Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

& 2006 ISCBFM All rights reserved 0271-678X/06 $30.00

www.jcbfm.com

Experimental stroke induces massive, rapid

activation of the peripheral immune system
Halina Offner1,2,3, Sandhya Subramanian1, Susan M Parker2, Michael E Afentoulis1,
Arthur A Vandenbark1,3,4 and Patricia D Hurn2
1

Neuroimmunology Research, Veterans Affairs Medical Center, Portland, Oregon, USA; 2Department of
Anesthesiology and Perioperative Medicine, Oregon Health and Science University, Portland, Oregon, USA;
3
Department of Neurology, Oregon Health and Science University, Portland, Oregon, USA; 4Department of
Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, USA

Clinical experimental stroke induces injurious local brain inflammation. However, effects on the
peripheral immune system have not been well characterized. We quantified mRNA and protein levels
for cytokines, chemokines, and chemokine receptors (CCR) in brain, spinal cord, peripheral
lymphoid organs (spleen, lymph node, blood, and cultured mononuclear cells from these sources),
and blood plasma after reversible middle cerebral artery occlusion (MCAO) or sham treatment in
male C57BL/6 mice. Middle cerebral artery occlusion induced a complex, but organ specific, pattern
of inflammatory factors in the periphery. At both 6 and 22 h after MCAO, activated spleen cells from
stroke-injured mice secreted significantly enhanced levels of TNF-a, IFN-c, IL-6, MCP-1, and IL-2.
Unstimulated splenocytes expressed increased chemokines and CCR, including MIP-2 and CCR2,
CCR7 & CCR8 at 6 h; and MIP-2, IP-10, and CCR1 & CCR2 at 22 h. Also at 22 h, T cells from blood and
lymph nodes secreted increased levels of inflammatory cytokines after activation. As expected,
there were striking proinflammatory changes in postischemic brain. In contrast, spinal cord
displayed suppression of all mediators, suggesting a compensatory response to intracranial events.
These data show for the first time that focal cerebral ischemia results in dynamic and widespread
activation of inflammatory cytokines, chemokines, and CCR in the peripheral immune system.
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665. doi:10.1038/sj.jcbfm.9600217; published online
24 August 2005
Keywords: chemokines; cytokines; peripheral immunity; receptors; stroke

Introduction
Clinical stroke and experimental cerebral ischemia
induce local inflammatory processes that undoubtedly contribute to total cerebral injury (Allan and
Rothwell, 2003; del Zoppo et al, 2001). Within
hours, transcription factors are activated locally in
brain tissue (e.g., nuclear factor-kB; ONeill and
Kaltschmidt, 1997) that upregulate proinflammatory
genes, including the cytokines tumor necrosis factor
a (TNF-a) (Liu et al, 1994), interleukin 1b (IL-1b)
(Liu et al, 1993; Wang et al, 1994), IL-6 (Wang et al,
1995a, b), and IL-1 receptor antagonist (IL-1ra)

Correspondence: Professor H Offner, Neuroimmunology Research

R&D-31, Portland VA Medical Center, 3710 SW US Veterans
Hospital Road, Portland, OR 97239, USA.
E-mail: offnerva@ohsu.edu
This work was supported by US Public Health Service NIH Grants
NS33668, NR03521, NS49210, and the Biomedical Laboratory
R&D Service, Department of Veterans Affairs.
Received 5 May 2005; revised 20 June 2005; accepted 14 July
2005; published online 24 August 2005

(Wang et al, 1997), and chemokines such as IL-8

(Liu et al, 1993), interferon inducible protein-10 (IP10) (Wang et al, 1998) and monocyte chemoattractant protein-1 (MCP-1) (Kim et al, 1995; Wang et al,
1995a, b). These factors promote expression of
adhesion molecules by vascular endothelial cells
that allow infiltration into the brain of blood
neutrophils, monocytes, macrophages, and T cells
that promote further brain injury (Barone and
Feuerstein, 1999). Moreover, inflammatory and
antigenic products derived from brain (e.g., myelin
basic protein) may leak across a damaged blood
brain barrier and produce reciprocal systemic
activation.
While postischemic inflammation within brain
has been well studied in models of focal stroke,
systemic inflammatory responses have been poorly
characterized. In patients with stroke, C-reactive
protein, white blood cell counts, and plasma IL-6
levels were increased on admission and persisted
for > 7 days (Emsley et al, 2003). A later study from
this group found a significant correlation in peak
plasma IL-6 levels measured within the first week

Stroke induces peripheral immunity

H Offner et al
655

after the stroke with brain infarct volume, stoke

severity, and long-term clinical outcome (Smith et
al, 2004). Additionally, experimental stroke in mice
caused a reduction in immune cells in peripheral
lymphoid organs and decreased secretion of TNF-a
and IFN-g that contributed to spontaneous bacterial
infections, a leading cause of mortality in stroke
patients (Prass et al, 2003). Gendron et al (2002)
recently showed that occlusion of the left or right
hemispheres caused a reduction in total splenocytes
and CD8 + T cells, and increased splenocyte
proliferation to mitogens. These results suggest that
there may be systemic repercussions in lymphoid
organs that occur in response to postischemic
inflammation in the brain. However, the relationship between such repercussions and CNS pathology is unclear, both from the perspective of the brain
and the peripheral immune system.
To initiate a more comprehensive study of this
problem, the present study quantified mRNA and
protein levels for cytokines, chemokines, and chemokine receptors (CCR) in brain, spinal cord,
peripheral lymphoid organs (spleen, lymph nodes,
and blood), and blood plasma 6 and 22 h after
middle cerebral artery occlusion (MCAO) in C57BL/
6 mice. We found that in addition to previously
described inflammatory changes in the brain, stroke
induced a complex, but organ specific, pattern of
inflammatory factors in the periphery as early as 6 h
after occlusion. These findings indicate that the
drastic inflammatory changes occurring in the
damaged brain are dynamically reflected in the
peripheral immune organs.

Materials and methods

Animals
The study was conducted in accordance with National
Institutes of Health guidelines for the use of experimental
animals, and the protocols were approved by the Institutional Animal Care and Use Committee. Age-matched,
sexually mature male mice (C57BL/6J; Charles Rivers;
body weight 20 to 25 g) were used in all experiments.

Ischemic Model
Focal cerebral ischemia was induced by 90 mins of
reversible MCAO of the right hemisphere under halothane
anesthesia, as previously described (McCullough et al,
2003; Sawada et al, 2000). In brief, mice were anesthetized
with 1.5% to 2.0% halothane in O2-enriched air. The
common carotid artery was exposed and the external
carotid artery was ligated and cauterized. Unilateral MCA
occlusion was performed by inserting a 6 to 0 silicone
coated, nylon monofilament surgical suture with heatblunted tip into the internal carotid artery via the external
carotid artery stump. The tip was positioned at a distance
of 6 mm beyond the internal carotid/pterygopalatine artery
bifurcation, and occlusion was confirmed by a laser-

doppler flow (LDF; Moor Instruments) probe positioned

over the ipsilateral hemisphere at the mid ear-to-eye
distance. The suture was then secured in place, and the
animal was awakened and assessed for intraischemic
neurologic deficit, that is, the presence or absence of
forelimb weakness; torso turning to the ipsilateral side
when held by tail; circling to affected side; inability to
bear weight on affected side; or spontaneous locomotor
activity or barrel rolling. Any animal without a visible
deficit was excluded from the study. At end-ischemia
(90 mins), the animal was briefly reanaesthetized and
reperfusion was initiated by filament withdrawal. Our
MCAO studies involved three separate experiments, each
involving a minimum of three replicate mice per group.

Isolation of Mononuclear Cells from Spleen,

Lymph Nodes, and Blood
Spleen and inguinal LN were isolated from sham and
MCAO mice and a single-cell suspension was prepared by
passing the tissue through a 100 mm nylon mesh screen.
The cells were washed using RPMI and the red cells lysed
using red cell lysis buffer (8.3 g NH4Cl in 0.01 mol/L TRISHCl, pH 7.4) and incubated for 8 mins. The cells were then
washed twice with RPMI, counted and resuspended in
stimulation medium containing 10% FBS for cytokine
detection by CBA and enzyme-linked immunosorbent
assay (ELISA). For real-time polymerase chain reaction
(PCR), splenocytes were pelleted, snap-frozen, and stored
at 801C until tested.
Cardiac blood was collected in 3 mg/ml EDTA. Cells
were then pelleted and the supernatant (plasma) was
collected and stored at 801C until tested for cytokines by
CBA and ELISA. Red cell lysis buffer was added to the cell
pellet and incubated for 10 mins. Cells were washed twice
using RPMI, counted and resuspended in stimulation
medium containing 10% FBS for CBA and ELISA assays.

Preparation of Spinal Cord for Polymerase Chain

Reaction
Spinal columns were dissected out of the mice and the
cords were purged using a 10 cm3 syringe containing
RPMI. The cords were then snap frozen using methylbutane over dry ice and stored at 801C until further testing
with reverse transcription (RT)-PCR.

Terminal Histopathology
The brains were harvested after 22 h of reperfusion and
sliced into five 2-mm-thick coronal sections for staining
with 1.2% triphenyltetrazolium chloride (TTC, Sigma, St
Louis, MO, USA) in saline as previously described
(McCullough et al, 2003). Infarction volume was measured
using digital imaging (MTI Series 68 Video Camera) and
image analysis software (Sigma Scan Pro, Jandel). The area
of infarct was measured on the rostral and caudal surfaces
of each slice and numerically integrated across the
thickness of the slice to obtain an estimate of infarct
volume in each slice.
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

Stroke induces peripheral immunity

H Offner et al
656

Cytokine Determination by Cytometric Bead Array

Spleen, lymph node and blood mononuclear cells were
cultured in a 24-well flat bottom culture plate with 5 mg/ml
plate-bound anti-CD3 and 2 mg/ml anti-CD28 antibodies at
4  106 cells/well in stimulation medium containing 10%
FBS for 24 h. Supernatants were then harvested and stored
at 801C until tested for cytokines. Also, plasma was
collected and frozen at 801C until tested for cytokines.
The mouse inflammation CBA (Cytometric Bead Array) kit
was used to detect IL-12p40, TNF-a, IFN-g, MCP-1, IL-10
and IL-6 simultaneously (BD Bioscience, San Diego, CA,
USA). Briefly, 50 ml of sample was mixed with 50 ml of the
mixed capture beads and 50 ml of the mouse PE detection
reagent. The tubes were incubated at room temperature for
2 h in the dark, followed by a wash step. The samples were
then resuspended in 300 ml of wash buffer before acquisition on the FACScan. The data were analyzed using the
CBA software (BD Biosciences). Standard curves were
generated for each cytokine using the mixed bead standard
provided in the kit and the concentration of cytokine in
the supernatant was determined by interpolation from the
appropriate standard curve.

Enzyme-linked Immunosorbent Assay for Detection

of Interleukin 1b and Interleukin 2
Plasma and culture supernatants from anti-CD3/CD28
antibody-activated spleen, lymph node and blood mononuclear cells were obtained as above. In total, 96-well
plates were coated with 100 ml of anti-mouse IL-1b or IL-2
capture antibody (4 mg/ml) in 1  PBS or sodium bicarbonate coating buffer. Plates were incubated at 41C overnight, washed with buffer (1  PBS/0.05% Tween-20), and
treated with blocking buffer (1  PBS, 2% BSA) for 2 h at
room temperature. Plates were then washed and 100 ml of
sample or standard was added to each well. Interleukin 1b
plates were incubated at room temperature for 2 h whereas
IL-2 plates were incubated at 41C overnight. Plates were
then washed and 100 ml of biotinylated cytokine-specific
antibody was added. Interleukin 1b plates were incubated
at room temperature for 2 h while IL-2 plates were
incubated at room temperature for 45 mins. Plates were
then washed and 100 ml of 1:250 (IL-1b) or 1:400 (IL-2)
diluted HRP was added. Plates were incubated at room
temperature for 30 mins followed by a wash step. This was
followed by addition of 100 ml TMB chromogen (KPL Cat
#52-00-2). The color was allowed to develop for approximately 30 mins, and the reaction stopped by adding 100 ml
stop solution (KPL Cat # 50-85-05). Optical density was
then measured at 450 nm.

Ribose Nucleic Acid Isolation and Reverse

Transcription-Polymerase Chain Reaction
Total RNA was isolated from brains and spinal cords using
the RNeasy mini kit protocol (Qiagen, Valencia, CA, USA)
and then converted to cDNA using oligo dT, random
hexamers and Superscript RT II enzyme (Invitrogen,
Grand Island, NY, USA). Real-time PCR was performed
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

using Quantitect SYBR Green PCR master mix (Qiagen)

and primers (synthesized by ABI). Reactions were conducted on the ABI Prism 7000 Sequence Detection System
(Applied Biosystems, Foster City, CA, USA) to detect
mRNA quantified as relative units (RE). Primer sequences
for the following genes are:

L32:

(F: GGA AAC CCA GAG GCA TTG AC;

R: TCA GGA TCT GGC CCT TGA AC),
IFN-g:
(F: TGC TGA TGG GAG GAG ATG TCT;
R: TGC TGT CTG GCC TGC TGT TA),
TNF-a:
(F: CAG CCG ATG GGT TGT ACC TT;
R: GGC AGC CTT GTC CCT TGA),
IL-10:
(F: GAT GCC CCA GGC AGA GAA;
R: CAC CCA GGG AAT TCA AAT GC),
IL-6:
(F: CCA CGG CCT TCC CTA CTT C;
R: TGG GAG TGG TAT CCT CTG TGA A),
TGF-b1:
(F: CCG CTT CTG CTC CCA CTC;
R: GGT ACC TCC CCC TGG CTT),
IL-13:
(F: ACT GCT CAG CTA CAC AAA GCA ACT;
R: TGA GAT GCC CAG GGA TGG T),
FoxP3:
(F: GGC CCT TCT CCA GGA CAG A;
R: GCT GAT CAT GGC TGG GTT GT),
IL-1b:
(F: TTG ACG GAC CCC AAA AGA TG;
R: TGG ACA GCC CAG GTC AAA G),
RANTES: (F: CCT CAC CAT CAT CCT CAC TGC A;
R: TCT TCT CTG GGT TGG CAC ACA C),
MIP-2:
(F: TGG GCT GCT GTC CCT CAA;
R: CCC GGG TGC TGT TTG TTT T),
IP-10:
(F: CGA TGA CGG GCC AGT GA;
R: CGC AGG GAT GAT TTC AAG CT),
CCR1:
(F: GGG CCC TAG CCA TCT TAG CT;
R: TCC CAC TGG GCC TTA AAA AA),
CCR2:
(F: GTG TAC ATA GCA ACA AGC CTC AAA G;
R: CCC CCA CAT AGG GAT CAT GA),
CCR3:
(F: GGG CAC CAC CCT GTG AAA;
R: TGG AGG CAG GAG CCA TGA),
CCR5:
(F: CAA TTT TCC AGC AAG ACA ATC CT;
R: TCT CCT GTG GAT CGG GTA TAG AC),
CCR6:
(F: AAG ATG CCT GGC TTC CTC TGT;
R: GGT CTG CCT GGA GAT GTA GCT T),
CCR7:
(F: CCA GGC ACG CAA CTT TGA G;
R: ACT ACC ACC ACG GCA ATG ATC),
CCR8:
(F: CCA GCG ATC TTC CCA TTC TTC;
R: GCC CTG CAC ACT CCC CTT A).

Statistical Analyses
Means7s.d. of cytokine and chemokine concentrations
and RE of chemokines and CCR were calculated for groups
of MCAO versus sham treated mice and differences were
evaluated for significance (P < 0.05) using Students t-test.

Results
Physiologic Measurements and Histology

Intraoperative rectal temperature was controlled in

all animals (36.91C70.61C and 36.71C70.61C in
MCAO and shams, respectively). Occlusion was
confirmed in all MCAO animals, and intraischemic
LDF was 13%71% and 11%72% of baseline signal
in the 6 and 22 h MCAO groups. Infarction at 22 h
was present in all animals, and damage was
consistent with previous work in this model (cortex:
48%710% of contralateral cortex; striatum: 76%7

Stroke induces peripheral immunity

H Offner et al
657

12% of contralateral striatum; total: 47%710% of

contralateral hemisphere).

(TNF-a, IL-1b, IL-6, Figure 1A) and chemokines

(RANTES, IP-10, MIP-2, Figure 1C), as well as
noninflammatory factors (TGF-b1, IL-10, IL-13,
Figure 1B), but not IFN-g or FoxP3 (Figures 1A and
1B). In most instances, there was already substantial
basal expression of CCR, and MCAO further enhanced expression of only CCR3 (right hemisphere
only) and CCR8 (both hemispheres) (Figure 1D).
After 22 h of reperfusion, ipsilateral tissue showed
nearly the same exact pattern but generally lower
levels of expression of cytokines and chemokines,
with the exception of IL-6 (Figure 1A) and MIP-2

Middle Cerebral Artery Occlusion-Induced Changes in

Cytokines and Chemokines in Brain and Spinal Cord

As expected, there were striking differences in

cytokines, chemokines, and chemokine receptor
levels in postischemic brain (Figure 1). At 6 h,
ipsilateral cortex and striatum showed pronounced
increases in expression of inflammatory cytokines

Brain

TNF-
10000

15000

6000

RE

RE
5000

4000

0
6h

L R

40000

16000

22h

10000

30000

7500

8000

10000

0
R

6h

22h

2000
RE

RE

RE

600
*

6h

50000

RE

RE

10000
5000
0
L

6h

22h
IL-6

15000

0
L R

L R

6h

40000

10000

30000

7500

20000

5000

10000

2500

0
L R

22h

L R

22h

12500

RE

500

0
L

1500
1000

400
200

22h

2500

800

1000

*
6h

IFN-

5000

0
R

1000

2000

22h

2500
L

3000

12500

40000

20000

6h

IL-1

RE

24000

2000

L R

50000

32000

SHAM
MCAO

0
L R

RE

L R

3000

1000

2000

RE

4000

8000
RE

10000

Spinal Cord

*
6h

22h

Figure 1 Effects of stroke on expression of cytokines and chemokines/receptors in CNS tissue. Brains and spinal cords were collected
from sham and MCAO-treated mice 6 and 22 h after occlusion, and mRNA prepared from ipsilateral (right) and contralateral (left)
hemispheres of brain and spinal cords tissues for RT-PCR analysis. Relative expression (RE) of message levels are presented for (A)
inflammatory cytokines; (B) regulatory cytokines and FoxP3; (C & D) chemokines and chemokine receptors. * Indicates a significant
difference in expression in stroke mice versus sham-treated mice. indicates significant change in expression in ipsilateral versus
contralateral brain hemispheres.
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

Stroke induces peripheral immunity

H Offner et al
658

TGF-1

RE

200000
100000
0
L R

6h

0
6h

1000

2000
1500
1000

500

500
0

0
L R

6h

L R

L R

22h

2500

RE

RE

RE

1500

100000

L R
22h
IL-10
10

3000
2500
2000
1500
1000
500
0

150000
50000

L R

L R

SHAM
MCAO

200000

400000
320000
240000
160000
80000
0

RE

300000
RE

Spinal Cord

Brain

6h

L R

22h

22h
IL-13

1000

200

250

RE

150
*

500

RE

RE

750

100
50

0
L R

6h

L R

L R

500
400
300
200
100
0

L R

22h

6h

22h

6h

22h

FoxP3

RE

RE

150
100
*

50
0
L R

6h

100

100
80
60
40
20
0

L R

80
*

RE

200

60
40
20
0

L R

22h

L R

Figure 1 Continued

(Figure 1C), which were notably increased. However, more widespread changes in expression of CCR
were evident at 22 h, including five-fold additional
increases in intensity of CCR1 and CCR2 (Figure 1C),
and a 40-fold increase in intensity of CCR5 (Figure
1D), and lower but significant changes in CCR3,
CCR7, and CCR8 (Figure 1D).
Changes in expression of inflammatory factors
were not limited to the brain. In spinal cord, many
mediators were decreased at 6 h MCAO (relative to
sham), with a striking reduction in mRNA for most
of the same inflammatory cytokines (TNF-a, IL-1b,
IL-6) and chemokines (IP-10, MIP-2) as were
increased in injured ipsilateral brain tissue (Figures
1A and 1C). The exception was an increase in
expression of IFN-g in spinal cord. Interestingly,
there was a similar reduction of expression at 6 h of
CCR1 and CCR2, but enhanced expression of CCR5
(Figures 1C and 1D). By 22 h after occlusion,
expression of most mediators and CCRs was reduced
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

or normalized, with the exception of TNF-a and

CCR1. These findings suggest an early compensatory
effect within the uninvolved spinal cord tissue in
mice with MCAO that was attenuated by 22 h after
occlusion.

Changes in Peripheral Cytokine Levels Induced by

Stroke

Although induction of inflammatory factors has

been documented in stroke-injured brain tissue,
little is known about these factors in the circulation
or peripheral immune organs. Previous reports
noted a significant increase in IL-6 in blood plasma
from patients with stroke (Emsley et al, 2003; Smith
et al, 2004). We confirmed plasma elevation of IL-6
in MCAO mice at both 6 and 22 h after occlusion, as
well as IFN-g and MCP-1 at the 6 h time point
(Figures 2A and 2B). Other cytokines and chemo-

Stroke induces peripheral immunity

H Offner et al
659

L R

6h

RE

30000
15000
0
6h

MIP-2
*

L R

10000

22h

40000
32000
24000
16000
8000
0

L R

1250
1000
750
500
250
0

22h

6h

22h

450
300

RE

RE

RE

L R

15000

6h

RE

RE

22h

CCR1
300
240
180
120
60
0

20000

L R

250000
200000
150000
100000
50000
0

L R

25000

0
L R

RE
6h

22h

5000

L R

L R

6h

75000
60000
45000
30000
15000
0

100000
80000
60000
40000
20000
0

L R
IP-10

45000

L R

SHAM
MCAO

500
400
300
200
100
0

22h

60000
RE

L R

L R

75000

RE

RANTES
*

500
400
300
200
100
0

RE

500
400
300
200
100
0

Spinal Cord

RE

RE

Brain

150
*

L R

L R

6h

6h

L R

*
22h

22h

450

2400

300

1800
1200

150

L R

600

0
L R

900

300

600

1200

RE

3000
RE

RE

CCR2
600

L R

6h

L R

6h

22h

22h

Figure 1 Continued

kines tested in blood plasma were unchanged at

either time point.
We envisioned that ischemia might also induce
cytokine changes in distant peripheral immune cell
populations. Thus, mononuclear cells were isolated
from various organs 6 and 22 h after MCAO or sham
treatments, and cytokines were assessed by CBA and
ELISA in supernatants of cultures stimulated for an
additional 24 h with plate-bound anti-CD3/CD28
antibodies. The most striking and consistent

changes induced in the stroke mice versus shaminjured mice were observed in the spleen. At both
the 6 and 22 h time points, activated spleen cells
from stroke-injured mice secreted significantly enhanced levels of the inflammatory factors TNF-a,
IFN-g, IL-6, MCP-1, and IL-2 (Figures 2A and 2B),
with increased secretion of the anti-inflammatory
factor, IL-10, only at the 22 h time point. Levels of
IL-12p40 were low and did not change significantly
at either time point after occlusion (not shown).
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

Stroke induces peripheral immunity

H Offner et al
660

Brain

Spinal Cord
CCR3

RE

RE

75
50
25
0
L R

6h

100
80
60

50

L R

22h

7500

300

200
RE

100

0
L R

200

150

150

100

50

0
6h

CCR6

L R

0
22h

RE

RE

RE

300
200
100

0
6h

1500

L R

L R

22h

CCR8

* *

600
RE

RE

300
150

300
250
200
150
100
50
0

L R

6h

22h

6h

22h

1600
1200
800
400

0
L R

2000
*

450

1000
500

L R

RE

L R

22h

40

L R

CCR7

250

6h

60
20

400

500

22h

80

L R

750

6h
100

100

50
L R

22h

L R

RE

200
RE

RE

6h

60

0
L R

22h

120

50
L R

6h

180

2500

240
RE

5000

150

0
L R

CCR5

250

RE

MCAO
100

40
20
0

L R

SHAM

150
RE

100

0
L R

6h

L R
22h

Figure 1 Continued

Moreover, unstimulated spleen tissue from stroke

mice had increased expression of message for MIP-2,
CCR2, CCR7, and CCR8 at the 6 h time point, and
MIP-2, IP-10, CCR1 and CCR2 at the 22 h time point
(Figure 3). Similar increases in secretion of TNF-a,
IL-6, IL-2, and IFN-g (LN only) were observed only at
the 22 h time point in activated lymph node and
blood mononuclear cells (Figures 2A and 2B).
Interestingly, IL-1b was not detected at either time
point in any of the peripheral lymphoid organs or in
plasma (not shown), suggesting that the source of
this cytokine was solely from injured brain. These
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

data show that focal cerebral ischemia produced

local inflammatory effects within the recovering
brain, and distal effects in lymphoid organs. In
contrast, early suppression of inflammatory mediators was observed in spinal cord.

Discussion
It is now well established that the initial insult from
stroke is followed by an early induction of inflammatory cytokines and chemokines that attract mono-

Stroke induces peripheral immunity

H Offner et al
661

TNF-
250

40

Sham
MCAO

75
30

60

pg/ml

100

45
30

50

pg/ml

150

pg/ml

200

pg/ml

90

20

10

15

6h

6h

22h

Spleen

6h

22h

22h

6h

22h

Blood

LN

Plasma

IFN-
2500

60

1500

5
0

6h

20
5
4
3
2
1
0

6
3

22h

6h

Blood

LN

Spleen

6h

22h

6h

22h

pg/ml

500
10

pg/ml

40

pg/ml

pg/ml

12

80

22h

Plasma

IL-6
150

20

120

50

10
5

22h

Spleen

60

40
20
0

6h

100

80

pg/ml

pg/ml

pg/ml

15

pg/ml

100

6h

22h

LN

150

100

6h

22h

Blood

50
15
12
9
6
3
0

6h

22h

Plasma

Figure 2 Effects of stroke on cytokines secreted from stimulated splenocytes, lymph node cells and blood cells, and from blood
plasma. Spleens, lymph nodes, blood, and blood plasma were collected 6 h and 22 h after vascular occlusion and immune cells were
stimulated for 48 h with plate-bound anti-CD3/CD28 antibodies. Supernatants and blood plasma were evaluated for levels of
secreted factors, including (A) TNF-a, IFN-g, and IL-6; and (B) MCP-1, IL-2, and IL-10. * indicates a significant difference in
expression in stroke mice versus sham-treated mice.

nuclear cells and granulocytes, which cause further

damage to the ischemic and surrounding areas of
brain tissue. The results presented above confirm
and extend these previous observations in brain and
are the first to document the additional rapid and
profound effects of focal cerebral ischemia on
systemic immune responses in lymphoid organs.
As early as 6 h after cerebral vascular occlusion,
activated splenocytes released significantly elevated
levels of inflammatory cytokines on stimulation
through the T-cell receptor (TCR). In addition,
unstimulated splenocytes expressed increased message levels for the chemokine, MIP-2, and CCR, and
blood plasma had increased levels of several
inflammatory cytokines, including IL-6 that had
been reported previously in stroke patients (Emsley
et al, 2003; Smith et al, 2004). Later, 22 h after stroke
induction, T cells from spleen as well as blood and
lymph nodes secreted increased levels of inflammatory cytokines and IL-10 (spleen only) after activation. In contrast, spinal cord tissue from mice with

cerebral vascular occlusion had reduced levels of

inflammatory factors, suggesting a compensatory
response to brain injury.
There is consensus that the major cytokine
players that contribute to postischemic inflammation include IL-1, TNF-a, and possibly IL-6 (Zheng
and Yenari, 2004). Interleukin 1 and TNF-a are
pleiotrophic factors that can be detected as early as
1 h after onset of stroke, even before significant
neuronal death (Allan and Rothwell, 2001), but later
may have neuroprotective effects. Both cytokines
promote early-stage inflammation by increasing
expression of chemotactic factors and adhesion
molecules by vascular endothelium leading to early
infiltration of monocytes and macrophages (within
18 h), neutrophils (within 48 h) and T lymphocytes
(within 72 h) (Stevens et al, 2002). These early
inflammatory factors in combination are neurotoxic,
and also induce the production of additional
cytokines and chemokines by other brain cells. In
the damaged brain, IL-1 and TNF-a are largely
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

Stroke induces peripheral immunity

H Offner et al
662

MCP-1
40

300

50

pg/ml

20
10

6h

30
20

6h

30
20
10

22h

22h

Spleen

Sham
MCAO

40

10

50

40

30

200
100

pg/ml

pg/ml

50

pg/ml

400

6h

22h

LN

6h

22h

Blood

Plasma

IL-2
350

30

60

100

pg/ml

250
150

pg/ml

20

40

pg/ml

pg/ml

300

10

20

50

0
6h

22h

6h

Spleen

22h

6h

LN

22h

6h

Blood

22h

Plasma

IL-10
*

70

30

80

20

60

30

40

pg/ml

40

pg/ml

20

pg/ml

pg/ml

15

60

50

10

10

20

20

10

6h

22h

Spleen

6h

22h

LN

6h

22h

Blood

6h

22h

Plasma

Figure 2 Continued

produced by activated microglial cells, but may also

be secreted by other brain cells, including astrocytes, endothelial cells, and neurons (del Zoppo et
al, 2001), and later by infiltrating mononuclear cells
from blood. Studies of IL-6 have produced conflicting results. Interleukin 6 and IL-6R expression
paralleled cell death by neurons after ischemic
insult (Vollenweider et al, 2003), and peak levels
of IL-6 in plasma from stroke patients had prognostic value in predicting long-term outcome (Smith et
al, 2004). However, mice deficient in IL-6 did not
have more severe cerebral damage after stroke than
wild-type mice (Clark et al, 2000), suggesting that
this cytokine may not be critical for stroke pathogenesis.
Other less well-studied proinflammatory chemokines have also been reported in stroke-damaged
brain tissue, including IL-8, MIP-2, and MCP-1 (for
attracting neutrophils and monocytes to the site of
damage), and IP-10, MIP-1a, and RANTES, along
with their respective CCR (Cartier et al, 2005),
particularly CXCR3 (that binds IP-10) and CCR5
(that binds MIP-1a/b, RANTES, and MCP-2). Chemokines induce neuronal death either directly
through neuronal receptors or indirectly via microglial activation and killing, and previous work
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

indicates these factors are induced after MCAO, for

example, IP-10, CXCR3 (Wang et al, 2000), and
CKR5 (Spleiss et al, 1998). Furthermore, intraventricular MIP-1a injection enhances cortical infarction in mice, while pharmacological chemokine
receptor antagonists reduce damage (Takami et al,
2001, 2002). In contrast, the anti-inflammatory and
neuroprotective factors, IL-10, TGF-b, and IL-1ra,
were produced in synchrony with the first wave of
inflammatory cytokines (Allan and Rothwell, 2001;
Stoll, 2002). It is noteworthy that we detected
selectively enhanced expression within the postischemic right brain hemisphere of TNF-a, IL-1b, IL-6,
TGF-b1, IL-10, RANTES, IP-10, MIP-2, and various
CCR at both 6 and 22 h after vascular occlusion, a
pattern that is entirely consistent with previously
published literature.
In stark contrast, the pattern of cytokine expression in spinal cord tissue from stroke-damaged mice
was nearly the reverse image of changes observed in
the damaged right hemisphere, suggesting that
cytokine expression is organ-specific within the
CNS. Whether the apparent immunosuppression in
spinal cord represents a compensatory response to
intracranial events remains to be shown. However,
previous studies in rat show neuronal degeneration

Stroke induces peripheral immunity

H Offner et al
663

RANTES

MIP-2
8000

14

Sham
MCAO

IP-10
4000

12

6000

4000

RE

RE

RE

10

3000

2000

2000
1000

2
0

0
6h

22h

6h

CCR1

6h

22h

CCR2

160

300

CCR4

CCR5
10

90

120

40

6
4

30

100

RE

RE

RE

80

60

200

RE

22h

6h

22h

6h

CCR6

600

0
6h

22h

CCR7

2000

22h

6h

22h

CCR8

300

*
1500
400

RE

RE

RE

200
1000

200

100

500

*
0

0
6h

22h

0
6h

22h

6h

22h

Figure 3 Effects of stroke on expression of chemokines/receptors in spleen tissue. Spleens were collected from sham and MCAOtreated mice 6 and 22 h after occlusion and mRNA evaluated by RT-PCR for expression of chemokines and chemokine receptors. *
indicates a significant difference in expression in stroke mice versus sham-treated mice.

of lumbar sacral spinal cord after permanent MCAO,

accompanied by enhanced tissue immunolabeling
for c fos and OX-42 (a microglial marker), and
increased local levels of TNFa and IL-1b (Fu et al,
2004; Wu and Ling, 1998a, b). Disagreement between these earlier findings and the present ones
may be related to the greater severity of a permanent
MCAO model, species differences (rat versus
mouse), or tissue sampling (lumbar-sacral cord as
compared with the total cord sample in our study).
In this regard, it is noteworthy that Smith et al
(2004) found a correlation between peak levels of
plasma IL-6 and stroke size, severity, and long-term
outcome. These data would support the possibility
that less severe focal ischemia might induce less
pronounced changes in systemic cytokine secretion.
The major finding in this study was the rapid and
widespread increase in production of inflammatory
factors (TNF-a, IL-6, IL-2, MCP-1, and MIP-2) by
basal and activated splenocytes that occurred as
early as 6 h after stroke, with similar changes
occurring later in the spleen as well as in lymph

nodes and blood. Our experiments were performed

in male C57BL/6 mice, exclusively, and both genetic
strain and sex influence innate immunologic responses to injury. For example, C57BL/6 and Balb/c
differ greatly in stress sensitivity and display of
dominant immune characteristics (Chiodini and
Buergelt, 1993) However, the C57BL/6 strain is a
common strain employed in experimental stroke
and is well-characterized in terms of lymphoid and
leukocyte populations and immunocompetence
(Kajioka et al, 2000).
While there were many similarities in the pattern
of expression of splenic versus brain cytokines and
chemokines, there were also striking differences. In
particular, splenic T cells activated with anti-CD3/
CD28 antibodies produced a significant increase in
IFN-g (not observed in brain), but no levels of IL-1b
(observed only in brain). Others have evaluated the
effects of MCAO on lymphoid tissue, demonstrating
extensive loss of lymphocytes in spleen and thymus,
a shift from T helper cell (Th1) to Th2 cytokine
production and increased lymphocyte apoptosis by
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

Stroke induces peripheral immunity

H Offner et al
664

12 h of reperfusion (Gendron et al, 2002; Prass et al,

2003). We observed enormous splenic T-cell cytokine/chemokine production that was readily measured by early reperfusion (minimum 6 h). These
observations are significant in that they may play a
role in the adaptive immune response to stroke.
Recent data in humans suggest that T cell mediated
immune responses are important to both stroke
pathogenesis and outcome (Nadareishvili et al,
2004). Interestingly, induction of T cell tolerance to
brain antigens reduced postischemic brain damage
(Becker et al, 1997). Moreover, data from neuronal
models of traumatic injury suggest that T cells
primed to respond to myelin basic protein can
enhance recovery (Hauben et al, 2000; Moalem et al,
1999). One possibility that must be considered is
that a dysfunctional bloodbrain barrier leads to
exposure of normally cloistered brain structural
elements, initiating autoimmune-like processes
and cell-mediated immune defenses in the periphery that may have varying effects on stroke progression. However, autoimmune responses to newly
released brain antigens would require days, rather
than hours, to manifest.
Given the location of the splenic T cells distant
from the site of cerebral stroke, and low level of
infiltrating mononuclear cells yet present in the
injured brain (Stevens et al, 2002), it is unlikely that
inflammatory cells found in the spleen at 6 h
represent brain-infiltrating cells that had emigrated
out of the damaged brain. However, a second
intriguing possibility is that the rapid inflammatory
response observed in the spleen and the subsequent
spread of activated lymphocytes to lymph nodes and
blood resulted from sympathetic neural stimulation
initiated from within the damaged brain tissue.
Norepinephrine and b2-adrenergic receptor (b2AR)
stimulation from infection and injury has been
strongly implicated in the regulation of the immune
response (reviewed in Kohm and Sanders, 2001),
and it is conceivable that brain injury might also
transmit sympathetic signals to the spleen. This
possible explanation has a number of merits: (1)
cytokines such as IL-1b can directly activate sympathetic neurons, which are known to express IL-1R,
thus allowing for the possibility that the early
induction of IL-1 after stroke might induce efferent
sympathetic signals to peripheral lymphoid organs,
including spleen; (2) sympathetic stimulation results
in the local release of norepinephrine in lymphoid
organs including spleen; (3) the b2AR for norepinephrine is selectively expressed by CD4 + T cells
and B cells; (4) norepinephrine drives Th1 cell
differentiation and secretion of IFN-g through stimulation of b2AR on nave CD4 + T cells by augmenting
the IL-12 signaling pathway (Swanson et al, 2001);
(5) norepinephrine increases the number of circulating lymphocytes, and thus might account for the
later appearance of T cells in the lymph nodes and
blood, which also produced inflammatory cytokines
on stimulation through the TCR.
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665

Lastly, our novel demonstration of a drastic and

rapid release of inflammatory cytokines from activated splenocytes and lymphoid tissue may represent only the initial challenge for the peripheral
immune system imperiled by stroke. In a murine
model similar to the present study, b-adrenoreceptor
mediated systemic immunodeficiency was readily
manifested at 3 days after cerebral ischemia,
characterized by defective IFN-g production, failure
of lymphocyte activation, septicemia, and pneumonia (Prass et al, 2003). We speculate that the
ischemic brain uses sympathetic neural signaling
to trigger, and ultimately exhaust, endogenous
immune defenses against injury, leading to deleterious and organ-specific consequences.

Acknowledgements
The authors thank Ms Eva Niehaus for assistance in
preparing and submitting the manuscript.

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Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654665