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Experimental stroke induces massive, rapidactivation 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

1Neuroimmunology Research, Veterans Affairs Medical Center, Portland, Oregon, USA; 2Department ofAnesthesiology and Perioperative Medicine, Oregon Health and Science University, Portland, Oregon, USA;3Department of Neurology, Oregon Health and Science University, Portland, Oregon, USA; 4Department ofMolecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, USA

Clinical experimental stroke induces injurious local brain inflammation. However, effects on theperipheral immune system have not been well characterized. We quantified mRNA and protein levelsfor 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 inmale C57BL/6 mice. Middle cerebral artery occlusion induced a complex, but organ specific, patternof inflammatory factors in the periphery. At both 6 and 22 h after MCAO, activated spleen cells fromstroke-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 andlymph nodes secreted increased levels of inflammatory cytokines after activation. As expected,there were striking proinflammatory changes in postischemic brain. In contrast, spinal corddisplayed 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 widespreadactivation of inflammatory cytokines, chemokines, and CCR in the peripheral immune system.Journal of Cerebral Blood Flow & Metabolism (2006) 26, 654 665. doi:10.1038/sj.jcbfm.9600217; published online24 August 2005

Keywords: chemokines; cytokines; peripheral immunity; receptors; stroke

Introduction

Clinical stroke and experimental cerebral ischemiainduce local inflammatory processes that undoubt-edly contribute to total cerebral injury (Allan andRothwell, 2003; del Zoppo et al, 2001). Withinhours, transcription factors are activated locally inbrain tissue (e.g., nuclear factor-kB; ONeill andKaltschmidt, 1997) that upregulate proinflammatorygenes, including the cytokines tumor necrosis factora (TNF-a) (Liu et al, 1994), interleukin 1b (IL-1b)(Liu et al, 1993; Wanget al, 1994), IL-6 (Wang et al,1995a, b), and IL-1 receptor antagonist (IL-1ra)

(Wang et al, 1997), and chemokines such as IL-8(Liuet al, 1993), interferon inducible protein-10 (IP-10) (Wang et al, 1998) and monocyte chemoattrac-tant protein-1 (MCP-1) (Kim et al, 1995; Wanget al,1995a, b). These factors promote expression ofadhesion molecules by vascular endothelial cellsthat allow infiltration into the brain of bloodneutrophils, monocytes, macrophages, and T cellsthat promote further brain injury (Barone andFeuerstein, 1999). Moreover, inflammatory andantigenic products derived from brain (e.g., myelinbasic protein) may leak across a damaged bloodbrain barrier and produce reciprocal systemicactivation.

While postischemic inflammation within brainhas been well studied in models of focal stroke,systemic inflammatory responses have been poorlycharacterized. In patients with stroke, C-reactiveprotein, white blood cell counts, and plasma IL-6levels were increased on admission and persistedfor > 7 days (Emsleyet al, 2003). A later study fromthis group found a significant correlation in peakplasma IL-6 levels measured within the first week

Received 5 May 2005; revised 20 June 2005; accepted 14 July2005; published online 24 August 2005

Correspondence: Professor H Offner, Neuroimmunology ResearchR&D-31, Portland VA Medical Center, 3710 SW US VeteransHospital Road, Portland, OR 97239, USA.E-mail: [email protected] work was supported by US Public Health Service NIH GrantsNS33668, NR03521, NS49210, and the Biomedical LaboratoryR&D Service, Department of Veterans Affairs.

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

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after the stroke with brain infarct volume, stokeseverity, and long-term clinical outcome (Smith etal, 2004). Additionally, experimental stroke in micecaused a reduction in immune cells in peripherallymphoid organs and decreased secretion of TNF-aand IFN-g that contributed to spontaneous bacterialinfections, a leading cause of mortality in stroke

patients (Prass et al, 2003). Gendron et al (2002)recently showed that occlusion of the left or righthemispheres caused a reduction in total splenocytesand CD8 + T cells, and increased splenocyteproliferation to mitogens. These results suggest thatthere may be systemic repercussions in lymphoidorgans that occur in response to postischemicinflammation in the brain. However, the relation-ship between such repercussions and CNS pathol-ogy is unclear, both from the perspective of the brainand the peripheral immune system.

To initiate a more comprehensive study of thisproblem, the present study quantified mRNA andprotein levels for cytokines, chemokines, and che-mokine receptors (CCR) in brain, spinal cord,peripheral lymphoid organs (spleen, lymph nodes,and blood), and blood plasma 6 and 22h aftermiddle cerebral artery occlusion (MCAO) in C57BL/6 mice. We found that in addition to previouslydescribed inflammatory changes in the brain, strokeinduced a complex, but organ specific, pattern ofinflammatory factors in the periphery as early as 6 hafter occlusion. These findings indicate that thedrastic inflammatory changes occurring in thedamaged brain are dynamically reflected in theperipheral immune organs.

Materials and methods

Animals

The study was conducted in accordance with NationalInstitutes of Health guidelines for the use of experimentalanimals, and the protocols were approved by the Institu-tional 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 90mins ofreversible MCAO of the right hemisphere under halothaneanesthesia, as previously described (McCullough et al,2003; Sawadaet al, 2000). In brief, mice were anesthetizedwith 1.5% to 2.0% halothane in O2-enriched air. Thecommon carotid artery was exposed and the externalcarotid artery was ligated and cauterized. Unilateral MCAocclusion was performed by inserting a 6 to 0 siliconecoated, nylon monofilament surgical suture with heat-

blunted tip into the internal carotid artery via the externalcarotid artery stump. The tip was positioned at a distanceof 6 mm beyond the internal carotid/pterygopalatine artery

bifurcation, and occlusion was confirmed by a laser-

doppler flow (LDF; Moor Instruments) probe positionedover the ipsilateral hemisphere at the mid ear-to-eyedistance. The suture was then secured in place, and theanimal was awakened and assessed for intraischemicneurologic deficit, that is, the presence or absence offorelimb weakness; torso turning to the ipsilateral sidewhen held by tail; circling to affected side; inability to

bear weight on affected side; or spontaneous locomotoractivity or barrel rolling. Any animal without a visibledeficit was excluded from the study. At end-ischemia(90mins), the animal was briefly reanaesthetized andreperfusion was initiated by filament withdrawal. OurMCAO studies involved three separate experiments, eachinvolving 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 andMCAO mice and a single-cell suspension was prepared by

passing the tissue through a 100m

m nylon mesh screen.The cells were washed using RPMI and the red cells lysedusing red cell lysis buffer (8.3 g NH4Cl in 0.01 mol/L TRIS-HCl, pH 7.4) and incubated for 8 mins. The cells were thenwashed twice with RPMI, counted and resuspended instimulation medium containing 10% FBS for cytokinedetection by CBA and enzyme-linked immunosorbentassay (ELISA). For real-time polymerase chain reaction(PCR), splenocytes were pelleted, snap-frozen, and storedat 801C until tested.

Cardiac blood was collected in 3 mg/ml EDTA. Cellswere then pelleted and the supernatant (plasma) wascollected and stored at 801C until tested for cytokines byCBA and ELISA. Red cell lysis buffer was added to the cell

pellet and incubated for 10mins. Cells were washed twiceusing RPMI, counted and resuspended in stimulationmedium containing 10% FBS for CBA and ELISA assays.

Preparation of Spinal Cord for Polymerase ChainReaction

Spinal columns were dissected out of the mice and thecords were purged using a 10cm3 syringe containingRPMI. The cords were then snap frozen using methylbu-tane over dry ice and stored at 801C until further testingwith reverse transcription (RT)-PCR.

Terminal Histopathology

The brains were harvested after 22 h of reperfusion andsliced into five 2-mm-thick coronal sections for stainingwith 1.2% triphenyltetrazolium chloride (TTC, Sigma, StLouis, MO, USA) in saline as previously described(McCulloughet al, 2003). Infarction volume was measuredusing digital imaging (MTI Series 68 Video Camera) andimage analysis software (Sigma Scan Pro, Jandel). The areaof infarct was measured on the rostral and caudal surfacesof each slice and numerically integrated across thethickness of the slice to obtain an estimate of infarctvolume in each slice.

Stroke induces peripheral immunityH Offner et al

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Cytokine Determination by Cytometric Bead Array

Spleen, lymph node and blood mononuclear cells werecultured in a 24-well flat bottom culture plate with 5 mg/mlplate-bound anti-CD3 and 2 mg/ml anti-CD28 antibodies at4 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 wascollected and frozen at 801C until tested for cytokines.

The mouse inflammation CBA (Cytometric Bead Array) kitwas used to detect IL-12p40, TNF-a, IFN-g, MCP-1, IL-10and IL-6 simultaneously (BD Bioscience, San Diego, CA,USA). Briefly, 50 ml of sample was mixed with 50 ml of themixed capture beads and 50 ml of the mouse PE detectionreagent. The tubes were incubated at room temperature for2 h in the dark, followed by a wash step. The samples werethen resuspended in 300ml of wash buffer before acquisi-tion on the FACScan. The data were analyzed using theCBA software (BD Biosciences). Standard curves weregenerated for each cytokine using the mixed bead standardprovided in the kit and the concentration of cytokine in

the supernatant was determined by interpolation from theappropriate standard curve.

Enzyme-linked Immunosorbent Assay for Detectionof Interleukin 1band Interleukin 2

Plasma and culture supernatants from anti-CD3/CD28antibody-activated spleen, lymph node and blood mono-nuclear cells were obtained as above. In total, 96-wellplates were coated with 100 ml of anti-mouse IL-1bor IL-2capture antibody (4mg/ml) in 1 PBS or sodium bicarbo-nate coating buffer. Plates were incubated at 41C over-night, washed with buffer (1 PBS/0.05% Tween-20), and

treated with blocking buffer (1 PBS, 2% BSA) for 2 h atroom temperature. Plates were then washed and 100 ml ofsample or standard was added to each well. Interleukin 1bplates were incubated at room temperature for 2 h whereasIL-2 plates were incubated at 41C overnight. Plates werethen washed and 100 ml of biotinylated cytokine-specificantibody was added. Interleukin 1bplates were incubatedat room temperature for 2 h while IL-2 plates wereincubated at room temperature for 45 mins. Plates werethen washed and 100 ml of 1:250 (IL-1b) or 1:400 (IL-2)diluted HRP was added. Plates were incubated at roomtemperature for 30 mins followed by a wash step. This wasfollowed by addition of 100ml TMB chromogen (KPL Cat

#52-00-2). The color was allowed to develop for approxi-mately 30 mins, and the reaction stopped by adding 100mlstop solution (KPL Cat # 50-85-05). Optical density wasthen measured at 450 nm.

Ribose Nucleic Acid Isolation and ReverseTranscription-Polymerase Chain Reaction

Total RNA was isolated from brains and spinal cords usingthe RNeasy mini kit protocol (Qiagen, Valencia, CA, USA)and then converted to cDNA using oligo dT, randomhexamers and Superscript RT II enzyme (Invitrogen,Grand Island, NY, USA). Real-time PCR was performed

using Quantitect SYBR Green PCR master mix (Qiagen)and primers (synthesized by ABI). Reactions were con-ducted on the ABI Prism 7000 Sequence Detection System(Applied Biosystems, Foster City, CA, USA) to detectmRNA quantified as relative units (RE). Primer sequencesfor the following genes are:

Statistical Analyses

Means7s.d. of cytokine and chemokine concentrationsand RE of chemokines and CCR were calculated for groupsof MCAO versus sham treated mice and differences wereevaluated for significance (P< 0.05) using Studentst-test.

Results

Physiologic Measurements and Histology

Intraoperative rectal temperature was controlled inall animals (36.91C70.61C and 36.71C70.61C inMCAO and shams, respectively). Occlusion wasconfirmed in all MCAO animals, and intraischemicLDF was 13%71% and 11%72% of baseline signalin the 6 and 22 h MCAO groups. Infarction at 22 hwas present in all animals, and damage wasconsistent with previous work in this model (cortex:48%710% of contralateral cortex; striatum: 76%7

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).


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12% of contralateral striatum; total: 47%710% ofcontralateral hemisphere).

Middle Cerebral Artery Occlusion-Induced Changes inCytokines and Chemokines in Brain and Spinal Cord

As expected, there were striking differences incytokines, chemokines, and chemokine receptorlevels in postischemic brain (Figure 1). At 6 h,ipsilateral cortex and striatum showed pronouncedincreases in expression of inflammatory cytokines

(TNF-a, IL-1b, IL-6, Figure 1A) and chemokines(RANTES, IP-10, MIP-2, Figure 1C), as well asnoninflammatory factors (TGF-b1, IL-10, IL-13,Figure 1B), but not IFN-g or FoxP3 (Figures 1A and1B). In most instances, there was already substantialbasal expression of CCR, and MCAO further en-hanced expression of only CCR3 (right hemisphere

only) and CCR8 (both hemispheres) (Figure 1D).After 22 h of reperfusion, ipsilateral tissue showednearly the same exact pattern but generally lowerlevels of expression of cytokines and chemokines,with the exception of IL-6 (Figure 1A) and MIP-2

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Figure 1 Effects of stroke on expression of cytokines and chemokines/receptors in CNS tissue. Brains and spinal cords were collectedfrom 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 significantdifference in expression in stroke mice versus sham-treated mice. indicates significant change in expression in ipsilateral versuscontralateral brain hemispheres.


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(Figure 1C), which were notably increased. How-ever, more widespread changes in expression of CCRwere evident at 22 h, including five-fold additionalincreases in intensity of CCR1 and CCR2 (Figure 1C),and a 40-fold increase in intensity of CCR5 (Figure1D), and lower but significant changes in CCR3,CCR7, and CCR8 (Figure 1D).

Changes in expression of inflammatory factorswere not limited to the brain. In spinal cord, manymediators were decreased at 6 h MCAO (relative tosham), with a striking reduction in mRNA for mostof the same inflammatory cytokines (TNF-a, IL-1b,IL-6) and chemokines (IP-10, MIP-2) as wereincreasedin injured ipsilateral brain tissue (Figures1A and 1C). The exception was an increase inexpression of IFN-g in spinal cord. Interestingly,there was a similar reduction of expression at 6 h ofCCR1 and CCR2, but enhancedexpression of CCR5(Figures 1C and 1D). By 22 h after occlusion,expression of most mediators and CCRs was reduced

or normalized, with the exception of TNF-a andCCR1. These findings suggest an early compensatoryeffect within the uninvolved spinal cord tissue inmice with MCAO that was attenuated by 22 h afterocclusion.

Changes in Peripheral Cytokine Levels Induced byStroke

Although induction of inflammatory factors hasbeen documented in stroke-injured brain tissue,little is known about these factors in the circulationor peripheral immune organs. Previous reportsnoted a significant increase in IL-6 in blood plasmafrom patients with stroke (Emsleyet al, 2003; Smithet al, 2004). We confirmed plasma elevation of IL-6in MCAO mice at both 6 and 22 h after occlusion, aswell as IFN-g and MCP-1 at the 6 h time point(Figures 2A and 2B). Other cytokines and chemo-

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kines tested in blood plasma were unchanged ateither time point.

We envisioned that ischemia might also inducecytokine changes in distant peripheral immune cellpopulations. Thus, mononuclear cells were isolatedfrom various organs 6 and 22 h after MCAO or shamtreatments, and cytokines were assessed by CBA andELISA in supernatants of cultures stimulated for anadditional 24 h with plate-bound anti-CD3/CD28antibodies. The most striking and consistent

changes induced in the stroke mice versus sham-injured mice were observed in the spleen. At boththe 6 and 22 h time points, activated spleen cellsfrom stroke-injured mice secreted significantly en-hanced 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-inflammatoryfactor, IL-10, only at the 22 h time point. Levels ofIL-12p40 were low and did not change significantlyat either time point after occlusion (not shown).

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Moreover, unstimulated spleen tissue from strokemice had increased expression of message for MIP-2,CCR2, CCR7, and CCR8 at the 6 h time point, andMIP-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 atthe 22 h time point in activated lymph node andblood mononuclear cells (Figures 2A and 2B).Interestingly, IL-1b was not detected at either timepoint in any of the peripheral lymphoid organs or inplasma (not shown), suggesting that the source ofthis cytokine was solely from injured brain. These

data show that focal cerebral ischemia producedlocal inflammatory effects within the recoveringbrain, and distal effects in lymphoid organs. Incontrast, early suppression of inflammatory media-tors was observed in spinal cord.

Discussion

It is now well established that the initial insult fromstroke is followed by an early induction of inflam-matory cytokines and chemokines that attract mono-

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nuclear cells and granulocytes, which cause furtherdamage to the ischemic and surrounding areas ofbrain tissue. The results presented above confirmand extend these previous observations in brain andare the first to document the additional rapid andprofound effects of focal cerebral ischemia onsystemic immune responses in lymphoid organs.As early as 6 h after cerebral vascular occlusion,activated splenocytes released significantly elevatedlevels of inflammatory cytokines on stimulationthrough the T-cell receptor (TCR). In addition,unstimulated splenocytes expressed increased mes-sage levels for the chemokine, MIP-2, and CCR, andblood plasma had increased levels of severalinflammatory cytokines, including IL-6 that hadbeen reported previously in stroke patients (Emsleyet al, 2003; Smithet al, 2004). Later, 22 h after strokeinduction, T cells from spleen as well as blood andlymph nodes secreted increased levels of inflamma-tory cytokines and IL-10 (spleen only) after activa-tion. In contrast, spinal cord tissue from mice with

cerebral vascular occlusion had reduced levels ofinflammatory factors, suggesting a compensatoryresponse to brain injury.

There is consensus that the major cytokineplayers that contribute to postischemic inflamma-tion include IL-1, TNF-a, and possibly IL-6 (Zhengand Yenari, 2004). Interleukin 1 and TNF-a arepleiotrophic factors that can be detected as early as1 h after onset of stroke, even before significantneuronal death (Allan and Rothwell, 2001), but latermay have neuroprotective effects. Both cytokinespromote early-stage inflammation by increasingexpression of chemotactic factors and adhesionmolecules by vascular endothelium leading to earlyinfiltration of monocytes and macrophages (within18 h), neutrophils (within 48 h) and T lymphocytes(within 72 h) (Stevens et al , 2002). These earlyinflammatory factors in combination are neurotoxic,and also induce the production of additionalcytokines and chemokines by other brain cells. Inthe damaged brain, IL-1 and TNF-a are largely

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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 werestimulated for 48 h with plate-bound anti-CD3/CD28 antibodies. Supernatants and blood plasma were evaluated for levels ofsecreted factors, including (A) TNF-a, IFN-g, and IL-6; and (B) MCP-1, IL-2, and IL-10. * indicates a significant difference inexpression in stroke mice versus sham-treated mice.


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produced by activated microglial cells, but may alsobe secreted by other brain cells, including astro-cytes, endothelial cells, and neurons (del Zoppo etal, 2001), and later by infiltrating mononuclear cellsfrom blood. Studies of IL-6 have produced conflict-ing results. Interleukin 6 and IL-6R expressionparalleled cell death by neurons after ischemicinsult (Vollenweider et al, 2003), and peak levelsof IL-6 in plasma from stroke patients had prognos-tic value in predicting long-term outcome (Smith etal, 2004). However, mice deficient in IL-6 did nothave more severe cerebral damage after stroke thanwild-type mice (Clark et al, 2000), suggesting thatthis cytokine may not be critical for stroke patho-genesis.

Other less well-studied proinflammatory chemo-kines have also been reported in stroke-damagedbrain tissue, including IL-8, MIP-2, and MCP-1 (forattracting neutrophils and monocytes to the site ofdamage), and IP-10, MIP-1a, and RANTES, alongwith their respective CCR (Cartier et al , 2005),particularly CXCR3 (that binds IP-10) and CCR5(that binds MIP-1a/b, RANTES, and MCP-2). Che-mokines induce neuronal death either directlythrough neuronal receptors or indirectly via micro-glial activation and killing, and previous work

indicates these factors are induced after MCAO, forexample, IP-10, CXCR3 (Wang et al, 2000), andCKR5 (Spleiss et al, 1998). Furthermore, intraven-tricular MIP-1a injection enhances cortical infarc-tion in mice, while pharmacological chemokinereceptor antagonists reduce damage (Takami et al,2001, 2002). In contrast, the anti-inflammatory andneuroprotective factors, IL-10, TGF-b, and IL-1ra,were produced in synchrony with the first wave ofinflammatory cytokines (Allan and Rothwell, 2001;Stoll, 2002). It is noteworthy that we detectedselectively enhanced expression within the postis-chemic right brain hemisphere of TNF-a, IL-1b, IL-6,TGF-b1, IL-10, RANTES, IP-10, MIP-2, and variousCCR at both 6 and 22 h after vascular occlusion, apattern that is entirely consistent with previouslypublished literature.

In stark contrast, the pattern of cytokine expres-sion in spinal cord tissue from stroke-damaged micewas nearly the reverse image of changes observed inthe damaged right hemisphere, suggesting thatcytokine expression is organ-specific within theCNS. Whether the apparent immunosuppression inspinal cord represents a compensatory response tointracranial events remains to be shown. However,previous studies in rat show neuronal degeneration

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of lumbar sacral spinal cord afterpermanentMCAO,accompanied by enhanced tissue immunolabelingfor c fos and OX-42 (a microglial marker), andincreased local levels of TNFa and IL-1b (Fu et al,2004; Wu and Ling, 1998a, b). Disagreement be-tween these earlier findings and the present onesmay be related to the greater severity of a permanentMCAO model, species differences (rat versusmouse), or tissue sampling (lumbar-sacral cord ascompared 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 ofplasma IL-6 and stroke size, severity, and long-termoutcome. These data would support the possibilitythat less severe focal ischemia might induce lesspronounced changes in systemic cytokine secretion.

The major finding in this study was the rapid andwidespread increase in production of inflammatoryfactors (TNF-a, IL-6, IL-2, MCP-1, and MIP-2) bybasal and activated splenocytes that occurred asearly as 6 h after stroke, with similar changesoccurring later in the spleen as well as in lymph

nodes and blood. Our experiments were performedin male C57BL/6 mice, exclusively, and both geneticstrain and sex influence innate immunologic re-sponses to injury. For example, C57BL/6 and Balb/cdiffer greatly in stress sensitivity and display ofdominant immune characteristics (Chiodini andBuergelt, 1993) However, the C57BL/6 strain is acommon strain employed in experimental strokeand is well-characterized in terms of lymphoid andleukocyte populations and immunocompetence(Kajioka et al, 2000).

While there were many similarities in the patternof expression of splenic versus brain cytokines andchemokines, there were also striking differences. Inparticular, splenic T cells activated with anti-CD3/CD28 antibodies produced a significant increase inIFN-g(not observed in brain), but no levels of IL-1b(observed only in brain). Others have evaluated theeffects of MCAO on lymphoid tissue, demonstratingextensive loss of lymphocytes in spleen and thymus,a shift from T helper cell (Th1) to Th2 cytokineproduction and increased lymphocyte apoptosis by

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Figure 3 Effects of stroke on expression of chemokines/receptors in spleen tissue. Spleens were collected from sham and MCAO-treated 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.


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12 h of reperfusion (Gendronet al, 2002; Prasset al,2003). We observed enormous splenic T-cell cyto-kine/chemokine production that was readily mea-sured by early reperfusion (minimum 6 h). Theseobservations are significant in that they may play arole in the adaptive immune response to stroke.Recent data in humans suggest that T cell mediated

immune responses are important to both strokepathogenesis and outcome (Nadareishvili et al ,2004). Interestingly, induction of T cell tolerance tobrain antigens reduced postischemic brain damage(Becker et al, 1997). Moreover, data from neuronalmodels of traumatic injury suggest that T cellsprimed to respond to myelin basic protein canenhance recovery (Haubenet al, 2000; Moalemet al,1999). One possibility that must be considered isthat a dysfunctional bloodbrain barrier leads toexposure of normally cloistered brain structuralelements, initiating autoimmune-like processesand cell-mediated immune defenses in the periph-ery that may have varying effects on stroke progres-sion. However, autoimmune responses to newlyreleased brain antigens would require days, ratherthan hours, to manifest.

Given the location of the splenic T cells distantfrom the site of cerebral stroke, and low level ofinfiltrating mononuclear cells yet present in theinjured brain (Stevenset al, 2002), it is unlikely thatinflammatory cells found in the spleen at 6 hrepresent brain-infiltrating cells that had emigratedout of the damaged brain. However, a secondintriguing possibility is that the rapid inflammatoryresponse observed in the spleen and the subsequentspread of activated lymphocytes to lymph nodes and

blood resulted from sympathetic neural stimulationinitiated from within the damaged brain tissue.Norepinephrine and b2-adrenergic receptor (b2AR)stimulation from infection and injury has beenstrongly implicated in the regulation of the immuneresponse (reviewed in Kohm and Sanders, 2001),and it is conceivable that brain injury might alsotransmit sympathetic signals to the spleen. Thispossible explanation has a number of merits: (1)cytokines such as IL-1bcan directly activate sympa-thetic neurons, which are known to express IL-1R,thus allowing for the possibility that the earlyinduction of IL-1 after stroke might induce efferentsympathetic signals to peripheral lymphoid organs,including spleen; (2) sympathetic stimulation resultsin the local release of norepinephrine in lymphoidorgans including spleen; (3) the b2AR for norepi-nephrine is selectively expressed by CD4 + T cellsand B cells; (4) norepinephrine drives Th1 celldifferentiation and secretion of IFN-gthrough stimu-lation ofb2AR on nave CD4 + T cells by augmentingthe IL-12 signaling pathway (Swanson et al, 2001);(5) norepinephrine increases the number of circulat-ing lymphocytes, and thus might account for thelater appearance of T cells in the lymph nodes andblood, which also produced inflammatory cytokineson stimulation through the TCR.

Lastly, our novel demonstration of a drastic andrapid release of inflammatory cytokines from acti-vated splenocytes and lymphoid tissue may repre-sent only the initial challenge for the peripheralimmune system imperiled by stroke. In a murinemodel similar to the present study, b-adrenoreceptormediated systemic immunodeficiency was readily

manifested at 3 days after cerebral ischemia,characterized by defective IFN-g production, failureof lymphocyte activation, septicemia, and pneumo-nia (Prass et al , 2003). We speculate that theischemic brain uses sympathetic neural signalingto trigger, and ultimately exhaust, endogenousimmune defenses against injury, leading to deleter-ious and organ-specific consequences.

Acknowledgements

The authors thank Ms Eva Niehaus for assistance in

preparing and submitting the manuscript.

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