immunological aspects of ischaemic stroke

Immunological Aspects of Ischaemic StrokeTherapeutic Implications

Guido Stoll,1,2 Sebastian Jander,1 Mario Siebler1 and Michael Schroeter1

1 Department of Neurology, Heinrich-Heine-Universität, Düsseldorf, Germany2 The Center for Biological and Medical Research, Heinrich-Heine-Universität,

Düsseldorf, Germany

ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2131. Atherosclerosis, Inflammation and Cerebral Ischaemia . . . . . . . . . . . . . . . . . . . . . . . . 2142. Inflammation, Immune Mediators and Development of Ischaemic Brain Lesions . . . . . . . . . . 216

2.1 Mechanisms of Neuronal Death and Experimental Stroke Models . . . . . . . . . . . . . . . . 2162.2 Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

2.2.1 Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2172.2.2 Monocytes/Macrophages and Activation of Microglia . . . . . . . . . . . . . . . . . . 2192.2.3 T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

2.3 Cell Adhesion Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2202.4 Transcription Factors, Cytokines and Nitric Oxide . . . . . . . . . . . . . . . . . . . . . . . . . 221

2.4.1 Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2212.4.2 Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2212.4.3 Nitric Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

Abstract There is increasing evidence that inflammatory processes play a central rolein atherosclerosis and in secondary infarct growth after focal cerebral ischaemia.Focal cerebral ischaemia is often the result of arterio-arterial thromboembolismarising from plaques in the internal carotid artery (ICA). In the ICA, the extentof inflammatory infiltration by T cells and macrophages, and the expression ofmatrix metalloproteinase-9 in high grade stenoses, correlate with clinical featuresof plaque destabilisation.Within theCNS, focal ischaemia induces a strong inflammatory response,with

recruitment of granulocytes, T cells andmacrophageswhich is facilitated by earlyupregulation of cell adhesion molecules. In experimental animals, anti-adhesionstrategies have led to a dramatic reduction of stroke volumes; however, thesestrategies have failed to be effective in humans.‘Immunological’ transcription factors and inducible nitric oxide synthase are

upregulated in focal ischaemia and contribute to secondary infarct growth be-tween 24 and 72 hours after the initial insult. The cytokines interleukin-1β andtumour necrosis factor-α are induced prior to inflammation. Functionally, these

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cytokines exert both neurotoxic and neuroprotective effects after cerebral isch-aemia.At present, immunological strategies targeted at a single immunomodulator

for the treatment of stroke are hampered by an incomplete understanding of thecomplex cellular and molecular interactions that lead to divergent functionaleffects of inflammatory cells and immunological mediators after focal ischaemia.

Cerebrovascular disorders are a major health-care problem. Stroke is the third leading cause ofdeath in industrialised countries.Cerebral ischaemia is often the result of embol-

isation from an atherosclerotic plaque in the carotidartery. There is increasing evidence that inflamma-tion plays a pathogenic role in the development ofatherosclerosis and in destabilisation of carotidplaques that convert chronic atherosclerosis into anacute disorder. Emboli from symptomatic carotidplaques transiently or permanently occlude majorintracranial arteries and thereby induce focal isch-aemia with ensuing infarction of brain tissue.These infarcts, which develop within minutes tohours of cessation of cerebral blood flow, oftencontinue to grow during the first week after isch-aemia. Cells and mediators of the immune systemsuch as cytokines, adhesion molecules and nitricoxide (NO) have been implicated in the pathophys-iology of this secondary infarct growth. This re-view focuses on the contribution of the immunesystem to these events.

1. Atherosclerosis, Inflammation andCerebral Ischaemia

Atherosclerosis is a systemic disease of largearteries, which has for a long time been consideredto simply reflect the pathological accumulation oflipids in the vessel wall. Atherosclerosis, however,has an inflammatory component consisting of Tcell and macrophage infiltration in areas of lipiddeposition.[1]Stemme and colleagues[2] isolated T cells from

human atherosclerotic plaques and found that a sig-nificant proportion recognised oxidised low den-sity lipoproteins as antigens. A pathogenic role ofT cells in atherosclerosis is supported by the find-ing that the increased lipid accumulation which oc-

curs in apolipoprotein E (ApoE)–deficient mice(animals that rapidly develop atherosclerosis) is as-sociated with massive T cell infiltration.[3]Arterio-arterial thromboembolism from extra-

cranial stenoses of the internal carotid artery (ICA)is an important pathogenic mechanism of isch-aemic stroke. However, even high grade ICA ste-noses (≥70% luminal narrowing) carry a highlyvariable annual risk of stroke. This can be as highas 13% following a recent occurrence of transientcerebral or retinal ischaemia[4,5] or as low as 1 to2% in clinically asymptomatic patients.[6,7]In many patients with high grade ICA stenosis,

long term transcranial Doppler ultrasonography(TCD) can reveal microemboli passing through theipsilateral middle cerebral artery (MCA). Thenumber of microemboli is higher in recently symp-tomatic than in asymptomatic patients, predicts theoccurrence of future ischaemic symptoms, and de-clines after carotid endarterectomy.[8,9]We performed a quantitative immunocytochem-

ical analysis of inflammatory infiltration (T cells,macrophages) in endarterectomy specimens from37 consecutive patients undergoing surgery forhigh grade ICA stenosis (fig. 1A and B).[10] Wefound that the percentage of macrophage-rich areasand the number of T cells per mm2 section areawere larger in recently symptomatic patients thanin asymptomatic patients (macrophages: 18 ± 10%vs 11 ± 4%, p = 0.005; T cells: 71.2 ± 34.4 vs 40.5 ±31.4 mm2, p = 0.005). The presence of micro-embolism was likewise associated with an increasein macrophage-rich areas. Macrophage (19 ± 10% vs9 ± 3%, p = 0.0009) and T cell (71.5 ± 39 vs 46.4 ±22 mm2, p = 0.045) infiltration was more pro-nounced in predominantly atheromatous than infibrous plaques, but did not correlate with the pre-sence of surface ulceration or luminal thrombosis.

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Similarly, Bassiouny and coworkers[11] found 3times more macrophages infiltrating the region ofthe fibrous cap in symptomatic versus asymptom-atic carotid plaques (1114 ± 1104 vs 385 ± 622).Taken together these studies suggest a role forplaque-infiltrating macrophages and T cells in theclinical destabilisation of plaques in patients withhigh grade ICA stenoses. In accordance with afunctional role in the recruitment of inflammatorycells, expression of intercellular adhesionmolecule-1(ICAM-1) was significantly increased in plaquesfrom symptomatic compared with asymptomaticpatients and predominantly located in regions withsevere narrowing of the lumen.[12]

How could infiltration by immune cells contri-bute to arterial thromboembolism and subsequentcerebral ischaemia? T cell–derived cytokines suchas interferon-γ (IFNγ), tumour necrosis factor-α(TNF-α) and interleukin-1 (IL-1) can activatemacro-phages that in turn release matrix-degrading metal-loproteinases (MMPs) [fig. 1D] and prothromboticmolecules such as tissue factor (TF)[13] [fig. 1C].MMPs degrade connective tissue. In support of

a pathophysiological role for MMPs in plaquedestabilisation, Loftus and colleagues[14] recentlyfound significantly increased levels of MMP-9 incarotid plaques of patients who had been symptom-atic (transient cerebral or retinal ischaemia) within

Fig. 1. Plaque infiltration by macrophages (A) and T cells (B), and expression of prothrombotic tissue factor (TF) [C] and collagen-degrading matrix metalloproteinase-1 (MMP-1) [D] in an endarterectomy specimen from a symptomatic patient with high gradeinternal carotid artery stenosis. Note that inflammation is predominantly located in the fibrous cap in the immediate vicinity of theatheromatous core of the lesion (star in A), while TF and MMP-1 expression extends to the luminal surface (top of each plate). Barrepresents 100μm.

Immunology of Ischaemic Stroke 215


1 month before surgery (125.7 vs <32 μg/L for asym-ptomatic andsymptomaticpatients; p=0.003).MMP-9immunoreactivity was concentrated in areas of in-flammatory infiltration and was higher in plaquesshowing microemboli as revealed by TCD and his-topathological changes in the form of intraplaquehaemorrhage, plaque necrosis and plaque rupture.TF, another macrophage product, is a glycopro-

tein which initiates blood coagulation by bindingactivated coagulation factor VII, with ensuingthrombus formation.[15] Proinflammatory cytokinessuch as TNF-α and IL-1 activate TF gene transcrip-tion in endothelial cells and macrophages. In ac-cordance with a putative pathophysiological rolewe were able to localise TF in the fibrous cap ofcarotid plaques (Jander S, Stoll G, unpublished ob-servations) [fig. 1C]. CD40-CD40-ligand (CD40L)signalling could ‘bridge’ T cell inflammation andTF production by macrophages in atheroscleroticplaques. In vitro stimulation of human macro-phages through CD40, by either membranes fromactivated T cells or recombinant CD40L, inducedexpression of TF protein and activity, and MMPs.[16]Moreover, genetic disruption of CD40L in ApoE-deficient mice which are prone to accelerated ath-erosclerosis led to reduced T lymphocyte/macro-phage inflammation and smaller atheroscleroticplaques.[17]The key role of TF in thrombogenicity of human

atherosclerotic plaques was further substantiatedby Badimon and colleagues.[18] In vitro blocking ofTF activity by antibodies decreased platelet and fi-brinogen deposition and thrombus formation onatherosclerotic plaque specimens.Although our understanding of the molecular

basis of plaque destabilisation is still limited, theresults to date point to inflammation and macro-phage-derived factors such as TF and MMPs aspotential therapeutic targets in patient with symp-tomatic ICA disease. It is well established that as-pirin (acetylsalicylic acid) can reduce the risk offurther episodes of cerebral ischaemia in patientswith transient ischaemic attacks or mild strokes[19]and the rate of perioperative complications afterthromboendarterectomy.[20] This has mainly been

attributed to the inhibitory effects of the drug onplatelet aggregation, but might well include inhib-itory effects on the inflammatory cascade[21] withinthe plaques.Likewise, HMG-CoA reductase inhibitors

(statins) have been shown to lower stroke risk byapproximately 30%, an effect attributed to theircholesterol lowering action.[22] However, statinsalso modulate immune functions[23] and inhibit theinduction of NO synthase (NOS) and cytokines[24]putatively involved in plaque destabilisation.MMP inhibitors are attractive therapeutic agents.

Interestingly, the antibiotic doxycycline exerts aninhibiting effect on MMP activity and thereby wasable to reduce aneurysm growth in an experimentalsetting.[25]

2. Inflammation, Immune Mediatorsand Development of Ischaemic Brain Lesions

2.1 Mechanisms of Neuronal Death andExperimental Stroke Models

Focal impairment or cessation of blood flow tothe brain restricts the delivery of substrates, mostimportantly oxygen and glucose, and thereby im-pairs maintenance of ionic gradients. This is fol-lowed by depolarisation of neurons and glia whichrelease excitatory amino acids (e.g. glutamate) intothe extracellular space and accumulate Ca2+.[26,27]Ca2+ is a universal second messenger which leadsto the production of proteolytic enzymes, free-radical species and further activation of glutamatereceptors. In the centre of the ischaemic territory,where the blood flow reduction is most severe,these processes induce rapid necrotic cell death.A significant proportion of neurons, however,

die by an internal programme of self-destruction,called ‘apoptosis’or ‘programmed cell death’. Thisis regulated by proteins of the Bcl-2 family.[28-30]In focal ischaemia, apoptotic neurons are intermin-gled with necrotic neurons in the core of infarctwithin hours of the insult. Surprisingly, they arestill present during the first week after focal isch-aemia, preferentially in the boundary zone.[31] The

216 Stoll et al.


relationship between proapoptotic Bax and its anti-apoptotic homologues Bcl-2 and Bcl-X-L seems tobe a critical determinant of the relative resistanceof neurons to apoptotic cell death.[32-34] This is il-lustrated functionally by findings in mice over-expressing Bcl-2, which developed smaller in-farcts after MCA occlusion (MCAO) than micewith normal expression of Bcl-2.[35]Considering that, in vitro, the process of apopto-

sis from the initial structural changes to completecellular fragmentation takes only hours, the pres-ence of apoptotic neurons days after ischaemiasuggests that cell death after ischaemia is a dyn-amic process extending well beyond the initial is-chaemic insult. The molecular mechanisms thatentertain progressive neuronal loss during the firstpostinfarct week await further elucidation, butthere is indirect evidence that immune mediatorsare involved.Several animal models have been established

for the study of stroke pathomechanisms that moreor less reflect the human situation.[36] Surgical oc-clusion of major arteries is widely used. PermanentMCAO at proximal sites leads to infarctions of thebasal ganglia and the neocortex (fig. 2A). Asexemplified by Garcia and colleagues[37] in theWistar rat, ischaemic injury after permanent MCAOevolves through different stages: necrosis of se-lected and scattered neurons becomes apparent at3 to 6 hours; necrosis of large numbers of neuronsoccurs in the caudate nucleus and putamen be-tween 6 and 12 hours followed by pannecrosis after24 to 48 hours; and pannecrosis incorporating theentire area supplied by the MCA appears 72 to 96hours after MCAO (fig. 2A). Statistical analysishas revealed that the infarct volume changed sig-nificantly between 6 and 72 hours, but not there-after; this was confirmed later by experiments inknock-out animals lacking interferon regulatoryfactor (IRF).[38]Early reperfusion significantly modifies the

chronology of these events.Withdrawal of an intra-luminal thread advanced into the proximal MCAreconstitutes perfusion (transient MCAO) andleads to variable infarct extension, depending on

the duration of ischaemia.[39] Short-lasting isch-aemia (i.e. 30 minutes) leads to a restricted infarc-tion with pannecrosis of neurons and glial cells inthe caudate nucleus and putamen, but only partlyaffects the neocortex (fig. 2B). Developing infarctsare surrounded by a large ischaemic penumbra thatcan be salvaged by reperfusion.[40] The ischaemicpenumbra refers to cortical areas where the bloodflow is reduced but is still above a threshold thatmaintains a reversible state of ischaemic neuronalinjury.[41] Depending on the timing of reperfusion,neocortical infarctions are moreover surroundedby areas of selective neuronal death characterisedby loss of large pyramidal neurons but preservationof endothelial and glial structures.[42]Selective neuronal death is an ongoing process

over several weeks and can be pharmacologicallyattenuated, in animal models, by caspase inhibi-tors.[43] Caspases are enzymes that are sequentiallyactivated and promote apoptotic cell death.In human thromboembolic stroke, emboli from

the carotid system or of cardiac origin occludemajor intracranial arteries. Some of these embolispontaneously resolve within minutes (a situationthat is mimicked by experimental transient MCAO),others persist for hours or become permanent(mimicked by permanent MCAO).

2.2 Inflammation

Focal cerebral ischaemia leads to local activa-tion ofmicro- and astroglia and to a dramatic influxof haematogenous leucocytes.[44,45]

2.2.1 GranulocytesGranulocytes are the first haematogenous cells

that appear in the brain in response to focal isch-aemia (fig. 2I). In experimental animals and in hu-mans, granulocytes accumulate in cerebral vesselswithin hours of ischaemia before they invade theinfarct and its boundary zone.[44,46,47] This processpeaks at 24 hours after infarction, thereafter thenumber of granulocytes rapidly declines. Withinthe second week after infarction, granulocyteshave mostly disappeared.Granulocytes are attracted by chemokines re-

leased from ischaemic tissue.[48] In transient, but



Fig. 2. Cerebral infarction after permanent (A) or transient (B) occlusion of the middle cerebral artery (MCA) in the rat (margins aremarked by arrowheads). Note complete infarction of the MCA territory in plate A. In contrast, the volume of infarction is much lessafter transient MCA occlusion (MCAO) [B], indicating that reperfusion rescued most of the neocortex [B]. Plates C, D and E show thenormal appearance of neocortex, with neurons and glia revealed by cresyl violet staining (C) and the distribution of astroglia (D) andramified microglia (E) revealed by immunocytochemical staining for glial fibrillary acidic protein (GFAP) [D] or the microglia markerOx42 (E). Plates F, G and H represent an area of ischaemic pannecrosis with the border zone on the left bottom at 7 days aftertransient MCAO. Note complete loss of neurons and hypercellularity which is mostly due to microglial activation and macrophageinfiltration as shown by immunocytochemistry in plate H. GFAP-positive astroglia (G) are absent in the infarct core, but activated inthe border zone (left bottom). Plates I, J and K show inflammatory cells in ischaemic lesions: (I) granulocytes as revealed by theirtypical polymorphic nuclei; (J) T cells stained for CD5; and (K) phagocytes derived from activated microglia and infiltrating macro-phages stained by the ED1 antibody. Bar in B represents 1mm for plates A and B, in E represents 50μm for plates C-H, and in Krepresents 25μm for plates I-K.

218 Stoll et al.


not permanent, ischaemia models, intravasculargranulocyte accumulations probably reduce bloodflow in the reperfusion phase and thereby contrib-ute to the extension of infarctions.

2.2.2 Monocytes/Macrophages and Activation of MicrogliaThe most abundant leucocytes that enter the

brain after focal cerebral ischaemia are mono-cytes/macrophages (fig. 2H and J).[44,45] The CNS,moreover, contains a resident glial population, theramified microglia, that upon activation transforminto round phagocytes, and share functions andphenotypic characteristics with blood-derivedmacrophages.[49,50]Haematogenous macrophages are attracted pre-

ferentially into areas of pannecrosis probably dueto the local upregulation of the chemokine mono-cyte chemoattractant protein-1 (MCP-1).[51] Thisprocess starts about 12 hours after infarction andthere is a further increase in macrophage numbersduring the first 2 weeks.[46,52-55] Thereby, the infil-trates demarcate necrotic brain tissue from normaltissue, and rapidly remove debris leaving a glialscar.Activated resident microglia significantly con-

tribute to this phagocytic response.[56] In rat focalischaemia, two different microglia/macrophagepopulations can be distinguished phenotypi-cally.[53,54,56,57] Conventional CD4+ macrophagesthat are also present in other CNS lesion para-digms[50,58] gradually increase from day 2 on andpeak at day 14, at which point they cover the entirearea of infarction. In addition, a separate and un-usual population of microglia/macrophages ex-press the T cell surface molecule CD8.[57] CD8+microglia/macrophages were transiently presentbetween days 3 and 6 after infarction and by day14 have almost disappeared. They were exclusiv-ely located in the border zone and core of pann-ecrotic brain tissue.[57]The functional role of CD8+ microglia/macro-

phages in brain ischaemia is unclear at present.CD8+ cells could contribute to exacerbation ofischaemic brain damage as well as to tissue re-modelling and healing processes. In vitro studies

performed independently on alveolar macrophagesshowed that signalling via the CD8 molecule ledto activation of TNF-α, IL-1β and inducible NOS(iNOS) expression with concomitant enhanced cy-totoxicity toward Leihmania major.[59]In transient, as in permanent, MCAO, pan-

necrotic ischaemic tissue is infiltrated by haema-togenous leucocytes. Depending on the onset ofreperfusion, however, in addition during transientMCAO, areas with selective neuronal death can bedelineated in which only microglia and astrocytesare activated. These areas lack T cell and macro-phage infiltration (Schroeter M, Janders S, Stoll G,et al. unpublished observations).[60] Likewise, fi-bre tracts undergoing antero- or retrograde second-ary degeneration after focal ischaemia show signsof microglial activation by increased expression ofCD4 and major histocompatibility complex class Iand II molecules.[56] This is similar to the micro-glial response to mechanical injury to CNS fibretracts.[58,61] The newly described population ofCD8+ microglia/macrophage seems to be rela-tively unique to areas of ischaemic pannecrosissince no such cells were seen in areas of selectiveneuronal death and during fibre tract degenera-tion.[57]

2.2.3 T CellsT cell recruitment into the CNS is usually only

observed in autoimmune and inflammatory dis-eases. However, in both experimental and humanstroke a significant number of T cells preferentiallyinfiltrate the border zones of infarctions (fig. 2J)[Jander S, Brück W and Stoll G, unpublished ob-servations].[53,54] In experimental autoimmune en-cephalomyelitis (EAE), systemic immunisationwith myelin proteins such as myelin basic protein(MBP) creates CD4+ helper/inducer T cells thatare antigen-specific and enter the CNS after 10 to12 days to induce myelin destruction by macro-phages.[62] In cerebral ischaemia, the period be-tween lesion induction and T cell infiltration is tooshort for the generation of such a systemic, antigen-specific immune response. Therefore, the T cellresponse is likely to be antigen-nonspecific. Thesignal that attracts T cells and keeps them in the



CNS parenchyma for many days is unknown atpresent.Although not yet proven, there is circumstantial

evidence that T cell infiltration in ischaemic anddegenerative CNS lesions might be beneficial. Inan elegant study Becker and colleagues[63] orallyadministered naive rats with MBP, a procedureknown to induce immunological tolerance. Theseanimals were subsequently resistant to the induc-tion of EAE. Surprisingly, MBP-tolerant animalshad dramatically reduced infarct volumes at 24 and96 hours post-occlusion when subjected toMCAO.This effect was attributed to the infiltration ofevolving ischaemic brain lesions with T cells,producing transforming growth factor (TGF)-β.TGF-β is neuroprotective in vitro and in vivo andis a potent immunosuppressant.[64-66]This finding points to immunomodulation as a

new therapeutic strategy and, in particular, to afunctional role of T cells in stroke development. Totherapeutically exploit T cell inflammation it is es-sential, however, to clarify which population oflymphocytes is responsible for neuroprotectionand whether this effect can be cellularly transferredto naive animals.The potential beneficial role of T cell inflamma-

tion in CNS disorders was recently corroborated.Injection of autoimmune T cells against MBP intorats with optic nerve lesions dramatically reducedsecondary apoptotic cell death of retinal gangliacells.[67] Although the mechanisms of this benefi-cial effect are currently unknown, T cell–derivedneurotrophins could be involved. Autoreactive Tcells have recently been shown to secrete biologi-cally active brain-derived neurotrophic factor uponantigenic stimulation.[68]

2.3 Cell Adhesion Molecules

The inflammatory response described in section2.2 involves multiple cell adhesion steps that pro-vide the ‘traffic signal’ for leucocytes to enter thebrain through the endothelial wall.[69,70]Early upregulation of P-selectin and E-selectin

on endothelial cells in the ischaemic brain reducesthe velocity of circulating leucocytes in the blood-

stream, by binding to the ligand L-selectin that isconstitutively expressed on the surface of theseblood cells.[71] The weak binding of leucocytes tothe vessel wall is strengthened under the influenceof chemokines that are released from the site ofinjury. The ensuing adhesion process is mediatedby the integrin family of adhesion molecules.Different molecules direct the adhesion of leu-

cocyte subsets:• lymphocytes constitutively bear theCD11a/CD18(leucocyte function associated antigen-1; LFA-1)and the very late antigen-4 complex (VLA-4) ontheir surface

• monocytes bear LFA-1, VLA-4 and the CD11b/CD18 complex (synonyma:MAC-1, complementtype 3 receptor, CR3)

• granulocytes bear LFA-1 and the CD11b/CD18complex.The corresponding endothelial counter-receptors

that require active stimulation for expression areICAM-1 for LFA-1 and CD11b/CD18, and vascu-lar cellular adhesion molecule-1 (VCAM-1) forVLA-4.Cellular adhesion molecules are upregulated on

microvessels early after permanent and transientfocal cerebral ischaemia. Several groups demon-strated upregulation of ICAM-1 messenger RNA(mRNA) and protein as early as 3 hours after isch-aemia on endothelial cells of intraparenchymalblood vessels in the ischaemic cortex. ICAM-1 ex-pression peaked at 6 to 12 hours and persisted forseveral days, even in human stroke.[47,53,54,72-74]Similarly, mRNA for endothelial leucocyte adhe-sion molecule-1 (ELAM-1) and VCAM-1 was up-regulated.[72,75]The functional relevance of cell adhesion pro-

cesses in stroke development has been establishedin transient focal ischaemia. Treatment with anti-bodies directed against the CD11b/CD18 complexon granulocytes and monocytes/macrophages ofrats subjected to 2 hours of transient MCAO led toa significant reduction in infarct volume[76-78] andto a decrease in numbers of apoptotic cells.[79] Inparallel, infiltration by granulocytes was reduced.Similar results were obtained when a recombinant

220 Stoll et al.


neutrophil inhibiting factor directed against theCD11b/CD18 complex was used.[80] Blocking ofthe corresponding ligand on endothelial cells,ICAM-1, had a stronger mitigating effect and re-duced stroke volumes in rats at day 2 by 80%.[81,82]Moreover, ICAM-1 knockout mice showed a 5-fold decrease in infarct size.[83,84]In contrast, treatment with anti-leucocyte anti-

bodies was ineffective in models of permanentMCAO.[82,85] The most likely explanation for thisdiscrepancy is that granulocytes adhere to themicrovascular endothelium via the ICAM-1/CD11badhesion pathway, thereby mechanically discon-necting dependent parenchyma from reperfusion(‘no reflow phenomenon’). Prolonged hypoxialeads to an expansion of the infarct area into thepenumbra zone.

2.4 Transcription Factors, Cytokines andNitric Oxide

Cytokines have been described as soluble fac-tors that orchestrate immune responses.[86] In thenervous system, cytokines have been primarilyimplicated in the pathogenesis of autoimmunediseases such as EAE, and multiple sclerosis inhumans.[87,88] In recent years it has become clearthat cytokines, in particular IL-1β and TNF-α, aswell as immunological transcription factors andother mediators such as NO, are markedly up-regulated in focal cerebral ischaemia. Their ex-pression represents a double-edged sword – on theone hand these immune mediators contribute to anexpansion of ischaemic lesions, on the other handthey are neuroprotective.

2.4.1 Transcription FactorsIn a seminal paper, Iadecola and colleagues[38]

formally proved that immune cascades contributeto significant infarct growth beyond 24 hours afterfocal cerebral ischaemia. IRF is a transcription fac-tor that can be activated by the proinflammatorycytokines TNF-α and IL-1β. IRF induces gene trans-cription of IFNα and -β, iNOS and IL-1 convertingenzyme (ICE; caspase 1). The latter is involved insignalling cascades leading to apoptosis and in thecleavage of precursor molecules into the biologi-

cally active proinflammatory cytokines IL-1 andIL-18.[89]After focal ischaemia IRF gene expression was

markedly upregulated at 12 hours and reached apeak at day 4. Knockout mice lacking the IRF genewere protected from ischaemic brain damage anddeveloped smaller infarctions which were accom-panied by a substantial attenuation of neurologicaldeficits.[38] Although the molecular mechanismsunderlying the beneficial effects in IRF knockoutmice have not yet been identified, lack of NO in-duction is a likely candidate (see section 2.4.3).Accordingly, macrophages from IRF knockoutmice produced virtually no NO and synthesisedonly low levels of iNOS mRNA in vitro.[89]Likewise, nuclear factor-κB (NF-κB) is an im-

portant transcription factor that is involved in theinduction of immune molecules including iNOS,IL-1, TNF-α and ICAM-1 and in the regulation ofischaemic cell death.[90] Neuronal NF-κB can beactivated by a variety of ischaemia-associatedstimuli such as glutamate, hypoxia, reactive oxy-gen species and TNF-α.[91] Early activation ofNF-κB has recently been demonstrated in focalischaemia.[91] In support of a pathophysiologicalrole in stroke development, targeted disruption ofthe p50 subunit of NF-κB led to reduced infarctsafter transient MCAO in transgenic mice.[92] Onthe other hand, NF-κB activation leads to the in-duction of various antioxidant and antiapoptoticfactors and thus can equally contribute to neu-roprotection and plasticity after focal brain isch-aemia.[93]

2.4.2 Cytokines

Interleukin-1βSurprisingly, transcripts for cytokines are rap-

idly induced after focal cerebral ischaemia andprecede haematogenous cell infiltration. Levels ofIL-1β mRNAwere elevated in the ischaemic cortexwithin 1 hour and up to day 4 following permanentMCAO.[94,95] Concomitant with the upregulationof IL-1β, messages for IL-1 receptors were in-creased.[96]Endothelial cells, microglia and macrophages

are considered major sources of IL-1β in focal



brain ischaemia,[95,97] while the contribution ofneurons, astrocytes and oligodendrocytes is stillcontroversial.[98,99]The most compelling evidence that IL-1β is

involved in ischaemic brain damage derives frompharmacological studies and from stroke inductionin genetically manipulated laboratory animals.Intracerebroventricular injection of IL-1β dramat-ically exacerbates brain damage after focal isch-aemia.[100,101] Theeffectsof IL-1β canbe antagonisedby its natural antagonist, IL-1 receptor antagonist(IL-1ra). In ischaemic lesions IL-1ra mRNA wasgreatly increased at 12 hours after permanentMCAO and remained elevated for up to 5 days,concomitant with increased IL-1β expression.[96]Currently, it appears that in vivo the agonistic ef-fects of IL-1β predominate over the antagonisticeffects of IL-1ra.Exogenous administration of IL-1ra either in-

tracerebroventricularly or systemically reducedfocal ischaemic brain damage.[102-104] Betz andcolleagues[105] transformed recombinant adeno-virus vectors carrying the human IL-1ra cDNAintorodent brain and were able to ameliorate brain in-jury after permanent focal ischaemia in these ani-mals.ICE cleaves pro–IL-1β to generate biologically

active IL-1β. Inhibition of ICE decreased infarctvolumes in mice[106] and rats,[107] further support-ing a potentially noxious role of IL1-β in isch-aemia. Moreover, transgenic mice with a mutantICE gene developed smaller infarcts, less neuro-logical deficits, lower IL-1β levels and decreasedDNA fragmentation after transient and permanentMCAO than normal animals.[108,109] Althoughthese results suggest a pathological role for IL-1β,the effects might not solely be due to reduction ofIL-1β production, since ICE additionally plays acritical role in activating apoptotic pathways andmodulating the release of the proinflammatorycytokine IL-18.IL-1βmRNA induction was not restricted to the

ischaemic lesion, but also occurred in the entireipsi- but not contralateral cortex.[110] Davies and

colleagues[98] found IL-1 expression also con-tralaterally by immunocytochemistry.Although not yet formally proven, remote IL-1

induction could be involved in postischaemic brainplasticity. Schneider and colleagues[111] recentlydemonstrated that IL-1 critically contributes to themaintenance of long term potentiation (LTP) with-out affecting its induction. LTP is an importantmechanism of brain plasticity and is facilitated inthe surround of ischaemic brain lesions.[112]The mechanisms and mediators of the various

actions of IL-1β in focal ischaemia, which can bedetrimental or beneficial depending on cofactors,timing and location, are largely unknown and needfurther elucidation. One deleterious mechanismprobably involves the induction of cell adhesionmolecules on endothelial with ensuing enhancedinfiltration of granulocytes.[100,103,113] Other cur-rent hypotheses have been thoroughly reviewedelsewhere.[99]

Tumour Necrosis Factor-αSimilar to IL-1β, TNF-α is rapidly induced

within the first 3 hours after MCAO, and persistsduring the following few days.[114-116]In the study by Liu and co-workers,[114] TNF-α

immunoreactivity was confined to neurons in theevolving infarct at 6 and 12 hours and was prim-arily associated with macrophages located withinthe infarcted tissue at day 5. Botchkina and col-leagues[117] described TNF-α in neurons, astro-cytes, microglia and infiltrating granulocytes ofrats after focal cerebral ischaemia. Concomitantwith the expression of TNF-α, the TNF receptorsp55 and p75 were upregulated within 6 and 24hours, respectively. TNF receptor p55 has been im-plicated in transducing the cytotoxic signalling ofTNF. The presence of TNF protein and TNF p55receptors therefore suggested an injurious role forexcessive TNF produced during the acute responseto cerebral ischaemia. Accordingly, intracerebro-ventricular injection of TNF-α 24 hours prior toMCAO significantly exacerbated the size of infarc-tion, probably by activating capillary endotheliumto a proadhesive state.[115,116] Moreover, TNF-αcan induce apoptosis in a variety of target cells[118]

222 Stoll et al.


and thereby could contribute to delayed neuronaldeath.TNF-α, however, also protects neurons.[119] In

support of a neuroprotective role of TNF in cere-bral ischaemia, mice lacking both TNF receptorsor selectively p55 TNF receptors developed largerinfarcts than those with these receptors.[120,121] Thedivergent actions of TNF-α have further been elu-cidated in a model of brain percussion injury byScherbel and colleagues.[122] Mice lacking TNF-αdeveloped smaller memory deficits in the acutepost-traumatic period at 48 hours postinjury, but inthe long term showed persistent motor deficitsafter 4 weeks, when wild type animals had com-pletely recovered. These data suggest that TNF-αis deleterious in the acute phase, but that these neg-ative effects are outweighed by an essential role ofTNF-α in long term recovery and repair. As dis-cussed in great detail elsewhere,[123] the context ofmediators at a given time after brain ischaemiamaydetermine whether the effect of TNF-α is neuro-toxic or protective.Several pharmacological approaches to block-

ing TNF-α induction or activity during cerebralischaemia have been assessed. Systemic treatmentof rats within 1 to 2 hours after onset of MCAOwith CNI-1493, a selective inhibitor of TNF-αsynthesis, led to a 72% reduction of brain infarctvolume.[124] This therapeutic effect was associatedwith decreased TNF-α levels in the ischaemicbrain as revealed by Western blot analysis. Similarneuroprotective effects due to TNF-α inhibitionwere obtained with dexanabinol (HU-211), a non-psychotropic cannabinoid analogue.[125] TNF-αactivity can also be neutralised by antibodies orsoluble receptors. Pretreatment of rats with intra-venous anti–TNF-α antibody reduced infarct sizeand improved neurological outcome after transientMCAO.[126] In permanent MCAO, application of adimeric form of the type 1 TNF receptor which waslinked to polyethylene glycol, neutralised circulat-ing TNF-α and reduced cortical infarct volume ina dose-related manner.[127]

2.4.3 Nitric OxideNO is a small molecule that exerts pleiotropic

actions. NO is synthesised by oxidation of L-arginine by the enzyme NOS. NOS exists in 3 iso-forms: neuronal NOS (nNOS), endothelial NOS(eNOS) and iNOS.NO production is increased at all stages of

cerebral ischaemia.[128] Early during ischaemia,NO produced through eNOS activation in endothe-lial cells is beneficial and induces an increase ofcerebral blood flow by vasodilatation and exertsanti-adhesion effects on leucocytes.In contrast, nNOS and iNOS appear to be detri-

mental. nNOS activity peaks within the first hourafter ischaemia and rapidly decreases due to lossof NOS-containing neurons by tissue necrosis.After MCAO in mice, iNOS mRNA expression inthe post-ischaemic brain began between 6 and 12hours, peaked at 96 hours, and subsided after 7days.[129] Cytokines can induce iNOS transcriptionby activation of the transcription factors NF-κB orIRF (mentioned in section 2.4.1). Disruption of theiNOS gene in mice led to smaller infarcts and lessmotor deficits after focal ischaemia.[129] Most im-portantly, such reduction in ischaemic damage andneurological deficit was observed 96 but not 24hours after ischaemia, providing strong evidencethat iNOS expression is one of the critical factorsthat contributes to the delayed expansion of braindamage. iNOS induces synthesis of large amountsof NO continuously for long periods, which thenreacts with superoxide to form peroxynitrite, acytotoxic agent.There are many pharmacological agents avail-

able that inhibit NO synthesis. Application of non-specific NOS inhibitors such as nitro-L-arginineand L-nitro-arginine methyl ester led to contra-dictory results. Non-selective inhibition of NOSwas found to reduce, enhance or not affect isch-aemic brain damage.[128] In contrast, the selectivenNOS inhibitors 7-nitroindazole, ARL17477 ands-methylisothioureido-L-norvaline reduced cere-bral ischaemic damage.[130-132] Similarly, the iNOSinhibitor aminoguanidine attenuated post-ischaemiciNOS activity and reduced infarct size after



MCAO.[133,134] Importantly, aminoguanidine didnot influence nNOS enzymatic activity. Takentogether these pharmacological studies add strongevidence for a detrimental role of nNOS and iNOSin stroke development.

3. Conclusions

Therapeutic options for the treatment of strokeare currently limited.[26,135-137] Most measures areaimed at secondary prevention of stroke, and in-volve carotid surgery and inhibition of platelets.As outlined in the first part of this review,

immune processes might become new targets insymptomatic carotid artery stenoses. Patients withneurological symptoms of acute ischaemia benefitfrom treatment with alteplase (recombinant tissueplasminogen activator; rt-PA) administered within3 hours of the onset of symptoms. This drug facil-itates partial or full reperfusion.[138,139] However,recently the beneficial effects of alteplase havebeen challenged by animal experiments. UsingtPA-deficient mice, Wang and colleagues[140] dem-onstrated that, although useful in reconstitutingcerebral blood flow after intravenous application,endogenous tPA in the brain is neurotoxic.Modern imaging techniques have provided evi-

dence that human infarcts continue to grow duringthe first days after ischaemia, similar to the find-ings in experimental animals.[141,142] Based on theencouraging animal experiments (see 2.3) anti–ICAM-1 antibodies were administered to patientswho had had a stroke. Although this treatment waswell tolerated,[143] it failed, for unknown reasons,to show benefit in an acute stroke trial.[144]It has been further suggested that neuropro-

tective agents be combined with strategies to re-establish normal blood flow and to avoid cloggingof reperfused microvessels, in an attempt to expandthe time window and increase effectiveness.[70,137]These therapeutic concepts, however, are vague atpresent and need verification by clinical trials.As outlined in this review, cells and mediators

of the immune system are important participants inthe complex interplay of cellular and molecularprocesses leading to neuronal death, infarct demar-

cation, tissue remodelling and plasticity after focalischaemia. The signalling cascades leading to de-layed neuronal death are incompletely understood.Likewise, the molecular mechanisms underlyingfunctional recovery in the brain are obscure.[145]The often contradictory role of an individual cyto-kine and the ill-defined functions of infiltratingleucocytes in the ischaemic brain hampers the de-velopment of straightforward immunological strat-egies for stroke treatment. Yrjanheikki and collea-gues,[146,147] however, recently demonstrated thattetracycline derivatives reduced inflammation andprotected neurons in experimental focal and globalischaemia, a nonspecific approach that appears ap-plicable to human stroke. Nevertheless, effectivetreatment will require a more exact knowledge of neteffects of immunological processes at any giventime and location in the brain.

Acknowledgements

The authors’ work cited in this review was supported bythe Deutsche Forschungsgemeinschaft (SFB 194, B6). DrStoll holds a Hermann- and Lilly-Schilling professorship.

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Correspondence and offprints: Dr Guido Stoll, Departmentof Neurology, Heinrich-Heine-Universität, Moorenstr. 5,40225 Düsseldorf, Germany.E-mail: [email protected]

228 Stoll et al.


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