Review

Inflammatory mechanisms in ischemic

stroke: role of inflammatory cells
Rong Jin,* Guojun Yang, and Guohong Li*,1
*Department of Neurosurgery, Louisiana State University Health Science Center, Shreveport, Louisiana, USA; and Department
of Internal Medicine, Ningbo University School of Medicine, Ningbo City, China
RECEIVED NOVEMBER 30, 2009; REVISED JANUARY 11, 2010; ACCEPTED JANUARY 17, 2010. DOI: 10.1189/jlb.1109766

ABSTRACT
Inflammation plays an important role in the pathogenesis of ischemic stroke and other forms of ischemic
brain injury. Experimentally and clinically, the brain responds to ischemic injury with an acute and prolonged
inflammatory process, characterized by rapid activation
of resident cells (mainly microglia), production of proinflammatory mediators, and infiltration of various types
of inflammatory cells (including neutrophils, different
subtypes of T cells, monocyte/macrophages, and other
cells) into the ischemic brain tissue. These cellular
events collaboratively contribute to ischemic brain injury. Despite intense investigation, there are still numerous controversies concerning the time course of the
recruitment of inflammatory cells in the brain and their
pathogenic roles in ischemic brain injury. In this review,
we provide an overview of the time-dependent recruitment of different inflammatory cells following focal cerebral I/R. We discuss how these cells contribute to
ischemic brain injury and highlight certain recent findings and currently unanswered questions about inflammatory cells in the pathophysiology of ischemic stroke.
J. Leukoc. Biol. 87: 779 789; 2010.

Introduction
Stroke is the third leading cause of death and the most frequent cause of permanent disability worldwide [1], and inflammation appears to play an important role in the pathogenesis
of ischemic stroke and other forms of ischemic brain injury.
Clinically, the susceptibility of the patients to stroke and the
subsequent prognosis are influenced by systemic inflammatory
processes [2, 3]. Stroke patients with systemic inflammation
exhibit clinically poorer outcomes [4 6]. Experimentally, focal
cerebral ischemia induces a time-dependent recruitment and

Abbreviations: BBBblood-brain barrier, CTcomputed tomography,

DCdendritic cell, DCPDC precursor, ECendothelial cell, I/Rischemia
and reperfusion, LFA-1lymphocyte function associated antigen 1, Mac1leucocyte integrin CD11B/CD18, MCmast cell, MCAmiddle cerebral
artery, MCAOMCA occlusion, mDCmyeloid DC, MMPmatrix metalloproteinase, MPOmyeloperoxidase, MRImagnetic resonance imaging,
pDCplasmacytoid DC, PSGL-1P-selectin glycoprotein ligand-1,
ROSreactive oxygen species, tPAtissue plasminogen activator, Treg
cellT regulatory cell

0741-5400/10/0087-779 Society for Leukocyte Biology

activation of inflammatory cells, including neutrophils, T cells,

and monocytes/macrophages, and inhibiting the inflammatory
response, decreases infarct size and improves neurological deficit in experimental stroke [7, 8]. Although anti-inflammatory
approaches have proven successful in animal models [9 11],
attempts to translate this into clinical application have been
unsuccessful [12, 13], likely as a result of the heterogeneity in
mechanisms underlying postischemic brain inflammation and
the uncertain time window at which inflammation could be
targeted in the human disease situation [13]. Thus, a comprehensive understanding of the time-dependent recruitment of
inflammatory cells following focal cerebral I/R and how these
cells differentially (and synergistically) contribute to ischemic
brain injury is a prerequisite for developing effective therapeutic interventions for the treatment of acute ischemic stroke by
targeting inflammatory pathways in a time-dependent manner.
Despite intense investigation, there are still numerous controversies concerning the time course of the recruitment of
inflammatory cells in the brain and their pathogenic roles in
ischemic brain injury. In the present review, we provide an
overview of the time-dependent recruitment of different inflammatory cells following focal cerebral I/R. This review focuses on the potential contribution of these cells to ischemic
brain injury and highlights recent findings and currently open
questions regarding inflammatory cells in the pathophysiology
of ischemic stroke.

EXPERIMENTAL STROKE MODELS AND

LEUKOCYTE RECRUITMENT
Ischemic stroke results from transient or permanent reduction
in regional cerebral blood flow. In humans, ischemic stroke
occurs most often in the area perfused by the MCA [14]. Studies in animal models of stroke have provided an invaluable
contribution to our current understanding of the pathophysiology of ischemic stroke [15]. One of the most relevant stroke
models involves transient or permanent MCAO in the rats and
mice [14, 15]. Rats are one of the most suitable species for
stroke study because of the pathogenetic similarities of strokes
1. Correspondence: Vascular Biology and Stroke Research Laboratory, Department of Neurosurgery, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA. E-mail: gli@lsuhsc.edu

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Journal of Leukocyte Biology 779

in rats and humans [16]. In recent years, the importance of

mouse MCAO models has increased rapidly with the development of transgenic or knockout techniques for a targeted single gene [15]. Currently, there are three main categories of
transient MCAO: intraluminal MCAO with thread or wire filaments (the most widely used model in the literature); abluminal application of potent vasoconstrictor endothelin-1 to the
MCA; and thromboembolic models, including photochemically
induced thrombotic MCAO and the introduction of emboli
into the cerebral circulation. The details of the design and
operation of these animal stroke models have been described
elsewhere [14, 15]. The results of the comparisons between
transient and permanent MCAO models in rats and mice are
summarized in Table 1.
Emerging data indicate that transient MCAO models may
better mimic the pathophysiology of human stroke compared
with permanent occlusion models in rats and mice (Table 1).
In human stroke, cerebral vessel occlusion is seldom permanent, as most cases of human ischemic stroke have spontaneous or thrombolytic therapy-induced reperfusion [14, 15].
Leukocyte infiltration into the ischemic brain in transient
MCAO models is more prominent, and antileukocyte strategies
(including antiadhesion molecule strategies) have generally
proven to be more effective in animal stroke models of transient but not permanent ischemia [8, 15]. For this reason, experimental studies about leukocyte recruitment and ischemic
brain injury are now performed mostly using transient cerebral
I/R models in rats and mice [8, 15].

TIME-DEPENDENT RECRUITMENT OF
INFLAMMATORY CELLS DURING
CEREBRAL I/R
The brains inflammatory responses to postischemia are characterized by a rapid activation of resident cells (mainly micro-

glial cells), followed by the infiltration of circulating inflammatory cells, including granulocytes (neutrophils), T cells, monocyte/macrophages, and other cells in the ischemic brain
region, as demonstrated in animal models [2225] and in
stroke patients [26 29]. In the acute phase (minutes to hours)
of ischemic stroke, ROS and proinflammatory mediators (cytokines and chemokines) are released rapidly from injured tissue
[22, 23]. These mediators induce the expression of the adhesion molecules on cerebral ECs and on leukocytes and thus,
promote the adhesion and transendothelial migration of circulating leukocytes [8]. In the subacute phase (hours to days),
infiltrating leukocytes release cytokines and chemokines, especially excessive production of ROS and induction/activation of
MMP (mainly MMP-9), which amplify the brain-inflammatory
responses further by causing more extensive activation of resident cells and infiltration of leukocytes, eventually leading to
disruption of the BBB, brain edema, neuronal death, and
hemorrhagic transformation [22, 23] (Fig. 1). However, many
of these proinflammatory factors have a dual role at early and
late stages of stroke. For instance, regardless of the cellular
origin, MMP-9 has been shown to affect early ischemic brain
damage detrimentally but promote brain regeneration and
neurovascular remodeling in the later repair phase [22]. Thus,
a thorough understanding of the time course of events leading
to inflammation in ischemic brain injury is critical [23].

Resident microglia and blood-derived macrophages

Microglial cells, the resident macrophages of the brain, are
activated rapidly in response to brain injury [30, 31]. Experimental data have shown that resident microglia are activated
within minutes of ischemia onset and produce a plethora of
proinflammatory mediators, including IL-1 and TNF-, which
exacerbate tissue damage [3234] but may also protect the
brain against ischemic and excitotoxic injury [3537]. Postischemic microglial proliferation peaks at 48 72 h after focal cere-

TABLE 1. Comparison of Transient and Permanent MCAO Stroke Models in Rats and Mice
Transient MCAO
Reperfusion
Ischemic damage

With MCA reperfusion after a defined period

of focal cerebral ischemia.
Lesions primarily in the ipsilateral cortex and
striatum but also shown in hippocampus.

Leukocyte
infiltration

Inducing adhesion and infiltration of a large

number of leukocytes in the ischemic brain
tissue.

Antileukocyte
(including
antiadhesion
molecule)
therapy
Clinical
relevance

Immunoblocking or genetic deletion of a

number of adhesion molecules (e.g.,
CD11b/CD18, ICAM-1, P-selectin)
effectively reduces ischemic brain injury.
Generally appreciated, as most cases of
human ischemic stroke have spontaneous
or tPA-induced reperfusion.

780 Journal of Leukocyte Biology

Volume 87, May 2010

Permanent MCAO

References

Without reperfusion.

[14, 15]

Lesions primarily in the ipsilateral cortex

but also shown in striatum. Lesion size
in the cortex comparable with or
larger than transient MCAO.
Although inducing a significant amount
of leukocyte rolling and adhesion in
pial venules, only a small number of
leukocytes infiltrated into ischemic
tissue.
Less effective.

[810, 15]

Limited, as human ischemic stroke is

seldom permanent.

[14, 15]

[810, 15]

[911, 1721]

www.jleukbio.org

Jin et al. Inflammatory cells and ischemic stroke

Cerebral I/R

Expression of cytokines (IL-1, IL-6,

TNF-
), chemokines (MCP-1, MIP-1),
and ROS from injured brain tissue

Expression of leukocyte adhesion molecules

both on brain microvascular ECs (ICAM-1, E/P-selectin) and on leukocytes (Mac-1, LFA-1,
L-selectin, and PSGL-1)

Alternation of BBB tight junctions, and adhesion

and transendothelial migration of circulating
cells (neutrophils, monocytes, and T cells)

Amplifying cerebral inflammatory responses,

via production/release of ROS through NADPH
oxidase and MPO, MMPs (especially MMP-9),
cytokines and chemokines
cytokines,

Activation of resident cells

(microglia, astrocytes,
endothelial cells)

Expression of
neurotrophic and
protective factors

Brain regeneration
and neurovascular
remodeling at late
stages of I/R

MMP-9
Severe BBB breakdown, brain edema,
neuronal death, and hemorrhage
transformation

Stroke recovery

Figure 1. Potential inflammatory pathways that respond to cerebral

I/R. Mac-1 is a 2-integrin (CD11b/CD18); PSGL-1 actually functions
as a ligand for E-/P-/L-selectins.

bral ischemia and may last for several weeks after initial injury
[38, 39]. In contrast to the rapid resident microglia response,
blood-derived leukocytes are recruited to the brain tissue, usually with a delay of hours to a few days [24, 25, 40].
However, reactive microglia are morphologically and functionally similar to blood-derived monocyte/macrophages [24,
25]. To date, it has been difficult to distinguish these cells in
the brain, as there is a lack of discriminating cellular markers
[24, 41]. Blood-derived macrophages are able to acquire a
ramified morphology indistinguishable from resident microglia, and reactive resident microglia can develop into a phagocytic phenotype indistinguishable from infiltrating macrophages. Fortunately, the use of chimeric mice with the GFP
bone marrow provides a powerful tool to distinguish their
roles and contributions in ischemic brain injury [24, 25, 41].
Most current data have shown that blood-derived macrophages
are recruited into the ischemic brain tissue, most abundantly
at Days 37 after stroke (but not significant prior to 3 days after cerebral ischemia) [41 44]. Schilling et al. [24, 41, 42]
show that resident microglial activation precedes and predominates over blood-derived macrophage infiltration after transient
MCAO in a chimeric mouse model. These studies demonstrated
that neutrophils are the first blood-derived leukocytes seen at
Day 1 in the damaged brain, whereas blood-derived macrophages (GFP-positive) were rarely observed at Day 2 but
reached peak numbers at Day 7 and decreased thereafter. In
contrast, resident microglial cells (GFP-negative) are already
activated rapidly at Day 1 after focal cerebral ischemia. Intriguingly, even at Days 4 and 7, most macrophage-like cells remain
GFP-negative, indicating that they are resident microglia-derived; however, in mouse models of transient MCAO [45] and
permanent MCAO [25], others demonstrate that blood-de-

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rived macrophages (Iba1-positive) are infiltrated into the brain

24 48 h after focal cerebral ischemia, but the number of the
infiltrating macrophages remains much lower than activated
resident microglia. Together, most current data support the
hypothesis that the vast majority of macrophage-like cells in
the ischemic brain represents activated resident microglia, especially during the first few days following cerebral I/R injury.

Neutrophils
Of the various types of leukocytes, neutrophils are among the
first to infiltrate ischemic brain (30 min to a few hours of focal
cerebral ischemia), peak earlier (Days 13), and then disappear or decrease rapidly with time [8, 23]. However, the infiltrating neutrophils may remain more than 3 days or longer in
the ischemic brain after focal cerebral I/R, but most likely,
their existence is largely masked after 3 days by large-scale accumulation of activated microglia/macrophages in the inflammatory site [46]. In the rat model of endothelin-1-induced cerebral ischemia, Weston et al. [46] observed that neutrophil
infiltration into the brain increases at 1 day, peaks at 3 days,
and although reduced, continues through 7 and 15 days after
cerebral ischemia.
A recent study seems to challenge the current view, as it
provides evidence demonstrating that the recruitment of other
inflammatory cells may precede neutrophil infiltration in response to cerebral ischemia [47]. In a mouse transient MCAO
model, flow cytometric analysis of cell samples isolated from
the ischemic brains shows that the majority of leukocyte cells
in the ischemic hemisphere at 3 days after MCAO includes
neutrophils [47], which is consistent with most reports in the
literature [43, 48, 49]. However, an interesting observation is
that the infiltration of other inflammatory cells, including
macrophages, lymphocytes, and DCs, in the ischemic hemisphere precedes the neutrophilic influx [47].

T lymphocytes
Earlier studies suggest that lymphocyte recruitment into the
brain is involved in the later stages of ischemic brain injury
[50 52]. In a rat model of the photochemically induced focal
ischemia, immunocytochemistry reveals that numerous T cells
infiltrated the border zone around the infarct by Day 3, and
the number of infiltrating T cells increased further between
Days 3 and 7 [50]. In a mouse model of transient MCAO, flow
cytometeric examination of the inflammatory cell infiltration
in the ischemic brain reveals that (CD3) T cells increased
relatively late (3 4 days) postischemia, whereas activated
(CD11b) microglia/macrophages and (Ly6G) neutrophils
increased significantly at earlier times postischemia [52]. However, more recent studies in rodent models demonstrate that T
cells accumulate in the ischemic brain within the first 24 h
after focal cerebral I/R and may influence the evolution of
tissue inflammation and injury prior to their appearance in
the extravascular brain compartment [40, 53]. In recent years,
increasing research efforts have been devoted to the roles of
specific T cell subtypes in ischemic stroke. There are many
subtypes of lymphocytes, and several subtypes of T cells have
been implicated in the pathogenesis of ischemic stroke [8,
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Journal of Leukocyte Biology 781

In addition to the above leukocytes, several other types of inflammatory cells such as DCs and MCs have been implicated
recently in ischemic brain injury. These inflammatory cells are
considered as early responders to act in the first-line defense
in response to cerebral ischemia. In a mouse model of transient MCAO, Felger et al. [54] show that DCs accumulated in
the ischemic hemisphere at 24 h after focal cerebral ischemia,
particularly in the border region of the infarct where T cells
accrued. MCs in the brain are typically located perivascularly
and contain potent, fast-acting vasoactive and proteolytic substances. In a rat model of transient MCAO, Strbian et al. [55]
show that brain MCs regulate early brain swelling and neutrophil accumulation at 4 h after ischemia.
In summary, our current knowledge about the time-dependent infiltration of inflammatory cells into the brain is based
on immunohistochemistry and especially on flow cytometry of
brain samples [47, 52]. However, there are important limitations of these approaches. For flow cytometric analysis, there is
a need to isolate cells from brain tissue using enzymatic digestion ex vivo. The surface antigens for specific types of inflammatory cells may be modulated after the enzymatic digestion.
In addition, immunohistochemistry and flow cytometry cannot
examine dynamic alteration in the same animal as a result of a
nonsurvival procedure. Similarly, our current knowledge about
adhesive interactions of inflammatory cells with cerebral microcirculation after cerebral I/R is based on optical imaging
technologies (especially on intravital microscopy), which allow
for observation and quantification of cell adhesion to the walls
of intact cerebral microvessels [8, 40]. There are important
limitations of these approaches, including the need to examine microvessels on or near the brain surface, labeling the total leukocyte population, and being unable to assess early and
late adhesive events in the same animal as a result of a nonsurvival procedure. Of note, there are many inconsistencies in the
literature about the time course of the recruitment of various
inflammatory cells in the brain following focal cerebral ischemia, even in the very same experimental animal models [47,
52] (Fig. 2).
With improvements in imaging technology and labeling
methods, such as positron emission tomography/single photon
emission tomography and functional MRI, it has now become
possible to examine accurately inflammatory cell trafficking
and the molecular activity (e.g., MPO and oxidative activity)
noninvasively in ischemic brain parenchyma in living animals.
Advanced imaging techniques and experimental approaches
will provide the opportunity to visualize and assess more directly the dynamic profiles of specific inflammatory cell trafficking, adhesive interactions, and molecular activity of these
inflammatory cells with cerebral microcirculation and with
each other in the brains of living animals at early and late
stages of cerebral I/R. The application of such imaging technologies and approaches should help to address some impor782 Journal of Leukocyte Biology

Volume 87, May 2010

10

Fold incrrease in ischemic

m
vs. c
contralateral

Other inflammatory cells

7.5

macrophages /
activated microglia

neutrophils
2.5

T cells

3/4

Days after cerebral I/R

B
70

here
X103 cells/ischemic hemisph

40]. However, the time course of the recruitment of different

subtypes of T cells into the ischemic brain remains largely undetermined.

60
50
40
30
20
10

T cells
0

Days after cerebral I/R

Figure 2. Schematic showing a time-dependent recruitment of inflammatory cells into the brain following focal cerebral ischemia in mice.
The figure, adapted from (A) ref. [52] and (B) ref. [47] with permission. Note that a transient 60-min MCAO model in C57Bl6 mice was
used in both reports.

tant unanswered questions about how these cells contribute to

ischemic brain injury differentially and collaboratively.

ROLE OF ACTIVATED
MICROGLIA/MACROPHAGES IN
CEREBRAL I/R DAMAGE
Resident microglial cells are major inflammatory cells in the
brain that are among the first cells to respond to brain injury,
and multiple lines of evidence have shown that activated microglia play a dual role in ischemic stroke. Microglia exert

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Jin et al. Inflammatory cells and ischemic stroke

neurotoxic functions through the production of ROS via

NADPH oxidase [56], cytokines (IL-1, IL-6, TNF-) [30, 31],
and MMP-9 [57]. These events precede leukocyte infiltration
into the brain and may play a crucial role in mediating the
initial increase in BBB permeability and the early infiltration
of circulating leukocytes into the brain [56 58]. Microglia activation potentiates damage to BBB integrity, whereas inhibition
of microglial activation may protect the brain after ischemic
stroke by improving BBB viability and integrity in vivo and
in vitro [58].
In contrast, activated microglia also appear to play a neuroprotective role after cerebral ischemia [59 62]. Production of
neurotoxic and neuroprotective factors emphasizes the complex role of resident microglia in the process of tissue damage,
neuronal survival, and regeneration in the response to cerebral ischemia. The protective role of microglia is possibly mediated by their ability to eliminate excess excitotoxins in the
extracellular space, in part through phagocytosis of infiltrating
neutrophils [39]. Further, accumulating evidence indicates
that microglia can produce various neurotrophic factors such
as neurotrophins and growth factors (fibroblast growth factor,
TGF-1), which are involved in neuronal survival and brain
tissue repair in cases of brain injury [59 62]. Intriguingly, recent work [63] has identified a neuroprotective role for microglia-derived TNF in cerebral ischemia through TNF-p55R in
mice.
Experimentally, TNF has neuroprotective and neurotoxic
effects. Although TNF can be produced by microglia and infiltrating leukocytes in the brain, the neuroprotective effects of
TNF appear to be attributed to microglia- but not leukocytederived TNF. These findings may have clinical relevance and
potential applications. TNF is implicated in ischemic stroke
and trauma in humans [64], where similar to the mouse [65,
66], it is produced by microglia and infiltrating leukocytes
[67]. In addition, abundant evidence indicates a neuroprotective role of proliferating microglial cells in cerebral ischemia
in vivo [38, 39]. Selective ablation of proliferating microglial
cells exacerbates ischemic brain injury associated with a decrease in insulin-like growth factor-1 and an increase in cytokines (IL-1, IL-6, TNF-) [38].
As discussed above, activated microglia are morphologically
and functionally indistinguishable from blood-derived monocyte/macrophages in the brain. Thus, it has been difficult to
determine their distinct contribution to the pathogenesis of
ischemic stroke. Nevertheless, the difference of the time
course of their recruitment in the brain suggests that they contribute to ischemic brain injury in different time-dependent
manners. Experimental studies using GFP bone marrow chimera mice indicate that blood-derived macrophage infiltration
into the brain occurs at a later time after focal cerebral I/R
[24, 25, 41]. These studies have revealed significant differences in terms of the ratio and contribution of resident microglia versus exogenous infiltrating macrophages to early postischemic cerebral injury. Resident microglia dominate over
blood-derived macrophages during the first 3 4 days of cerebral I/R [24, 25, 41]. In the absence of blood-derived
monocytes, brain microglia is able to differentiate into macrophages [56].

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Regardless of their origin, activated microglia/macrophages

seem to be critical in the clearance of infiltrating neutrophils
after cerebral I/R. As discussed above, neutrophil infiltration
occurs in the first 3 days after cerebral I/R, and thereafter,
macrophage-like cells replace them as the dominant inflammatory cells in the ischemic site. The major pathway for clearance of infiltrating neutrophils and their potentially cytotoxic
substances from the inflammatory sites is apoptosis followed by
engulfment by activated microglia/macrophages [68 71]. Macrophages can resolve neutrophils and therefore, reduce neuronal injury by triggering neutrophil apoptosis, engulfing them,
and thereby preventing the release of cytotoxic substances into
the surrounding tissue [68, 69]. Induction of apoptosis and
phagocytosis of apoptotic neutrophils by reactive microglia/
macrophages is a critical step in the resolution of the inflammatory response and in preventing further exacerbation of the
ischemic injury [69, 71]. In a rat model of endothelin-1-induced cerebral ischemia, Weston et al. [46, 72] demonstrate
that large-scale emigration of neutrophils into the ischemic
region occurs during the first day and peaks at 3 days after
cerebral ischemia. Double immunostaining clearly shows that
macrophages (stained by ED-1) engulf neutrophils (stained by
anti-polymorphonuclear neutrophil sera) in the brain and that
this engulfment of invading neutrophils increases with time
(50% of neutrophils in the brain are engulfed at 3 days and
85% at 15 days) [46]. Nevertheless, it is unclear whether the
ED-1-stained cells in the brain represent activated resident
microglia or/and infiltrating macrophages.

ROLE OF NEUTROPHIL INFILTRATION IN

CEREBRAL I/R DAMAGE
Despite intense investigation, the exact role of neutrophils in
the pathogenesis of ischemic stroke remains under debate.
Most experimental and clinical studies support the importance
of neutrophil infiltration in ischemic stroke. In animal models
of focal cerebral I/R, recruitment of neutrophils in the ischemic brain occurs within 30 min to a few hours and peaks in
the first 3 days [8, 23]. Genetic deficiency or antibody blockade of leukocyte adhesion molecules (e.g., ICAM-1, CD11b/
CD18, P-selectin) [8 11, 1721, 49] has been shown to reduce
infarct volume, brain edema, neurological deficits, and mortality in animal models of ischemic stroke. These protective effects appear to be more effective in the transient but not permanent MCAO models in rats and mice. Clinical studies have
confirmed that neutrophils accumulate intensively in the regions of human cerebral infarction, and this accumulation is
correlated with the severity of the brain tissue damage and
poor neurological outcome after ischemic stroke [28, 29, 73].
Furthermore, total leukocyte and neutrophil counts are increased in the first 3 days after symptom onset in stroke patients, and this is associated with larger final infarct volumes
(on CT and MRI) and increased stroke severity [27, 29]. A
number of potential mechanisms may explain how activation
and accumulation of neutrophils contribute to the pathogenesis of ischemic stroke. These mechanisms include: excessive
production of ROS, such as superoxide and hypochlorous acid
via NADPH oxidase and MPO, respectively; release of a variety
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Journal of Leukocyte Biology 783

of proinflammatory cytokines (IL-1, IL-6, TNF-) and chemokines (MCP-1, MIP-1, IL-8); release of elastase and MMPs
(mainly MMP-9); and enhancing expression of leukocyte 2integrins (Mac-1, LFA-1) and adhesion molecules (PSGL-1,
L-selectin). By these mechanisms, infiltrating neutrophils amplify a cerebral inflammatory response that may exacerbate
ischemic brain injury further [8, 22, 23] (Fig. 1). Nevertheless,
the pathogenic role of neutrophils in ischemic stroke remains
uncertain, and some studies fail to demonstrate a clear correlation between neutrophil infiltration and infarct formation
[74 79].
Recent studies suggest that neutrophil infiltration may play a
more prominent role in the pathogenesis of ischemic stroke in
individuals with elevated systemic inflammation. In stroke patients with prior infection, total leukocyte and neutrophil
counts and the extent of leukocyte-platelet adhesion and activation are elevated in the circulation [29, 80, 81]. Recent experimental studies have shown that systemic inflammation exacerbates neutrophil infiltration in the brain and thus, alters
the kinetics of the BBB tight junction disruption after experimental stroke in mice [4, 82]. These studies clearly demonstrate that infiltrating neutrophils are the primary source of
increased (fivefold) MMP-9 activity in the ischemic brain of
IL-1-challenged mice at 4, 8, or 24 h after MCAO. A transformation from transient to sustained BBB disruption caused by
enhanced neutrophil-derived neurovascular MMP-9 is a critical
mechanism underlying the exacerbation of ischemic brain injury by systemic inflammation, mediated through conversion
of a transient to a sustained disruption of the tight junction
protein, claudin-5, and markedly exacerbated disruption of the
cerebrovascular basal lamina protein, collagen-IV [82]. These
molecular mechanisms may contribute to the poor clinical outcome in stroke patients presenting with antecedent infection.
Stroke patients presenting with an elevated systemic inflammatory status may be at increased risk of MMP-9-mediated neurovascular proteolysis and hemorrhagic transformation [83], particularly when recombinant tPA is administered for thrombolytic therapy, as tPA is known to promotes neutrophil
degranulation and MMP-9 release [84, 85]. In this regard, it is
critical to better understand the exact roles of neutrophils in
the pathogenesis of ischemic stroke under clinically relevant
conditions that are linked to an elevated systemic inflammatory status, such as prior infection, atherosclerosis, type 2 diabetes, obesity, and rheumatoid arthritis.

ROLE OF DIFFERENT SUBTYPES OF T

LYMPHOCYTES IN CEREBRAL I/R
DAMAGE
In recent years, considerable research efforts have been devoted to understanding the roles of lymphocytes in ischemic
brain injury. Several subtypes of T cells have been implicated
in the pathogenesis of ischemic stroke, and accumulating evidence indicates that different subtypes of T cells play differential roles in response to cerebral I/R injury.
784 Journal of Leukocyte Biology

Volume 87, May 2010

CD4 and CD8 T cells

Experimental evidence indicates that in the vascular bed of
other organs (e.g., intestine, liver, and kidney), CD4 and
CD8 T cells contribute importantly to the pathogenesis of
I/R injury [86 88]. Recent work has shown that CD4 and
CD8 T cells are major contributors to brain inflammation in
a mouse model of transient MCAO. Studies using intravital
video microscopy show that Rag1(/), CD4 T cell(/),
CD8 T cell(/), and IFN-(/) mice have comparable,
significant reductions in cerebral I/R-induced leukocyte and
platelet adhesion in cerebral microcirculation, compared with
wild-type mice after exposure to focal cerebral I/R [40]. Futhermore, data indicate that CD4 and CD8 T cells contribute
to the inflammatory and thrombogenic responses, brain infarction, and neurological deficit associated with experimental
stroke [40]. Moreover, experimental studies have shown that
CD4 TH1 cells may play a key role in the pathogenesis of
stroke through releasing proinflammatory cytokines, including
IL-2, IL-12, IFN-, and TNF-, whereas CD4 TH2 cells may
play a protective role through anti-inflammatory cytokines
such as IL-4, IL-5, IL-10, and IL-13 [89]. It is important to
note that some of these cytokines, especially IFN-, are known
to be critical in prevention of infections, which are a leading
cause of death in stroke patients, especially in the postacute
phase of stroke [90], which results mainly from immunodepression caused by depletion of circulating T cell and NK cell
populations and therefore, the antibacterial cytokine IFN- in
the early reperfusion period [90]. Therefore, treatment of
stroke patients by targeting T cells must be designed carefully
to evaluate and reduce deleterious and enhance protective
actions of specific T cell subtypes.

Treg cells
Treg cells come in many forms, including CD4CD25 forkhead box p3 T cells (Tregs) and other subsets. Treg cells play
a key part in controlling immune responses under physiological conditions and in various systemic and CNS inflammatory
diseases [9193]. Experimental data have shown that Treg cells
are capable of modulating effector T cell function and secreting anti-inflammatory cytokines (IL-10, TGF-) [94, 95]. These
actions enable Treg cells to be pivotal players in the fields of
self-tolerance, immunologic homeostasis, and damage control
at the site of inflammation [96]. More recently, an elegant
study by Liesz et al. [97] reveals that the Treg cells are key cerebroprotective immunomodulators in acute experimental
stroke in mice. They found that Treg cells prevent secondary
infarct growth by counteracting excessive production of proinflammatory cytokines and by modulating invasion and/or activation of lymphocytes and microglia in the ischemic brain. Depletion of Treg cells increases delayed brain damage profoundly and deteriorates functional outcome, and Treg cells
antagonize enhanced TNF- and IFN- production, which induce delayed inflammatory brain damage. Also, Treg cell-derived secretion of IL-10 is the key mediator of cerebroprotection via suppression of deleterious cerebral cytokine (TNF-,
IFN-) production. Absence of Treg cells augmented postischemic activation of resident and infiltrating inflammatory cells

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Jin et al. Inflammatory cells and ischemic stroke

including microglia and T cells, the main sources of cerebral

TNF- and IFN- [97, 98], respectively. TNF- expression is
elevated early after ischemia in the brain, where it is generated
predominantly by microglia. Whereas IFN- is almost absent in
normal brain tissue, its expression increases at a later timepoint after cerebral ischemia than does TNF- expression, and
its expression is strongly induced after Treg cell depletion.
Taken together, these findings reveal a previously unknown
role of the Treg cells as cerebroprotective immunomodulators
after stroke, thus potentially providing new insights into the
endogenous adaptive immune response after acute brain ischemia.

T cells
T cells represent a small subset of T cells that possesses a
distinct TCR on their surface. A majority of T cells has a TCR
composed of two glycoprotein chains, called and TCR
chains. In contrast, in T cells, the TCR is made up of one
-chain and one -chain. This group of T cells is usually much
less common than T cells [99]. The conditions that lead to
responses of T cells are not fully understood, and current
concepts of T cells as first line of defense, regulatory
cells, or bridge between innate and adaptive responses [99]
only address facets of their complex behavior. A recent study
by Shibata et al. [100] demonstrates that resident T cells
control early infiltration of neutrophils in the peritoneal cavity
of mice after Escherichia coli infection. They indicate that a
rapid and transient production of IL-17 after i.p. infection
with E. coli precedes the influx of neutrophils. Flow cytometric
analysis of intracellular cytokine demonstrates that the T
cell population is the major source of IL-17. Neutralization of
IL-17 results in a reduced infiltration of neutrophils and impaired bacterial clearance. Mice depleted of T cells by antiTCR- mAb treatment have diminished IL-17 production and
reduced neutrophil infiltration after E. coli infection [100].
More recently, an elegant study by Shichita et al. [101] reveals
a pivotal role of cerebral IL-17-producing T cells in the delayed phase of ischemic brain injury. In a mouse model of
transient MCAO, they demonstrate that the infiltration of T
cells into the brain as well as the production of cytokines IL-17
and IL-23 play pivotal roles in the evolution of brain infarction
and accompanying neurological deficits. Blockade of T cell
infiltration into the brain by the immunosuppressant FTY720
reduced cerebral I/R damage. The expression of IL-23 (most
likely derived from activated microglia/macrophages) [102,
103] increases on Day 1 after I/R, whereas IL-17 levels are elevated after Day 3, and this induction of IL-17 was dependent
on IL-23. Immunohistochemistry shows that T cells are localized in the infarct boundary zones at 4 days after cerebral I/R.
Intracellular cytokine staining confirms that T cells are a
major source of IL-17. Further, gene knockouts demonstrate
that IL-23 functions in the immediate stage of cerebral I/R
injury, whereas IL-17 is an important role in the delayed phase
of cerebral I/R injury, during which apoptotic neuronal death
occurs in the penumbra. A significant reduction in infarct volume is observed in TCR- knockout mice, as well as in mice
treated with TCR--specific antibody [100]. These findings
reveal a previously unknown role of the T cells in the pathowww.jleukbio.org

genesis of ischemic stroke. Therefore, the T cells could be a

novel, therapeutic target for limiting the inflammatory events
that amplify the initial damage during cerebral I/R.

ROLE OF OTHER INFLAMMATORY CELLS

IN CEREBRAL I/R DAMAGE

DCs
DCs are immune cells that form part of the mammalian immune system and constitute key elements in the control of immune activation or immune tolerance [104]. Their main function is to process antigen material and present it on the surface to other cells of the immune system, thus functioning as
effective APCs [105]. There are at least two major lineages of
DCs [106]: mDCs, which respond to bacteria and fungi, releasing IL-12, and pDCs, which release IFN- upon viral infection.
Both lineages are detected as DCPs in blood, patrolling
through the circulation and invading the tissue in response to
a local infection or other inflammatory situation. mDCs
and/or pDCs appear to play a role in several proinflammatory
diseases, especially atherosclerosis [104, 107]. In multiple sclerosis, mDCs invade the human brain, subsequently triggering
cerebral inflammation [108].
Several clinical and experimental studies suggest the potential importance of DCs in cerebral inflammation and tissue
injury during ischemic stroke [54, 109, 110]. Using flow cytometeric analysis of blood samples, Yilmaz et al. [109] found that
acute stroke leads to a significant but transient decrease in circulating DCPs within 24 h after symptom onset in stroke patients, and patients with large stroke size in CT scan have significantly lower mDCP, pDCP, and total DCPs than those with
smaller stroke. Follow-up analysis shows a significant recovery
of circulating DCP in the first 2 4 days after stroke. Double
immunohistochemical staining demonstrates colocalization of
mDCs and T cells and a high expression of HLA-DR close to
mDCs observed, suggesting that mDCs are mature and able to
activate T cells in the infarcted brain [109]. Thus, circulating
DCPs may be recruited into the infarcted brain and thereby
trigger cerebral immune/inflammatory reactions in the brain.
This view is also supported by previous findings that have
shown that DCs are present in the ischemic brain in a rat
model of permanent MCAO [110]. Immunohistochemistry
showed that numbers of DCs are low in nonischemic (sham)
brains but are elevated in the ischemic hemispheres at 1 h
(11-fold increase) and increase further in the 6-day observation period with an 84-fold increase at 6 days after MCAO. Activated DCs expressing MHC-II remain elevated at 6 days after
MCAO in the ischemic versus nonischemic hemispheres [110].
More recently, Gelderblom et al. [47] demonstrate that DCs
are increased by 20-fold on Day 3 and 12-fold on Day 7 and
thus, constituted a substantial proportion of infiltrating cells.
DCs exhibit a significant up-regulation of MHC-II, and the increase of DCs is even more pronounced if only MHCII highexpressing DCs are analyzed (100-fold increase). To date,
there is no direct experimental evidence showing the correlation between the increase of DC numbers and brain infarction
in cerebral ischemia. Nevertheless, these previous observations
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Journal of Leukocyte Biology 785

may constitute a basis for further studies about DCs in the

pathogenesis of ischemic stroke.

MCs
MCs reside in a variety of locations in the brain of different
species, including humans, where they appear to be concentrated in the diencephalic parenchyma, thalamus, and cerebral
cortex [111, 112]. Their subendothelial and perivascular location at the boundary between the intravascular and extravascular milieus and their ability to respond rapidly to blood- and
tissue-borne stimuli via release of potent vasodilatory, proteolytic, fibrinolytic, and proinflammatory mediators render MCs
with a unique status to act in the first-line defense in various
pathologies [55]. Experimental evidence indicates an emerging role of mast cells in cerebral ischemic injury and hemorrhage [55]. In experimental cerebral I/R, MCs regulate BBB
permeability, brain edema formation, and the intensity of local
neutrophil infiltration [55]. Strbian et al. [113] demonstrate
that cerebral MCs regulate early ischemic brain swelling and
neutrophil accumulation in a rat model of transient MCAO.
Pharmacological MC-blocking (sodium cromoglycate) leads to
a 39% decrease in brain swelling, and compound 48/80 (MCdegranulating agent) elevates it by 89%. Early ischemic BBB
leakage and postischemic neutrophil infiltration are significantly lower in MC-deficient rats than in the wild-type. In addition, MCs appear to play a role in the tPA-mediated cerebral
hemorrhages after experimental ischemic stroke and to be involved in the expansion of hematoma and edema following
intracerebral hemorrhage [113, 114]. MC stabilization was reported to reduce hemorrhagic transformation and mortality
after administration of thrombolytics in experimental ischemic
stroke [114]. Thus, MC stabilization may provide an adjuvant
therapy in treatment of acute ischemic stroke in patients.

ANTI-INFLAMMATORY THERAPY
The pathologic processes after ischemic stroke can be separated into acute (within hours), subacute (hours to days), and
chronic (days to months) phases [115, 116]. Clinical and experimental data show an acute and prolonged inflammatory
response in the brain after stroke, and leukocyte recruitment
is a hallmark feature of the prolonged inflammatory response
that occurs over hours to days after cerebral ischemia [117,
118]. Experimental stroke studies demonstrate that reperfusion represents an especially vulnerable period for the brain
[8 11], as it provides the potential benefits of restoring blood
flow to an ischemic region and simultaneously opens the flood
gates for a massive influx of activated leukocytes into ischemic
tissue. Thereby, the subacute reperfusion period after a stroke
is considered more amenable to treatment than acute neurotoxicity [116 118]. It is hypothesized that stroke outcomes
may be improved by antileukocyte strategies (including antiadhesion molecule strategies), which are targeted specifically to
the reperfusion period. This hypothesis is supported by numerous experimental findings [8, 15]. As discussed above, inhibition of leukocyte infiltration into the ischemic brain via
antiadhesion molecules (e.g., CD11b/CD18, ICAM-1, P-selec786 Journal of Leukocyte Biology

Volume 87, May 2010

tin) has been shown to reduce infarct size, edema, and neurological deficits in transient MCAO stroke models in rats and
mice [9 11, 1721, 72], but the benefits do not extend to permanent MCAO [9, 10]. Further, experimental studies demonstrate that antileukocyte strategies may extend the therapeutic
time window of tPA reperfusion therapy in acute stroke [8,
15]. For example, in a rat thromboembolic stroke model, UK279276 treatment reduces infarct size only in combination
with tPA and prolongs the efficacy time window for tPA from
2 h to 4 h [11]. UK-279276 is a recombinant glycoprotein and
is a selective antagonist of the CD11b integrin of Mac-1
(CD11b/CD18) and has been shown to reduce neutrophil infiltration and infarct volume in the transient MCAO model in
rats when administered within 4 h after onset of ischemia
[119]. These results raise the question of whether antileukocyte strategies provide an effective therapy for stroke patients.
Clinically, several drugs that target neutrophil recruitment
have been developed as potential therapies for ischemic
stroke. Three such drugs were tested in clinical trials: a mAb
to ICAM-1 (Enlimomab, R6.5) [12], a humanized antibody to
the CD11b/CD18 (Hu23F2G or LeukArrest) [13], and the
UK-279276 [120]. All clinical trials with these drugs have been
unsuccessful as a result of lack of neuroprotective efficacy and
side-effects such as leukopenia and immunosuppression. These
clinical outcomes further intensify the debate over the role of
neutrophils in ischemic stroke [74 79] and raise the question
of whether inflammation in general and neutrophils in particular may serve as useful therapeutic targets in treatment of
human stroke.
Despite intense investigation, it remains unclear why antiinflammatory therapy succeeded in animal models but not in
clinical application. Can animal models truly replicate human
stroke? The main limitations of the most current animal studies include at least the following: limited clinical relevance of
the experiments in animal stroke models that are performed
in young and healthy animals and normal physiological conditions and targeting single-cell type (mainly neutrophils) and
single adhesion molecule (e.g., ICAM-1 or CD11b/CD18). It is
widely acknowledged that no single animal model replicates
human stroke perfectly, and the current animal models do not
replicate the complexities of human stroke. Nevertheless, animal models can provide mechanistic insights that have correlated quite well with clinical findings in terms of the pathophysiology of stroke [15].
In addition to neutrophils, in recent years, considerable research has been devoted to understanding the roles of other
cell types, in particular, T lymphocyte subtypes in ischemic
brain injury. Many relevant questions remain largely unanswerable, at least at present; for example, how different inflammatory cells work together in the brain after stroke (in temporal
and spatial domains with different time-dependent manners)
and whether (and how) these cells function in a common
pathway contributing to the pathogenesis of ischemic stroke.
There are no definitive answers to questions such as these, because of the complexity and multiplicity of the mechanisms by
which inflammatory cells contribute to ischemic brain damage.
Not only do different types of inflammatory cells contribute
differentially to the pathogenesis of ischemic stroke, but also,

www.jleukbio.org

Jin et al. Inflammatory cells and ischemic stroke

the same cell type may play different roles in different stages
of ischemic stroke. Moreover, the same molecule produced by
different cells (e.g., microglia- and leukocyte-derived TNF-)
may play different roles [63, 64]. Nevertheless, oxidative stress
might serve as a common pathway for different inflammatory
cells [56]. Oxidative stress is an important mediator of tissue
injury in acute ischemic stroke. During ischemic stroke, ROS
are generated by various types of inflammatory cells and trigger the expression of a number of proinflammatory genes,
including cytokines and adhesion molecules, which play an
important role in leukocyte-endothelium interactions and secondary brain damage after cerebral ischemia. These proinflammatory genes are regulated by oxidant-sensitive transcription factors (e.g., NF-B) [56].

CONCLUSIONS
Emerging data suggest that inflammatory cells play complex
and multiphasic roles after ischemic stroke, and most of the
cell types display beneficial and adverse effects. There is a
growing body of evidence that inflammatory cell infiltration is
predominantly deleterious in the early phase after ischemic
stroke. Antileukocyte strategies (including antiadhesion molecule strategies) reduce ischemic brain injury in animal models;
however, attempts to translate experimental findings into clinical therapies have been unsuccessful. Most likely, targeting a
single cell type or single adhesion molecule is not a feasible
way to treat human stroke. Many relevant questions remain to
be answered; for example, how different inflammatory cells
work together in the brain after stroke; whether (and how)
these cells contribute to the pathogenesis of ischemic stroke
via a common pathway; and how to evaluate and reduce deleterious and enhance protective actions of specific types of inflammatory cells. By addressing these questions, future research might provide novel, alternative stroke mechanisms and
develop new therapeutic directions for ischemic stroke. Future
efforts should be directed toward defining the time-dependent
interactions between inflammatory cells and their interactions
with cerebral vasculature with advanced brain imaging technologies and other approaches in animal models and human
stroke patients. Future basic research should be performed
under clinical relevant conditions linked to elevated inflammatory states, such as prior infection, atherosclerosis, and type 2
diabetes. More sophisticated therapies with pleiotropic beneficial effects and more sophisticated targeting of potential inflammatory cells (and molecules) will increase the likelihood
of successful clinical translation [116].

AUTHORSHIP
The concept, design, and writing of the manuscript: Guohong
Li; the literature search and discussion of the manuscript:
Rong Jin and Guojun Yang, who equally contributed to this
work.

ACKNOWLEDGMENTS
The work was supported by the National Institutes of Health
grant HL087990 (G.L.) and by a Scientist Development grant

www.jleukbio.org

(0530166N) from American Heart Association (G.L.). We give

special thanks to Dr. Michael Wyss for critical review of this
manuscript.

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KEY WORDS:
inflammation leukocytes brain ischemia

Volume 87, May 2010

Journal of Leukocyte Biology 789