplicated text of one or more published papen. These texts
must be bound as an integral part of the thesis.
If this option is chosen, connecting texts that provide logical
bridges between the different papers are mandatocy. The thesis must be
written in such a way that it is more than a mere collection of manuscripts;
in other words, resuits of a senes of papen must be integrated.
The thesis must still confonn to al1 other requirements of the
"Guidelines for Thesis Preparation". The thesis must include: A Table of
Contents. an abstract in English and French, an introduction which
clearly states the rationale and objectives of the study, a review of the
literature, a final conclusion and summary, and a thorough bibliography
or reference list.
Additional material must be provided where appropriate (e.g. in
appendices) and in sufficient detail to allow a clear and precise
judgement to be made of the importance and originality of the research
reported in the thesis.
ln the case of manuscripts CO-authored by the candidate and
others, the candidate is required to make an explicit statement in the
thesis as to who contributed to such work and to what extent.
Supervisors must attest to the accuracy of such statements at the doctoral
oral defense. Since the task of the examiners is made more difficult in
these cases, it is in the candidate's interest to make perfectly clear the
responsibilities of al1 the authors of the CO-authored papers.
ABSTRACT
Experimental autoimmune encephalomyelitîs (EAE) is a central
nervous system (CNS) disease, with symptorns reminiscent of Multiple
Sclerosis. During disease, the CNS is infiltrated by immune cells, with
increased expression of MHC antigens and Th1 cytokines.
We asked whether antigen presentation in the CNS controls the
bias towards Th1 cytokine in mice. We examined the capability of
microglia to act as antigen presenting cells. We found that they
stimulated T cell responses in vitro, but supported increased production
of Th1 cytokines only. We also found that 11-12 mRNA was upregulated
during peak disease, produced by resident microglia. We therefore
propose that microglia, through their production of 11-1 2, influence the
cytokine response in the CNS to produce the obseived Th1 bias.
We investigated the effect of deletion of l F N ~ o n the ability to
induce EAE in genetically-resistant (BALB/c) mice. IFN-y -1- mice were
susceptible to induction of EAE, and cellular infiltrates in the CNS
showed increases in message for CD3 and TNFa. Therefore, IFN-y
converts an otherwise EAE-susceptible mouse strain to become EAE-
resistant.
We examined whether the negative effect of IFN? on generation of
encephalitogenic T cells in the periphery might differ from its role in the
CNS. We interbred IFNq -1- mice to mice transgenically overexpressing
IFN-y in the CNS. Levels of IFN-y in CNS of the resulting mice were
insufficient to induce spontaneous disease. These mice will be useful
tools for studying the role of IFN-y in CNS disease.
During EAE, T cells of many specificities infiltrate the CNS. The
function of those that do not recognize CNS antigens ('bystanders') is
unclear. We transferred Ovalbumin-reactive (bystander) T cells to mice
prior to EAE onset. Flow cytometry analysis showed that the vast majority
of myelin basic protein-reactive T cells in the CNS were memory/effector
cells, but only very few bystander T cells showed an
activated/memory/effector phenotype. As a whole population, the
bystander cells did not upregulate their expression of CD44 or IL-2Ra, or
production of IFN-y, thus are probably not contributing to the ongoing
immune response during disease.
L'ABSTRACT
L'EAE est une maladie du système nerveux central (SNC) qui
peut être induite chez la souris et qui présente des symptômes proches
de ceux de la Sclérose En Plaques. Pendant la maladie, le SNC est
infiltré par des cellules immunes, il y a augmentation de I'expression des
antigènes du CMH et de la production de celtaines cytokines,
démontrant la présence d'une réponse immune de type Thl. Pour
déterminer si cette orientation de la réponse vers la voie Th1 est le
résultat de cellules présentatrices de l'antigène propres au SNC, la
capacité des cellules microgliales à présenter l'antigène a été étudiée.
Ces cellules sont capables de stimuler des clones T spécifiques du SNC
et provoquent la production de cytokines de type Th1 uniquement.
L'expression de I'ARNm de l'IL12 est également augmentée dans la
phase aigüe de la maladie. Ainsi, la microglie, par la libération d'lL12,
pourrait agir sur la production centrale de cytokines et orienter la
réponse immune vers la voie Th1 .
Le rôle de I'IFN y dans l'induction de I'EAE a été étudié chez une
souche de souris genetiquernent résistantes à I'EAE (souche BALB/c).
Après délétion du gene de I'IFN y, une EAE a pu être induite chez ces
souris, qui présentaient alors des lésions infiltrantes dans le SNC ainsi
qu'une augmentation de l'expression des ARNm du CD3 et du TNF a.
Ces résultats indiquent donc que l'absence d'lFN yest capable de
convertir un phenotype résistant à I'EAE en un phénotype sensibleLe
rôle central de I'IFN y a alors été comparé à celui qu'il joue à la
périphérie pour la production de cellules T encephalitogènes. Pour cela,
les souris knock-out pour le gene de I'IFN y ont et6 croisées avec des
souris transgéniques sur exprimant cette cytokine dans le SNC. Chez les
animaux hybrides, le niveau d'expression de I'IFN y dans le SNC était
insuffisant pour produire une maladie spontanée, cependant cette
nouvelle souche constitue un outil précieux pour poursuivre l'étude du
rôle de I'IFN y dans les maladies du SNC.
Au cours de I'EAE, plusieurs catégories de cellules T infiltrent le
SNC mais parmi celles-ci, certaines ne reconnaissent pas d'antigènes
centraux. Ces cellules T « spectatrices 3, ont été transférées, avant
l'apparition de la maladie, à des souris immunisées contre la MBP. Une
analyse par cytometne en flux démontre que la grande majorité
descellules T réactives contre la myéline trouvées dans le SNC sont de
type T effecteurn mémoire alors que seulement quelques unes des
cellules spectatrices sont de ce type. Ces résultats indiquent donc que
les cellules T spectatrices ne présentent pas une augmentation de
l'expression des marqueurs d'activation des cellules T ni de la
production des cytokines. Elles ne participeraient donc pas à l'extension
de la réponse immune au cours de la maladie.
Ab
APC
A51 9
BBB
CD25
CD44
CD45
CD45RB
CNS
CSF
CFA
d
DO1 1.10
EAE
FSC
GKO
G9
G91
HSP
IFN-y
IFN+
IL-1 2
rfL-12
iv
LN
MBP
MHC
MOG
antibody
antigen presenting cell
1FN-y transgenic mouse with 1.3 Kb MBP promoter
blood brain barder
interleukin-2 receptor alpha
T cell activation rnarker, also known as Pgp-1
common feukocyte antigen
T cell activation rnarker of varying mw isoforms
central nervous system
cerebral spinal fluid
cornplete Freund's adjuvant
day ova-peptide specific TCR transgenic mouse
experimental allergic encephalomyelitis
forward scatter
IFN-y knock out mouse
(GKO x A51 9 IFN-y transgenic) mouse
(GKO x VT1 IFNy transgenic) mouse
heat shock protein
interferon gamma
interferon gamma receptor
interleukin 12
recombinant IL-1 2
intra venous
lymph node
myelin basic protein
major histocompatibility complex
myelin oligodendroglia protein
MRI
mw
MS
NK
neomycin-R
NA
PLP
PNS
OVA
SSC
SC
TCR
TN F-a
VT1
magnetic resonance imaging
molecular weight
Multiple Sclerosis
natural killer cell
neomycin resistance gene
not applicable
proteolipid protein
peripheral nervous system
ovalburnin
side scatter
sub cutaneous
T cell receptor
tumor necrosis factor alpha
1FN-y transgenic mouse with 9.6 Kb MBP promoter
For al1 of the work presented within this thesis, the author is the
primary researcher and experimenter. The following are the daims to
originality contained within:
T cell encephalitogenicity and Antigen Presentation in the
C N S
1. I have shown that encephalitogenic CD4+ T cells within the CNS
are induced to express a Th1 cytokine profile. Encephalitogenic,
peptide-specific T cells that had the ability to produce both Th1 (IFN-y)
and Th2 (11-4) cytokines when stimulated by lymphoid organ-derived
antigen presenting cells, were shown to be restricted to the production of
Th1 cytokines when re-isolated frorn the CNS. This detailed analysis
significantly increases our understanding of encephalitogenic T cells, as
the rnajonty of work published on this subject analyzed the entire CNS
but did not determine the cell type that was making the message.
2. CNS antigen presenting cell populations, the majority of which
were microglia, were found to act as antigen presenting cells for
functional T cell responses in vitro and to only induce mRNA for ml, but
not Th2 cytokines, reminiscent of the in vivo observations (see section
1). This is in contrast to the non-biased T cell response elicited from
peripheral lymphoid organs such as the spleen (completed in vivo and in
vitro). Currently appearing publications on this issue found microglia to
induce incomplete T cell responses. My work directly demonstrates the
ability of these CNS antigen presenting cells to induce the proliferation of
T cells in an antigen specific manner and shows a unique ability of
antigen presenting cells within the CNS to direct the cytokine expression
patterns of disease-causing T cells.
3. Microglia and infiltrating macrophages isolated from CNS tissue of
mice with EAE upregulated mRNA for the p40 (inducible) subunit of the
switch cytokine 11-12. Thus, I have identified a mechanism whereby
antigen presenting cells endogenous to the CNS can bias the T cell
cytokine production towards a Th 1 response.
Actions of Interferony in EAE
4. 1 have directly demonstrated that 1FN-y can have counter-
inflammatory effects on the immune response during EAE. The
genetically EAE-resistant BALB/c strain of mouse was converted to a
susceptible phenotype by the removal of IFN-y. Therefore, IFN-ycan act
to inhibit the induction of EAE. The same trend towards increased
susceptibility was also obsewed in a genetically susceptible strain of
mouse (SJUJ) as an increase in disease incidence and severity
indicating that this is a general phenomenon. This greatly extends Our
understanding of the role of IFN-y in EAE.
5. This is the first demonstration that TNF-a cytokine production can
be induced in the CNS during EAE in the absence of IFNy. It was
previously thought that the major action of lFNq in the CNS was to
induce the production of TNF-a, which in tum determined
encephalitogenicity.
6. 1 generated two lines of mice in which the endogenous IFN-y gene
has been knocked out and the only functionaf IFN-y gene is a transgene,
controlled by MBP promoters. This is the first report where the
expression patterns of an IF N-y transgene could be determined and
differentiated from that of the endogene. Using IFN y as a reporter gene
for a 1.3 Kb MBP promoter and a 9.6 Kb MBP promoter, I showed that
expression was not lirnited to CNS tissue. I have found that for the line
G91 (1.3 Kb), the expression of lFNy while again not lirnited to the CNS,
penpheral expression was much lower than that seen in the G9 (9.6 Kb)
mouse. Transgenic expression of IFNq under the control of either
prornoter in the absence of endogenous IFN-y was insufficient to cause
spontaneous CNS disease. This is most probably due to an insufficient
level of expression of IFNy.
Bystander T cells in EAE
7. 1 showed that Bystander T cells, (both ovalbumin-reactive T cell
lines and T cells transgenically expressing a TCR specific for an
ovalbumin peptide) can enter the CNS of mice with active EAE. The
transgenic systern is unique and an improvement on existing systems for
the study of bystander cells as they have not been manipulated in any
manner (ie. isolated, cultured, labeled). For bystander T cells isolated
from the CNS, I showed that the majority of them were not activated. by
virtue of their lack of surface expression of T cell activation proteins and
small size. This is the first such analysis. Thus, Bystander T cells do not
appear to be contributing significantly to ongoing CNS immune
responses.
INTRODUCTION
Although multiple sclerosis (MS) presents as a spontaneous
autoimmune syndrome, we do not understand the mechanisms or even
many of the components of disease initiation and propagation. Our goal
was to investigate aspects of central nervous system (CNS) inflammatory
responses in order to improve our understanding of this disease process.
Specifically, we set out to investigate the role of infiammatory cytokines in
experimental allergic encephalornyelitis (EAE), and this work led to
questions regarding antigen presentation and Bystander T cells.
We began our investigation with interferon gamma (IFN-y) as this
cytokine is known to be upregulated during disease and to have the
ability to mediate much of the tissue inflammation we see in EAE. We
were curious to know the mechanism for the specific upregulation of this
cytokine in T cells in the CNS, and this work led us to analyze the antigen
presenting cells endogenous to the CNS. It now appears that the T cells
that cause disease (encephalitogenic T cells), receive signals from
antigen presenting cells endogenous to the CNS (eg microglia) which
induce the T cells to differentiate along the T helper 1 (Thl) cytokine
pathway. We propose that this propagates the disease process by
upregulating MHC antigens on the antigen presenting cells (APCs),
inducing leukocyte extravasation to the CNS, upregulating interleukin 12
(IL-1 2) production and inducing other inflammatory cytokines such as
tumor necrosis factor alpha (TNF-a). In this manner, disease progresses
(Chapter 2).
There are conflicting reports in the literature regarding the role of
IFN-y. Our initial proposal had been that it was intimately involved in the
progression of disease. Work with mice lacking endogenous IFN-y
demonstrated that the role of this cytokine is multi-fold. We observed that
removal of IFN-y led to an increase in disease seventy for mice
genetically resistant to the induction of disease. From this work, we
proposed that IFNT may have divergent functions in the periphery and
CNS. In the periphery, the dominant role of IFN-y would be to regulate T
cell proliferation, but in the CNS, the cytokine may act primarily to
promote inflammation. The T cell anti-proliferative effect of the cytokine
still functions in the CNS, but the proliferation of T cells within the CNS
does not appear to be necessary for the induction of EAE (Chapter 3).
Based on this hypothesis, we interôred mice lacking endogenous IFNq
to mica which transgenically express IFNy under the control of a
promoter of a gene limited in expression to the CNS (MBP). We
hypothesized that the removal of the down-regulatory peripheral effect of
IFN-y combined with the overexpression of the cytokine in the CNS
would lead to spontaneous disease. While these mice do not exhibit
symptoms of EAE, we believe they will be a useful tool to investigate
differential effects of peripheral and CNS-specific IFN-y on T cell
migration and encephalitogenicity (Chapter 4).
Our work had also confirmed previous demonstrations that while
there are T cells specific for CNS antigens in the CNS of mice with
disease, many endogenous T cells also enter. We proposed that not al1
these cells would be specific for CNS antigens (bystander T cells),
however they might have the potential to become activated by the fact
that they are in an infiamed milieu. We have found that T cells specific for
non-CNS antigens are able to traffic to the CNS of mice with EAE and
that these bystander cells do not upregulate their expression of T cell
activation marken and inflarnmatory cytokines (Chapter 5).
Frorn this work, we propose a model whereby T cells are activated
in the periphery, then traffic to the CNS in search of the appropriate
antigen. Endogenous APCs in the CNS (microglia) present antigen to
these cells and bias them along the inflammatory (TM) cytokine
pathway. This leads to the induction of chemokines, TNF-a, MHC
antigens and 11-12 al1 of which propagates the inflammatory response
causing disease. While the role of IFN-yin the CNS is to mediate the
inflammatory response, the production of IFN-y in the periphery acts to
modulate or down-regulate T cell proliferation of encephalitogenic T
cells. In genetically susceptible strains of mice, the encephalitogenic T
cell response overwhelms this down-regulatory action of IFN-y, and the T
cells traffic to the CNS to cause disease. The removal of this anti-
proliferative action in mice genetically resistant to disease allows the
proliferation of the somewhat less robust encephalitogenic T cell
response, and again these cells traffic to the CNS to cause disease.
Finally, once disease has been induced, the CNS and blood brain
barrier become more amenable to the extravasation of leukocytes. This
creates a situation in which many T cells not specific for CNS antigens
may enter the CNS during an ongoing immune reaction. We did not find
that these cells were contributing to the immune response as they did not
upregulate their expression of T cell activation markers or production of
inflammatory cytokines. Therefore, any treatment designed for MS, does
not seem to have to consider the role these bystander T cells play
(Chapter 6).
CHAPTER 1
LITERATURE REVIEW
Exper~mental Allergic Encephalomyelitis :
A model for lmmunological Responses in the CNS
EAE is considered to be the best avaifable animal model for the
human disease MS because they share the following clinical and
pathological charactedstics:
1) Loss of motor control in lirnbs (including clinical paralysis)
For both EAE and MS, a lack of muscle tone precedes the
complete paralysis. The typical progression begins with inco-ordination,
paresis, trembling, ataxia and finally leads to overt paralysis. The chronic
relapsing model of EAE closely mimics the clinical features of MS with
multiple relapses and remissions.
2) CNS infiltration by T cells and macrophages
In both cases. C04+ T cells are found in the CNS along with
infiltrating activated macrophages, and demyelination occun with time
(Brown et al., 1982).
3) Inflammation and cytokine production
The infiltration of the CNS by the immune system also correlates
with the induction of infiammatory cytokines which are nonally absent or
expressed at low levels in the CNS. The increased expression of IFN-y
and TNF-a has lead to theories linking these cytokines to both disease
induction and tissue damage (discussed in Sobel et al., 1984. Owens et
al., 1 994 , Renno et al., 1 995 and Okuda et a!., 1 996).
EAE is induced with an injection of purified CNS protein or CNS-
reactive T cells. Thus, we have the ability to control and manipulate the
entire system. This makes it scientifically attractive for those wishing to
test individual variations and factors influencing disease. Although EAE
and MS are not identical diseases, the similarities such as loss of motor
control. immune cell infiltration and inflammatory responses, have been
shown to have analogous mechanisms in both EAE and MS. This
supports both the studies of EAE to fuither Our understanding of MS, and
general inflammatory responses within the CNS. I have used EAE as a
model system for these studies, but woufd like to begin this review with a
description of MS. 1 will then detail the aspects of EAE which are relevant
to the work presented in chapters two through six.
Multiple Sclerosis
Multiple Sclerosis (MS) is one of the most common demyelinating
diseases, but its cause remains unknown (reviewed in Kurland, 1994). In
North America, approxirnately 300, 000 people have MS (values based
on estimates from the Canadian and National Multiple Sclerosis
Societies) (CMSS, 1997 and NMSS, 1997). MS is thought to be an
autoimmune disease, because of an ever-expanding database of
evidence describing its etblogy and immunopathology. It is primarily
observed in Young, fernale adults (ages 25-40) more than in any other
sex or age group (Sweeney et al., 1986). There is no cure. although a
large number of potential treatments are currently under study (reviewed
in Weinstock-Guttman & Cohen, 1996). The progression and clinical
manifestation varies with the worst cases involving severe, acute
progressive neurologic deficits resulting in loss of motor function,
blindness, and sensory disturbances (see Martin et al., 1992, Sobel,
1995, Steinrnan, 1996). The disease typically cycles through stages of
recovery (lack or lessening of symptoms) and acute attacks. Although
remissions may be frequent, on the whole, the disease nonnally
progresses towards worsening disability. MS is not always observed at
its most destructive, and for many people, disease management is
possible. But on the whole, MS is progressive and the disability both
hinders the patient's quality of life and life expectancy.
lntriguing data regarding the epidemiology indicates that the
disease has a much higher prevalence in northem, temperate climates
and that the risk of disease inherent in these geographical locations can
be maintained after moving (discussed in Kurtzke, 1985). This supports
an environmental cause for the disease; for example, a pathogen such
as virus or bacterium endemic to northern climates. Potentially, the as-
yet unidentified pathogen could cause MS through the mechanism of
molecular mimicry (Goswami et al., 1987, Wucherpfennig et al., 1995 and
Talbot et al., 1996). Although a causative agent has yet to be identified,
there has been much discussion as of late supporting this theory,
especially in the related autoimmune field of diabetes (Atkinson et al.,
1994). Altematively, an infection may activate the host immune system in
such as manner as to uncover anergized T cells, or activate bystander T
cells, which then cause disease (discussed in McRae et al., 1995 and
Miller et ab, 1995). Definitive proof remains elusive (Vandvik 8 Norrby,
1989), therefore, we continue to discuss these theories because they
describe a potential pathway for disease induction.
The risk factors for disease are not limited to environment, and
there exist data (especially from studies of mono and dizygotic twins),
supporting the contribution of a series of recessive genes (reviewed in
Sadovnick 8 Ebers, 1995). Additionally, certain HLA alleles have been
linked to autoimmune disorders, and not surprisingly, also to MS. The
mechanism leading to disease may be hypothesized as follows. As the
HLA class II genes are responsible for antigen presentation to T cells,
they exert control an the T cell repertoire which develops in an individual
and could thus bias the immune response. Consistent with this, limited
TCR usage has also been cited as a disease-linkage factor (for example
Utz et al., 1993 ). Although this link to the HLA or TCR genes is extremely
attractive, "to date no component of the human MHC had been identified
to be both necessary and sufficient for the development of MS."
(Sadovnick & Ebers, 1995). Neverthelesç, the cause of MS is most Iikely
to be a combination of both environmental and genetic factors.
It is believed that the induction event may occur far before any
clinical symptoms are observed. This theory is supported by the findings
using magnetic resonance imaging (MRI) which have shown significant
lesions (CNS damage), can be found in the absence of clinical
symptoms, or subclinical disease (Grossman et al., 1988 and Kermode et
al., 1990). The myelin sheath which surrounds and insulates axons
enhances the conduction of nerve impulses and is destroyed with the
progression of the immune response, as are the cells responsible for
myelin production and maintenance (the oligodendrocyte). The
development of the lesion results in a plaque which is a visible area of
demyelination. Plaques are found throughout the CNS. Demyelination
results in a wide range of clinical symptoms as the specific neurons
affected Vary (ie. sensory versus motor). The production of CNS lesions
can be visualized using MRI and are seen as da*, atypical CNS tissue
(MRI reviewed in Paty, 1989 and Filippi & Miller, 1996). Correlative
studies have shown these radiologic lesions as areas of blood brain
barrier (BBB) breakdown and inflammation (discussed in Katz et al.,
1993). Recently, the sites where activated microglia are found in the
CNS of patients with MS have also been shown to correlate with both
infiammatory lesions and chronic active plaques (Vowinckel et al., 1997).
Plaques can be characterized as either active or inactive or "bumt-our,
with the acute or active ones containing macrophages, lymphocytes and
red blood cells (Raine, 1983, Harris et al., 1991, Sobel 1995). Inactive
plaques result from repeated immune responses in the CNS involving
cycles of de- and re-myelination with the final consequence of
repair/exhaustion and irreversible destruction of tissue and gliotic scars.
Cellular and Molecular lmmunology of EAE
wifh cornparisons to MS
Historical Perspective
Originally, experimentation began with the injection of crude
mixtures of CNS tissue which caused symptoms of paralysis in outbred
primates (Rivers et al., 1 933). This level of investigation was rapidly
superseded when inbred species with increased susceptibility were
identified (reviewed in Martenson, 1984). encephalitogenic (or disease-
causing) proteins were found, and adjuvants which reduced the number
of immunizations necessary to induce disease were utilized (reviewed in
Fritz & McFariin, 1989). Subsequently, the immunodorninant peptides
for individual species and strains were identified (reviewed in Martin et
al., 1992). EAE can be induced in a wide variety of rodents and primates.
Certain proteins and amino acid sequences are commonly used for
experimental induction due to their favourable presentation by the major
histocompatibility complex (MHC) molecules, with precise combinations
being dictated by species and strain.
Adoptive Transfer
A major advance in the study of EAE was gained with the
demonstration that the transfer of CD4+ T cells specific for the CNS
antigens such as myelin basic protein (MBP) could itself cause disease
(Pettinelli, et al., 1981, Zamvil et al.. 1985). Previousiy, it had been
deterrnined that 1 was the transfer of immune cells and not senim (Ab)
that was capable of causing disease (Paterson, 1960 and Stone, 1961).
These initial experiments did not rule out the contributions of other cell
types, but now easily allowed one to control and modify the disease-
causing cells.
There is a lack of evidence supporting a physiological relevance
for restriction of TCR usage for encephalitogenic T cells. Initial studies
showing biased VP usage (reviewed in Heber-Katz 8 Acha-Orbea, 1989)
were followed by variable results in both EAE and MS (reviewed in
Wilson et al.. 1993). Although any identification of limited TCR usage
within the CNS would support the theory that T cells are involved in the
MS disease process, it has yet to be shown that such cells have the
ability to cause disease or even that CNS-reactive T cells are involved in
the initiation of MS. Finally, the concept of restricted TCR usage has
failen out of favour, especially as theories supporting antigenic
determinant spreading (Lehmann et al., 1993 and Vanderlugt & Miller,
1 996) and chronic macrophagelmicrogliaI activation (discussed in
Sriram & Rodriguez, 1997) gain acceptance.
lmm unological Privilege
The CNS has long been considered to be immunologically
privileged and immune responses disallowed from occurring there "in
order to rnaintain homeostasis' (discussed in Baker & Billingham, 1977).
The lack of MHC expression was used as evidence for this theory
(discussed in Yong & Antel, 1992). Wlh advances in the sensitivity of
techniques for the detection of immune components such as T cells and
cytokines, we now understand that normal immune surveillance of CNS
tissue appean ta occur regulariy, and that activated T cells have Iittle to
no difficulty in crossing the tight junctions of the endothelial barrier which
makes up the blood brain bamer. Furthemore, resident cells of the CNS
(microglia) have been shown to actively participate in the immune
responses that occur in the CNS (Ford et al., 1995 and Krakowski 8
Owens, 1997). The specialization of the BBB appears to be in inhibiting
small proteinaceous molecules, some pathogens, unactivated leukocytes
and antibodies from freely crossing into the CNS from the blood
(Broadwell, 1989 and Derrnietzel & Krause. 1991). lntriguing data
indicate that the CNS abnormalities observed by magnetic resonance
imagine (MRI) closely follow the tirnecourse of clinical symptoms (Namer
et al., 1992), thus establishing a direct correlation for BBB disniption in
the pathogenesis of EAE.
Lymphocyte Tra fficking
Lymphocytes must recirculate throughout the body on a
continuous basis and also specifically migrate to sites of immune
responses. This trafficking is a highly regulated, multi-step process
dependent upon the expression of adhesion molecules and their
receptors (reviewed in Bradley & Watson 1996, Butcher & Picker, 1996
and Romanic et aL, 1997). Much work has gone into characteriring the
adhesion molecule expression of different leukocyte subsets and
subsequent unique patterns of recirculation and migration (discussed in
Janeway & Golstein, 1993). Interestingly, tissue tropism for effector CD4+
T cells appears to be regulated by both initial activation environment of
the T cell, and by the T cell itself as it interacts with the endothelium of the
tissue at the site of the immune response. Specifically, the production of
IFN-y and TNFa by activated T cells. along with IL-1 produced by
monocytes, has been shown to upregulate both selectins and integnns
on the surrounding endothelium (reviewed in Pober 8 Cotran, 1990). It is
interesting to speculate that these finding might have widespread
implications, especially for organs in which these cytokines are not
normally expressed. With specific respect to our understanding of the
CNS not being an immunologically privileged organ (discussed in
Brosnan et al.. 1992 and Fabry et al., 1994), much research regarding
the expression of adhesion molecules has been camed out in order to
understand the mechanisms of immune cell traffic to the CNS. Adhesion
molecules allow and direct the trafficking of leukocytes have shown in
many instances, to be regulated in similar manners as cytokines in both
EAE and MS (Raine, 1994, Brosnan et al., 1995 and McMurray, 1996).
Importantiy, these molecules are 'rarely noted in normal and
noninflammatory conditions whereas regulatory cytokines [and adhesion
molecules] were readily detectable in the same diseases" in the CNS
(Brosnan et al., 1995).
Immunological Components of trafficking
Initial work characterized the CNS as being infiltrated principally
by T cells, with subsequent identification of macrophages, and 8 cells
(Hickey et al., 1983, Trotter & Steinman, 1984 and Cross et al., 1990). In
the CNS environment, the expression of adhesion molecules has been
characterized (Cannella et al., 1991) and is indeed implicated in the
disease process (Baron et al., 1993, Hulkower et al., 1993 and Glabinski
et al., 1997). Trafficking involves three steps. Tethering, activation and
transmigration al1 involve interaction with adhesion molecules and the
activation step can be specifically promoted by the interaction of
circulating cells with chernokines. If any one of these steps are blocked,
cell migration is inhibited completely rather than partially (discussed in
Springer, 1994). For example, a4 integrin is required for the entry of
encephalitogenic T cells to the CNS (Baron et al., 1993). Thus, the
molecular interactions which control the entry of effector immune cells to
the CNS were initially attractive to those wishing to design intemention
therapies for organ-specific diseases. Unfortunately, trafficking appears
to be controlled by combinations of commonly utilized adhesion
molecules, thus disallowing inhibition of any single receptor/ligand
interaction due to their global expression patterns.
Chemokines are a subgroup of cytokines which attract circulating
leukocytes to the site of immune responses, thereby aiding inflammatory
responses. This family of related small MW proteins which tend to act in
combinations with adhesion molecules to control recruitment of
leukocytes. The initial expression of chernokines such as MCP-1
(Hulkower et al.. 1993) , during EAE appean to coincide with the earliest
appearance of clinical symptoms (discussed in Glabinski et al., 1997),
and the inhibition of certain chemokines (MIP-la) can prevent the
induction of EAE (Karpus et al., 1 994).
lmmunopathology
The pathology of EAE as viewed from a neuroimmunological
viewpoint focuses on the infiltrating cells within the CNS (Traugott et al.,
1985 and Matsumoto 8 Fujiwara, 1987). Perivascular infiltrates of
mononuclear cells of the immune system are observed at the onset of
symptoms. Dunng initial episodes, focal infiltrates are most commonly
found in the spinal cord and are largely composed of CD4+ T cells and
large numbers of Mae l+ cells, although CD8+ T cells and B cells can
also be found. Infiltrates have been charactenzed in MS and found to be
similar to those seen in EAE (Hofman et al., 1986, Cuzner et al., 1988,
Boyle & McGreer, 1990, Estes et al., 1990, Raine, 1991 and Prineas et
al., 1993). It is difficult to find activated T cells within an uninffamrned
CNS. Therefore, the presence of large numbers of T cells during disease
is implicated in symptoms and disease progression. Leukocyte infiltrates
are found localized around blood vessels. The entry of these cells to the
CNS is certainly from vessels. Initially, the cells of the immune system
appear in the perivascular space, which is a region between the
endothelium of the blood brain bamer, but still remaining without the
parenchyma of the CNS. The eventual loss of the integrity of the blood
brain barrier with disease enhances the flow of leukocytes into the CNS
(Namer et al., 1993). Over time, chronic lesions rnay develop in chronic-
relapsing EAE models. The infiltration of the CNS changes character
and is more accurately described as dispened throughout the spinal
cord and brain (including the parenchyma).
Potential roles for infiltrating T cells
There are a number of theories on the functions T cells have within
the CNS. Activated T cells found within the brain may potentially mediate
immune responses and tissue destruction through the production of
certain T cell-specific cytokines that could induce resident or other
infiltrating cells to directly or indirectly damage CNS tissue. For example,
activated encephalitogenic T cells are known to produce IFN-ywhich can
induce the production of TNF-a, thus leading to an inflammatory
response cascade. Secondly, the action of Fas-ligand, CD56 and
perforin (Vergelli et al., 1996, Vergelli et al., 1997 and Rubesa et al.,
1997) have been implicated in cell death in the CNS. The death of
oligodendrocytes (the rnyelin-producing cell of the CNS), may be caused
by Fas-ligand, CD56, perforin mechanisms. Altematively, these cells
rnay be killed by cytotoxic CD8+ T cells. The actual mechanism by which
CD8+ cytotoxic T cells might kill an oligodendrocyte is less certain as
oligodendrocytes do not express MHC Class I in many instances,
therefore could not be the direct targets of the cytolytic activity of these T
cells. In certain systems, this is not true and the upregulation of Class I
MHC was shown when the CNS is infected with Theiler's virus
(Rodriguez et al., l987), and in transgenic systems (Evans et al., 1996
and Howih et al., 1997). Although the focus of much research rests on
the numerous ap T cells within which the CD4 and CD8 subsets lie, the
y6 T cells have also been proposed to play a role in MS pathology. This
minor subset of potentially cytolytic T cells are limited in their specificity to
heat shock proteins (HSP), bacterial lipids and in special cases MHC-
restricted peptides. A role for these cells was supported by the discovery
of HSP within the CNS of MS patients (Selmaj et al., 1 991 , Selmaj et al.,
1992, Aquino 8 Selmaj, 1992 and Brosnan et al., 1996).
Much energy has gone into the characterization of T cells that are
specific for myelin basic protein and which can be easily found within the
circulating blood and CSF of MS patients. This work is based on the fact
that EAE c m be induced with short peptides from MBP, and led to the
hypothesis that MS results from MBP-specific T cell activation (Allegretta
et ab, 1 990 Olsson et al., 1 992). Recently, this hypothesis has been
questioned as there is no evidence that MS patients have higher
frequencies of these cells in their peripheral blood over healthy
individuals (Carter & Rodriguez, 1991, and Utz 8 McFarland, 1994).
Additionally, the potential for other CNS antigens such as MOG or PLP to
be involved in T cell activation is too great to be ignored, as they have
been shown to cause EAE.
An tigen Presenta tion
The identity of the antigen presenting cell(s) within the CNS has
been a controversial issue. During disease, the CNS is infiltrated by
large numbers of macrophages, and the resident microglial
(macrophage-like cells of the CNS) (Ford et al., 1995) cells are often
activated dunng disease. These cells have inherent APC capabiliües,
constitutively express MHC antigens, and as such are candidates for the
APC function necessary to activate T cells (Unanue, 1984 and Patarroyo,
1 994). Both micro- and astroglial cells are activated during disease.
These changes in phenotype correspond with both the clinical and
histological stages of EAE (Hickey & Kimura, 1 988, Matsumoto et al.,
1992b). Macrophages also phagocytose myelin (Powell & Lampert,
1983, Esiri et al., 1987, and Esiri et al., 1991), and may contribute to the
ongoing inflammatory response in this manner directly (by the production
of cytokines, and antigen presentation), or indirectly (by diversifying the
CNS antigens presented). Afthough both macrophages and microglia
share many characteristics, including their hemopoietic origin (Hickey &
Kimura, 1988. Hickey et al., 1992, and Myen et al., 1993), the
assumption that microglia are "brain macrophages" is not entirely
supportable. For example, we do not know whether microglia
phagocytose myelin as macrophages do. Further investigation of the
role of human microglia is necessary to understand their contribution to
MS (reviewed in Sriram & Rodriguez, 1997).
Macrophages and microglia are not the only cells of the CNS to
be considered for APC function, as astroyctes have proven competent in
some situations (Fontana et al., 1984). The contribution of astrocytes to
the disease process in vivo is a topic for debate as some groups were
unable to find the necessary CO-stimulatory molecules for T cell activation
(Sedgwick et al., 1991 and Matsumota et al., l992a), while another did
(Nikcevich et al., 1997). The role of astrocytes may not be as primary
APCs, as they have been shown to produce iNOS and TNF-a which are
al1 implicated in disease (Bo et al., 1 994, Brosnan et al., 1994, Tran et al.,
6 cells are found in the CNS although in srnaller numbers relative
to the other infittrating immune cells (Prineas, 1985). Although they have
historically been associated with the production of Abs, they are also
considered to be professional APCs as they express a full complement of
T cell CO-stimulatory molecules. The primary contribution of these cells to
the disease process may be the production of antibodies and potentially
of significance for tissue destruction in Ab-rnediated demyelination
(Genain et al., 1995, and Genain et al., 1996). Recent evidence has
highlighted the potential of Ab-rnediated demyelination to be a major
factor in CNS disease (Genain et al., 1 996). Finally, one diagnostic
symptorn of MS is the observation of 'oligoclonal" bands of antibodies in
the cerebral spinal fluid (CS0 (Ebers, 1 984 and Farrell, et al.. 1 985).
Co-Stimula tion
Co-stimulatory molecules are necessary for appropriate signalling
between the APC and T cell. These molecules act secondarily to the
TCR:MHC:peptide interaction, but without CO-stimulation, the T cell is not
activated, but becomes anergized. Immune responses are severely
reduced in mice lacking B7-1 (knockouts) demonstrating the overall
importance of this molecule in the immune system (Freeman et al., 1993).
CD80 (87.1) and CD86 (87.2) and their respective ligands, CD28 and
CTLA-4, are of critical importance in T cell activation, and can be used as
examples for this growing family of molecules. 87 expression is linked to
the biased cytokine expression obsewed in EAE (Kuchroo et al., 1 995
and Perrin et al., 1996). Similar functions for 87.1 and 87.2 have been
shown in other systems (Freeman et al., 1995 and Lenschow et al.,
1996). Blockade of this pathway inhibits the induction of chronic-
relapsing EAE (Perrin et al., 1996) and autoantigen epitope spreading,
which is neceçsary for relapses (Miller et al., 1 995). A differential
expression pattern for the two 67 molecules may predispose one for
disease, as the T cell cytokine production following activation can be
biased towards an inflammatory response depending upon the 87 co-
stimulation received. In support of this theory, the genetically susceptible
mouse strain SJL, has increased expression of 87-1 (Miller et al., 1995)
and Windhagen and colleagues (1995) have shown the sarne molecule
in MS lesions. This single systern of CO-stimulation molecules is only one
example. Many others (some of which remain to be described), most
certainly regulate T cell activation, and act to influence disease outcome.
Cytokines
Th lnh2 Decision Making
At the beginning of a section describing the roles Th1 and Th2
cytokines play in the EAE disease process, I would like to caution that our
understanding of the discreetness of these subsets has been questioned.
The hypothesis that individual T cells produce an array of cytokines in
response to stimulation appears to be much more valid (Kelso, 1995).
Considering this, there is a vast amount of data supporting biased
cytokine responses in various immune responses. The cytokines made
by CD4+ T cells can generally be divided into Th1 (IFN-y, TNF-a), those
promoting inflammatory responses and cell-mediated immunity, and Th2
(1 14, 11-1 0, IL-5), those promoting humoral and allergenic responses.
The production of one subset generally inhibits the production of the
other (reviewed in Fitch et al., 1993 and Myers et al., 1993). Originally,
Mosman and colleagues described Th1 and Th2 subsets from murine T
cell clones (Mosman 8 Coffman, 1989, and Fiorentino et al., 1989), but
recent work has provided evidence that these definitions may have
physioiogical relevance in hurnans (Manetti et al., 1993). Naive T cells
leave the thymus with the pluripotent capacity to produce both Th1 and
Th2 cytokine upon stimulation through TCRMHC interactions, and can
be designated Th0 in phenotype (Bradley et al., 1993). Development
along either pathway is controlled at many stages including the initial
APC interaction (discussed in Swain, 1994).
CD4+ T cells are involved in most aspects of immune responses at
least at a minor level. Additionally, they appear to develop along biased
pathways towards either Th1 or Th2 cytokine producing activated T cells.
Although many of the functions of T cells are 'mediated directly through
intercellular contact", (discussed in Fitch et al., 1993), cytokines, as the
soluble messengen of activated T cells, are responsible for detennining
the charactenstics of a particular immune response. Our understanding
both the physiological significance and moiecular regulation of the
development of these subsets impacts directly on our undentanding of
general immune responses. The cytokines present at the initial
activation stage of naïve T cells are of critical importance to the
subsequent development and contribution that T cell will have in the
ensuing immune response (Hsieh et al., 1992, Seder et al., 1992, Seder
et al., 1993). The role of the antigen presenting cell at this stage is
inextricably Iinked to this process (discussed in Seder et al., 1994).
Recently, our understanding of the development of Th1 and Th2
immune responses has been expanded with the discovery that adhesion
molecules may direct the recruitment of cell subsets into inflamed tissues.
The adhesion molecules P- and E-selectin were shown to bind only Th1
type CD4+ T cells (Austrup et al., 1997 and Borge et al., 1997).
Therefore, the selective recruitment of a single subset of CD4+ T cells
may determine the "character of a local immune response" (Austrup et
al., 1997). Therefore, the transcriptional regulations which control the
expression of functional selectin ligands on Th1 and Th2 subsets
regulate the distinct effector functions of CD4+ T cells.
The role of cytokines in MS has been recently reviewed
(Benveniste & Benos, 1 995, Navikas & Link, 1 996). Eariy woik
highlighted the potential of inflammatory cytokines in the disease process
and lad to a flurry of investigations. The initial observation that
differentiation of Th precursor cells into Th1 or Th2 effector cefls had
biological importance in the field of Leishmania research (Heinzel et al.,
1989), lead to investigations in a number of different systems including
EAE. The immune response within the CNS during EAE has been
characterizad as Th1 in phenotype and there appean to be a
concomitant lack of Th2 cytokines. Therefore, a bias in cytokine
production is considered to be a clue to the mechanism of disease
induction and progression. Furthemore, much of the work previously
completed in the rodent EAE system regarding the increased expression
of inflarnmatory cytokines in the CNS has been shown to be similar for
patients with MS: TNF-a (Hofman et al., 1989 and Selmaj et ai., 1 991 b),
IL-1 and IL-2 (Hofman et al., 1986), IL-1 2 (Nicoletti et al., 1996), and
iNOS (Merrill et al., 1 993, Cross et al., 1 994, Tran et al., 1 997).
lnterleukin- 12
Interleukin-12 (11-1 2) is a regulatory cytokine which acts
predominantly on T and NK cells. It promotes the production of IFN-y and generafized Th1 T cell responses, and a lack of 11-12 results in deficient
IFN-y responses (Magram et al., 1 996). It's role in EAE has been
confimed by antibody blocking studies which showed that when
otheiwise encephalitogenic T cells are cultured with anti-IL12 Ab,
symptoms were dramatically lessened (Leonard et al., 1995).
IL-1 2 and lFNy appear to mutually prornote one anothers'
production, although the molecular mechanisrns through which such
activations are camed out appear to be indirect. It has recently been
shown that interferon regulatory factor4 (IRF-1) promotes IFN-y
production by promoting the production of 11-1 2 in antigen presenting
cells (Lohoff et al., 1 997). Indeed, IRF-1 does not bind the IFNy promoter
or even function within the T cell itseff. It has been shown that the
presence of IFN-y elevates the expression of 11-12 by macrophages
(Manetti, et al., 1994 and Flesch et al., 1995), and it has been proposed
that lFNq signals macrophages and microglia to upregulate IL-1 2
production. Therefore, although IL-12 induces IFNq, IFN-y also
promotes 11-12 production, thereby ensuring a Th1 biased response.
Finally, genetic resistance to the induction of EAE can be the result of a
lack of sufficient IFN-y production by MBP-specific T cells and this
suboptimal T cell response can be made encephalitogenic by exposure
to IL-12 (Segal 8 Shevach, 1996).
ln terferon- y
l FN-y is secreted by activated T cells and NK cells. The receptors
for IFN-yappear to be expressed on almost al1 cell types (Aguet et al.,
1988 and Farrar & Schreiber, 1993), therefore, the cytokine exerts its
immunomodulatory effects within a wide range of organs and tissues.
One of the major functions of IFNq is to activate macrophages, thus
promoting antigen presentation, TNF-a and N O S production and
generally mediating cell-mediated host defense (Belosevic, et al., 1988
and Liew et al., 1990). Secondarily, the production of IFN-y regulates the
proliferation and stimulates the activation status of T cells (Gajewski et
al., 1 989 and Dalton et al., 1 993). IFNq is a potent inducer of MHC
antigen expression, acting directly at the transcriptional level (Penek &
Benveniste, 1995). Consequently, the normal regulation of MHC
induction is disturbed in mice lacking IFN-y, although MHC expression is
not abrogated (Goes et al., 1995). IFN7 affects many of the components
of the immune system, and much effort has concentrated on determining
the physiological role of IFNq during normal development and immune
responses.
Neither IFN-y, nor IFN-y receptor are necessary for normal murine
development, as IFN-y-deficient mice grow normally to become healthy,
fertile adults (Dalton et a/., 1993 and Huang et al., 1993). Although
animals lacking these cytokines do have impaired immune responses
(Dalton et al., 1993, Huang et al., 1 993), compensatory mechanisms
within the immune system appear to at least partially counteract the loss
of this cytokine.
Through clinical trials, it was discovered that the role of IFN-y
indeed appeared pro-inflammatory in that the "administration of IFN-y
promotes exacerbations of MS" (Panitch et al., 1 987 and Panitch &
Bever, 1993). lnflarnmatory responses were also observed with the
direct injection of IFNq to the CNS in rodent experiments (Simmons &
Willenborg and 1990 Sethna & Larnpson, 1991). IFN-y has been directly
implicated in the induction and propagation of CNS disease and recent
evidence from transgenic mouse systern supporting this theory is
dramatically convincing (Corbin et al., 1996, Horwitz et al., 1997, and
Renno et al.. 1997). As described, the roles of IFNq are many-fold, and
include a regulatory role for immune responses within the CNS (Billiau et
al., 1987, Duong et a/.,1994, Ferber et al., 1995 and Willenborg et al., 1996). Model systems which integrate the regulatory and activating
functions of IFN-y are useful in interpreting the function of IFNq
(Krakowski & Owens, 1996).
The regulatory functions of 11-4 with respect to EAE are biased
towards inhibiting the inflammatory response. Therefore, although the
promotion of a Th2 response is typically associated with the production of
antibodies, I L4 also has an important function in lirniting inflammatory
Th1 responses, which have the capability of leading to tissue damage
and pathology if directed against self-antigens.
A significant molecular mechanism through which such regulation
is seen is by the selective loss of 11-12 receptor 82. While IFNq
promotes the expression of this molecules, IL4 directly inhibits it, thus
shutting off the Th1 response (Szabo et al., 1997 and Rogge, et al.,
1997). This is another example of the mechanism through which
molecular and cellular signalling control the development of
physiological immune responses.
In a similar fashion to afmost al1 other cytokines, IL-4 is not present
in the normal, healthy CNS. The only time this down-regulatory cytokine
appears to be expressed is during the late, or remission stages of
disease (Kennedy et al., 1992 and Khoury et al., 1992). Additionally, 11-4
production has the ability to modify the overall immune response and
protect against EAE (Falcone & Bloom, 1997).
Tumor necrosis factor-a
With a lesser degree of ambiguity, the role of TNF-a in EAE has
best been described as being required for encephalitogenicity of T cells
(Baron, et al., 1993) and antibodies to both TNF-a and -p prevent the
transfer of EAE (Ruddle et al., 1990a). Additionally, the expression of
TNF-a in the CNS leads to CNS pathology and wonening of clinical
phenotype (Taupin et al., 1997). The majority of TNF-a is made by
macrophages and microglial cells during disease, not T cells (Renno et
al.. 1 994). thus implicating the CNS environment and the infiltration of
monocytes as major culprits in TNF-a pathology. The evidence
supporting a pro-inflamrnatory and disease-promoting role of TNF-a is
substantial. Nevertheless, knockout mice have shown that neither TNF-a
nor -p are required for the induction of EAE (Frei et al., 1997). This work
does not discount the role TNF mofecules have in the induction and
pathology of disease. Instead. experiments using genetically
manipulated mice have been recently reinterpreted since the
development of the mouse which lacks these cytokines almost certainly
involves the induction of compensatory proteins (discussed in Steinman,
1 997).
Bystander T Cells
A healthy CNS contains few T cells, little MHC (Baker, &
Billingham, 1977 and Craggs & Webster, 1985) and has an intact BBB.
The view of the immune privileged CNS has changed as we now know
that even the unaltered BBB allows the traffic of activated T cells. The
666 does differentiate between naive and activated T cells, allowing
passage of only the latter (Hickey. 1991 a and Hickey et a/.. 1991 b).
Hickey and colleagues (1 991 b) were able to specifically show that the
antigen-specificity of the T cell did not determine ability to migrate across
the BBB, into the CNS. Additionally, stimulation of the endothelial cells
which comprise the BBB drastically increases its pemieability to T cells of
al1 activation States. Changes in the exclusion abilities of the BBB may
affect the course of disease and potential relapse risk. MS patients are
challenged many times throughout their illness, by virus or bacterial
infections. This elevates the circulating levels of activated T cells.
Patients with long-standing disease also have disruption of their BBB.
Therefore, the CNS is both amenable to the traffic of T cells and
frequently, activated T cells are circulating and may find their way to the
CNS. Do T cells specific for non-CNS antigens contribute to the ongoing
immune response in MS?
The proposed function for T cells in the CNS is to mediate
disease, but in the situation where the BBB allows the entry of T cells of
any specificity and activation level, especially of those reactive to non-
CNS antigens, do such cells contribute to the ongoing immune
response? Cells that are not specific for a CNS antigen are defined as
"bystandenn with respect to EAE and MS studies. Bystander T cells have
been studied in many systems, but the data have yielded an incomplete
picture of the contribution these T cells make to ongoing immune
responses. It has been shown that perturbation of the blood brain barrier
may indeed be mediated in great part by bystander T cells (Namer et al.,
1993 and Seeldrayers et al., 1993). Therefore, one potential contribution
of these cells to disease may be to the disrupt the integrity of the BBB. In
one viral study, the production of type I interferons did appear to have a
role in activating T cells in a non-antigen specific manner. Therefore.
bystander T cell activation in viral immune responses may have a
physiological function (Tough 8 Sprent, 1996). Using a different viral
system which focussed on diabetes, the authors did not find any
evidence to support a role for bystander T cells in the disease process
(Ehl et al., 1 997). For the study of EAE and MS, we want to understand
the potential function of bystander T cells in order to detemine whether
we must direct therapy strategies against al1 T cells within the CNS or just
those specific for CNS-antigens.
Genetic Resistance and Susceptibility
For rodent systems, the strains susceptible or resistant to the
induction of EAE were described soon following the discovery that brain
homogenate caused symptorns of paralysis when injected SC. For the
mouse systems, fernale mice were noted for being mare susceptible to
the induction of disease than males, and this gender-related difference
was confirmed using T cell transfers by Voskuhl and colleagues (1 996).
The increased susceptibility of female mice was not only exploited by
researchers, but also considered to support the argument that EAE is a
relevant model for MS as there is longstanding evidence for a gender
bias for this human disease. Further studies were carried out in rodent
systems to find susceptibility loci in hopes of discovering the same for
MS. Both MHC (Linthicurn & Frelinger, 1 982, Jemison et al., 1 993 and
Mustafa et al., 1994) and non-MHC genes (Driscoll et al., 1 985) were
shown to be criticafly involved. What has been observed with respect to
human disease is a higher frequency of certain MHC genes among
patient populations, especially HLA DR2,DQl (Francis et al., 1986 a and
b, Haegert 8 Francis, 1992 and Hillert & Olfenip, 1993). Genetic
susceptibility at most makes up a component of disease, and no single
gene has been shown to be necessary or sufficient for MS. When one
mentally steps back from the details of this body of work, the polygenic
nature of EAE with its numerous potential susceptibility loci on many
chromosomes is inescapable (Baker et al., 1995). Recently, a series of
reports have indicated that the phenotype of genetic resistance may in
part be the result of suboptimal T cell responses to CNS antigens, and
that manipulating both the antigen and T cell results in autoimmunity
(Zhao et al., 1992, Abromson-Lehman et al., 1993, Nicholson et al., 1994,
Abromson-Lehman, et al., 1995, Pullen et al., 1995 and Krakowski &
Owens, 1 996). As stated previously, the cause of MS remains unknown,
and the work with animal models must be used for comparisons with
caution.
Trea tmen ts
An astonishing anay of treatments have been shown to effectively
change the course of EAE. Their relevance to designing viable
treatments for MS has often been criticized as the majority of these
experiments examined the onset of EAE, whereas MS is a progressive
disease and treatment would almost certainly begin long after the initial
immune response. There are a number of different types of immune
intemention therapies being designed and each targets one aspect of the
immune system involved in EAE. For example, Abs to the CD4 molecule
have been used successfully in preventing EAE (Brostoff & Mason, 1984,
Waldeor et al., 1985, Sriram & Roberts, 1986 and O'Neill et al., 1993)
and allowed for use in human trials, and this work led to the design of
CD4 analogues with fewer side effects than anti-CW Abs (Jarneson et
al., 1994). The range of treatments includes those based on inducing
immune deviation through cytokines (Ruddle et al., 1 990b and Racke et
al., 1994), depletion of macrophages (Huitinga et al., 1990). altered
peptides (Kardys & Hashim, 1981, Urban et al.,1989, Lamont et al., 1990,
Kennedy et al, 1990 and Samson & Smilek, 1995), oral tolerance to CNS
antigens (Higgins & Weiner, 1988, Khoury et al., 1 992), T cell recognition
(reviewed in Wraith et al., 1989 and discussed in Zeine et al., 1993), and
altered peptide ligands (Vandenbark et al., 1989, Offner et al., 1991,
Kuchroo et al., 1994, Karin et al., 1 994 and Nicholson et al., 1 995). This
is not an exhaustive list.
Validity of EAE as a mode/ for MS
It has been attractive to consider EAE an instructive model for the
human disease MS, because much information regarding the
immunology of EAE is known and there exist many successful treatments
proven to prevent or reverse disability. Additionally, many useful and
complementary observations have been made while studying EAE for
our understanding of MS. Whether al1 the comparisons between the
model and the disease that are made are valid is often questioned. The
danger lies in any attempt to coerce MS to f i the EAE paradigm.
EAE is an extremely useful system to study CNS inflammation.
The injection of purified MBP peptide with CFA or MBP-specific CD4+ T
cell clones allows researchers exquisite control over the resulting
immune responses within the CNS. It is still debatable whether MBP acts
as an autoantigen in MS, although there is evidence to support this
theory (Allegretta et al., 1990). One might predict that by the time MS is
diagnosed, antigen deteminant spreading has occurred to such an
extent as to make it impossible to establish the identity of a single
autoantigen. Additionally, for the sake of treatment, the identity of an
inducing autoantigen becomes much less relevant.
Why is EAE considered to be a highly instructive model for MS?
EAE is a model which is similar in many aspects to MS. fmportantly, the
T cell composition of lesions in bath EAE and MS have been found to be
remarkably similar. The activation of microglia and influx of circulating
macrophages correlated with symptorns and antigen presentation are
also comparable for both diseases. Additionally, there is little MHC
expressed in the normal CNS of either humans or laboratory animals,
and the increase in MHC antigens seen dunng disease for both MS and
EAE is interpreted to support a common immunopathological
progression. Considering the prevailing emphasis on the neuro-
immunology of MS. the number of immunological similanties between
EAE and MS gives credence to the use of the animal model for study.
EAE allows us to study the fundamental features of immunological
responses in the CNS. Technologically, the inbred mouse allows us to
directfy question issues such as cytokine availability, and T cell traffic, as
well as giving us such powerful tools as transgenic and gene knockout
lines. From this type of work, we are able to make cautious comparisons
to the human disease MS.
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PREFACE TO CHAPTER 2
Although scientiste have been able to categorize many immune
responses as favounng either Th1 or Th2 cytokines (this is especially
true for sorne diseases caused by parasites), the mechanisms directing
these biases are generally poorly understood. Many groups have noted
the lack of Th2 cytokines during episodes of EAE and the upregulation of
Th1 inflammatory ones. Therefore, almost by default, the disease has
been grouped as a 'Th1 disease".
The steps that are involved in the activation of a T cell and
subsequent cytokine production includes antigen presentation. From
this, we predicted that the CNS environment was unique in some manner
as compared to the penpheral lymphoid system, and that the control of
the observed cytokine bias rnight be regulated at the level of antigen
presentation.
For the work cornpleted in Chapter 2, the author is the primary
expenrnenter.
76
CHAPTER 2
The central nenrous system environment controls effector
CD4+ T cell cytokine profile in exparimental allergic
encephalomyelitis
Michelle L. Krakowski* and Trevor Owens*$
'Department of Microbiology & tmmunology, tDepartment of Neurology & Neurosurgery, Montreal Neurological Institute, Montreal, Quebec, Canada.
European Journal of Immunology, 1997. vol 27:
In Press
Published by: VCH Verlagesellschatt mbH, Germany
(copyright waiver obtained, see page 85)
Short Title: Mouse CNS APC induce Th1 responses
Keywords: EAEIMS, Th1 Kh2, monocyteslmacrophages, T
lymphocytes, Brain
This work was supported by the Multiple Sclerosis Society of Canada and the Medical Research Council of Canada. MK is suppoited by the National Science & Engineering Research Council of Canada and by a McGill Faculty of Medicine Scholarship.
Abbreviations: APC: antigen presenting cell, CNS: central nenrous system , CFA: corn plete Freund's adjuvant, EAE: experimental allerg ic encephalornyelitis, MNC: mononuclear cell, MBP: myelin basic protein, MS: multiple sclerosis, PLP: proteolipid protein
Michciie L. Krakorrriri and 'Ikevor Owens
Department of Ncurology and Neurosurgery, Montreal Neurological M t u t e , Montreal, Quebec, H3A 284, Canada
The centrai nervous system environment controis effector CD4+ T ce11 cytokine profile in
h uperimentai akrgic cacqhiomyeiïtïs w). CM+ T dis inaltrate the centrai nervous systcrn (CNS). We d a i m î CD4' T ccll linu m m SJUJ mict that wtre specific for enccphalitogcaic myetin buic protein (MBP) peptides and produced both Thl and Th2 cytokines. Thcst lines transftrred EAE to naive mice- Peptide-specific cells IC-isolatcd hm tbc CNS only producd 'hl cyto- kines, whemas T c& in the l p p h nodes produced both Th1 and Tb2 cytokines. Mononuciear ocils blated h m the C M , the majonty of which werc mimglia, prcsented antigen to and stimuiatcd MBPlspeeinc T ceU liaes in viao. Although CNS aatigen-pfesentbg ceils (APC) supportcd maeased production of mtcp fcron m - y -A by these T cclfs, thcm ans no in- in the interieufàn @L)4 signai. wherta~ spltaic APC induccd inexcases in both IFNq and I L 4 mRNA for IL-K @40 suùunit) was upregulatcd in both infiltrathg ma- phages and rcridcnt miaoglia h m micc with EAE. We have t h ~ showu that a Th1 cytokine bisr within the CNS can be i n d d by CNS APC, and that I L 1 2 ir up-regulated in microghl ccils within the CNS of micc with EAE. Microgiïa may thercforc contml'Ihl cytokine rrspaiwr within the CNS.
1 Introduction
6 EAE is a CD4' T KU-dependent discase characterizcd by central nervous system (CM) b5nmation and perivas- cular infiltrates of î D 4 + T alis and macrophages. It is the most frequently used mode1 for the human disease, multi- ple scierosis (MS) [l]. Discase can be induccd in susccp tible strains of rodents by îmmunitation with myelin basic proteins (MBP) or enaphalitogenic peptides- Discase can also be induccd by the passive transfer of myelin-reactive CD4' T ce& [2]. nie onset of EAE in MBP-immunked SJUJ micc corniates with the inneazed expression of the in fiammatory cytokines IFN-y and TNF-a 13-51. In con- junction with the iack of detectable iL-4 and IL-10 during disease induction, this cytokine expression pattern is typ ical of a Th1 type CD4'T c d response 13, 41.
-- -- - - - - - - - - -
Rcuived Febniary 11. l997; in h e d form July 21, 1941; acccpred Augurt 8. W97.
Rcsent addrtu: M. L. Krakmki. Depanment of Immunology. The Scripps Rucarch Institutc, k Soih CA. 92037. USA.
Coctcspaadcace: Michelle L. ICrakowski, Immunology Deput- ment. IMM-23. The Saipps Rcsearch ïnstinrte. 10550 N. Tomy Fines Rd., La Solla. W o r n i a . USA, 92307 Fax: +1-619-784-9096; c-mail: [email protected]
Abbreviations: CIYS: O n d nervous systcm &WC: Mon* nucleu ccii .MBP: Myelin basic protein .WS: Multiple sclem sis PLPs Protcoiipid protein
Kcy wards: Expcnmcntal allergic encephalomyclitis / Thl/T)iZ 1 ' . l i~ni is . .~=:z:~~rt-nh~cc !TI?-rnphocyte 1 Bmm
ForT œlls to producc cytokines, they must bc activatcd by recognition of antigca on antigen-prcsenting œb (APC). Tbo candidate for this function that are hsident in the CNS are anrocytes, which have becn show to prcscnt MBP to enccphalitogenicT ctD lines h via0 [q, and mi- crogiid dis. Miaogiia art maauphage-likt & that arc rcsidcnt in the CNS, which phenotypicdiy are disthguith- able h m macrophages by thcir Iow levcl of CD45 upres- sion [7]. Microglia sh the principal MHC class II' popula- tion in the CNS. Although microglia have b e n shown to prcscnt antigen to, and gentrate signincant proliftrative rtsponscs from, T ccll Iints in v iao [8, 91, the= is not yet a con se^ on the role microfia play in antigen presenta- tioa in the CNS.
CD4+ T ah leave the thymus with the pluripotent capa- biiity to produce both Tb1 and Th2 qtokincs (rcviewed in [IO]). Thcir differcntiation to Th1 (IFN-y-pducing) or Th2 (iL4produwg) is rcgulatcd by sipals that they receivc h m othcr Icukocytes, especiaiiy APC [Il]. Among thc signais that APC elaborate which conval cyto- kine production, the "suritch" cytokine IL12 (made by activated macrophages and B cc&) has bcea shown to be aitical for the devclopment of Th1 T cc& (12-U] . IL12 appears to act on T celt that arc aaivated by antigen to induce their production of EN-y [16. 171. IL-= has bcen dirtctly implicatcd in EAE, as anti-IL-= mAb =duce the incidence of EAE and r L i 2 both promotcd and Icngth- cncd the duration of discase [18,19]. IL-12 is composcd of an inducible 40-kDa @40) and a constitutively cxprtssed 35-kDa @35) subunit [20].
We have addressed the rni.rhnriicrn of the tnccphaiitogenic Th1 bias in EAE by adoptively m e r r i n g peptide- specïfic Th0 (Th1 + ThZ) T d h c s 10 induce distase in mice. We find that these T d i s exprtss a Th1 biascd cyto- kine profile only in the CNS. but not in LN- CNS-derivcd APC specificaiiy induad peptide-specific T cclls to express Th1 cytobcs in vino. Wt al50 h d that microglia producc IL-= dunng EAE. We proposc chat IL-12- producing XPC within the CNS. probabiy microglia. arc
iikeiy candidates for conmiüng the observedTh1 bias dur- ing inflammatory autoimmune disease in the CNS. Simiiar rnechanisrns of regulating cytokine production may oper- ate in other t issue-spSc autoimmune cikases.
2 Materiais and metbods
Fernale W J m i a bctween 5 and 8 wttks old wcre obtained from EWhSprague Dawley (Indianapolis, ïN) and housed undcr sp&c pathogen-the conditions. EAE was induccd in donor mice by two s.c, injections 7 days apan at the base of the tail and fianks with eithcr a total of 400 pg of whole MBP (Sigma, Monmal, Que, Canada) or 100 pg N-acetylated MBP peptides 89-101 (WEFKMWPRTP) or 91-103 (FFKNIVTPRTPPP) (Multipk Peptide Systems, San Diego, CA) in CFA (Difco, Detroit, MI) containhg 50 ~ r g H37RA Mywbuae- ritun tubcrculosis (Difco). Symptoms were 6rst observed 14 days after the initiai injection. Mice wae rnonitomd and assigned clinical scores as foilows: O (no symptoms), 1 (flaccid tail, clurnsincss), 2 (moderate hind b b parcsis), 3 (severe paresis or unilateral hind limb paralysis), 4 (hind and fore Iirnb paralysis), 5 (mon%und). AU cxperimental pmtocols were approved by the McGill University Animal Care Cornmittee.
Superficial inguinal, axüîary, brachiai and mesentcric LN ceiis werc isolated h m MBP-immunittd micc and cul- tured in RPMX 1640 (G!'bco/BRL, Burlington, Ont, Canada) supplemented with 10 % FBS (UBI, Lake Placid, NY) , 50 ph4 2-ME (Sigma), 100 U / d peniciïiin ( G i b d BRL), 100 &ml s t rcptorny~ (GibcolBRL) and 2 m M L- glutamine (GibcofBRL) in 24-weU plates (Fdcon, Mon- treaI, Que, Canada) at 4 x Id cc Wrnl with the appropri- ate .MBP peptide (5 &ml) in a total volume of 1.5 ml. Responsiveness to MBP peptide was assescd in p d c l microcuiturcs by ['HJthymidine (XCN Biochemicals, Mis- sissauga, Ont, Canada) incorporation at 4 days foliowing an overnight pulse (0.5 pCiIWeii). Celis werc culturcci for 14 days, then coliected by centrifugation on FicoU- Hlpaque (Phamacia, Montreal, Que, Canada), and re- cul tured for another 14 days with MBP peptide and irradi- ated (3000 rad) autologous splenocytcs as APC. Within 6-8 weeks ( 3 4 rc-stimulation), ccll Lues lost rcactivity to components of the adjuvant (PPD), and showed stimula- tion indices to MBP peptide of 10-50-fold. Thrce days fol- lowing rc-stimulation, activated T ceb were mUectcd, labeled with the lipophilic dye f KHZ-GL (Sigma) [21] and injeaed into naive SJUJ mice (1 x IO7 blastdmouse). The use of PKH2 has bccn previously o p t i . for our system (211. AU animais developcd symptom of EAE 10 days later.
23 Isolation of mononudear cells from CNS and LN
CD:' Iynphoqzes wcrc coiiccted €rom CNS and LN as soor. x, EAE was obscrvcd. Micc were maesthetized with . . CR. .r:i! !:vdratc (7.5 dkr , pnor ;O pcr5sion through thc
h e m with icccold PBS. ïngumal LN were collected fim, the mouse perfused, thcn the brains aud spinal cords har- vestcd. Pooled CNS and secondary lymphoid tissue were dwociated by passing tbxough a wirc mes&. Aftcr cenui- fuging at tOO X g for 10 min, dirsociated CNS tissue was nsuspended in 70 % Penoli (Phatmacia) and œntnfugcd for 20 min at 500 x g on a 30 % : 37 % :7O % Percofl gradi- ent. Mononucicar ccllr were obuincd h m the 37% <LW8 %ml): 70% ( L W g/ml) interface, washed and sorteci using a £fwhx;tnœ-aaivatcd ceii sorter (E:ACS). Betwten 0.8 x 1- x 101 mononudear celis wert isol- ated h m each mouse. A1I œiIs wert maintairied at 4°C until staining.
CcUs wert stained with PEcoupled anti-CD4 (PE: CD4) (Becton Dickinson, MisPssauga, Ont, Canada), anti- MACI1 ( M m (221) or biotinyiatcd anti-CD45 (My89 [22D for u) min at 4'C. PEsonjugated goat anti-rat (Sou- theni Biotecbnology, Birmingham, AL) followed ami- MAC-1 while streptavidin :Spectxai Red (Southcrn Bio- technology) was used as a ~condary magent for CD45 rtaining. Fluortsccoœ was analyzcd on a FACScan using =SIS ï I software (Becton Dickinson). Dead ce& wert mludsd by gating on a combination of forvud and side scatter. CD4' c e k were sorud (FACStar, Benon Dickui- son) on the basis of PKHZ sraning (sec Sea. 2.2 for pro- cedure). Sortcd cc& were microccntrifuged at 200 x g for 2 min, snap-frotcn in liquid nimgen and n o r d at -70°C und mRNA was isolated
2.5 RNA iroïatioli amd reverse byrrcnbYrrcnption-PCR
mRNA was isolatcd using QuickRep micro mRNA puri- fication kits (Phannach). Total RNA was isolatcd from homogenizcd tissue using TRXZOL (Gibw/BRL) and the yicld quantificd by spectrophotorneuy. cDNA was synthe- sizcd using the Gr'bco/BRL Superscript Prc-amplification systcm (Gi'bco/BRL). PCR conditions uscd werc previ- ousiy optimauf for lincar amplincation to d o w cornpari- son between samples [5]. Spccificaüy, wc have pcwiously shown the diminution of the KWCR signai with the titra- tion of input RNA indicating the scnsitivity of the system [23]. For PCR anaiysis, cqivalent amouno of &NA @ascd on original input cc11 numben for sortcd celis) wcre mpiüïed using 2 5 UTaq DNA polyrncrase (GibdBRL), 1 mM of each dNïP, 50 p o t of cadi prirner and a PCR b a r mixture containhg 50 mM Ki, 10 mM Tris (pH 8.3). LS m M M g Q and 0.01 % gelatia. The ptimcrs u ~ t d were: 1FN-y sense 5'-CGACTCCI1-I-ICCGCIT- CCT-3 ' ; antisense ~ ' - A C A G T G C A T ~ G G C ~ ~ ' T G C - 3'; $-a- sense 5'-TGGGTCAGAAGGACKCTATC- 3 ' ; antiscnse 5'-CAGGCAGCTCATAGcT~CT-3 * ; IL-4 xnse 5'-ATGGGTCTCAACCCCCAGmA-3 * ; anti- scnse ~*-GATG~CAT~AGGTAAACGTA-~ ' ; and L i 2 (p40 subunit) senst 5'-GCTGCTCATGGCTGGTG- CAAAG-3 ' ; 5'-GAAGTAGACGTTC- AAGAGCCCG-3' and CD3-y (for primer sequtncc, sce [SI), PCR rwctions were canied out in a Perkin-Ehcr Cetus 9600 Gene amp thermal cydtr (Norwalk. CI') for 28 q d c s (denaturation, 30 s 3t 94'C; rinneshg. 30 s at 55°C and clongation for 30 s at 72°C). N'e have p r c v i o ~ ~ l ~
I Min- m Werk~aU GmbH 25192 Plankstldt UiUI
A-#799
SCHWARZ
shown the absence of a plateau &ect in the amplification of these producu [q. 40-cycle, non-linear PCR was aiso carried out for I L 4 amplification in some instances. PCR products (80 YO of amplified rample) w a c then separated in 1% agame gels with ZAE buffer, transftraasfarcd to Hybond-N membranes (Amersharn, Baie d'Ur&, Que, Canada) by capillary action and hybridiscd with =P- Iabeled cDNA probes in Rapid-hyb buffer (Amcnham) for 18 h at 42°C. &NA probes used w a e as foiluwr: EH- y: a 643-bp hgment digestcd by Pst ItHind III mm done pSP65-IFN-Y; actin: a 1.3-kb Pst1 bgncnt of plasmid pHpPr-1-nco; I L 4 a 2tXLbp fragment h m PstUEooRI digestion of pSPWIL4 (cDNA insms purifiecl h m p b mids dcscn'bed by Kelso and Gough, [24D, and CD3y a 700-b~ HindIIl+ EcoRI hgment fmm done pBlO.AT33g-1 [q, The IL-= probe was a synthesizcd 22- mer (5 ' -TCîGTCTGGîGAGMGGTCACA-3 '1. IL12 primen and probe wert kindly provided by Dr. Wkync Lapp (McGiII University). Bloa waic wasilcd micc for 15 min in 1 x SSC, 0.1 % SDS at room temperature fol- lowcd by two U-min washcs in 0.1 x SSC, 0.1 % SDS at 65 OC, except for p40 L U btots which wcrc washed twice at room temperature in 6 x SSC, OJ% SDS. Each blot was exposcd ovcrnight to a phosphorimager scrcai (Moïe- cuIar Dynamics, Sunnyvaie, CA), and the intcnsity of cach band quantificd uMg ImageQuant software (MoIecular f ynamics). AU cytokine signals wcre normaikd to & actin or CD3-y (forT œii sampics) and expresscd in ditr- ary phosphorimager units.
CNS mononucIcar cc& wcrc isolatcd h m naïve micc as described. Cells were irradiateci (3000 rad) and dtured at 1 x ld ceWweil in 96-weii round-bottom piatu (Falcon, Montreal. Que). Tccll-depleted spleen ails [26] were uscd at the same ceii density as wntrol APC. MBP p89-101- rencrivc T tells (1 x IV) wcrc added in the prtsena or absence of exogcnous antigen (50 pg/rnl for whole protein, 5 &ml for peptide) to a total volume of U)O pl, and pmlif- eration assayed ùy an 8-h pH)ùymidine p S c aftcr 2 riays of culture. Parailel cultures were harvested for mRNA isolation to determine cytokine production pm- acs.
2.7 Miaoytokine assay for bioacaivc Tt4
MBP p89-101-mctive T celis (1 x 10') werc addcd to anti-CD3 antiiody (145-2Cll)aated 24wcll plates in a total volume of 1 ml of RPMI with 10 % FBS, supplc- rnented as descriid above, and incubatcd at 37'C mer- night. Supcrnatant was thcn coilectcd for bioassays, CTQS ce&, which arc dependent on E4, w u t cultucd in 96 weii fiat-bottom plates at 2 x 10. ceiWwell with twofold dilutions of T ceil supernatant in a total volume of 100 pl for 36 h. Repiicate weiis also containcd the inhibitory l lB 11 antibody (5 pg/mi) which is specific for bioactive IL- 4. The numbcr of viable tek was rneasured by 3443- dimethyitfuaz01-2-~1)-2 ~aipenyltetrazolium brornîde aswy. and rcad at 550 nm (refcrenœ 690 m).
Miaogüa werc culhucd in a total volume of UW) pl at 1 x 1@ c c ~ U in 96weiï round-ùottorn w c b (Fidon) üt tither mcdiurn abne or medium containhg supcrnataat &om the Tbl done, E9.D4 (261, with additional LPS (1 w) (SaLnonrlto obomu, Difco). Celis w c h culh~cd for 4 days, colkcted, pooled and a smaü aliquot taken for FACS anaiysïs. RNAwas irolated h m the rcmnining sam- pres as dercn'kd,
To establish whether the previously descrtbed Th1 qto- kizie bias in EAE reflccts a seleetion p- acting on enaphzlitogenicT ce& withm the CNS ratha than at the levcl of T ccii induction in tht periphcry, we galeratcd T œli h c s spcci6c h r MBP peptides p89-101 and p91- 103, which arc cnctpbalitogmic in SJUJ micc [ml. Lines werc tt-stimuiated with antigen and splenic APC e a c n t to remove rcsponsivemcss to tuberculin PPD (on average, three times) (Fig. la, b). Ail lines sclected in vitro weric homoge~~cousiy CDP (data not shawn). Reverse traa- scription 0 - P C R d y s i s 3 days aher re-rtimulation showcd mRNA for both Itd and EN-y for ail ccD lines (n = 3) mg- 2a and b show two rcprcsentative Iincs). The &tedon of message by KI-PCR rcficcts production of ôioauk protcin [BI. It is gencrally acceptai that cncephrlitogenic T cclls maire FN-y, and we have shown the up-ccguhtion of IFN-y message for our peptide- rcactive all lines (Fig. 1). To confinn that our cncephaii- togenic Lines aIso made IL-4, we carried out bioasays on supematants h m T ccii ihes stimulated in vitro wîth MBP peptide. Data in Table 1 show nvo independent MBP p89-101-speçific Lines (n = 2) which produccd bioactive ïL4, ~)nsistcnt with the observations of Swain and coIIcagucs and O'Garra and coiieagucs for activated
Figure t TùymidW iacoiporrtion by p89-101-wcùve T a& aftu isolation h m an immuoited m o w (a) and aftcr ont round of testimulatiom @) witb APC a d peptide. Parallcl cuitures m m puised wiwitb pwthymi&t t o monitor ceil Line rcaerinty CO both the MBP peptide and PPD. Data art sbown as thymidine iacor- poraaon by uipiiate mierociLtmrrs ( ~ p m * SE). p89-101: p c g tide 89-101 (5 pg/ml), PPD a d MBP: (both at 50 (ighi). Rcprc- scntative data are shawn of five expcrimcnrs.
-le L Production of bioactive It4 by MBP m d e - u e T c d Lacs" - -
ld ~~d in t incorparr t ion IL4 produccion h e (cpm) in rrsponse to: (UM) in m p o u e to:
Medium MBPp89-101 PPD MBPp89-101 MBP p69-101 + m i l
a) MBP p89-101-CUCtiVe T œIIs wen assaycd for rpceifiPty to MBP p89-101. or Itimdated with aaa-CD3 aatibody rad supernatant colleaed for bioassays- CT4S a~say~ wext euricd out with and without the addition of uiti-IL4 Ab (IlBtl) which is spesc for bibaCtiPt IIA. 'Ihe Iimit of dLtCCtjOll for lbainrhisassaywu>0-5Ulml.
Wpm L PCR aqi;t;at;aa of cytolMt gmc ufrrrso~ by m-103 (a) rnd -101 (b) T d Lintr- Celh wtrr ~ U c e t d 2 days rftu rr-ltimuïaïon, immtdiittly prior to injection iato naive mice, and mRNA holucd for and@. AU wcre nonnrl- Ltd to ci* PoctiD or CD3-y. V'ues aolrmlited to the W3-y signai ut shown (crpresred in vbiaaxy phosphorinuger Pairs). No fL4 signai wu det#ted h m aThl clone and no EN-y signal vu deteaed from r 'Ib2 doae.
memory T cek in viao [28,29]. Tbis production was iohi- bited by the addition of anti-IL4 antibody- T tells wcre labefed with the lipophilic dye PKH2 in v i a 0 and tram- ferrtd into S W I mice. After 10 days, di micc &'bitcd symptoms of EAE (n = 3). of iabef retention in vivo (Fig. 3a). PKH2 (+) labelcd
CD4' T œiis werc also found in the CNS of mice with EAE. PKHZ-labeled ( tndcnrd encephalitogenic T aii iine) and unlabeled (endogrnous rccmits) CD4' œlls were sorted, and cytokine mRNA d y z e d and cornparcd to production by the originaI c d popuiation imrnediateiy prior to injection (n = 3). Fig. 4a shows that CD4'T alls m m both the LN or CNS produccd readiIy dctectabk IFN-y message. By conuast, IL4 =RNA was not detect- able in the CNS under the samc amplification conditions (data not shown), whtrcas iL-4 message was detcctcd in
W e have prcviousiy demonstxated that T ccll infiltration to t!!e CNS coinades with tk onset of W (211. Tbereforc. as soon as mict showcd syrnptoms, T celis were soncd h m CNS and LN on the basa of CD4 (FL2) and PKH2 (FLI) staining. Prcvious wo* bas validated that only CD3' T ceh arc containcd within CD4' populations [5]. The fact that no CD4+, PKH2 (+) labeled d i s (Iowa left quadrant), but only CD4'. PKX2 (+) iabelcd c& (upper Ieft quadrant. Fig. 3b) wcrc found in LN indicatcs fidclity
LYNPH NOOE
ripure 3. FACS soniag of CD-i*. PKH2 (i) Iabclcd cck fmm LN (a) and CNS (b) of micc wirh EAE. Lymphocytes wcre iso- latcd fmrn inguinal LN pnor to perfusion m d Erom the QiS afier perfusion. and naincd for the uprasion of C D 4 CDI*. P K X - IA-tIx! ;:nd -unhbcicd cclls u-crc WCS-sortcd for mRKA analysts.
PW-labeled =Us b r n tbc LN Tag. 4b). The cytolgne pkcnotypc dinemacc for the PKH2-labeled and -unla- belcd œiis fwther CO- the separate idcntity of these populations. Using *de RT-PCR, barcly deteaable Icveis of IL-4 mRNAwere found in the CNS (Fiji- 4b). R r this non-lincar P a , the differcn~cs in cytokine produc- tion between cell populations is stiii evidcnt, emphsking thtir dissimilarity. We thertforc intcrpret thejc resuits to show selcction for Th1 cytokine production in the CNS.
CNS APC + T + PPD p CNS APC + T + MBP
T *lm
Spina + T PPD IIi Spleen + T + MBP
6
O 10000
Thymidina incorpotalion
figure 5. Rcsponsa of p89-lOl-ceacti~c T œUs ta antigen prc- sented by eithcr T celldepleted splecn crb or APC h r u the CNS. APC were irradiatcd (3000 rad) beforc use. p89-101- rcacrive T ells wcre tddcd. in the prcsencc or absence of u o p - nous sntigcn and pmlifention assayed afrcr 2 da- of c~lntrr. AU Jntircns wcrc addcd at CO u g h l cxccpt pcptidc (5 ug/ml)-
3.3 MkrqH APC direc! r Ihl qîokîae biu
Mictoglial APC hlated îkom unmanipulatcd mict were cuituhd with antigcn and MBP peptide p89-101-reactive Ta&. Cultures wcre harvcstcd aftcr 2 &ys for RNA k h t i o n (n = 4). Fig. 6a shows that IFN-y but not IL4 (c) levels wcrt marued when T uk were prcscnud rhe appropriate antigeai by APC h m the CNS of nive mice. Neirhu cytobe mRNA signai incnascd over background when the T œil lirie wu cuitaucd with CNS APC and an irrcievant antigen (PPD) (data not shown). By contast, splenic APC inckrced incmscs in both IL4 (d) and IFN-y @) message over background leveis mg. 6b). Note that, with rqm% to Fa. 6, the absolute Ievels of cytokine can- aot k M y wrnpahd betwecn p p b - Each -ph rcphsents separate Southern blou, tbus the d e s arc not comparable- Iastead, one can analyre the changes or dif- ferences in cytokine production. Thercforc, APC h m the CNS induccd a Tb1 biascd cytokine profile by T ceil liner in *o.
Convol (unperfused) spleen and pedked CNS tisnie were isolatcd h m animab that wcre cithcr unmanipu- Iatcd or cxpcricncing severt EAE (grade 3). and IL-12 mRNA lcvcis mcasured. Message for :hc inducible pJO
figure 6. RT-PCR anai* of IFN-y and IL-4 message produœd by p89-101-rraai~e T œüs whtn prrscnted antigen by either spIcnic M C (B. D) or mononucicar ah Whtcd from the CNS (A, 9 of an unmanjpulated mouse. Coaûol wcib (roiid bars) contaured APC aad anagen. but no T œUs. Crus-àatched bus s k w rcsulu h m weiïs conuiniog APC. aaugea aadT cellr. CM- turc conditions a m idcatiai to thoK of Rg. 5. but for cytokine uralysis. aiis werc hmcsted md =RNA Wh-, followed by RX-PCWSoutb~ blottbg.
subunit of L i 2 was incrcased in spinal cord in mice with EAE (data not shown). To determine which c e U type was responsible for the production of IL-= in the CNS, we isolated microglia and infiltratiog macrophages h m the CNS of mice a t peak distasc. lbis time point was spc&- caiïy chosen, as our own data [23] and that of others [3i, 321 show it to concspond to maximal T ccU activation as judged by cytokine production and phcnotype. Fig. fa shows a reprcsentative (n = 4) ceii staining of Mac-1' œlis £rom the CNS of micc a t peak discase. Tbese were soned on the basis of differential expression of CD45 The CD4Sb popdation (R3 gate, Fïg. 7a) corresponds to thosc ccUs used as APC in Figs. 5 and 6. Fïg. 7b shows that IL-12 message was svongIy erprtsstd in both miaugiia and inaltrating macrophages. Thercforc, IL-l2 is made by microgiia isolated h m the CNS of micc with EAE.
4 Discussion
We have invcstigated the CNS environment and the rolc it p lay in iduencing Thl/lb2 dccisions- WC found that whiie T ceii lines spedc for encephalitogenic peptides produced both Th1 a d ni2 cytokines in vitro, the cyto- kine profile divcrgcd in a target organ-speàfic manncr within the animal. Strikingly, encephalitogenic T ceils pro- duccd only EN-y in the CNS, and no IL4 message was apparent a t peak EAE. This is consistent with and utends the 6ndings of Kennedy ct al. [33] who showed that IL4 appcrus late in diseasc coinadent with remission and Khoury ct al. [34] who showed a condation of IL4 mRWA with suppression of EAE in rats. Given our dcmonsrntion rhat sdection for the wokinc profile of cr.rc~h:iii!o~snlc T ccils occurrcd in thc C ' I S . thcn bot5
Figure Z C i 2 plodiicaou by micmgiia aad maauphages h m tbe CNS. FACS 5ortâ1g of Mac-l* -5- (gate R2) and 1P (gatc R3) ceils from tbc C N S of mice at peak disase (a). CD4P. Mac- 1' and CD4Sh, Mac-1' & were soncd and mRNA wiarrd. KT-PCR amplimas (3U bp) for L U message is sbown 0). Luie 1: CD4SU, Mac-1' œRs, ianc 2 CD45*, Mac-l' œlls. lane 3: no mRNA*
antigcn prcsentation and the action of L i 2 (a known Th1 switch cytokine) becorne of potential importance-
Although our muits do not formally exdude the possiiil- ity that the entry of Th2 ceils to the CNS is prohibitcd, daka h m Cua ct al. [3q indicate that Thî ceiis can indecd enter the CM. Austrup et al. [36) report that puri6cdThl popdations selcctively home to delayed-type hypenensi- avity sites. Our experirncnû involvcd hetcrogeneous, non- cammitted Th1 and TU (Tho) populations in t r a d e r s t a unimrnunùed animais. Whethcr or not E- and P-selecrin recognition might operate to direct uncornmittcd ni1 CD4' T cclls to cross the blood-brin bamcr as suggcstcd by Aunnip e t ai. (361, our data identify an additional mechanism whcrebyT celi populations within the CEiS arc induccd to a Th1 phenotype.
Antigen prcsentation in the CNS may be a critical factor in rtgdating cytokine bias by inntuaùag T ccb. We have found that populations of cciis h m the CNS of unmanip ulated SJUS micc which arc prcdominanùy mimglia prcs- ent antigen to CD4' T c d fines. Our dernonmation that microglia can a a as APC supports the rcsults of Marnr- moto et al. [37] and Frci et al. [38] who showed that EN-y-induced microglia and neonatally-dtrived rniqo- glia, respcdvely, acted as APC in vitro. Ford et al. [39] found that n t microglia induce T ccil prolifcntion, but find that cytokine production and T cc11 fatc wcrc selcc-
tively idiuenced, Our &ta wnfinn that antigcn prrstnta- tion by microgiia induccs seleabc T œil responses.
A number of ccli lineagcs that arc prtsent in the C M dur- h g disease, inciuding m e s , perivascuk macru- phages and B cck, have been hplicated in antigcn prcs- entation and so become candidate APC to inducc a Tbt bias. The ability of rat amoqta to preseat antigcn to T c d d o n u has been shown in vibo [q, but rince our CNS mononudear c d prcparations do not conlirin astm- c y t u 17, their contn'bution ta the mgulation of cytokine production in viiro was not asescd The CD4Sb* pexhras- cuIar macrophage ù also capable of antigen prcscntatim 191, but analogous celis are abundant in the peripbcrai lymphoid system, inducihg the splten, and s p h k APC did not &a am1 bias, Stockinger et aL [40] havc shown that B ceih can induce a switch to theTh2 subset. Because WC do not xe'lh2 cytokines in the CNS of micc witb EAE. the number of ianlaating B ah E cicher insufncicnt 1411 or their effea is ovcrcome by the CNS environment.
This argues cither that the environment h m which an APC dcrivcs influences iu abiüty to bias a cytokine rrsponse, or that CNS APC populations contain a subset of celis distinct h m thow in LN or spleen, We favar mî- dent mimglia as the APC that stimuIate the TctU r sponse and cantrol the Thl-biascd qtofcine production in the CNS. ActivatedT ceils cross the bfood-brah bacrier by vinue of their expression of adhcsion m o l d e s and inhercnt circulaihg capabilities (3,421. In passively trans- ferrd EAE, the host cndothclium is not endogenousiy inaamcd, and the activation state of the T ccii dixce& cntry to the CNS. W c pro- that prcsentation of C N S antigen by microglia then dirccu the Th1 bias. We cannot formally exciude asfrocytcs or ptrivasdar macrophages £rom involvemcnt in thk proccs in sinr. but prcvious stud- ies [9,31], and o u . own data 123 1 favor micmglia as the ini- tiaiIy dominant APC in the CNS.
The stimulus that a T ccll rcdvcs barn an APC is the combintd result not ody of TCR: WC-peptide interac- tion, but also signais receivtd through co-stimulatory molecules, The molecules that dominate our undentand- ing so far art B7-1 (CD8O) and B7-2 (CD86), which arc ligands for CD28 and CTLA-4 onT cclis. I b o groups have linked B7 m o l d e expm-on with biased cytokine pro- duction, one of them in EAE [43, W. 87 expmsïon is also impticated in rcgulation of autoantigcn epitopc recog- nition by T celis in EAE 145). Prcfcrcntiai expression of one B7 subfamily membcr might çontn'bute to the biased cytokine rcsponsc in the CNS. Consîstent with this, Miller et al. [45] noted incrcascd expression of B7-1 in the CNS of S U S micc with EAE and Wmdhagen et al, [46) showcd B7.1 expression in MS Icsions. Additionally, CïLA4 Ig trcatment inhibitcd the induction of EAE [47, 481.
It is noteworthy that the increased IL-i2 expression which we observed correlated with the previously described up- rcgulation of IFN-y message [3, 4). A complu interplay between IL-= and PN-y has becn descn'bed. The phs- encc of IFN-y contributes to the induction of L i 2 expres- sion by macrophages (11, 49. SOI, and it is likcly that PN-y feeds back on macrophages and microglia to upregulate IL-12 production. AdditionaUy. IL-= may very well be made by orhcr ccils of thc CNS. for cxrirnple. =troc)-tes.
Such produdon, if found, would mtngthen our hypothe- sir that the CNS environment modulates the cytokine pm- me of enceph?LitoguicT d i s . We have not aamhcd this question as our protocol Utludes aftmcytes, and adult murine astrocytcs arc notorioiuly difncult to culturc. Con- sensus suggests that fGi2 induccs EN-y prodrrction and a Thl switch, but that IFNy can sïpnifiirntly augment IL42 priming for mbqtmt IFN-y pmduccion by T œiis [q. Consistent with our data, EAE was prevented by a d m h b uatioxt of anti-&î2 a n t i î c s in vivo [B, î9J. Aithough wc have dete1a1iDcd tlmt miemglia are induccd to inaeasc expression of It12 during EAE, our &ta do not defini- tively crtablish that this cytokine ù solely responsible for the Thl bias- Howcver, it is dcar that the obrervcd T ce11 bias if dcpcndtnt upon initiai intcraaion wîth the APC in the CNS anid cytokines that tbey produœ, and we favor a mle for IGU in Lhit v, W bave thdore shown Ut APC wnhin the CNS direct ytokine secretion by innltratïag CD4' T cc&, and pm- vide a W y mecb?nimi for tbir envhxtmental regdation of a T ccii rcsponse. Thu cffkct is not sbown by lymphoid APC, thus dcmonstrsting a unique aspect of CNS inunune d v i t y . APC h m othcr non-lymphoid tissues may iikc- wire direct cytokine bïascs, and this would p h d e a mech- aaism for tissuc-spcQfiaty of T ccll rrspoofcs thai mduce ninammation vasus humoral rcsponsu. A 'Ihl bias is observeci in many organ-spccifc autoimmune disase, as weiï as in o thu inflammatnry rcspoascs. It is of fundamen- ta1 immunologicd importane to understînd the mecha- nism for thu. Uk have k e n able to link tissue-specSc APC to the 'lhl switch, which has implications for other tissues and diseases.
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VCH VerlagsgeseUschaft mbH Buschatrasse I t D-69469 Germany
To whorn it may concem,
According to the regulations of McGU University. 1 must "obtain official copyright wrivers f m the copyright holder(a)" in order to use the foilowing material in m y doctoral dioserration. May I plwe have such a waiver from y ou for my paper. European Joumai of Immunology, 'The cenual nervous system environment controls effeccor CD4+ T ceIl cydeinc profile in experimental d lergic cncephalornyelitis". volume 27.
Please do not hesitate to contact me if you have any questions. Thank you in advance for your tirne.
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PREFACE TO CHAPTER 3
As previously stated, EAE is mediated by CD4+ T cells. Those
encephalitogenic, CNS antigen-specific effector, cells have been
analyzed by many groups in an attempt to understand how they cause
disease. We now know that they upregulate expression of molecules
necessary for extravasation from the blood into various tissues, CO-
stimulatory molecules and certain cytokines. Cytokines are the soluble
means of communication T ceils use to signal other cells and effect
changes in the immune response.
The appearance of clinical symptoms of EAE correlates with the
upregulation of infiammatory cytokines such as TNF-a and IFN-y.
Because these cytokines are coincidently expressed in the CNS during
disease and are known to mediate inflammation (a hallmark of EAE),
they are believed to play a role in disease induction.
We have shown that during disease, CNS-specific antigen
presenting cells have the ability to promote T cell production of IFN-y,
thus enhancing CNS inflammation. Butl as it might be expected, it
appears that the role of IFNq dunng disease may not be entirely
straightforward. It has long been known that IFN-y may have anti-
proliferative effects on T cells and there are reports in the literature where
the removal of IFN-y promoted disease. In order to better understand the
role(s) IFNq has during EAE, we have studied rnice in which the gene
encoding IFN-y has been disrupted disallowing expression.
For the work completed in Chapter 3, the author is the primary
experimenter.
CHAPTER 3
1FN-y Confers Resistence to Experimental Allergic
Encephalomyelitis
The European Journal of Immunology,
vol: 26: 1 641 -1 646. 1996.
Published by: VCH Veilagesellschaft mbH, Germany
(copyright waiver obtained, see page 1 23)
Michelle Krakowski't and Trevor Owens's
Department of Microbiology & Immunology* and Neurology 8 Neurosurgerys, Montreal Neurological Institute, McGiII University, Montreal Quebec, H3A 2B4, Canada
Running title: Enhanced EAE in IFN-y knockout mice Keywords: IFN-y, Experimental allergic encephalomyelitis, mouse, knockout, resistance
t please address reprint requests to Michelle Krakowski, Neuroimmunology Unit, Room 033, Montreal Neurological Institute, 3801 Rue University, Montreal Quebec, H3A 264. phone: (51 4) 3984937, fax: (51 4) 398-7371, e-mail: Michelle@ rclvax.medcor.mcgill.ca.
Eur. J. Immunot. 1%. 26: 1641-1646
' Deparunent of Miuobiotogy and Immunology, McGill University. Mon treai, Canada Department of Neurology and Neurosurgery. McGill University, Montreal. Canada
ïnterferon-y confers resistance to experimental
In experîmental ailergic encephalomyelitis (EAE), T ceils infiltrate the e n t r a i nervous system (CNS) and induce inflammation. These CD4' T ceik secrete interferon (EN)-y, levels of which correlate with dis- severity, and which is proposed to play a key role in disease induction- Many strains of rnice are tesis- tant to EAE- We have nudied the effect of deletion of EN-y on the ability to induce EAE in resistant BALWc-backcrossed mice. As expccted, only û-6 % of BALB/c o r BALBlc-backc~ossed miœ developed EAE when imm* with myelin basic protein in adjuvant. Strikingly, abrogation of IFN-y expre&on by targettd dismption of the EN-y gene (GKO mice) converted thcm to a susctpt- ibb phenotype. As many as 71 % of these IFN-ydeficient mice developed EAE, a frequency comparable to that seen wïth the susceptible SnB strain. Ln addi- tion, EAE was of unusuaüy high severity in mice lacking IFN-y, h u n o l o g i d chamck&ks of discase in E N - y - d e f i k t mict wcre comparable to those seen in susceptible ( S J W ) mice with EAE, including perivascuiar i d t r a t i o n in the CNS and order-of-magnitude incrcases for both CD3 y chain and TNF-a mRNA IeveIs in the spinal cord. We thus demonstrate chat lack of IFN-y converts an othemise EAE-rcsistant mouse strain to becorne susceptible to discase. There- fore, in BALBlc mice, IFN-y confers resistance to EAE.
1 Introduction
EAE is a CD4' T celi-dependent disease characterized by inflammation of central nervous system (CNS), which can be induced in susceptible anirnals by immunization with myelin proteins such as myelin basic protein (MBP) (reviewed in [Il).
Hitologicaiiy, EAE presents with perivascufar infiltrates in the CNS largely composed of CD4' T ϟs and mac- rophages. In rodent models of EAE, animals remit from initial episodes of EAE and kequently subsequently relapse. EAE is the paradigrnatic animal mode1 for the human disease multiple sclerosis (MS) because they share many clinical and pathological aspects (reviewed in [Z]).
Each episode of EAE has been shown to correlate with increased expression of infiammatory cytokines such as IFN-y and TNF-a [3, 41. These cytokines are proposed to play a role in disease initiation [SI, Indeed, innathecal injections of iFN-y induced inflammation in Lewis rats [6, 71. This is consistent with a general role for IFN-y in induc- ing inflammation as has been shown by a number of naas- genic systems in which aberrant organ-speciuc expression of IFNy in mice led to autoimmune diseases modelling diabetes, myasthenia gravis and uveitis [&IO].
Correspondcoce: Michelle Kralromki, Neuroimmunology Unit, Room 033, Monueal Neurological Institutc. 3801 Rue University, Montreal, Quebec H3A 284. Canada Fax: + 1-514-3987371; c-maü: [email protected]
Abbrevhtions: CNS: Cenual nervous systcm GKO: EN-y knockout MBP: Myelin basic protein MS: Multiple sderosis
Key words: interferon-y / Expcrimental dlcrgic cnccphalomyeii- tis / Mouse / KnOdcout / Rcsistancc
O VCH Verlag~gestllxhaft mbH. D-69451 Weinheirn, 1996
However, the piaure has been complicatcd by a number of conuary observations. Administration of anti-EN-y mAb to g e n e t i d y EAE-mistant mice induced an EAE- susceptibie phenotype [ll, U]. Vourthuis e t al- [U] showed that intravenmcuiar injection of IFN-y prevents EAE in rats. And h d y , Ferber e t al. [14j demonstrated chat E N - y was not necessary for the induction of EAE using genetic knockout mice in which the single gene encoding IFN-y was disrupted [q and backmssed to an EAE-susceptible strain (BIO.PL). Due to these uncertain- ties about the role of IFN-y in EAE, we have duectly tested its importana using IFN-y knockout (GKO) mice of an EAE-rrsistant gcnotype- Our experiments show that abrogation of IM-y exp-on in mice on a genetic back- ground known to be resistant to the induction of EAE (BALBlc) convened them to a susceptible phenotype- For mice of both resistant and suscepabIe (WJ) genetic backgrounds, there was a negative correlation between the number of IM-y alleles prescnt and severity, incidence and duration of disease. These data indicate that F N - y confen resistance against EAE, and this activity is most strongly apparent in othemise disease-resistant mice.
2 Materiais and methods
Interferon-y knockout (GKO) mice [SI were obtained from Gcnentech. They were generated by the insertion of a ntomycin gcne within the single-copy gene for IFN-y. These mice werc originally generated on a 129Sv (H-Sb) background, then crossed to C57BUbl (H-2') and back- -d five tirna to BALB/c (H-2d). Hemiygous (GKO+/-) mice that we received were intercrossed and progeny screened by DNA PCR for neornycin and IFN-y [16]. This generated m i a that were homozygous (GKO-/-) o r hemizygous (GKO+/-) for the ncomycin
1642 M. Krakowski and T. Owczls Eur. J. Immunol- 19%. 26: 1641-1646
integrand, or that lacked neomycin and so expresscd IFN-y normaliy (GKO+/+), al1 on the same background. GKO mice were also crossed with (H-2') (Hadan- Sprague Dawley, Iadianapolis. Dl). BALBIc mice were purchased €rom Charles River Canada (St-Constant. Canada). Mice were housed under specific pathogen-Eiee conditions and given acidified Wacer. Housing and experi- mental protocots were approved by the McGiII 'University Animal Care Cornmittee,
2 2 Induction of EAE
EAE was induced by two s.c injections, 1 week apart, of 400 pg bovine MBP [17] in camplete Freund\ adjuvant (CFA) (Difco, Detroit, MI) containhg 50 pg Mywbacze- rùun rubendosis pcr injection (Difco). fa SWJ m i a . symptorns are usually first obscrved 14 âays ahcr the initiai injection. Miœ were mooitorcd, weigised daily and assigned severity scores as foilows: O (no symp- toms), 1 (flaccid tail), 2 (moderate hind b b parcsis, clurnsiness), 3 (severe paresis o r unilateral hind lirnb paral- ysis), 4 (complete hindlfore Limb paraiysis), 5 (moribund) Pl-
23 Histological analysis
Animals were lethaiiy anesthecized (Somnotol, 4.45 mYkg body mas) (MT Ptiarmaœutical, Cambridge, Canada) then perfused through the heart with iœ-çold, sterile PBS and CNS tissue rernoved, Sagittal sections (10 gm) h m brain and spinal cord were stained with hernatoxylin and eosin. A second group of GKO-1- micc were perfused under anesthesia with 4% paraformaldehyde, and sec- tioned as before. Anti-CD4 mAb (GK15) or isotype- matched primary mAb staining was reveaied using the Vectastain ABC kit (Dimension Labs, Missiaauga, Canada).
2.4 Reverse transcriptase ( m - P C R analysis for CD3 and TNF-a
TotaI RNA was isolated from homogenized tissue using TRTZOL (GibcofBRL, Burlington, Canada) and yield quantified by specuophotometry. cDNA was synthesized using the Gibco/BRL Superscript Pre-amplification systern (GibadBRL). PCR conditions used were optimized for Iinear amplification to aüow direct cornparison between samples, and both primcrs and cDNA probes have been published previously [3]. For PCR analysis, equivalent
amounts of &NA were amplificd using 2 5 U Taq DNA polymerase (Gibccdi3R.ï). 10 mM of each dNTP, 50 pmol of each primer and a 10 x PCR b e r mixture containhg U) m M Ka, 100 mM Tris-HC1 pH 8.3, 15 m M MgQ and 0.1 % gelatin. PCR reactions were carried out in a Perlan- Elmer Cetus %O0 Gene amp thermal cycler (Norwalk,
for 28 cycles (denaturation, 30 s at 94"C, annealing, 30 s at 55°C and elongation for 30 s at 7î°C). PCR prod- ucts (80% of amplified sample) were then separated in 1.5 % agarose gels wirh Tris-acetate-EDTA (TAE) buffer. Separated amplimers were vansferred to Hybond-N mern- branes (Amersham, Baie d'Urfe, Canada) by capillary action and hybridized with 32P-labeled cDNA probes in Rapid-hyb buffer (Amersham) for 18 h at 42°C. BIots were washed twiœ for 15 minutes in 1 x SSC, 0.1 % SDS at room temperature foliowed by 15-min washes in 0.1 x SSC, O J % SDS at 65°C- Each blot was enposcd overnigfit to a phosphorimager screen (Molecular Dynam- ia, Sunnpde, CA), and the intensity of cach band quanti- fied using ImageQuant software (Molecular Dynamics) - Cytokine agds were normaliLed to chat of &actin. Resulu are expressed in arbiaary phosphorimager units.
3.1 Genet idy tesisent mice lacking 1- develop severe EAE
We used GKO mice obtained h m Genentech that had been backcrossed for five generations to BALBlc fiom the original- C57BU6 x i29Sv founding liae and so were pre- dominantly of the BALBlc background (H-2d). Hemizy- gous GKO+/- miœ were intercrosseci to generate (GKO-1-, +/- and +/+ mice on the same genetic back- ground, BALB/c (H-29, which is known to be &tant to EAE induction- No ïFN-y protein was detectable in mice with both alleles of the IFN-y gene disnipted (GKO-/-), while it was readily detectable in wild-type (GKO+/+) lit- termates [El. Hemizygous (GKO+/-) mice produced les (approximately 60 % ) IFN-y protein than wild-type litter- mates in response to Con A stimulation (personal commu- nication, Dr. T. Stewart, Genentech). To investigate directly the effect of targeted disruption of the EN-y gene on susceptibility to EAE, we irnmunized homozygous (GKO-1-), hemïzygous (GKOH-) and wild-F lit- termate (GKO+/+) mice of predorninantly H-2 haplo- type with MBP in CFA and assessed the incidence and progression of EAE.
Table 1 shows that mice whicé expresscd both copies of IFN-y (GKO+/+) were resistant to the induction of dis- ease, as only li16 presented with clhical symptorns. This
Table 1. GKO-/- m i a exhiibit increascd susccptibility to EAE cornparcd with GKO+I- and GKO+/+ miccmJ
Genotype Haplotype Inadcnce Mean day Seventy IFN-y H-2 of onset
a) All micc in thii table had bccn badrcmsscd to BALWc for five generations. Micc wcce immunkd wïth MBP as in Sect. 2.2. and monitorcd daily for syrnptoms. For cadi gcnorype, thrce xparatc immunizations of agc- and scx-marched groups wece carricd out. The values are prcsentcd as mcan f SEM. Sevcrity was calculatcd as the mean of maximal d i i i n d u for each mouse.
Eur. J. Immunol. 1996.26: 1641-1656 Enhanccd W E in IM-y knockout mice La3
Iow frequency contrasts with SJUJ mice fo r which inci- dence n n g e s between 3û-70 % . with a n average o f 55 YO . depending o n the experiment [M. 19). T h c atypical. very late onset (day 26) of t h e o n e mouse that devcloped symp toms, is also consistent with a resistant phenotype.
Strikingly. mice in which IFN-y expression had becn dis- rupted had a 71 % (17QJ) incidcncc rate, with a day of onset typical o f susceptible s t n i n s such as SSUJ (day 19.6 f 2.1) and a high mean mrixirnril severity o f diseasc (4 2 0.6) (Table 1). In c o n t n s t to E A E in thc susceptible mouse s t n i n SSUJ for which monality rates a r c cxtrcrnely low ([181 and Krakowski and Owens. unpublished observations). this immunization criused a 30 % rnortality n t e rimong GKO-/- micc.
Thc Jclction o f o n e copy of t h e IFN-y gene (GKO+/-) causeci n o s ignif iant diffcrcncc in discase incidence o r onscr (day 23.Y & 4.8) frorn GKO+/+ mice. Howcvcr. m c m maximai scvcnty of diseasc was rcIativcly high ( 3 . 3 iz 1.3) for thosc micc that did show symptoms.
In i i I I c:isiss. symptoms were accompanicd by wcight loss. Discrisc incidcncc did not di fier significmtly bctwcen in;ilcs and fcmales. Whcn GKO-/- micc wcrc immunizcd witli ovalbumin in CFA. nonc dcveloped EAE. ruling ou t tlic possibility thac CFA was cncephaiitogcnic in thcsc rnicc (data not shown). Thesc rcsults dcmonsrratc that dis- niption of thc IFN-y gcnc in EAE-rcsistant micc rcndcrs thcm susccptiblc to EAE induction. This su_cgcsts that in resistant s tnins . I M - y rnay be o n e of the factors inhibitins tlic inductiim of E A E .
3.2 Histopathology in GKO-I- micc with EhE
To cliaractcrizc funhcr the discase g e n e n t c d in IFN-y knockouts. six GKO-/- micc with sevcrc EAE wcrc cx- mnincd for CNS histopathology- AI1 six anirnals hrid histo- logical lesions chanctcr izcd by mcningeal and penvascu- iar inflammatory infiltrates in thc spinal cord and brain (Fig. 1). Many cells witliin the infiltrïtcs stained positively with anti-CD4 antibody (data not shown). T h c frequency
Fgiirc I. Pcrivriscular infiltnrion in GKO-1- micc with EAE. Micc wcrc killcd at peak discase. pcrfused and mgiiiat scctions of the spinal cord rind bnin staincd with hcmaroxylin rind cosin ( X 3 0 ) .
and degree of rnononuclear ce11 infiltration was typical o f EAE in S J U J rnice. Unmanipulated mice showed n o infil- t n t e s o r o ther pathological signs.
3 3 CD3 message in CNS of GKO-/- mice with dkase
T h e prcsence o f T cells in thc inf i l tnted tissue was con- firmcd by RT-PCR detection of m R N A for C D 3 -( chain in the spinal cord. Levels o f expression o f CD3 y were increased dnmatical ly in SSW micc with scvere E A E in comparison to the uninfiltnted CNS which contained background, rrlmost undetcctablc lcvcls of CD3 -[ rncssage (Fig. ZA). This samc order of magnitude of incrcasc in CD3 -1 rnRNA lcvcls wtis apparent in GKO-/- with EAE- dcspirc sornc variation betwccn individual mice. Thesc &tri i n d i a t c that T celis inf i f tntcd thc CNS of GKO-/- micc during EAE.
3.4 Intcrcrossing with SSUJ confers EGE susccptibility to GUO micc
Tiic micc in Tïblc 1 had bccn 'backcrosscd fo r fivc g e n c n - tions to BXLB/c. BALBIc micc a rc known ro bc rcsistant ro EAE and thc GKO*/ t micc also showcd this phcno-
GUO 4-
a
SJUJ
GUO 4-
SJUJ
3 i r r e 2. Incrcascd CD3 (A) and TNF-u (B) mRNA cxprcssion in GKO-/- and Sf UJ mice with EAE. RT-PCR \vas pcrformcd as dcs~ribcd in SCCL- 2-4- Each bar represcnts data fmm a scpa- rare mousc. normalizcd to the acrin signal from Phosphonmrigcr analysis. EAE: micc with sevcrc (gnde 3 4 ) EAE: unmanipu- larcd: unimmunized mice.
1644 M, Krakowski and T. Owens Eur- J- Immunoi. L M . 26: ldll-1646
Table 2. t n m s ~ d suxcptibility to EAE when GKO (H-2') rnicc arc intercrosscd wirh SJUJ (H-T) rnice"
Genotype IFN--[ Haplotype Incidence Mean day Sevcrity H-2 of onset
(CKO x SIUJ)F, +1- d'k 7/17 17 le 5 3 c0 .9 (GKO x SJUJ)Fl +I+ d'l~ SI33 17.8 le 6.8 2.8 L BALBlc +1+ d 0/24 - O SJUJ +/+ s 7/10 14 2 O 1.1 c 0.3
a) Mice were imrnunizcd a s describeci in Tabic 1. Data rcprescnt the sum of t h n x xpante experirnents except for the rcptescnrritivc SJUJ data shown. indicarcs prcdorninantly H - P genotype.
type. (BALBIc x SJUJ)F, mice are susceptible to EAE. To canlirrn that the b a i s for resistance of BALWc- backcrossed GKO+/+ (predominantly H-2') mice wris fundamentally similar to that of BALB/c (H-2') mice. we crossed [hem to SJUJ (H-?) and tested the F, genention for the predicted increase in susceptibility. T h e 1/16 nte of incidence of EAE in GKO+/+ (H-2") rnice (Table 1) was essentially identical to the OZ4 obtained for the resistant s t n i n BALBlc (H-2") (Table 2). In a single representative experiment using SJUJ. 70% of animals developed dis- case with a typical day of onset 17-23 5 6.8 and mean sever- ity of 1.1. These m e s of incidence and severity FaIl within the published n o m s Cor the SJL mouse [18. 191- The O '% incidence rate of resistant BALBIc mice was convertcd ro a 15 '36 (353) incidence with a mean maximal severity of 2.S 2 1 for GKO+/+ (H-2"') mice that hrid been bred wirh susceptible SJUJ mice. F, mice chat had o n c EN-;l allele knocked out (GKO +/-. H-2"') demonstnted an increased incidence in disease (4L 9'0. o r 7/17) as well as severity (3 & 0.9). This is consistent with a gcne dosage effect. A similar trend is apparent in Table 1. Thus. incer- crossing witli S JUJ confers susceptibility to E A E in GKO (H-Y) rnice. exactly as occurs for the BALBIc s tn in .
3.5 TNF-a message is up-reguloted in the CNS of GKO-I- rnice with EAE
EAE in S J U J and (BALBIc x SJUJ)F, mice is chancter- ized by an increase in TNF-u message in CNS 13. 41. We measured TNF-a mRNA levels in the spinal cor& of GKO-1- (H-2') mice with EAE. TNF-u production was significantly increased in GKO-I- mice with EAE (Fig. 28). with some variation between individual mice. The expected increase in TNF-a expression was also found in S J U J mice with E A E (Fig. 2B). Thus. elevated levels of TNF-a message are produced in the CNS of rnice deficient for IFN-y dunng EAE.
4 Discussion
The data presented here demonstrate that Iack of EN-y convens BALBIc rnice to an EAE-susceptible phenotype. Furthermore, mice of both resistant and susceptible back- grounds which Iacked one copy of the EN-y gene showed an increased incidence of EAE. Therefore. [FN-y acts as a resistance locus for E A E in BALBfc and S J U J mice. This disease-enhancing effect of IFN-y deletion contns ts with
previoudy described phenotypes in GKO mice, al1 of which manifestcd eithcr as immune deficiency or minimal effect [SO-241. Our data cornpiement but aiso extend the work of Ferber et al. [IJ]. who showed that IFN-y was not necessary Cor disease induction in the 610-PL. EAE- susceptible s tnin of mice.
Our data may clarïfy earlier suggestions of a d e for IM-'! in EAE resistance. When EAE-resistant mice were made susceptible by the systemic administration of mAb against I N - y [II. 121. it was not determined whether the Ab crossed the blood-bnin barn-er and entered the CNS. The authors of these studirs proposed that the effects of mAb treatment were due to selective removal of EN--1 from the periphery. One hypothesis to explain increased susceptibil- ity involved a cytokine gndient between the CNS and periphcry In our investigation of the rote of I M - ) in EAE-resistant mice. we show thar comptetc abrogation of the cytokine confew EAE susceptibility. chus makinl: unlikely hypothesec to explain diserise susceptibility that involve tissue-specific localization of IFN-y-
Although it may seem unexpected that a pro-infiammatory cytokine would exert apparently counterinfiarnmatory effecs. I M - y is known to have antiprolifentive infIuences [E . 261 on T ce11 responses and removal of [M-y leads to proIongedT ce11 responses in vitro [151. EAE is induced by myelin-specific CD4' T cells [27. ZS]. A minimal mode1 chat can account for ou r observations is that rncephalito- çenicT ce11 responses in BALBIc mice a re intrinsically les robust than in susceptible mice. Indeed. Abromson- Leeman and colleagues [29] have shown that the prolifer- ative capacity of LN T celIs from BALBIc mice immunized with MBP was significantly lower compared to that of T cells from (BALBIc x PUJ)F, mice- Rernoval of an immunomodulator (in this case IFN-y). would allow these suboptimal responses to surpass a threshold and so induce EAE. This predicts a low frequency of MBP-reactive T cells in BALBk mice and that experirnental elevation of this frequency would conven to a susceptible phenotype; indeed. adoptive transfer of MBP-reactive T cells induced EAE in BALBIc mice [29]. T h e immune modulatory role played by FN-y is Tikely to act in aIt strains of mice. so its removal should enhance disease severity. This suggests that the combination of genetic susceptibility and IM-Y deficiency in SJL-backcrossed GKO-/- mice would lead such mice to present with more severe disease than in BALBlc mice. Our resulu with respect to (GKO X S J U J)F, mice support this prediction. This hypothesis is atso
supponed by the d t s obtained by Ferber et al. [14], who showed increased scverity of EAE in suscepciile mice lacking both EN-y aiieles over those lacking o d y one.
Nevertheless, ïFN-y is not the o d y locus detcrmining rwistance: other Factors known to uiauenœ EAE- susceptibility include the pemeability of the blood brain banier [Ui] and the strength of enccphalitogenic T ce11 responses [31]. In addition, we cannot formally exclude that the susceptibility to EAE results Eiom effeas of gcnes other than IFN-y that wcrc carricd in the genetic crosses 132,331, aïthough we consider this unfilreiy. Sina multiple Littermates were evaluatcd, and aven the dcar-cut nature of the phenotypic ciifferma and its association with absence of the functionai EN-y gcne, the sirnplest and most direct interpretation is that the phenorype reflects an cffect of EN-y. It is probable that removal of IFN-y and its immunomodulatory effects from BAtB/c micc tips the balance of the anti-MBP T œll nsponse and so prcdis- poses towards autoimmunity.
IFN-y has been comlated with dkasc severity in EAE and is implicated in disease induction- It has k e n pro- posed b a t IFN-y acts to induce =-a production [31. Many groups have demonstrated the pmsence of TNF-a during EAE [3, 34-37] and MS [38, 391. It has k e n sug- gested chat the production of TNF-a within the'CNS of mice with EAE directly or indirectly mediates pathology [SI. This raises the question of how EAE can be induœd in the absence of EN-y. In the present study, we observed increased TNF- levels in the C N S of EN-y-deficient miœ with EAE. This demonstrates an EN-y-hdependent mechanisrn for the induction of TNF-a. Our resdts do not argue against a role for IFN-y in the induction of TNF-a. However, the present data demonsuate that IFN-y is not necessary for this induction, presumably because of com- pensatory mechanisms that induceTNF-a. During EAE in EN-y wild-type mice, both mechanisms are LikeIy to oper- ate. Our ability and that of Ferber e t al. [14] to induce EAE in IFN-ydeficient mice shows that the induction of EAE does not require IFN-ydependent mediators, and we additionaily pmvide a rnechanism for EAE via CFh-y- independent induction of TNF-a. From this, we predict that other autoimmune pathologies will be inducible in IFN-y-deficient rnice.
EAE is the preferred animal model for MS because of many clinical and pathological sunilaritics (reviewed in [2]), so it becomes of interest whether IFN-y might have analogous effects in MS. A trial of systemic, recombinant IFN-y in rclapsing-rtmitting MS patients [a, 411 was dis- continue. due to an incfea~ed ntunbcr of exaarbations. This observation in MS confiicts with the inhibition of EAE in rats by intraventricular administration of EN-y [13], and wouid not be predicted by those stuclits in which administration of anti-IFN-y Ab cxacerbated EAE [Li-U]. The apparcntly conuadictory effects of IFN-y between EAE and MS may be expiainecl by differential actions of IFN-y on responses in the periphery venus in the CNS. Thus, FN-y acts to inhibit proliferation of T cells, but induces inflammation in tissues. Which of these effects will domiaate wiU be influenœd by both the
- temporal andespatial expression of the cytokine. In long- standing MS patients, extensive blood-brain bamer dis- ruption is Iikely. Systemically administered IFN-y is likely
to have had dinct access to the CNS and so exaarbatcd innazimation within existing plaques. In the MS study, autoimmune T œii responses were not measund, but immunomodulatory effects of IFNy were not apparent [40,41]. ïnterestingly, immunorcgulation by CD8+ T alls previously d e s n i d in MS has k e n shown to be mediatcd by m - y [42j.
In the initial episode of EAE, blood-brain bamer integrity may not be as severeIy compromised as in late MS, so the effects of IFN-y may be biased towards immune modula- tion in the periphery. The f a a that intravenmtncular injcc- tion of IFN-y in rats inhibitcd EAE (13) can be intcrprctcd to show that CNS di- in those cxperiments was more strongiy infiuenced by T celi response than by tissue inflammation, so the net eff- of IFN-y was immunomod- data- In the samc study, and-EN-y mAb exacerbataï disease, consistent with this explanation. îh support of our model, d i . intrathecal injection of IFN-y into normal rat CNS produceci idammation 16.71. The EAE susceptiil- ity observed in GKO micc can also be explained by this model. T ceii proiüerative rcspoases iiz v i h o in GKO remain elevated many days aftcr T œ k from EN-y- producing littermates have returncd to baseline [15j, which suggests that GKO mice have a reduced ability to dom-regulate T c e U respoases. Our data show that the ability to i n d u e other idammatory cytokine nich as TNF-cL remains intact- This and long-lastingT ce11 respons- es may be at least partially responsible for the increased severity of the disease and the unusually hi& mortality rate.
Our results show that the cytokine EN-y operates in BALBk mice to d u c e encephalitogenic responses below a threshold required for oven disease. This reveals a modulatory role for IFN-y which may need to be factored into interpretation and design of immunotherapeutic pm- tocols for diseases such as MS and which conm'butes to the complex action of this pleiotropic cytokine.
nic auihors wouid üke fo rhrurk Genentcch Incorpomted for pro- vidùig Ihe GKO m i e for lhcre upvirmnrr and il Dodelet for helpful commenrt during the prepararion of d u manuscript. The auhors &O chOnk R. Varmn and & Bowbon&re for maulrouring the mouse colony, G. Vrrge for hirrology. anâ G. Chan for prepara- tion of MBt? Research in k o r Owens' lab K supportcd by rhe Medical Rucarch Cound of &nada. and the Multiple Sderosu Soaëy of Cimna'a. Micheüe Krakouski was supponed by a Nadonal Sciuice and EngUieering Rcrcar& Cowuil of CMO& and McGiU Faculty of Medicine Awqrds.
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.. .- iieinheim, Novambat 6 , 1996
.-Cc . , -*.&- 0.
PREFACE TO CHAPTER 4
The function of IFN y in the central nervous systam (CNS) dunng
expenmental allergic encephalomyelitis (EAE) was of interest as our
previous work had predicted that the outcome of action of this cytokine on
T cells in the periphery might differ from that in the CNS. Specifically, we
proposed that the dominant effect of IFN y expression in the CNS would
be to prornote inflammatory responses. To dissect the function of IFN y in
the CNS from that elsewhere, our goal was thus to create a mouse with
CNS-restricted IFN y expression, at levels equivalent to those seen in
EAE.
For the work completed in Chapter 4, IFN y transgenic mice were
generated by Toufic Renno and Veronique Taupin. IFN y knockout mice
were obtained from Genetech. Screening of interbred mice was carried
out by Lyne Bourbonniere and the candidate. Study of IFN y expression
patterns was camed out by the candidate.
CHAPTER 4
CNS Specific IFN y Expression
Michelle Krakowski, Véronique Taupin,
Toufic Renno, Alan Peterson and Trevor Owens
Keywords: IFN y, mouse, central nervous system, transgenic, knock-out
This work was supported by the Multiple Sclerosis Society of Canada and the Medical Research Council of Canada. MK was supported by a
McGill Faculty of Medicine Scholarship.
Abbreviations: CNS: central newous system, MBP: myelin basic protein,
PN S: peripheral nervous system
Abstract
We interbred IFN y knock out (GKO) mice to mice transgenically
expressing IFN y under the control of myelin basic protein (MBP)
promoters. Two promoters were used. The first contained 1.3 Kb of 5'
flanking sequences, which had previously been demonstrated to drive
CNS-specif ic B galactosidase expression. The second contained 9.6 Kb
of 5' flanking sequence, and while CNS expression was observed for the
reporter gene $ galactosidase driven by this prornoter, expression was
also found in Schwann cells of the peripheral newous system. The
transgenic mouse expressing IFN y under control of the 1.3 Kb promoter
is designated A519, while the transgenic mouse using the 9.6 Kb
promoter is called VT1-9.6. Transgenic mice were bred to GKO -/- mice
and progeny were intercrossed to obtain homozygosity for both the IFN y
transgene and disruption of the endogenous IFN ygene. The only IFN y
expressed in these mice was that transcribed from the transgene, which
was under the control of one of the two MBP promoter constnicts. The
resulting GKO x IFN y transgenic mice were designated 1%9" [GKO x
A519-1.3 Kb MBP:IFN y] and "G91n [GKO x VT1-9.6 Kb MBP:IFN y]. Both
lines developed nonnally in a pathogen-free facility, with no reduction in
reproductive capacity and no abnormal behaviour. IFN y mRNA was
detected by RT-PCR in both the brain and spinal cord of these animals,
but also in peripheral tissues in both fines of rnice. The cellular source of
the peripheral IFN y has not yet been determined. In the case of G91
rnice, it is likely to be in Schwann cells. The resulting IFN y expression in
the CNS of these mice, despita being equivalent to that seen in EAE,
appean insufficient to induce overt CNS pathology or symptoms, even
when the down-regulatory action of IFN y in the periphery is reduced.
Introduction
The onset of symptoms in EAE correlates with the upregulation
and expression of inflammatory cytokines in the CNS such as IFN y and
TNF a (Hofrnan et al., 1989, Renno et al., 1995 8 Okuda et al., 1995).
The potential role of IFN y in autoimmune disease is further supported by
evidence demonstrating the expression of this cytokine in multiple
sclerosis (MS) and diabetes (Beck et al ., 1988 and Jiang and Woda,
1991). Transgenic overexpression of IFN y in the pancreas, retina,
neuromuscular junction and CNS al1 induced symptorns and pathology
mirnicking autoimmune disease (Sarvetnick et al., 1988, Geiger et al.,
1 994, Gu et al., 1995 and Corbin et al., 1996). Therefore, these cytokines
have been proposed to be involved in disease initiation (Owens et al.,
1 994).
IFN y is well known for its pro-inflammatory actions in the immune
system (discussed in Owens et aL, 1994) and its ability to upregulate
MHC expression, macrophage and microglia activation and TNF a
induction have al1 implicated this cytokine in inflammatory responses in
the CNS. However, there is also evidence that IFN y can down-regulate
the immune response and inhibit T cell proliferation (Dalton et al., 1993,
Fitch et al., 1993 Krakowski & Owens unpublished observations). From
our previous work, we had proposed that the dominant action of IFN y
was dependent upon its location (periphery venus CNS). Specifically,
we hypothesized that IFN y acted in the periphery to limit the
encephalitogenic T cell response by controlling T cell proliferation
(Krakowski & Owens, 1996). However, once T cells were in the CNS and
recognized the correct antigen:MHC cornplex, the IFN y produced would
function to promote inflammation and disease. Presumably, the anti
profiferative effect of the cytokine on T cells does not alter the course of
disease once activated T cells are within the CNS. We directly tested the
role of IFN y in the CNS by generating mice which over-express IFN y in
the CNS, but have the endogenous gene disabled. Our expenments
show that the level of IFN y produced in the CNS is not suficbnt to
generate spontaneous disease.
Materials & Methods
Transgenic Mice
Mice were housed under specific pathogen-free conditions.
Housing and experirnental protocols were approved by the McGill
University Animal Care Cornmittee.
A5 1 911.3Kb MBP Promoter
A51 9 mice were generated by Renno (Renno 1993). Briefly,
Hindlll was used to excise a DNA fragment frorn the linearized PM2hFN y
plasmid that included the entire IFN y cDNA, flanked by 1.3 Kb upstream,
and 2.2 downstream MBP sequences. cDNA was micro-injected into
oocytes which were then re-implanted into pseudo-pregnant mice. The
integration of the transgene in the resulting litter was determined by
southem biotting analysis of Sstl digested tail DNA probed with (32P]-
labeled IFN y cDNA. Briefly, Sstl digestion excised the IFN y transgene,
but also the endogene. 60th hybridized the [32P] radioactive cDNA
probe, but the digested genomic products were of different sires, so they
could be differentiated on a southern blot. Mice which were non-
transgenic (wildtype) lacked the transgene and had only the endogene
on the southem blot. Phosphorimager analysis (Molecular Dynamics,
Sunnyvale. CA) was used to evaluate transgene copy number.
Transgene intensity / endogene intensity ratios of 1 -5 or lower defined
heterozygotes, 1.8 or higher defined homozygous mice. The A519 line
was backcrossed from the original (C3H x C576i/6)F2 to SJUJ (Harlan-
Sprague Dawley) for five generations.
VT119.6Kb MBP Promoter
VT1 transgenic mice contahing the murine IFN y cDNA with a 9.6
Kb MBP promoter were generated by Renno and colleagues and
screened in a simifar manner to that for A519 mice (Renno et al., In
Preparation). These mice had been backcrossed once to SJUJ mice
before they were used in these experiments. Levels of IFN y in the CNS
were equivalent to those seen in non-transgenic mice with EAE.
(3KO
Interferon y knock out (GKO) mice which had been backcrossed to
BALB/c for five generations (Dalton et al., 1993) were obtained from
Genentech. They were generated by the insertion of a neomycin
resistance (R) gene within the single copy gene for IFN y. Mice were
screened by DNA PCR for neomycin-R and IFN y (Goes et al., 1995). IFN
y PCR pimers were designed to span the junction between exon 2 and 3
of the IFN y gene. Targeted integration of the neomycin-R cassette into
this region disnipted the IFN y gene and disallowed the production of an
IFN y amplimer, as well as an IFN y transcript. Thus, DNA from GKO -1-
mice produced only neomycin-R amplimen (375 bp product), GKO +/- mice produced both the neomycin-R and IFN y products, and GKO +/+ mice produced only IFN y (220 bp) products.
9 & G91 double transaenicâ
GKO -/- were bred to either A51 9 +/+ or VT1+/+ IFN y mice and
progeny interbred to generate homozygosity for both transgenes. The
progeny of these GKO x IFN y transgenic mice were designated "09"
[GKO x A519-1.3 Kb MBP:IFN yi and "G9In [GKO x VT1-9.6 Kb MBP:IFN
y]. Mice were screened using a combination of techniques descnbed for
each individual transgene.
RNA isolation and RT-PCR
Total RNA was isolated from homogenized, perfused tissue using
TRIZOL (GibcoIBRL, Burlington, Ont) and yield quantified by
spectrophotometry. cDNA was synthesized using the GibcoIBRL
Supencript Pre-amplification system (GibcolBRL). PCR conditions used
were p reviousl y optimized for linear arnplif ication to allow exact
cornparison between samples (Renno et al., 1995). For PCR analysis,
equivalent amounts of cDNA were amplified using 2.5 U Taq DNA
polymerase (Gibco/BRL), 1 mM of each dNTP, 50 pmol of each primer
and a PCR buffer mixture containing 50 mM KCI, 10 mM Tris (pH 8.3), 1.5
mM MgCI2 and 0.01 % gelatin. The primers used were: IFN y antisense
5'-CGACTCCTmCCGCTTCCT-3 sense, 5'-
ACACTGCATCTTGGCTTTGC-3, $ Actin sense 5'-
TGGGTCAGAAGGACTCCTATC-3', antisense 5'-
CAGGCAGCTCATAGCTCITCT-3'. PCR reactions were carried out in a
Perkin-Elmer Cetus 9600 Gene amp thermal cycfer (Nowalk, CT) for 28
cycles (denaturation, 30 seconds at 94 OC, annealing, 30 seconds at 55
OC and elongation for 30 seconds at 72 OC). We have previously shown
the absence of a plateau effect in the amplification of these products
(Renno et ab, 1994). 40 cycle, non-linear PCR was also camed out in
sorne instances where confirmation of negative signals was desired.
Equal volumes of PCR products (80% of amplified sample) were then
separated in 1 % agarose gels with TAE buffer.
Screening of Transgeniic mice
Mice that overexpressed IFN y, under the control of an MBP
promoter, and which lacked endogenous IFN y were generated by
crossing IFN y transgenic mice to IFN y knockouts and interbreeding the
progeny. The generation of double transgenic mice from heterozygous
FI parentals is predicted to occur at a frequency of 111 6, which we
observed (1 11 5 for 0 9 and 111 8 for G91) indicating that in utero death
due to transgenesis did not occur. Mice developed normally in our
specific pathogen free facility with no outward signs of CNS disease.
Inter transgenic mouse lines were screened using a combination
of techniques specific for each transgene. Figure 4.1 shows a DNA PCR
which evaluated neomycin-R cassette insertion into the endogenous IFN
y gene. Lane 1 contains DNA from a heterozygous (GKO +/O) mouse
[both IFN y and neomycin-R amplimers], lane 2 shows a homozygous
knockout (GKO of-) [neornycin-R amplimers only], lane 3 a non-transgenic
(GKO +/+) [IFN y amplimen only], and lane 4 shows an A S 9 mouse
[non-transgenic for neomycin-R, but expresses endogenous IFN y]. This
PCR does not amplify a 220 bp product from the A519 transgene as the
primers were designed to hybridize to genomic DNA and not the cDNA
which was used for the transgenic mouse. Screening of the F2 litter of G9
mice is shown in lanes 5-17. Lane 6 shows DNA from a non-transgenic
F2 littermate, lacking a neornycin-R PCR product, but an IFN y amplimer is
observed at 220 bp. Lane 7 shows DNA from a heterozygous transgenic
G9 mouse with both neomycin-R (375 bp) and IFN y amplimen, while
lane 8 contains DNA from a knockout G9 mouse that lacks the IFN y
product, but contains the neomycin cassette. Similar screening was
carried out for the G9l Mers, and analogous PCR amplimer patterns
were found (data not shown).
Once mice had been screened for disniption of the endogenous
IFN y gene, southem blots of Sstl digested DNA from the same mice were
probed with radiolabelled IFN y cDNA to evaluate presence of the IFN y
transgene. Figure 4.2 shows the position of the transgene (large anow,
lane 1) with respect to the endogenous (small arrow, lane 1) IFN y gene
as well as the increase in band intensity of the IFN y when a
heterozygous G9 mouse (lane 5) is compared to a homozygote (lane 1).
Phosphorimager analysis was used to quantitate these bands and mice
with a ratio of between 0-1.5 were typed as heterozygotes, while those
with a ratio of 1.5 or greater were typed as homozygotes. Additional
controls shown on this blot are an A51 9 homozygous mouse (lane 7)
which shows the typical pattern for this transgenic mouse, and a GKO -/-
mouse (lane 9). The transgene expression pattern obsewed for the
A51 9 +/+ mouse is identical to that for the G9 +/+ mouse.
Figure 4.3 shows the screening for G91 mice. Note that the
positions of the endogenous (small arrow) and transgene (large arrow)
differ for G9 and G91 mice. We obsewed the transgene in Lanes 1 and 3
which contain DNA from G91 homozygous mice, and lanes 2 and 4
contain DNA from a heterozygous mouse. The transgene is not
observed in lanes 5 and 6 which contain DNA from non-transgenic F2
littermate mice that do not have the IFN y transgene. For cornparison,
lane 7 contains DNA from an A519 homozygous and lane 8, a
heterozygous AS1 9 mouse. Once again, Phosphorimager analysis was
used to quantitate the endogene and transgene bands. Mice with a ratio
of between 0-1.5 were typed as heterozygotes, while those with a ratio of
1.5 or greater were typed as homozygotes.
Importantly, no other bands appeared on the Southern blot during
this analysis indicating that the enzyme Sstl did not cut within the
promoter of the endogenous IFN y gene. Additionally, analysis of double
knockout mice (GKO -/O) in these litters showed that the PCR primers did
not amplify the cDNA constnict used to generate the transgene, but only
the endogenous genomic sequence.
iFN y mRNA analysis by UT-PCR
Previous work had shown that IFN y mRNA was undetectable by
Northern blot analysis in the CNS of unmanipulated transgenic (both
A51 9 and VT1) mice. Additionally, IFN y protein was undetectable by
histological and western blot analyses, so RT-PCR was used to analyze
IFN y mRNA. Animals were perfused to remove circulating lymphocytes
and RNA was isolated from the brain, spinal cord, Iiver, heart, and
spleen. No abnormalities in the gross structure of the intemal organs,
including the CNS, were observed.
G9 mice were shown to express IFN y in spinal cord, brain, spleen,
liver and heart by RT-PCR (figure 4.3 a). G91 mice were shown to
express IFN y in spinal cord and brain. as well as spleen and liver (figure
4.4 a). Levels of IFN y mRNA in the CNS were compared to those from
IFN y knockout mice (GKO -1-), wildtype (BALWc x SJUJ) F2 mice, SJVJ
mice with severe EAE, and to RNA from a negative control cell line
(plasmacytoma mll-4) (Karasuyama & Melcherç, 1 988). IFN y mRNA
levels in G9 mice were substantially higher than in G91 mica (figure 4.4
a). The low levels of IFN y expression in G91 mice was confirrned using
40 cycle RT-PCR (data not shown) and the relative difference in transcript
levels between G9 (higher) and G91 (lower) mice was again observed.
Message levels for f3 actin from the same samples were also analyzed to
ensure that approximately equal amounts of mRNA were being
compared. Thus, we have show that the transgenic expression of IFN y
under the control of the MBP promoter in a mouse lacking endogenous
IFN y expression is not lethal, nor does spontaneous ÇA€ develop.
Additionally, we have shown that MBP prornoter-driven IFN y expression
was not restricted to the CNS.
Discussion
The cytokine IFN y has been proposed to play a role in the
induction of EAE because it is known to induce inflammation and the
expression of the cytokine correlates with the onset of symptoms dunng
disease (Renno et aL, 1995 and Okuda et al., 1995, Hofrnan et al., 1988).
IFN y has been overexpressed in a number of organ-specific transgenic
systems, and this generated diseases mimicking autoimmune syndromes
(Sarvetnick et al., 1 988, Geiger et ai., 1 994, Gu et al., 1 995 and Corbin et
al., 1996). Our laboratoty has generated MBP promoter-driven
transgenic systems in which IFN y was overexpressed (Renno, 1993 and
Renno et a/. , in preparation). In some mice, the levels of IFN y expressed
were equivalent to those seen in non-transgenic mice with EAE, but the
transgenics did not develop spontaneous symptoms. Instead, it was
observed that once EAE was induced, mice continued to exhibit
symptoms and pathology, with no remission.
Our previous work with IFN y knockout mice demonstrated that the
role of IFN y in EAE is not only pro-inflammatory but also anti-proliferative
and so disease-inhibitory (Krakowski 8 Owens, 1 996). Conside ring this,
we hypothesized that the dominant role of IFN y in EAE may differ for the
periphery and the CNS. The pro-inflammatory effects of IFN y in the CNS
and in the periphery, are probably the same: to cause inflammation,
induce MHC antigens and TNF a. During the induction of EAE by
immunization with MBP and adjuvant, the generation of the
encephalitogenic T cell will be influenced by these functions of IFN y, but
it appears not to be critical to development of EAE in GKO mice. The
anti-proliferative effects of the cytokine may inhibit the induction of
disease when acting during the generation of the encephalitogenic T cell
response, but this same anti-proliferative effect does not affect the
function of those activated T cells that have already reached the CNS. It
may be that proliferation of T cells in the CNS is not necessarily required
for disease.
By this logic, the removal of endogenous IFN y with concurrent
transgenic overexpression in the CNS might promote disease. Thus, we
generated unique mice in which the only IFN y expression was
transgenic and under the control of an MBP promoter, in order to better
understand the relative effect of IFN y in the CNS and periphery. These
mice did not, however, show spontaneous symptoms. A likely reason
why we do not see spontaneous autoimmune disease in the G9 and G91
mice is that the levels of IFN y expression were not high enough to
induce disease. Spontaneous pathology had not been observed for the
A51 9 or VT1 transgenics, although in other transgenic mice in which IFN
y is overexpressed in the CNS under the control of MBP promoten,
spontaneous EAE was obsewed with pathology characteristic of
demyelinating disorden (Corbin et al, 1996 and Renno et al., in
preparation, Horwitz et al., 1 997).
Previous studies had showed that while spontaneous disease did
not occur in A519 or VTI mice, persistent non-relapsing symptoms and
pathology followed immunization. Considering this, it is probable that
immunization of our 09 and G91 mice with MBP to generate a T cell
response will induce an extremely severe, unremitting form of EAE with
upregulation of MHC antigens, TNF a. and rnicroglia activation.
The cellular sources of IFN y expression in the periphery of these
mice rernain to be determined. The expression pattern that we have
seen for G9 mice is not predicted by previous experiments using
promoters of a similar length with Lac-z as the reporter gene (Gow et al.,
1992). Either there has been a deregulation of the promoter (due to the
site of integration of the construct), or the transgene is somehow
influencing promoter activity. The influence of this peripherally
expressed IFN y may be to inhibit the generation of the encephalitogenic
T cell response, thus be a contributing factor to the lack of spontaneous
disease development of these mice. For G91 mice, the likely source of
IFN y is the Schwann cell of the PNS, as the same promoter induced Lac-
z expression in this cell type (Peterson et al., personal communication).
Such a mouse which did not express IFN y outside the nervous system
(peripheral or central), would be a useful tool to complement those
already available for studying what are likely to be differential roles of IFN
y acting on infiltrating T lymphocytes versus endogenous cells of the
CNS.
Acknowledgments
We thank Lyne Bourôonniere and Grace Chan for excellent
technical assistance in transgenic screening and mouse colony
maintenance.
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Figures
Figure 4.1 : DNA PCR for the neornycin cassette and endogenous IFN y
gene. PCR amplimen (IFN y = 220 bp, neomycin = 375 bp) were
analyzed on a 1% agarose gel stained with ethidium bromide (figure
4.1 ). Lane 1 : GKO +/- [220 & 375 bp products]; lane 2: GKO -/- [only
375 bp]; lane 3: GKO +/+ [only 220 bp]; and lane 4: A519 mouse [only
220 bp]; lane 6: non-transgenic littenate; lane 7: heterozygous G9; and
lane 8: knockout G9 mouse. Lanes 5, 9-17 show the screening for the
remaining mice of this F2 litter. Lane 18 contains the 100 bp DNA ladder.
The extremely bright band midway in the gel indicates the migration level
for 600 bp sized products. Note that this PCR does not identify the IFN y
transgene.
Figure 4.2: Genotype analysis of IFN y transgene integration in G9
mice. The positions of the transgene (large arrow, lane 1) and the
endogenous (small arrow, lane 1) IFN y genes are indicated. The
increase in band intensity of the IFN y transgene for homozygous G9
rnice (lanes 1 and 3) is seen when compared to a heterozygous G9
mouse (lane 5). Mice that did not contain the IFN y transgene (negative
littemates) are shown in lanes 2,4 and 6. Two A519 homozygous mice
(lanes 7 and 8) and a GKO 4- mouse (lane 9) were analyzed for
comparison. The chart below shows ratios of transgene intensity /
endogene intensity from the phosphorimager analysis of the transgene
and endogene. A ratio of between 0-1.8 indicated heterozygosity, while
a ratio of 1.5 or greater indicated hornozygosity. NA = not applicable.
Figure 4.3: Genotype screening for 091 mice.
The positions of the endogenous (small arrow) and transgene (large
arrow) are shown for the G91 mouse (lane 1). Lanes 1 and 3 contain
DNA from G91 homozygous mice, lanes 2 and 4 contain DNA from a
heterozygous mouse and lanes 5 and 6 contain DNA from G91 mice that
do not have the IFN y transgene. Lane 7 contains DNA from a VT1
homozygous and lane 8 a VT1 heterozygote mouse. The chart below
shows the phosphorimager analysis of the intensity of the bands for the
transgene and endogene and resuiting numerical evaluation to
determine transgenicity, as for figure 4.2.
Figure 4.4: RT-PCR analysis of IFN y mRNA ievels in G9 mice. RT-
PCR analysis of IFN y mRNA expression of three G9 mice is shown in
figure 4.4 a. Lanes 1-5, 6-1 0, and 1 1-1 5 show the RT-PCR analysis of
IFN y mRNA in 3 separate G9 mice. Results are shown for brain (lanes 1,
6, 1 l ) , spinal cord (lanes 2, 7, 12). heart (lanes 3.8, 13). liver (4, 9, 14)
and spleen (lanes 5, 10, 15). by RT-PCR. GKO 4- tissue (lanes 16-1 9)
did not amplify the 449 bp product for IFN y, although a faint,
inappropn'ately sized band is seen (lanes 16= brain, 1 ?'spinal cord,
18=iymph node, 19=sciatic nerve). IFN y amplimer was observed from
SJUJ rnouse with severe W (lane 24), and for some tissues from a
(BALBlc x SJUJ) FI naïve mice (22=heart, 23=sciatic nerve), but not al1
(lanes 2O=brain, or 21= spinal cord). Figure 4.4b shows the $ Actin
amplimers for al1 these samples (in the same numerical order), indicating
that relatively equal amounts of RNA were containad in al1 samples. The
last lanes of each gel contain 100 bp DNA ladder. A total of four G9 mice
( n 4 ) were anakjmd and the same pattern of IFN y expression was found
for all.
Figun 4.5: RT-PCR analysis of IFN y mRNA levels in G91 mica . Lanes 1-4 and 5-8 show RT-PCR analysis of IFN y mRNA in two separate
G91 mice. Results are shown for spinal cord (lanes 2 and 6) and brain
(lanes 1 and 5), as well as liver (lanes 4 and 8), but extremely faint signal
was observed for spleen samples (lanes 3 and 7) (figure 4.5 a). Levels
of IFN y were compared to two separate IFN y knockout mice (GKO -/O) in
lanes 9-1 1 and 12-1 4 (lanes 9 and 12=brain, spinal cord=l 0 and 13,
lymph node=l 1 and 14) Additionally, wildtype (BALBlc x ÇJUJ) F2 rnice (lane 19=brain, 2O=spinal cord, 21 =haart. 22=spleen), SJUJ mice with
severe EAE (lane 24). and a cell line known not to transcribe IFN y (the
plasmacytoma mlL4) (lane 23) were analyzed. In figure 4.5 a, IFN y
mRNA levels in a G9 mice (lane 1 S=brain, 1 Gispinal cord, 17=heart,
18=spleen) can be directly cornpared to two G91 mice (lanes 1-8) and
are seen to be higher in G9 mice. Figure 4.4 b contains the Actin
amplimers for al1 these samples in the same order, indicating that
relatively equal amounts of RNA were contained in al1 samples. The last
lanes of each gel contains 100 bp DNA ladder. A total of four G91 mice
were analyzed (n=4) and the sarne pattern of IFN y expression found in
all.
PREFACE TO CHAPTER 5
We have descnbed certain aspects controlling encephalitogenic T
cell activation with respect to antigen presentation and cytokine
influences. We know that dunng EAE, the activated immune system
releases a plethora of inflammatory cytokines and attractive chemokines.
Additionally, the T cells which enter an inflamed CNS are not necessarily
specific for CNS antigens. From our work, we theorized that the
production of IFN y and TNF a do not only act to cause inflammation in
the CNS, but might also activate these nonCNS specific cells which in
turn contribute to the ongoing immune response.
For the work completed in Chapter 5, much of the preliminary work
was completed by Jia-You Lin. For the data shown in this chapter, the
candidate completed the majority of the experiments herself.
CHAPTER 5
Bystander CD4+ T Cells do not contribute to Experimental Allergic Encephalomyelitis (EAE)
Michelle L. Krakowski*§ and Trevor Owens*?
Departrnent of Microbiology 8 Immunology*, Departrnent of Neurology & Neurosurgeryt
Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 284, Canada.
5 Current address: The Scnpps Research Institute, Dept. Immunology, IMM-23, LaJolla Califomia, 92037, USA.
Keywords: Central newous system, T lymphocytes, Bystander, Cytokines
This woik was supported by the Multiple Sclerosis Society of Canada and the Medical Research Council of Canada. MK was supported by the National Research Council of Canada and a McGill University Faculty of Medicine Scholarship.
Abbreviations: CNS: Central nervous system, CFA: Complete Freund's adjuvant, DO1 1.1 0: OVA-peptide specific TCR transgenic mouse, EAE: Experimental allergic encephalomyelitis, FSC: forward scatter, IL-2Ra: interfeukin-2 receptor alpha (also CD25), LN: Lymph node, MBP: Myelin basic protein, OVA: ovalbumin, PPD: purified protein derivative, SSC: side scatter,
Abstract
EAE is a CD4+ T cell-mediateci disease of the CNS. Symptoms
can be induced by immunization with myelin basic protein (MBP) or by
transfer of MBP-reactive CD4+ T ceils. Mice with EAE experience limb
paralysis and lymphocyte infiltration to the central newous system (CNS).
During disease, T cells of many specificities infiftrate the CNS but the role
played by those that do not recognize CNS-antigens is unclear. To
investigate the trafficking capacity and general phenotype of such cells,
we transferred OVA-reactive CD4+ T cells that were labeled with a
fluorescent dye, to MBP-immunized SJL mice 7 days pnor to disease
onset. T cells were isolated from CNS and LN at the time of disease
onset and analyzed by flow cytometry. Only 25% of the dye-labelled,
CD4+ cells in the CNS were blasts, of which only 20% were CD45RBiow.
Similar proportions and phenotypes of OVA-reactive CD45RBlow blasts
were found in the LNs. Previous work showed that almost no OVA-
reactive blasts entered the CNS of naïve mice, whereas almost al1 MBP-
reactive CD4+ cells were CD45RBJow blasts in the CNS of mice with EAE.
The blast and CD45RB phenotype of OVA-reactive T cells in inflarned
CNS suggested that the majonty of these cells were not engaged in an
active immune response. Tharefore, we investigated whether the
bystander cells in mice with EAE were making cytokines and contributing
to the inflammatory immune reaction. We used mice transgenic for the
expression of a TCR specific for a peptide of ovalbumin as a "bystander
mouse" (DO1 1 .IO) and injected encephalitogenic, fluorescently-labelled
T cells lines to induce EAE. We observed the trafficking of both the anti-
MBP T cell line and endogenous or "bystander" cells to the CNS. The
bystander cells were FACS sorted from the brain and found not to
u preg ulate expression of T cell activation molecules (IL-2Ra, CD44, and
IFNq) nor the production of the inflammatory cytokine IFN-y. From this
data, we conclude that it is unlikely that bystander T cells contribute to
ongoing, organ-specific immune responses.
Introduction
A general function of T cells is to circulate throughout the body in
order to maximize their encounter with pathogenic antigens. This is
facilitated by the expression of circulating and homing surface markers
which promote extravasation frorn blood into tissues (reviewed in Bradley
8 Watson, 1996 and Butcher & Picker, 1996). During an immune
response, T cells of many specificities may traffic to sites of inflammation,
driven in part by chemokine and inflammatory cytokine production. Many
of these do not encounter antigens for which they are specific and so are
defined as 'Bystander T cells". It is not well understood whether
bystander T cells at the site of an immune response can become
activated non-specifically (eg. by the individual or combined effects of
cytokines and cellular interactions), and then contribute to the ongoing
cellular response (discussed in Owens et al., 1994). In the case of
delayed type hypersensitivity or autoimmune response, this would
include mediation of tissue damage.
The potential contribution of bystander T cells to ongoing immune
responses has broad implications for al1 immune reactions, and design of
disease-intervention therapies. It is important to determine if bystander T
cells contribute to tissue damage in organ-specific autoimmune
diseases. The CNS-specific disease Multiple Sclerosis (MS) progresses
for many years. During this time, patients experience infections and
mount immune responses to them. thus generating elevated frequencies
of activated T cells of a variety of specificities. These are the potential
bystander T cells.
Experimental allergic encephalomyelitis (EAE) is an experimental
model for MS, (discussed in Traugott et al.. 1985 and Martin et al., 1992).
EAE can be induced in a wide variety of rodents and primates. The
central neivous system (CNS) infiltration that characterizes EAE is
primarily composed of CD4+ T cells and macrophages. During EAE, the
production of infiammatory cytokines, chemokines and expression of
adhesion molecules on brain endothelium are al1 increased (Renno et
al., 1995, Okuda et al., 1995, Cannella et al., 1991, Baron et al., 1993,
Hulkower et ai., 1993 and Glabinski et aL, 1997). The CNS thus
becomes more amenable to the entry of both activated and resting T cells
of al1 specificities. EAE is induced by either the immunization against
myelin cornponents or by transfer of CD4+ T cells reactive to these
proteins (Martin et al., 1992). The entry of endogenous, bystander T cells
to the CNS has been descnbed (Werkele et al., 1986 & 1987 and Zeine
& Owens, 1992), but the contribution, if any, that these bystander T cells
make towards the immune response remains unclear.
We have investigated the activation status of bystander T cells in
the CNS of mice with EAE. We show that a small minority of these cells
express an activated/memory phenotype. Upon further characterization,
we found that bystander cells do not appear to be participating in the
immune response by virtue of their lack of expression of other T cell
activation markers and production of cytokines. These data indicate
during an inflammatory immune response, antigen-non-specific
activation of T cells is not likely to occur.
Materials & Methods
Mice
Fernale SJUJ mice (5-8 weeks) were purchased (Harlan-Sprague
Dawley, Indianapolis, IN) and al1 animals were housed in our specific
pathogen-free facility.
00 1 1.1 0 transgenic mice which express a TCR specific for the
peptide (aa 323-339 ISQAVHAAHAEINEAGR) of the chicken protein
ovalbumin (OVA) (Murphy et aL, 1990) that had been backcrossed to the
BALBlc genotype, were obtained from Drs. Michael Julius and Philippe
Poussier (University of Toronto). Mice were bred to SJUJ mice (HSD),
and FI progeny interbred to homozygosity for expression of the TCR.
Expression of the transgene was determined by FACS analysis (Becton-
Dickinson , Mississauga, Canada) from a sample of peripheral blood
(see Murphy et al., 1 990 for procedure).
Mice that were not deliberately irnmunized were considered naive.
Induction of EAE
EAE was induced in one of two manners. (1) Naïve SJUJ mice
were immunized with two SC. injections one week apart of 400 pg myelin
basic protein (MBP) (Sigma, Montreal, Canada) emulsified in complete
Freund's adjuvant (CFA) (Difco, Detroit, MI) containing 50 pg of
mycobacterium tuberculosis H37RA (Difco). (II) Naïve 001 1.10 TCR
transgenic mice received T cell lines specific for MBP raised in non-
transgenic littermates (see method below) to ensure MHC compatibility.
Mice were monitored daily and assigned clinical scores as follows: O (no
symptoms), 1 (flaccid tail), 2 (moderate hind limb paresis, clumsiness), 3
(severe paresis or unilateral hind limb paralysis), 4 (complete hind/fore
limb paralysis), 5 (moribund).
T ce// /rires
Ovalbumin and MBP specific T cell lines were raised as follows.
Naive SJUJ or DO1 1-10 mice were immunized twice with the antigen of
interest ( 400 pg ovalbumin, or 400 pg MBP respectively) in CFA as
described above. Seven days after the last immunization, draining
superficial inguinal, axillary, and brachial lymph nodes were removed
and cultured in RPMl 1640 (GibcoBRL, Burfington, Canada)
supplemented with 10% FBS (UBI Lake Placid, NY), 50 pM 2-ME
(Sigma), 100 U/ml penicillin (GibcoBRL). 100 p g h l streptomycin
(GibcoIBRL) and 2 mM L-glutamine (GibcdBRL) in 24 well plates
(Falcon, Montreal, Canada) at 4 x 106 cells/ml with the appropriate
antigen (OVA 50 pg/ml, or MBP 50 pghnl) in a total volume of 1.5 ml.
Reactivity to the antigen of interest was assessed in parallel
microcult ures (96 well plate, Falcon) by [Wlthyrnidine (ICN Biochem icals,
Mississauga, Canada) incorporation at 4 days following an ovemight
pulse (0.5 pCüwell) immediately ex-vivo. Cells were cultured for 3 days,
collected by centrifugation on Ficoll-Hypaque (Pharmacia, Montreal,
Canada), labelled with PKH2 dye and transferred to naïve mice (see
below).
T ce// Transfers
Three days following antigen stimulation T cells were collected on
Ficoll-Hypaque and injected into mice (1-2 x 107 T ceIIS/mouse). T cells
were labelled with the lipophilic dye PKH2-GL (Zynaxis, Malvern, PA)
and injected into SJUJ mice that had been previously immunized with
M W in CFA. The lipophilic dye is know to be retained in cell membranes
over two weeks in vivo (Horan 8 Slezak, 1989). The SJUJ immunized
mice were injected with OVA-specific T ceils between 8-1 0 days after the
first MBP injection. In separate experiments, MBP-specific T cells were
injected into naive DO1 1.1 0 homozygous transgenic mice in order to
induced EAE.
Isolation of Mononuclear Cells from CNS and LN
Animals were lethally anesthetized (Somnotol, 4.45 mVkg body
mass) (MT Pharmaceutical, Cambridge, Canada), inguinal LNs collected,
then the animals were perfused through the heart with cold, sterïle PBS
and CNS tissue removed. Pooled CNS and secondary lymphoid tissue
were dissociated by passing through a wire mesh and centrifuged at 200
x g for 10 minutes at 4 OC. LN cells were resuspended in sterile PBS and
maintained on ice while dissociated CNS tissue was resuspended in
70% percoll (Pharrnacia) and centrifuged for 20 minutes at 500 g, 20 'C
on a 30%:37%:70% percoll gradient. Mononuclear cells were collected
from the 37% (1 .O48 g/ml): 70% (1 .O86 g/ml) interface, washed, stained
and sorted by FACS
Flow C ' m e t t y
Cells were stained with phycoerythrin-coupled anti-CD4 (PE:CD4)
(BD), biotinylated anti-OVA peptide TCR (KJ1.26:B) (Haskins et ai.,
1983). foiiowed by streptavidin spectral red (SRAV) (Southem
Biotechnology, Birmingham, AL), for 20 minutes at 4 OC. T cell activation
markers, anti-CO44 (PgP-l), anti IL-2R (CD25), anti-IFN-yReceptor (GR-
20)(Basu et al., 1988), pan antiCD45 and anti-CD45RB (Bottomly et al.,
1989) were each stained on the isolated cells. Fluorescence was
analyzed on a Vantage FAC Sorter (BD) and sorted cells micro-
centrifuged at 200 x g for 2 minutes, snap frozen in liquid nitrogen and
stored at -20 OC until mRNA was isolated.
Isolation of mRNA and RT-PCR
mRNA was isolated using the QuickPrep micro mRNA purification
kit (Phamacia), and cDNA was synthesized using the Superscript Pre-
amplification system (GibcoBRL). PCR conditions used for actin and
IFN y were previously optimized for linear amplification to allow
cornparison between samples (Renno et al., 1995). The amount of cDNA
used for each PCR was equalized based on the original number of cells
sorted. The specifics of the reactions and southem blotting techniques
are discussed in Krakowski & Owens (1996), with the exceptions of the
PCR for 11-4 (Renno et ai., 1995). Cytokine signals were norrnalized to
that of B actin. Results are expressed in arbitrary phosphorimager units.
Results
Bystander T cells trac to both the LN and CNS
In order to investigate the role of bystander T cells in EAE, T cell
fines reactive to the chicken egg protein ovalbumin (OVA), were raised in
SJUJ mice for passive transfer experiments. Short tenir culture of OVA-
prirned LN cells removed reactivity to components of the priming
adjuvant (PPD) and enhanced both the specificity and stimulation index
(SI) to the desired antigen (OVA) (Figure 5.1). Post in Mtro selection, the
activated line was found to be CD4+ (Figure 5.2 A) and
CD45RBîntemediate (int) (Figure 5.2 B), a T cell activation marker. CD45RB
staining is generally used to differentiate naïvelresting (low expression)
venus activatedlmemory T cells (high expression).
Four to seven days prior to the expected appearance of symptoms
in a group of MBP and CFA immunized mice, fiuorescently-labelled
(PKHP), OVA-specific T cells were injected iv (Figure 5.3 for protocol).
This experiment was repeated three times (na). It is known that the
onset of disease symptoms correlates with the infiltration of T cells to the
CNS (Zeine 8 Owens, 1992), therefore, as soon as paralysis was
observed, mice were sacrificed and cells isolated from both the
peripheral lymphoid system (LNs) and CNS. These cells were stained
with antibodies specific for the expression of CD4 and CD45RB. Within
the LN, not only were PKH2+ cells found (Figure 5.4 A), but they were
also CD4+ (Gate R2, Figure 5.4 A). This indicates that OVA-specific cells
had trafficked to this organ. The OVA-specific T cells within the LN were
fuithet analyzed by forward (FSC) and found to be predominantly (96%)
of a small, resting phenotype (Figure 5.4 B). The expression of CD45RB
was then examined for these same cells (Figure 5.4 C). We see in figure
5.4 C that the majority of the bystander cells in the LN had a CD45RBhioh
phenotype, consistent with a resting state. Very few bystander T cells in
the LN were large, CD45RBiow cells (1%). Therefore, only a small
minority of bystander T cells in the LN display characteristics of activated
T cells.
The infiltrating T cells of the CNS were similariy analyzed. Figure
5.5 A shows that OVA-specific bystander T cells entered the CNS along
with endogenous, disease-causing (encephalitogenic) T cells as we see
staining for both CD4+ T cells (endogenous) and CD4+ PKH2+
(bystanders). Of the dye-labelled T cells, 26.96 were blasts (Figure 5.5 B,
upper right quadrant,), whiie the majorÏty were srnall (67%. upper left
quadrant 5.5 B). Analysis of this same cell population showed that the
majority of the CD4+ OVA-reactive blasts in the CNS expressed
CD45RBhieh (figure 5.5 C) indicative of a resting state.
The fact that we found blasting CD45RBlow cells in both the LNs
and CNS of mice where no OVA antigen is predicted to be present may
be interpreted to mean that these bystander T cells were activated in
vivo. Altematively, the observed activation state of the transferred OVA-
specific blasts in the mice with EAE could potentially be the consequence
of residual in vitro stimulation rather than the result of in vivo activation.
In order to differentiate these possibilities, we changed our experimental
system from one in which we transferred bystander T celk to an
immunized mouse (Figure 5.3) to that where the mouse contained only
naïve bystander cells (Figure 5.6). Encephalitogenic T cells from non-
transgenic littermate mice were transferred to TCR transgenic mice
(DO1 1.1 0, (BALWc x SJUJ)F1) mice that are homozygous for the
expression of a T cell receptor specific for an OVA peptide (Figure 5.6).
Non-transgenic littemate mice were immunized with MBP to be
used as a source of T cell lines. These Iines displayed specificity for the
antigen of interest, were CD4+ and strongly labelled with the lipophilic
dye PKHP (data not shown). Pnor to injection, an aliquot of this anti-MBP
T cell line was taken for FACS analysis. In Figure 5.7 we see the
expression levels of CD44 (moderate), CD25 (moderate), pan CD45
(very strong), and IFN-yR (moderate) in comparison to the T cell
hybndoma control (DO1 1.1 0 T cell hybndoma (Bottomly et al., 1989).
These dye-labelled T cells were transferred to naïve 001 1.10
homozygous transgenic mice to induce EAE and disease symptoms
were observed 1 1 days after iv injection in one of the four mice injected.
Both the dye labelled and endogenous T cells were FACS sorted from
the LN and CNS of mice with disease and analyzed for T cell activation
markers and cytokine production.
We observed that the dye-labelled T cells were found in both the
LN and CNS of these bystander mice (see Figures 5.8 and 5.9 dot plots).
In the LN, the T cell activation marker profile of the endogenous
bystander T cells did not differ from that of a naïve mouse (Figure 5.8). In
contrast, the profile for the anti-MBP T cell line indicated that these cells
were activated blasts (Figure 5.8) with increased expression of CD25,
CD44 and FNyR (Figure 5.8).
Within the CNS, once again the bystander T cells that had entered
displayed a T cell activation marker phenotype identical to that of naïve T
cells (Figure 5.9), while the anti-MBP T cells were blasts (Figure 5.9) and
had the same activated phenotype as seen in the LN (Figure 5.9).
These T cells from both the LN and the CNS were FACS sorted
based on dye label and CD4 expression. T cells were re-soited to check
purity and were found to be greater than 94% pure.
RT-PCR analysis of these T cell samples revealed that MBP-
reactive T cells produce message for the inflammatory cytokine IFN? in
the CNS. IFNq message was not produced by bystander T cells (OVA-
peptide-specific) in either the LN or CNS of these bystander mice.
Discussion
W e have tracked and analyzed bystander T cells during EAE. The
surface phenotype and cytokine mRNA levels of these cells as they
accurnulated in the CNS and LN closely resembled that of
unmanipulated, naive T cells. This is in contrast to the encephalitogenic
T cells isolated from mice with EAE, which had a memory/effector
phenotype.
It is currently accepted that activated T cells may cross the blood
brain barfier (discussed in Owens et al., 1994 and Weller et al., 1996). At
the same time, the uninflamed CNS does not contain T cells. This
remains true, even in situations where there is a vigorous peripheral
immune response occurring. Trafficking into the CNS requires that either
the T cell or the BBB endothelium is activated to induce an upregulation
of adhesion molecules to mediate trafficking. According to this model,
the extravasation of an activated T cell has the ability to initiate the
activation of the endothdial cell it is crossing. Detailed charactenzation
of adhesion molecules (reviewed in Butcher 8 Picker, 1996 and Bradley
& Watson, l996), implied that trafficking is antigen-independent. In
support of this, we observed the entry of activated OVA-reactive T cells
into the CNS, consistent with other repoits of non-CNS antigen specific T
cells entering the CNS (Trotter & Steinman, 1984, Wekerle et ai., 1986,
1987, Hickey et al., 1991, Zeine & Owens, 1992, and Barton et a/.,
1995). It has been proposed that although entry to the CNS is activation-
dependent, accumulation and activation are conditional upon antigen-
recognition. Antigen presentation would lead to upregulation of T cell
markers and cytokine production, and lymphocyte activation which we
observed.
Endogenousfy derived T cells from the periphery have been
shown to constitute approxirnately 40070% of the CD4+ population in the
CNS during EAE (Trotter & Steinman, 1984, Wekerie et al., 1986, 1987,
Cross et al., 1 990). There is evidence that such rec~ i ted T cells
contribute to the process of blood brain barrier (BB8) breach. It was
shown using magnetic resonance imaging (MRI), that T cells specific for
non-CNS antigens caused more severe BBB breakdown than
encephalitogenic T cells (Namer et al., 1 993). PPD-specific T cells that
had been activated h vitro, were shown to cause greater rupture of the
blood brain barrier and oedema than MBP-specific cells (Namer et al.,
1993). Interestingly, EAE symptoms were not increased (Namer et al.,
1 993).
Phenotype changes for T cell activation/memory markers are
dynarnic. Specifically, reverse transitions from low to high molecular
weight (MW) CD45RB expression are known to occur (discussed in
MacKay, 1991 ). Such reversais are likely to contribute to the
heterogenous phenotype of bystander populations. The majority of OVA-
reactive bystander T cells in an EAE lesion are small in size and
CD45RBlow. Those bystander T cells that are blasts are mostly
CD4SRBWh. This implies that while most bystander cells in the CNS are
small resting T cells, some appear to have been activated at some point,
but have modulated their activation phenotype marker, CD45RB. A
similar CD45RBhioh non-blast phenotype was observed for OVA-reactive
T cells in the CNS of naïve rnice (Zeine & Owens, 1992). However, we
do observe a small number of bystander T cells that are blasts and
CM5RBiow in phenotype (maximally 5%). It is possible that these cells
are contributing to the ongoing inflammatory response. Within the
constraints of these experiments, we are unable to absolutely confirm or
disallow such a contribution. The role played by such a small number of
bystander T cells is unlikely to be significant.
The encephalitogenic T cells found in the CNS were unique in that
they were almost uniformly of the memoryleffector phenotype. In contrast
to the varied phenotypes observed in the bystander T cell population, the
near homogeneity of encephalitogenic CD4+ T cells further supports the
idea that these activated cells are responding to antigen.
In contrast to the widely-used protocol of transfemng in vitro
activated T cells, in the experiments using DO1 1 .IO transgenics, the
bystander T cells are quite naïve, as they have never been cukured or
manipulated in any manner prior to the induction of disease. Recently,
work completed in a separate system, has confimed the naïve
phenotype of these T cells (Ingulli et al., 1 997). Because there is no
expression of ovalbumin within the mouse the transgenic T cells will not
be activated through TCR:MHC:Peptide mechanisms. This experimental
system therefore allows study of truly naïve T cells. This report
demonstrates that naTve T cells may cross the BBB in certain instances.
We propose that the BBB was amenable to trafficking because
endothelial cells had upregulated their expression of adhesion
molecules due to the previous extravasation of in vitro activated
encephalitogenic T cells. Secondly, this work also demonstrates that T
cells of various activation states enter the perivascular space with relative
ease once activated T cells have done so. The mere presence of such
cells does not mean that they are contributing to the ongoing immune
response. Taken together, the naïve phenotype of the bystander cells in
both sets of experiments, argues against a bystander contribution to EAE.
Instead, our work supports a requirement for antigen recognition in the
CNS in order to recruit a T cell response.
The potential contribution of bystander T cells to human disease is
of clinical relevance. MS patients suffer "bystander" immune responses
regulariy, such as viral infections. In order to design effective therapies,
for diseases such as MS, one must understand which are the effector
cells of the disease. Lymphocytes traffic to the CNS from the peripheral
blood, and this occurs rapidly in patients with progressive MS (Halfer 8
Weiner, 1987). Additionally, BBB leakage allows the entry of unactivated
T lymphocytes (Oksaranta et al.. 1 993). This implies that T cells of many
activation states can traffic to the CNS during MS and potentially
contribute to disease. Our data suggest that these cells do not become
activated simply as a result of exposure to an inflamed environment.
Upregulation of costirnulatory molecules and presentation of CNS
peptides occurs following the initial CNS-specific T cell response in EAE
and this may also occur in MS. In this inflammatory environment,
infiltrating immune cells may therefore recognize their specific antigen.
Such antigen presentation would contribute to the activation of a wider
range of T cells, thereby promoting CNS disease. It is at this stage that
epitope spreading may also contribute to disease. The inflammation and
destruction of CNS tissue will make available a wider range of peptides
for presentation, thus increasing the potential for recruited T cells to find
their appropriate antigen.
There is precedent in the literature for a lack of bystander immune
contribution as it was seen that "bystander activation is not sufficient to
cause clinically manifest autoimmune disease in a transgenic mouse
model of diabetes" (Ehl et al, 1997). Experimental systems studying viral
responses have pinpointed soluble mediators of the immune response
as potentiators of bystander activation. Worù completed by Hickey and
colleagues (1 991) and Tough & Sprent (1 996), has shown activated
bystander T cells in viral immune responses. The mechanism proposed
to be responsible for 'bystander activation" was Interferon I (Tough &
Sprent, 1996). The major dissimilarities between immune responses
elicited by viral infection versus encephalitogenic T cells may be
responsible for this obsewed difference in bystander activation in these
systems. Finally, work completed using passive transfer of V w
encephalitogenic T cells into naive mice, did indeed show bystander T
cells with activated/memory phenotypes (Barton et ai., 1995). In these
experiments, the specificity of the bystander T cells was undetermined. It
is highiy probable that these bystander T cells were specific for CNS
antigens, and thus became activated by antigen presentation within the
CNS. This possibility was precluded in our system using transgenic mice
and no bystander activation was obsewed.
By studying bystander T cells during disease, we have assessed
their contribution to the ongoing inflammatory response. We have
investigated the expression patterns of CD44, CD25 and IFN*, none of
which were upregulated on bystander T cells, although al1 were
expressed on encephalitogenic T cells. Additionally, we analyzed the
expression of IFNq mRNA and found that it was not upregulated in
bystander T cells, in contrast to CNS-reactive, encephalitogenic T cells.
We conclude that mere presence within an inflammatory environment
does not change the activation phenotype of CD4+ T cells. Bystander T
cells do not appear to be contributing to the ongoing inflammatory
response in the CNS during EAE.
Acknowledgments
The authors would like to thank Drs. M. Julius and P. Poussier for
generously providing DO1 1.1 0 mice and Dr. A. Peterson for re-deriving
the line. We are grateful to V. Dodelet for the scientific discussions and
assistance in data acquisition. We also thank J-Y. Lin for completing the
initial experimental procedures, and acknowledge the expert technical
assistance of K. MacDonald, G. Verge, L. Boutbonnière, and G. Chan.
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Figures
Figure 5.1: Short terni culture of LN selects for T cells reactive to OVA
OVA-reactive T cells were generated from LNs of OVA-primed SJUJ
female mice. Culture conditions: MA = medium alone, OVA = ovalbumin
(50 pg/ml), MBP = myelin basic protein (50 pg/ml), PPD = purified protein
derivative (50 j@ml) , IL-2 = 11-2 (5 Ulml), Spleen = irradiated
spleenocytes alone, T alone = T cells alone.
Medium W A PPD MBP I L -2 SPLEEN T alone
Antigen
Figure 5.2: Short terni culture of LN selects for CD4+, CD45RBint T cells
OVA-reactive T cells were generated as described in 5.1 . Post in vitro
selection, activated OVA-reactive T cells stained with anti-CD4 (A) and
CD45RB (B) antibodies and analysed by FACScan. Dotted lines
represent CD4 and CD45RB staining in cornparison to the darkly shaded
histograms of either unstained or secondary alone controls respectively.
1 54
Figure 5.3: Protocol for bystander OVA-specific T cell transfer
Day O
SJUJ female mice were prirned with 400 pg of M W in CFA scat the base of the tail
- Day 7
L e n da- later, mice were boosted with the same emulsion, ' Y , in thenank
Day &IO
Between days 0-1 0, 1 -5-2-0 x 1 07 CD4+, OVA-rga~tk8, PKH2 fluorescently-labelled T cells were injected iv.
Day 14-17
Between days 14-17. miœ exhibited symptoms of EAE and were sacrifii . Inguinal LNs were taken, and miœ were perfuseâ to remove circulating lymphocytes. FACS s a n g was cafried out based on C04+ and PKH2+ expression. mRNA was prepared f t m ~ ~ r t e d -Ils fot RT-PCR analysis.
Figure 5.4: OVA-reactive T cells traffic to the LN, but the majonty have
low FSC
Prior to perfusion, inguinal LNs were removed and stained with anti-CD4
and anti-CD45RB antibodies. Al1 cells were analysed for both PKH2 and
CD4 (A) expression and cells gated as seen in gate R1 figure 5.4 A.
CD4+, PKH2 labelled and unlabelled cells were FACS sorted for future
mRNA analysis (A). The majority (approximately 96%) of CD4+ and
PKH2+ cells that entered the LNs of mice with EAE had low FSC (6).
These same cells were subsequently analysed for CD45RB expression
(C) and it was found that the major@ of CD4+, PKH2+ (OVA-reactive) T
cells in the LN that were blasts were CD45RBhish.
Figure 5.5: OVA-reactive T cells traffic to the CNS
Post-perfusion, CNS tissue was removed and stained with anti-CD4 and
anti-CD45RB antibodies. All CD4+ cells were analyzed (Figure 5.5 A).
Once again, CD4+, PKH2 labelled (+) and unlabelled (-) cells were FACS
sorted for future mRNA analysis. 26Y0 of the CD4+, PKH2+ (OVA-
reactive) T cells in the CNS that had high FSC (blasts) (Figure 5.5 B). Wiihin the CNS, cells with high FSC typically had high CD45RB
expression, while those that were srnall (low FSC) were CD45RBiow. The
majority (67%) of C04+ and PKH2+ cells that entered the CNS of mice
with EAE had low FSC (B). Of those double positive cells with high FSC,
the ovewhelming majority were CD45RBhioh (C).
Figure 5.6: Protocol for DO1 1.1 0 =bystander mouse" experirnent
Day O
Non-transgenic mice were primed with 400 pg of MBP in CFA SC at the base of the tail
Day 7
Seven days îater, mice were boasted with the same emulsion, SC, in the flank-
Day 1 O
Mice were sacrificecl, LNCs cultureci with 50 pghnl MBP for aime da*.
Day 14 -
Cells were coll8Cteû, fluorescent labelleci and injected iv to 0 0 1 1 - 10 transgenic mice.
Day 2 1-23
Between days 21 -23. mice exhibitecl syrnptoms of EAE and were sacrificecl. Inguinal LNs and CNS tissue were taken. FACS sorting was canied out based on CD& and PKH2+ expression. Cells were alsa anaiyzed for T œll activation marker expression. mRNA was prepared ftom s~r ted celk f~ RT- PCR analysis.
Figure 5.7: Characterization of the Anti-MBP T cell line prior to
Injection.
Prior to injection, an aliquot of the anti-MBP T cell Iine raised in non-
transgenic DO 1 1 .10 littermates was taken for FACS analysis. The
efficiency of staining with the lipophilic dye PKHP was assessed (very
strong, not shown) as well as the expression levels of CD44 (moderate),
CD25 (moderate), pan CD45 (very strong), and I FN-fi (moderate).
Expression levels were compared to that of an IL-2-independent T cell
hybridoma control (001 1.1 0).
Figure 5.8: Transgenic bystander T cells of the LN.
See figure 5.6 for expenmental procedure. Cells were isolated from the
LNs prior to perfusion and stained with anti-CD44, CD25 and IFN-yR
antibodies. Both dye-labelled (+) and unlabelled (0 ) CD4+ cells were
analyzed and sorted for future RT-PCR experiments (dot plot). The vast
rnajority of endogenous bystander T cells expressed identical levels of
CD44 CD25 and IFN* as naïve T cells from control, unmanipulated
mica. The anti-MBP T cells expressed high levels of CD44, CD25 and
IFN-yR. Thus, bystander T cells of the LN appear unactivated while the
injected, anti-MBP cells display an effectorlmemory phenotype.
Figure 5.9: Transgenic bystander T cells of the CNS.
Cells were isolated from the perfused CNS and stained as in figure 5.6.
Bystander T cells were found in the CNS (dot plot) and expressed
identical levels of CD44 CD25 and IFN-yR as control T cells. The anti-
MBP T cells expressed the same pattern of these markers in the CNS as
in the LN (CD44, CD25 and IFNqR). Thus the bystander T cells of the
CNS have an unactivated phenotype in contrast to the anti-MBP
memoryhffector cells.
Obl O
CHAPTER 6 Conclusions & Summary
Much of the data presented here has focussed on the role of IFN-y
during EAE. Aithough I have shown that this cytokine is not necessary for the induction of EAE, I do believe that it norrnally plays an important role in CNS inflammation. From the Iiterature and from the work presented
here, I propose that 1FN-y has two functiona It both acts to (A) inhibit
proliferation of T cells. but it also (B) promotes inflammation in tissues (Figure 6.1 ). Following immunization with MBP. the encephalitogenic T
cell response develops in the periphery. In this situation, the dominant
role of IFN-y is to limit the proliferation of such cells. In contrast. the
dominant action this cytokine has in the CNS is pro-inflammatory and
any anti-proliferative effect may be less critical. Indead, T cell proliferation within the CNS is most likely not necessary for the induction
of disease (Willenborg et al., 1988). Thus, the temporal and spatial
expression of IFN-y detemines which functional role dominates. Once
within the CNS, IFN-y made by activated T cells acts on antigen
presenting cells. such as macrophages and microglial cells. and induces
the upregulation of MHC molecules and other cytokines such as TNF-cx
and 11-12. This promotes the induction of an inflarnmatory. Th 1 T cell
response within the CNS. Additionally. IFN-y promotes CNS disease
through its ability to act on the blood brain barrier and make the CNS
more amenable to trafficking lymphocytes.
Strong evidence for this proposal was observed using IFN-y
knockout mice. The presence of IFN-y disallowed the development of
EAE in the BALWc mouse where the encephalitogenic T cell response
rnay be relatively weak. At a simplified level. one can describe the genetic resistance of this mouse as a combination of the anti-proliferative
effects of the IFN-y, and the poor anti-MBP response T cell (Figure 6.2).
Almost certainly. other factors also influence EAE-susceptibility. The
rernoval of the anti-proliferative effects of I FN-y in the GKO-/- revealed the
adequacy of the encephalitogenic T cell response. The resulting CNS
inflammation did not include lFNy obviously, but another classical
inflammatory molecule. TNF-a, was found. Additionally, the lack of IFNq
promoted temporally extended T ceil responses. The resuiting severity of EAE observed in this mouse, although initially surprising, demonstrated
the dual roles this cytokine plays. The anti-proliferative function of IFN-y
was not initially observed in the genetically susceptible mouse strain,
SJUJ, because the encephalitogenic T cell response is so robust.
No major differences in the symptoms of EAE or immune response
have been identified when the 1FN-y and IFN.yft-knock out mice are
compared to those with an undisrupted complement of cytokines.
Therefore. the question of what role does IFN-y have in EAE becornes
pertinent. Transgenic rnice from a number of different labs have
unequivocally demonstrated that the presence of IFN-y in the CNS can
both in itiate spontaneous disease and disallow the expected recovery phase of EAE (Corbin et a/., 1996, Horwitz et al., 1997, Renno et al.. 1997). The experiments with these mice can be criticised as there is no
control of the production of IFNq, resulting in constitutive expression,
which is unlike that observed in a non-transgenic mouse. Nevertheless,
these genetically engineered mice do provide direct evidence that the expression of this cytokine within the CNS can cause CNS inflammation
and EAE symptoms. In the knock out mice, compensatory mechanisms
within the immune system may fulfil critical roles of IFN-y, but in
genetically unmanipulated mice, IFN-y d o have signif icant. disease-
promoting functions during EAE. Finally. from ail of this work. we better
understand why it may be unwise to use IFN-yas a treatment for MS
patients (Pan itch et ai., 1 987).
The deterrninants of T cell encephalitogencity and eventual disease phenotype are intimately linked (Figure 6.3). For EAE, we induce the inlial T cell priming in the periphery. I have shown that this peripheral priming results in a population of activated T cells which have the capacity to produce both Th1 and Th2 cytokines. Activated T cells, regardless of their antigen specificity. are observed within the CNS. I
demonstrated that if the appropriate antigen is not presented, T cells do not mediate an immune response. regardless if other T cells in this CNS
milieu are promoting a general pro-inflammatory response or not. From
this work, I have concluded that bystander T cells do not contribute to EAE. If the appropriate antigen is recognized. typically presented by a cell of the monocyte lineage, then a series of mutual activations will
occur. Norrnally, the T cell will be activated and produce IFN-y. The
m icroglial antigen presenting cell will produce 11-1 2 and TN F-a, promote
the generation of a Th 1 T cell response and upregulate MHC molecules. This activation process continues and affects many ceils of the CNS. The blood brain barrier upregulates the expression of adhesion molecules.
Chemokines are produced within the CNS, and attract further leukocyte trafficking across the activated endothelium. ûther resident cells of the CNS may contribute to the inflammatory and tissue destructive response by making such molecules as nitric oxide. Together, these actions can result in the longtemi activation of microglia and macrophage cells within the CNS. These can then begin the longterm process of destroying the myelin sheath and oligodendrocyte cell.
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Figures
Figure 6.1 Role of IFN-y in EAE
Figure 6.2 T cell Encephalitogenicity vs IFN-y Actions
Periphery CNS lnhibits T cell Proliferation Upregulation of MHC &
lnflamrnatory cytokines
Cytokines MHC
Figure 6.3 Peripheral T cell Activation Leads to Infiammation within the
CNS
