NeuroMolecular Medicine

Copyright 2005 Humana Press Inc.

All rights of any nature whatsoever reserved.
ISSN1535-1084/05/07:229242/$30.00
doi: 10.1385/NMM:7:3:229

REVIEW ARTICLE

Stroke and T-Cells

Thiruma V. Arumugam,*,1 D. Neil Granger,2 and Mark P. Mattson1

1
Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, 5600 Nathan
Shock Drive, Baltimore, MD 21224; and 2Department of Molecular and Cellular Physiology, Louisiana
State University Health Sciences Center, Shreveport, LA 71130
Received August 10, 2005; Revised August 11, 2005; Accepted August 11, 2005

Abstract
The microvasculature of the brain region affected by a stroke assumes an inflammatory phenotype
that is characterized by endothelial cell activation and barrier dysfunction and the recruitment of
adherent leukocytes. Although most attention has been devoted to the possible role of neutrophils
in the tissue responses to ischemic stroke there is evidence that T-lymphocytes also accumulate in
the postischemic brain. Although comparable detailed analyses of lymphocyte involvement in
ischemic brain injury have not been performed, emerging findings suggest a role for T-cells in the
pathogenesis of ischemic stroke. The recruitment of T-cells to the site of brain injury is critically
dependent on the coordinated expression of adhesion molecules on the activated capillary endothe-
lium. Whether the recruited lymphocytes are acting directly on brain tissue or indirectly through
activation of other circulating blood cells and/or extravascular cells remain unclear. Cytotoxic
CD8+ T-cells may induce brain injury through molecules released from their cytotoxic granules.
CD4+ T-helper 1 (TH1) cells, which secrete proinflammatory cytokines, including interleukin-2 (IL-
2), IL-12, interferon-, and tumor necrosis factor-, may play a key role in the pathogenesis of stroke,
whereas CD4+ TH2 cells may play a protective role through anti-inflammatory cytokines such as
IL-4, IL-5, IL-10, and IL-13. T-cells should be considered as therapeutic targets for ischemic stroke.
However, because infection is a leading cause of mortality in the postacute phase of ischemic stroke,
and considering anti-inflammatory role of CD4+ TH2, treatment targeting T-cells should be carefully
designed to reduce deleterious and enhance protective actions of T-cells.
doi: 10.1385/NMM:7:3:229
Index Entries: Leukocyte; adhesion; stroke; T-cells; ischemia/reperfusion injuries; inflammation.

Introduction stroke and limiting ischemic damage are major goals

to decrease stroke mobility and mortality. Brain
Stroke is a leading cause of morbidity and mor- tissue is exquisitely sensitive to oxygen and glucose
tality in the world, resulting in the death of approx deprivation such that even brief ischemia can initi-
(5) 106 people per year. Enhancing recovery from ate a complex sequence of events that ultimately
*Author to whom all correspondence and reprint requests should be addressed. E-mail: arumugamt@mail.nih.gov

NeuroMolecular Medicine 229 Volume 7, 2005

230
culminates in cellular death. The pathophysiologi-
cal processes in stroke are complex and involve
disruption of the bloodbrain barrier (BBB), energy
failure, loss of cell ion homeostasis, acidosis,
increased intracellular calcium levels, excitotoxicity,
free radical-mediated toxicity, generation of arachi-
donic acid products, cytokine-mediated apoptosis,
activation of glial cells, and infiltration of leuko-
cytes. Evidence of an inflammatory component in
stroke comes from the observations that ischemic
injury can be attenuated by preischemic induction
of systemic neutropenia (Ishikawa et al., 2004a),
pharmacological blockage of adhesion molecules or
their receptors (DeGraba, 1998; Frijns and Kappelle,
2002), targeted disruption of genes encoding inflam-
matory cytokines, and interfering with the action
of inflammatory mediators (Dirnagl et al., 2003; Han
and Yenari, 2003). Recent studies reveal that brain
ischemia is a powerful stimulus to trigger a series
of events that lead to vasodilatation and increased
permeability of local blood vessels, and mobiliza-
tion and infiltration of circulating leukocytes, a
common process shared by all ischemia/reperfu-
sion (I/R) injuries.
In response to I/R, the microvasculature assumes
an inflammatory phenotype characterized by leuko-
cyteendothelial cell adhesion, leukocyte capillary
plugging, endothelial barrier dysfunction, activa-
tion of resident macrophages, and an enhanced gen-
eration of oxidants and inflammatory mediators
(Ishikawa et al., 2004b). The mechanisms that
underlie the microvascular dysfunction and brain
cell death that are associated with ischemic stroke
are currently an area of intense investigation.
Indeed, there are several lines of evidence that sup-
port a role for leukocyte-mediated inflammation in
the pathogenesis of ischemic stroke. The contention
that leukocyte-mediated inflammation is a cause
rather than merely an outcome of the brain injury
resulting from I/R is supported by reports that
administration of blocking antibodies against spe-
cific cell adhesion molecules (CAMs) that mediate
leukocyte recruitment can reduce infarct size and brain
edema in animal models of stroke (Matsuo et al., 1994;
Prestigiacomo et al., 1999). Although leukocyte
recruitment and activation are strongly implicated in
the microvascular dysfunction and tissue injury asso-
ciated with ischemic stroke, relatively little is known
about the contribution of specific leukocyte subpop-
ulations such as CD8+ and CD4+ T-lymphocytes
NeuroMolecular Medicine
Arumugam et al.
to this pathological condition. Most attention has
been devoted to the possible role of neutrophils
because this leukocyte subset accumulates early
after reperfusion. However, there is evidence that
T-lymphocytes also accumulate in the postischemic
brain within a few hours after reperfusion. Cytotoxic
T-cells can contribute to tissue damage by several
mechanisms including release of inflammatory
cytokines that recruit other leukocyte subsets.
Although there is a limited amount of evidence
supporting a role for T-lymphocytes in ischemic
brain injury, there is a large body of evidence that
invokes a major role for lymphocytes in the patho-
genesis of I/R injury in other vascular beds (Le
Moine et al., 2000; Shigematsu et al., 2002; Burne-
Taney et al., 2003). This article will piece together
recent findings related to T-cell involvement in
stroke injury pathogenesis.
Evidence for T-Cell Involvement
in Stroke
Immunohistochemical and flow cytometric stud-
ies of postischemic brain tissue have demonstrated
that the initial rapid recruitment of neutrophils is
followed over the subsequent hours to days by the
accumulation of other leukocyte subsets, including
lymphocytes and monocytes (Campanella et al.,
2002; Stevens et al., 2002). Intravital microscopic
studies of the postischemic brain microcirculation
have also revealed that the accumulation of leuko-
cytes in postcapillary venules is generally accom-
panied by the recruitment of adherent platelets,
suggesting that the venules assume both a proin-
flammatory and prothrombogenic phenotype after
I/R (Ishikawa et al., 2003; Arumugam et al., 2004).
Although the impact of the accumulated platelets
in the postischemic microcirculation and recruit-
ment of T-lymphocytes is not known, these cells
may enhance thrombosis and/or neurovascular
damage with hemorrhage. The binding of activated
platelets to endothelial cells and/or leukocytes
appears to be important during inflammation
because it can influence the intensity of an inflam-
matory response through the release of different
bioactive compounds. In our intravital microscopy
study of leukocyte adhesion in postischemic cere-
bral venules, treatment of mice with antineutrophil
serum to render them completely neutropenic only
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Stroke and T-Cells
reduced the number of adherent leukocytes by 60%,
suggesting that leukocyte subsets in addition to neu-
trophils are trafficking through the brain microcir-
culation 4 h after reperfusion (Ishikawa et al., 2004a).
The revelation that T-lymphocytes participate in the
early neutrophil recruitment and tissue damage fol-
lowing ischemic injury in other organs raises the
question of whether T-cells also contribute to stroke-
induced microvascular dysfunction and tissue
damage.
The role for lymphocytes in the development of
ischemic injury is supported by reports describing
significantly blunted injury responses in mice that
were genetically deficient in one or more lympho-
cyte subpopulations. It has been reported that CD8+-
or CD4+-deficient mice exhibit an improved kidney
function after renal I/R (Ysebaert et al., 2004; Burne-
Taney et al., 2005) and reduced hepatocellular injury
after liver I/R (Zwacka et al., 1997). There are sev-
eral reports describing a critical role for different
lymphocyte-specific (CD11a, CD11b/CD18) and
endothelial cell (intercellular adhesion molecule
[ICAM-1], P-selectin) adhesion molecules in medi-
ating the brain edema and necrosis, and resultant
neurological deficit, caused by transient cerebral
ischemia (Prestigiacomo et al., 1999; Arumugam
et al., 2004; Vemuganti et al., 2004). All of the above-
listed criteria used to implicate a role for adherent
T-cells in the pathogenesis of I/R injury have been
met in animal models of cerebral ischemia. In addi-
tion, evidence derived from studies employing
immunosuppressive agents supported the involve-
ment of these immune cells in the pathogenesis of
ischemic stroke. Together with granulocytes, many
T-cells identified by immuno-cytochemistry
invaded the infarct region (Schroeter et al., 1994;
Jander et al., 1995). After both permanent middle
cerebral artery occlusion (MCAO) and photochem-
ically induced ischemia, CD4+ T-cells infiltrated the
infarct region from day 1 onward. At day 3, their
number increased further and reached a peak
around day 7, followed by a substantial decrease
within the next 7 d. T-cells accumulated in the
boundary zone of the infarction, often in close vicin-
ity to blood vessels. Flow-cytometric analyses of the
inflammatory cells that accumulated in the ischemic
rat brain (using specific surface markers for differ-
ent leukocyte subsets) indicated that although there
is a fourfold larger number of neutrophils in ischemic
vs nonischemic hemispheres at 24 h following
NeuroMolecular Medicine
231
permanent MCAO, there is also a twofold larger
number of T-lymphocytes in the ischemic vs non-
ischemic hemispheres (Campanella et al., 2002). The
immunosuppressive agent FK506, which is used to
prevent T-cell-mediated allograft rejection as well
as other autoimmune diseases, significantly reduced
brain infarct size in a rat model of focal cerebral artery
occlusion (Brecht et al., 2003). However, another
widely used immunosuppressive agent, cyclosporine,
as well as the T-cell activation inhibitor rapamycin,
did not afford protection in the same model system
(Bochelen et al., 1999; Phillis et al., 2002).
Mechanism of T-Cell Recruitment
in Stroke
There are several potential mechanisms/path-
ways that T-cells can employ to promote the adhe-
sion of lymphocytes, neutrophils, and platelets, and
the subsequent tissue injury/organ dysfunction fol-
lowing ischemic stroke (see Fig. 1). Under quiescent
conditions, neutrophils circulate freely and do not
interact significantly with the endothelium. In con-
trast, monocytes and lymphocytes exhibit a
continuous, low-level, physiological traffic across
the vessel wall. Monocytes emigrate from the blood-
stream to mature into tissue macrophages (microglia
in the brain) that may develop tissue- or organ-spe-
cific functions (Guo et al., 2004). Immune surveil-
lance of tissue requires that lymphocytes recirculate
between blood and lymph nodes, gaining entrance
to the latter at the high endothelial venules of post-
capillary venules in lymphoid tissue. Studies using
intravital video microscopy have established a
sequence of events involved in leukocyte emigra-
tion to extravascular sites of inflammation (Ishikawa
et al., 2004b).
The initial, rapidly reversible adhesion of leuko-
cytes to the endothelium under conditions of flow
produces rolling, which is the consequence of shear
forces acting on the leukocyte and adhesive inter-
actions between selectin receptors and their glycol-
conjugate counterstructures. Rolling is mediated
primarily by the interaction of E- and P-selectin on
activated endothelial cells and leukocytes
L-selectins with sialylated and fucosylated carbo-
hydrate moieties such as those expressed on various
membrane glycoproteins, particularly P-selectin
glycoprotein ligand-1 (PSGL-1) (Tailor and Granger,
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232
Fig. 1. Mechanisms of T-cell involvement in stroke.
2000). Rolling is initiated primarily by activation of
the endothelium by extravascular stimuli such as
bacteria-derived products or by endogenous medi-
ators produced by the endothelium or tissue cells.
Early on, rolling is mediated by endothelial
P-selectin and leukocyte L-selectin. E-selectin is
involved only at later time-points, as it is not con-
stitutively expressed or mobilized but rather is
induced over hours by de novo synthesis. Leukocyte
integrin receptors must be minimally adhesive in
order for them to circulate freely, but are able to
NeuroMolecular Medicine
Arumugam et al.
increase adhesivity rapidly. Once lymphocytes are
tethered to the endothelium by selectin interactions,
the leukocyte integrin receptors are activated by
endothelial membrane-expressed platelet-activating
factor (PAF), endothelial membrane-bound
chemokines, or locally secreted chemoattractants
(Hogg et al., 2004). Activation of the leukocyte
integrins increases their affinity for their endothe-
lial ligands of the immunoglobulin gene super-
family (IgSF). The leukocyte integrins involved in
adhesion to endothelial cells include lymphocyte
Volume 7, 2005
Stroke and T-Cells
function antigen-1, (LFA-1 or CD11a/CD18),
macrophage antigen-1 (Mac-1 or CD11b/CD18), and
very late antigen-4 (VLA-4, CD49d/CD29). The IgSF
ligands are ICAM-1 and ICAM-2 for the 2-integrins,
and vascular CAM (VCAM)-1 for VLA-4. The pres-
ence of recruited T-cells at the site of inflammation
is critically dependent on the coordinated expres-
sion of these adhesion molecules and the activated
capillary endothelium.
Naive T-cells that are not activated generally do
not cross the BBB. T-cells readily cross the BBB if a
T-cell response against a central nervous system
(CNS) autoantigen is initiated in lymphoid organs.
T-cells are restimulated on an encounter with the
target immunogen presented by local antigen-
presenting cells (APCs). Thus, several types of CNS
cells that may act as APCs are activated following
stroke, including microglia, and produce proin-
flammatory cytokines such as tumor necrosis factor
(TNF)-, interleukin (IL)-1, and IL-6, which may
enhance the appearance of adhesion molecules
(Price et al., 2003). These cells could act to enhance
the recruitment of T-cells. One possibility is that the
T-cells adhere to venular endothelium, enter the
tissue, and directly elicit cell necrosis. This possi-
bility is supported by reports describing the pres-
ence of T-cells in the brain early after reperfusion.
T-cell recruitment into the CNS is usually observed
in autoimmune and inflammatory diseases such as
experimental autoimmune encephalomyelitis
(EAE) (Stoll et al., 1993; Chavarria and Alcocer-
Varela, 2004). In EAE, systemic immunization with
myelin proteins such as myelin basic protein (MBP)
creates CD4+ helper T-cells that are antigen-specific
and enter the CNS after 1012 d. Second, large num-
bers of nonspecific T-cells and macrophages infil-
trate the CNS and cause myelin destruction and
clinical disease. In cerebral ischemia, the period
between lesion induction and T-cell infiltration is
too short for generation of a systemic, antigen-spe-
cific immune response. Therefore, the T-cell
response is likely to be antigen-nonspecific. Alter-
natively, lymphocytes may initiate and/or propa-
gate the inflammatory response that occurs within
the brain following stroke by releasing mediators
that increase the expression of endothelial CAMs
and/or activate resident macrophages or other pop-
ulations of leukocytes. This possibility is supported
by the general view that lymphocytes enter the brain
in very small numbers early following reperfusion,
NeuroMolecular Medicine
233
and reports describing the presence of activated
circulating T-lymphocytes in blood within the first
9 h of reperfusion following stroke in humans
(Garlichs et al., 2003).
The recognition that leukocyteendothelial cell
adhesion is an important component of the injury
process in postischemic brain has resulted in an
effort to characterize the kinetics of leukocyte accu-
mulation in capillaries (plugging) and postcapillary
venules (adhesion) after I/R and to define the spe-
cific CAMs that mediate this recruitment process.
The leukocyte recruitment response to brain injury
has been studied systematically following acute
focal ischemia by several laboratories. Many com-
pounds released by activated neutrophils and
endothelial cells may act as platelet agonists (e.g.,
superoxide, hydrogen peroxide) or antagonists
(ADPases); conversely, platelets can release factors
that may either inhibit or activate neutrophils (Ward
et al., 1988; Flad et al., 1997; Carden and Granger,
2000). Platelets can also facilitate the formation of
inflammatory mediators by endothelial cells
through transcellular exchange of precursor
metabolites (Whatley et al., 1990). Furthermore,
platelets can enhance the recruitment of leukocytes
into inflamed tissue by serving as a P-selectin-rich
platform on endothelium for leukocyte adhesion
(Vandendries et al., 2004) and by reducing shear
rates in the microcirculation through the release of
potent vasoconstrictors (e.g., thromboxane A).
Similarly, although several intravital microscopic
studies have attempted to define the molecular
determinants of the adhesion of the general leukocyte
population in the postischemic cerebral microcircu-
lation, there have been no attempts to define the
adhesion molecules that account specifically for the
enhanced lymphocyte trafficking (T-cells) in the brain
microcirculation after transient focal ischemia. Such
molecular mechanisms of lymphocyte recruitment
have been assessed, however, in brain microvessels
of mice treated with either TNF- or bacterial endo-
toxin (Piccio et al., 2002), and in mice receiving CD4+
and CD8+ T-lymphocytes obtained from patients with
multiple sclerosis (MS) (Battistini et al., 2003). These
studies have implicated a role for PSGL-1, a major
counter-receptor for P-selectin, VCAM-1, and
CD11/CD18 in the lymphocyte recruitment process.
Whether the same adhesion molecules mediate the
recruitment of lymphocytes into the postischemic cere-
bral microvasculature remains unclear.
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The 2-integrins expressed on T-cells allow these
circulating cells to establish strong adhesive bonds
with vascular endothelium and enable leukocytes
to remain stationary on the vessel wall. The 2-inte-
grins and ICAM-1 are known to mediate the firm
adhesion of leukocytes, including CD4+ and CD8+
T-lymphocytes, in inflamed venules. There are sev-
eral lines of evidence that implicate the 2-integrins
in ischemic stroke. Expression of CD11a/CD18 on
circulating leukocytes is increased in patients with
ischemic stroke (Kim et al., 1995). Furthermore, pre-
treatment of mice with blocking antibodies directed
against CD11a, CD18, or CD11/CD18 reduce infarct
size, edema formation, and neutrophil accumula-
tion after cerebral I/R (Matsuo et al., 1994; Yenari
et al., 1998). Similarly, mice that are genetically defi-
cient in either CD11a, CD11b, CD18, or ICAM-1 are
protected against ischemic brain injury (Prestigia-
como et al., 1999; Soriano et al., 1999; Arumugam
et al., 2004), suggesting that the leukocyte recruit-
ment mediated by these adhesion molecules is crit-
ical for the subsequent tissue injury. It remains
unclear, however, whether the contribution of the
2-integrins in ischemic stroke is entirely related to
modulating the recruitment of neutrophils or
whether these adhesion molecules are linked to the
involvement of CD4+ and CD8+ T-lymphocytes in
this injury process. Our recent findings strongly
implicate both lymphocytes and the 2-integrins in
mediating the recruitment of leukocytes and
platelets in cerebral venules, tissue injury (infarct
volume), and organ dysfunction (neurological score)
that are associated with ischemic stroke (Arumugam
et al., 2004).
Several inflammatory cytokines such as TNF-
and IL-1 as well as chemokines (IL-8, CXCR3) have
been implicated in the pathogenesis of ischemic
stroke. Support for the involvement of these medi-
ators of inflammation is provided by evidence that
the expression of mRNA or protein for these agents
is increased in brain tissue after exposure to ischemia
(Liu et al., 1994; Kostulas et al., 1999; Wang et al.,
2000). The elevated expression of mRNA or protein
occurs shortly after occlusion (within 13 h) and
precedes the influx of leukocytes after reperfusion.
Because cytokines such as TNF- and IL-1 are
known to either prime or activate cerebrovascular
endothelial cells and thereby enhance the surface
expression of endothelial CAMs, it is widely
assumed that the elevated cytokine levels detected
NeuroMolecular Medicine
Arumugam et al.
in postischemic brain tissue contribute to the recruit-
ment of inflammatory cells that mediate some of
the I/R injury (Elneihoum et al., 1996). Further sup-
port for a modulating role of TNF- in ischemic
brain injury is provided by reports describing an
exacerbation of MCAO-induced brain injury when
TNF- is administered by intra-cerebroventricular
injection 24 h prior to the ischemic insult (Barone
et al., 1997), and the reversal of this injury by intra-
ventricular injection of an anti-TNF- monoclonal
antibody (MAb). Treatment with the TNF- block-
ing MAb or soluble TNF-R1 has also been shown to
reduce tissue damage and improve outcome in
animal models of ischemic stroke (Barone et al., 1997;
Nawashiro et al., 1997). Furthermore, selective inhi-
bition of TNF--converting enzyme, an enzyme that
releases TNF- from its inactive cell-bound pre-
cursor, effectively suppresses the ischemia-induced
elevation in brain tissue TNF- levels and reduces
the infarct size and neurological deficit induced by
focal cerebral ischemia in the rat (Wang et al., 2004).
However, the actions of TNF- in I/R injury are
complex, as this cytokine can also act directly on
neurons to promote their survival through a nuclear
factor (NF)-B-mediated mechanism (Gary et al.,
1998). Brain levels of IL-1 are also increased after
ischemia and may exacerbate ischemic brain injury
(Liu et al., 1993). Intra-cerebroventricular adminis-
tration of recombinant IL-1 receptor antagonist
markedly reduces brain damage induced by transient
ischemia (Mulcahy et al., 2003), and a similar level
of protection has been demonstrated in mutant mice
that are genetically deficient in the IL-1-converting
enzyme (Schielke et al., 1998). Finally, there is also
evidence implicating chemokines, such as interferon
(IFN)-inducible protein-10 (IP-10), IFN--induced
monokine, and CXC chemokines in acute ischemic
brain injury (Wang et al., 2000). All of these
chemokines are ligands for the CXCR3 receptor
(whose production is stimulated by IFN-) that is
expressed on T-lymphocytes. A major consequence
of CXCR3 receptor activation is the recruitment of
T-lymphocytes. Acute brain ischemia is associated
with an increased expression of CXCR3 and a con-
comitant accumulation of leukocytes in the affected
brain tissue.
IFN-, considered a key regulator of immune
and inflammatory responses and absent from normal
brain parenchyma, may play a role in T-cell recruit-
ment in stroke. During inflammatory conditions
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Stroke and T-Cells
such as MS, IFN- is produced by infiltrating T-cells
and natural killer cells. The role of IFN- in perma-
nent MCAO is controversial, with one group demon-
strating an elevated IFN- mRNAand another study
unable to corroborate this response. IFN- from a
non-T-cell source may promote T-cell interactions
with the vascular wall (e.g., via upregulation of
CD40 which leads to upregulation of endothelial
adhesion molecules and lymphocyte recruitment;
Omari and Dorovini-Zis, 2003). Adherent lympho-
cytes may in turn release IFN- that activates the
vascular endothelium to support direct platelet con-
tact. This contention is supported by our recent find-
ings that the CD40CD40L dyad is an important
component of the early leukocyte recruitment in a
stroke model (Ishikawa et al., 2005).
Possible Mechanisms
of T-Cell-Mediated Stroke Injury
Cytotoxic T-cells (CD8+) recognize their antigen
in the context of major histocompatibility (MHC)
class I antigens on the surface of respective target
cells. In normal human brain tissue, class I antigens
are constitutively expressed on all cerebral endothe-
lial cells and on some microglia cells (Traugott, 1987).
Under inflammatory conditions, these MHC mole-
cules may appear on any cell type of the nervous
system (Vass and Lassmann, 1990), as their expres-
sion is regulated by the intensity of the proinflam-
matory stimulus (e.g., IFN- and TNF-) (Vass and
Lassmann, 1990; Neumann et al., 1995; 1997) and
the integrity of the target cell. Thus, in principle, all
cells of the nervous system may become targets for
cytotoxic T-lymphocytes. Cytotoxic T-cells may kill
their target cells either through their cytotoxic gran-
ules, containing perforin and granzymes, or through
TNF family receptor-mediated cytotoxicity, as in the
interaction between Fas-receptor with Fas-ligand.
For these reasons, direct CD8+ T-cell-mediated cyto-
toxicity could play a major role in mediating inflam-
matory tissue damage in the CNS following brain
I/R. In experimental models, MHC class II-specific
CD4+ cells have been shown to be instrumental in
the clearance of virus infection in the CNS. CD4+
T-cells can be categorized into T-helper 1 and 2 (TH1
and TH2) subsets, based on their profiles of cytokine
expression. CD4+ TH1 cells, which mediate EAE,
secrete proinflammatory cytokines, including IL-2,
NeuroMolecular Medicine
235
IL-12, IFN-, and TNF-, whereas TH2 cytokines
IL-4, IL-5, IL-10, and IL-13 are thought to have down-
regulatory properties. IFN- is thought to have a
key role in the pathogenesis of MS, and in EAE, TH1
T-cells are also strongly associated with clinical dis-
ease (Neumann et al., 2002; Segal, 2003). In human
stroke or in experimental models of stroke, the pos-
sible pathogenic role of CD4+ TH1 cells has so far
received little attention, despite the abundance of
CD4+ TH1 lymphocytes within the lesions.
Although the CNS is isolated from the immune
system by the BBB, activated CD4+ cells easily infil-
trate the CNS following cerebral I/R (Hickey and
Kimura, 1988; Schroeter et al., 1994). Consequently,
microglia have the opportunity to retain and further
stimulate CD4+ cells already primed to differentiate
into TH1 cells producing proinflammatory cytokines
(IL-2, IFN-, TNF-) or into TH2 cells producing
cytokines that support antibody-mediated responses
(IL-4, IL-5, IL-10, lL-13) (Mosmann and Sad, 1996).
However, it is has only recently been shown that
antigen-inexperienced, and thus unprimed, T-cells
are also able to permeate the BBB following brain
inflammation. However, these T-cells do not express
other T-cell activation markers, such as CD40 ligand,
and thus cannot interact with microglia through
CD40, the receptor for CD40 ligand. A recent study
by Fee et al. (2003) using traumatic brain injury sug-
gests that CD4+ T-cells contribute to the acute brain
damage following trauma; however, as in the case
of stroke injury the precise mechanism involved
remains to be defined. One possible mechanism is
that CD4+ T-cells participate in inducing apoptosis
of brain cells by producing inflammatory cytokines,
such as IL-2, IFN-, and TNF-, or via FAS-depen-
dent mechanisms (McLaurin et al., 1995; Aktas et al.,
2005; Chaparro-Huerta et al., 2005). In addition,
unregulated access of CD4+ T-cells to the CNS fol-
lowing ischemic stroke can lead to the dysregula-
tion of CD4+ cells, which then results in inappropriate
tissue damage. This dysregulation might be a con-
sequence of the constant lack or temporary absence
of inflammatory mediators and costimulatory
molecules readily available to CD4+ T-cells in lym-
phoid tissues. There is now abundant evidence from
several laboratories that immune pathology in non-
lymphoid tissues, for example the intestine, involves
the development of a dysregulated TH1 response in
various cytokine-deficient mouse models (Groux
and Powrie, 1999). IFN- and TNF--secreting CD4+
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236
TH1 cells were shown to be involved in intestinal
lesions (Bregenholt and Claesson, 1998). Our work
and another recent study using traumatic brain
injury model (Fee et al., 2003) provide evidence for
the possibility of dysregulated access of activated
CD4+ T-cells to the CNS during traumatic injury or
cerebral I/R. Activated T-cells may also contribute
to the pathogenesis of ischemic stroke by accumu-
lating within the microcirculation and impairing cap-
illary perfusion and exacerbating the hypoxic stress.
Neuroprotective Role for T-Cells
Following Stroke
Our recent studies and the observations of others
in studies of experimental stroke, EAE, and in an
animal model for MS, clearly point to a primary role
for T-cells in the pathogenesis of brain disease. How-
ever, despite their pathogenic potential, a neuro-
protective role for T-cells after CNS injury has been
suggested. In different experimental models of brain
injury, it has been shown that T-cells are capable of
protecting nervous tissue from secondary injury
(Schwartz and Hauben, 2002). Autoimmune T-cells
specific for MBP could prevent neurons from the
spread of neuronal death caused by primary injury
to the spinal cord (Moalem et al., 1999). T-cells spe-
cific for Copaxone 1, a compound used for MS ther-
apy, decreases secondary degeneration after optic
nerve crush (Kipnis et al., 2000). Passive and active
immunization with myelin peptides is also protec-
tive against neuronal death in spinal cord injury
(Hauben et al., 2000). In addition, it has been shown
that MBP-specific T-cells prevent cyst formation in
a spinal cord injury model (Butovsky et al., 2001).
These findings imply that T-cell responses against
specific self-antigens are not necessarily detrimen-
tal, and may have a neuroprotective role following
cerebral ischemia.
Because cell death following stroke might take
several days, the prime goal of neuroprotection is
to salvage the ischemic inflammation at the penum-
bra in which most of the apoptotic death occurs.
Anti-inflammatory responses of T-cells can be medi-
ated by cytokines including IL-10 and transform-
ing growth factor (TGF)-1 (Yoshimura et al., 2003;
OGarra et al., 2004; Grutz, 2005). IL-10 is preferen-
tially produced by Tr1-type regulatory T-cells, and
TGF-1 is preferentially produced by TH2-type reg-
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Arumugam et al.
ulatory cells, which both suppress TH1 cells. It has
been reported that elderly patients with a history
of stroke have significantly lower median IL-10
levels compared with elderly without stroke (van
Exel et al., 2002; Vila et al., 2003). Within the brain,
IL-10 may deactivate macrophage-like cells and
astrocytes and thus limit their involvement in a
secondary inflammatory process. Furthermore, IL-10
limits the role of glutamate cytotoxicity by inacti-
vation of NF-B (Bachis et al., 2001), a transcription
factor that modulates inflammation and key regu-
latory proteins in cerebral ischemia. IL-10 also
prevents adhesion and extravasion of leukocytes by
targeting the interface between the BBB (Kubes and
Ward, 2000). Moreover, TGF-1 is expressed in brain
following ischemia (Buisson et al., 2003) and may
play a role as an anti-inflammatory cytokine with
neuroprotective properties. TGF-1 has been shown
to protect cultured neurons from hypoxic (Prehn
et al., 1993), excitotoxic (Prehn and Krieglstein,
1994), apoptotic (Prehn et al., 1994), and toxic
peptide-induced injury (Meucci and Miller, 1996).
In vivo, administration of recombinant TGF-1 or
gene delivery of TGF-1 potently protects animals
against brain injury mediated by ischemic (McNeill
et al., 1994), excitotoxic (Ruocco et al., 1999), and
transient global ischemic injury (Henrich-Noack et
al., 1996). Neuroprotection by TGF- 1 has been
shown to involve the upregulation of antiapoptotic
Bcl-2 family proteins Bcl-2 and Bcl-xL (Kim et al.,
1998). These are target genes of the transcription
factor NF-B and that neuroprotection elicited by
TGF-1 and TNF- requires NF-B activation (Matt-
son et al., 1997; Zhu et al., 2004). However, it is also
important to note here that NF-B activation fol-
lowing brain ischemia may also be involved in the
activation of proinflammatory genes. Other neuro-
protective mechanisms mediated by T-cells follow-
ing stroke may be explained by anti-inflammatory
cytokines and growth factors produced by TH2
T-cells such as IL-4, IL-5, and IL-13. However, there
is no well-documented evidence for this.
Therapeutic Strategies to Target
T-Cells in Ischemic Stroke
Many therapeutic strategies for autoimmune dis-
eases and neurodegenerative disorders are based
on induction of immune regulation with the aim of
Volume 7, 2005
Stroke and T-Cells
altering or downregulating the phenotype of the
T-cell-mediated response. According to the studies
involving T-cells and ischemic stroke, clinical treat-
ment protocols targeting T-cells would have to be
carefully designed to target both deleterious and
protective actions of T-cells and also on their nat-
ural susceptibility to disease development. There-
fore, regulation-based therapies targeting T-cells for
stroke-related pathologies should be designed and
administered with caution. There are many strate-
gies to target T-cell-mediated pathogenesis in stroke
such as T-cell receptor (TCR) antagonism, admin-
istration of antiadhesion molecules, and anticy-
tokine treatment. The TCR recognizes an antigen as
a short peptide bound to a major MHC molecule on
the surface of APCs. As pointed out earlier, inter-
action between the TCR and MHCpeptide com-
plex leads to activation of T-cells. T-cell responses
to MHC molecules during cerebral ischemia or
during reperfusion and its deleterious effects can
be inhibited by introducing subtle changes in the
antigenic peptide amino acid content. Certain
analogs of an antigenic peptide containing single
amino acid substitutions in the TCR contact residues
fail to stimulate T-cell responses on their own, but
are able to diminish, or even inhibit, T-cell activa-
tion induced by the original peptide. The inhibitory
actions of TCR antagonists have been shown not
only in vitro but also in vivo (Kuchroo et al., 1994;
Williams et al., 1998; Toda et al., 2000). However,
the effect of TCR antagonists on the primary
responses of T-cells following cerebral ischemia has
not yet been determined.
The crucial role of adhesion molecules in the
pathogenesis of stroke makes them interesting tar-
gets for drug development. Studies with antiadhe-
sion strategies have been undertaken against many
inflammatory diseases in which T-cells involve-
ment is crucial in the disease development. However,
there have been a remarkable number of therapeu-
tic failures involved with clinically introduced anti-
adhesion therapies such as anti-ICAM-1 antibody
(Enlimomab Acute Stroke Trial Investigators, 2001).
Treatment with a murine anti-ICAM-1 antibody was
investigated in patients with acute ischemic stroke
in the Enlimomab acute stroke trial but, unfortu-
nately, the case fatality rate was significantly higher
in the Enlimomab patient group than in the placebo
group. Adverseeffects of Enlimomab may have been
caused by immunological factors, such as a specific
NeuroMolecular Medicine
237
humoral or cytotoxic immune response against the
medication because of its murine origin or a non-
specific complement activation induced by presen-
tation of Fc fragments (Furuya et al., 2001).
Another way of targeting T-cell adhesion during
cerebral I/R is to administer molecules or antibod-
ies that target LFA-1. Our previous findings impli-
cate a role for LFA-1 (CD11a/CD18) and Mac-1
(CD11b/CD18) in the recruitment of leukocytes in
cerebral venules following ischemic stroke (Aru-
mugam et al., 2004). Efalizumab (Raptiva, anti-
CD11a) is a humanized form of a murine antibody
directed against CD11a, the -subunit of LFA-1. In
vitro studies demonstrated that by binding to
CD11a, Efalizumab inhibits multiple key pathogenic
steps: T-cell activation (Werther et al., 1996), cuta-
neous T-cell trafficking (Gottlieb et al., 2000), and
T-cell adhesion. These findings suggest that Efal-
izumab exerts its clinical activity via several distinct
mechanisms. A small molecule antagonist of LFA-1
(BIRT 377) that inhibits LFA-1ICAM-1 molecular
interactions has also been developed and tested
against different disease conditions that involve T-
cells (Kapadia et al., 2001). However, neither Efal-
izumab nor BIRT 377 has been used in preclinical
or clinical studies of stroke.
Clearly, proinflammatory cytokines such as
TNF-, IFN-, IL-1, and IL-6 produced by T-cells
are involved in the pathogenesis of stroke and some
TH2-specific anti-inflammatory cytokines such as IL-
10 and IL-4 might exert beneficial effects as explained
in the previous section. Targeting T-cell-specific
proinflammatory cytokines without affecting ben-
eficial cytokines might be another way of treating
stroke. However, it should be noted here that most
of these proinflammatory cytokines produced
during stroke are produced by macrophages and
glial cells rather than T-cells. Thus, targeting inflam-
matory cytokines may be an effective option for the
therapy of stroke. In this regard, TNF-, IL-1, IL-6,
IL-18, and IFN- may represent interesting targets
for the treatment of stroke.
In conclusion, the brain microvasculature after a
stroke assumes an inflammatory phenotype that is
characterized by leukocyteendothelial cell adhe-
sion, leukocyte capillary plugging, endothelial bar-
rier dysfunction, activation of resident macrophages,
and an enhanced generation of oxidants and
inflammatory mediators. T-lymphocytes also accu-
mulate in the postischemic brain and contribute to
Volume 7, 2005
238
tissue damage by several mechanisms. Cytotoxic T-
cells may play deleterious roles either through their
cytotoxic granules, containing perforin and
granzymes, or through TNF family receptor-medi-
ated cytotoxicity. CD4+ cells also infiltrate the CNS
following stroke and contribute to the acute brain
injury by producing proinflammatory cytokines.
On the other hand anti-inflammatory responses of
T-cells can be mediated by cytokines including IL-
10 and TGF-1 preferentially produced by TH2-type
regulatory cells. Clinical treatments targeting T-cells
may have beneficial effects against stroke injury, but
must be designed to differentially target the dele-
terious and protective actions of T-cells.
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