mechanisms of inflammation in ms tissue: adhesion molecules and chemokines

Ž .Journal of Neuroimmunology 98 1999 57–68

Mechanisms of inflammation in MS tissue: adhesion molecules andchemokines

Richard M. Ransohoff )

Department of Neurosciences, The Lerner Research Institute, and The Mellen Center for MS Treatment and Research, CleÕeland Clinic Foundation,NC30, 9500 Euclid AÕenue, CleÕeland, OH 44195-0001, USA

Received 23 October 1998; received in revised form 9 December 1998; accepted 9 December 1998

Abstract

Ž .Molecular mechanisms of inflammatory leukocyte accumulation in the central nervous system CNS have been addressed during thepast fifteen years, using small-animal model systems. Identification of the molecules responsible for leukocyte-endothelial adherence, andthe elucidation of the roles of chemokines, has promoted further understanding. These insights have become clinically relevant, as attested

Ž .by ongoing and contemplated multiple sclerosis MS treatment trials. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Adhesion molecules; CNS; leukocyte; chemokines

1. CNS immune-mediated inflammation: overview

Inflammatory cell recruitment to the central nervousŽ .system CNS is a critical step in the evolution of patho-

logical and host-defense processes as diverse as headtrauma, stroke, viral encephalitis and multiple sclerosisŽ .MS . The CNS constitutes a leukocyte-deficient environ-ment with limited regenerative capacity; these attributesrequire efficient response to pathogens and noxious stim-uli. However, the brain and spinal cord are rigidly con-fined within the skull and dura, so that inflammatorydistension is potentially fatal. Further, the anatomic isola-

Ž .tion imposed by the blood–brain-barrier BBB and lack ofevident lymphatic drainage has made it difficult to definehow inflammatory processes within the CNS are initiated.Not surprisingly, given these attributes, CNS inflammation

Ž .exhibits distinctly regional character Fabry et al., 1994 .Recently, progress has been made in addressing generalmechanisms of leukocyte extravasation into lymphoid and

Ž .parenchymal tissues Butcher, 1991; Springer, 1994 . Theseinsights have been applied to the problem of inflammationin the CNS, through the exploitation of varied models ofinflammation and immunopathogenesis, including experi-

Ž . Žmental autoimmune encephalomyelitis EAE Tani and.Ransohoff, 1994; Ransohoff and Tani, 1998 .

) Tel.: q1-216-4448939; Fax: q1-216-4447927; E-mail:[email protected]

2. Regional character of the CNS inflammatory re-sponse

Ž .The central nervous system CNS has long been con-sidered to be an ‘immunologically-privileged’ site, primar-ily because of observations involving delayed rejection of

Žxenografts and tumor allografts Leibowitz and Hughes,.1983 . Immunopathologic states such as experimental au-

Ž .toimmune encephalomyelitis EAE , multiple sclerosisŽ .MS and Theiler’s murine encephalomyelitis virusŽ .TMEV -induced demyelination all document unequivo-cally that the CNS is an immunologically-competent or-gan. Historically, the concept of CNS immunologic privi-lege served to focus attention on the early steps of the

Ž .CNS immune response Lassmann et al., 1991 . As men-tioned above, CNS inflammatory reactions are shaped inpart by the unique anatomic character conferred by theBBB and the absence of fully developed lymphatic anti-gen-presentation sites in the CNS. Recent work using theEAE model effectively addressed issues of CNS antigen

Žpresentation and highlighted unresolved questions Hinrichset al., 1987; Hickey and Kimura, 1988; Hickey, 1991;

.Hickey et al., 1991; Fabry et al., 1994 .

3. Multi-step process of leukocyte extravasation

Mammalian species possess an intricate and highlyspecialized system of immune cells and soluble factors that

0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0165-5728 99 00082-X

( )R.M. RansohoffrJournal of Neuroimmunology 98 1999 57–6858

respond rapidly and with admirable flexibility, to protectand restore the homeostatic integrity of the organism,through a process termed inflammation. Circulating leuko-cytes, which are non-adherent by definition, comprise themobile sentry unit of this defense system, which continu-ously patrols the manifold tissue spaces of a multi-com-partment organism.

Extravasation is preceded by strong adhesion of leuko-cytes to the luminal surface of the endothelium; thus,phenotypic alterations in leukocytes and endothelial cellsare indispensable for immune surveillance and inflamma-tory responses. These phenotypic changes take place withina time span of milliseconds and are remarkably selective,indicating a robust process of information transfer, inwhich the leukocyte continuously surveys the status of theendothelial bed. The endothelium, in turn, provides infor-

Žmation about the presence of inflammatory stimuli such.as microbial pathogens in parenchymal tissues.

The process of information transfer between endothe-lium and leukocyte is embodied in the multistep process ofextravasation, during which the leukocyte makes a seriesof ‘gorno-go’ decisions about leaving the vasculature.Extravasation occurs during a diversity of physiologicaland pathological processes: lymphocyte recirculationthrough the lymphatic system; thymocyte migration duringmaturation; inflammation; and developmental colonizationof many organs with fixed reticuloendothelial elements.The essential events that mediate leukocyte extravasationappear to be conserved, so that a comprehensive schematichas been proposed and substantially validated. In thisview, the molecular mechanism leading to migration ofleukocytes from the bloodstream involves a series of se-quential and overlapping interactions between ligand–receptor pairs of three classes: carbohydrate–selectin;chemoattractant–chemoattractant receptor; and integrin–

Ž . Žimmunoglobulin Ig superfamily member Butcher, 1991;.Springer, 1994 . Furthermore, the combinatorial diversity

of these various ligand–receptor pairs confers the capacityto specify a wide range of individual leukocyte extravasa-tion events, in this ‘area code’ model of leukocyte traffick-

Ž .ing Springer, 1994 . These events include inflammation,both acute and chronic.

Selectinrcarbohydrate binding initiates leukocyte–en-dothelial tethering, an interaction which is challenged byintravascular shear forces, resulting in a highly reversiblestate termed rolling, which has been documented at physio-logic flow rates. The second phase of leukocyte emigrationrequires firm leukocyterendothelial adhesion, mediated byinteractions between activated leukocyte integrins and im-

Ž .munoglobulin Ig superfamily members on endothelialcells. There is a requirement for chemokines to achieve theconformational alterations involved in integrin activation,

Ž .leading to arrest–adhesion Campbell et al., 1998 . Follow-ing arrest and firm adhesion, directional migration is drivenin part by the transendothlial chemoattractant stimulusprovided by chemokines.

4. Immune-mediated inflammation in the central ner-vous system: multiple sclerosis

MS is a disease of the human central nervous systemwhich results in permanent neurological dysfunction inmany patients, due to destruction of myelin and axons andimpaired regenerative capacity of oligodendrocytes. Al-though the primary cause of MS remains an enigma, weare beginning to understand many aspects of the pathogen-esis of the disease. Magnetic resonance imaging studies

Ž .indicate that blood–brain-barrier BBB disruption is anearly event in the formation of MS lesions. Regionsof BBB compromise can resolve without permanentneurological dysfunction, or they can transform into de-myelinating lesions, that may have significant functionalconsequences. Some demyelinated lesions in MS brainremyelinate and remyelination may play an important rolein clinical recovery in some MS patients. Histologically,MS lesions are characterized by chronic inflammation,glial cell activation, myelin destruction and loss of axons.While the cellular and molecular mechanisms responsiblefor tissue destruction in MS brain are still poorly under-stood, recent studies indicate that demyelination and oligo-dendrocyte death is mediated by immune cells and byactivated parenchymal CNS cells. Infiltrating immune cellsconsist mostly of T-cells and monocytes. B-cells andeosinophils are rare in MS lesions. Both CD4-positive andCD8-positive T-cells are present in MS lesions.

Variability in disease progression is a prominent clinicalŽ .feature of multiple sclerosis MS . Some patients have

initial attacks, complete recovery, and no further symp-toms, while others progress rapidly and die within monthsof initial involvement. Most patients have remissions andexacerbations that occur during an unpredictable course.The diverse clinical course implies diversity in pathogene-

Ž .sis Lucchinetti et al., 1996 . A critical and in-depthanalysis of the molecules responsible for inflammation andtissue destruction in MS brain will provide future direc-tions for the development of therapeutics to delay or stopthe progression of the disease.

Microglia are the major cellular constituent of the en-dogenous CNS immunerinflammatory defense systemŽ .Perry, 1994 . Reactive microglia can be distinguishedfrom resting microglia by changes in morphology and byupregulation of monocytermacrophage molecules. Recentstudies have established that microglia in and around MSlesions become activated and transform into macropha-ges which phagocytose myelin. Using double-labeling im-munocytochemistry and confocal microscopy, the 3-di-mensional relationship between activated microglia cellsand myelin internodes at the edge of chronic-active MSlesions has been examined. These studies identified acti-vated microglia cells which extend processes that contactmyelin internodes. These processes extend along myelinfor variable distances, and many of these myelin intern-odes appear to be relatively intact. Similar relationships

( )R.M. RansohoffrJournal of Neuroimmunology 98 1999 57–68 59

between microglia cells and myelin internodes are notobserved in normal human brain. Other microglia cells inMS lesions engulf myelin internodes which are degenerat-ing. These observations indicate that activated microgliaattack normal-appearing myelin internodes and mediate

Ž .demyelination Bo et al., 1994; Trapp et al., 1998 . Recentobservations also indicate that axon destruction is a cardi-

Žnal feature of inflammatory MS lesions Trapp et al.,.1998 . This observation was made through use of immuno-

histochemistry for nonphosphorylated neurofilaments, epi-topes which are abundant only in axons that are physio-

Ž .logically compromised Trapp et al., 1998 . Using confocalŽmicroscopy, it was shown that axonal dilatations previ-

.ously reported by others represented terminal swellings oftransected axons in more than 95% of instances. Theseterminal swellings were hugely abundant in active MSlesions, occurring at a mean frequency four orders ofmagnitude more than in control white matter. It is pro-posed that axon destruction is the primary pathologicalsubstrate of chronic progression and fixed disability in MSŽ .Waxman, 1998 . Strikingly, there was a significant rela-

Žtionship between tissue inflammation defined as presence.of activated macrophages and axon destruction in these

MS lesions.In summary, current studies indicate that much of the

tissue destruction that occurs in MS brain is mediated byparenchymal CNS cells. Crucial to the understanding ofthe pathogenesis of the MS lesion however, is the mecha-nisms by which these parenchymal cells are activated. Thisreview will focus on selected cellular and molecular mech-anisms implicated in inflammatory leukocyte trafficking inMS brain and relevant animal models.

5. Adhesion molecules

Inflammation is a major hallmark of the MS lesion.Leukocyte accumulation at sites of inflammation involvesa series of interactions between leukocytes and endothelialcells, via molecules that require induction or activation.These interactions usually require cell–cell contact and useof antibodies or small molecule therapy to block themolecular interactions that underlie leukocyte trafficking isviewed as a promising therapeutic approach for treating

Ž .human inflammatory diseases Yednock et al., 1992 .To identify potential molecular substrates for leukocyte

trafficking and activation in MS brain, we first determinedthe immunocytochemical distribution of the b2 integrin

Ž .lymphocyte function associated antigen-1 LFA-1 and itsmajor endothelial ligands, intercellular adhesion moleculeŽ . Ž .ICAM -1 and ICAM-2 in MS tissue Bo et al., 1996 .Co-localization of these adhesion molecules with lineagespecific markers was accomplished by dual labeling im-munocytochemistry and confocal microscopy. ICAM-1 andICAM-2 were detected on endothelial cells. ICAM-2 was

not upregulated in MS brains, consistent with prior reports.However, endothelial ICAM-1 was massively increased inMS lesions. In control brain, 10% of glucose transporter-1-positive vessels contained ICAM-1 immunoreactivity onthe luminal surface. ICAM-1-immunoreactivity was de-tected on 81% of vessels in MS lesions and at significantly

Ž .increased frequency 37% of vessels in non-lesional areasof the MS brain. LFA-1 was detected on the vast majorityof infiltrating lymphocytes and monocytes, and these cellswere often closely apposed to each other. These resultssuggest a role for ICAM-1, -2, and LFA-1 in the

Žtransendothelial migration of leukocytes into MS brain Bo.et al., 1996 .

VLA-4 is an a1 integrin which is expressed on thesurface of lymphocytes, monocytes, and eosinophils. Anumber of studies have linked the expression of VLA-4 bylymphocytes to the pathogenesis of inflammatory demyeli-

Žnating diseases Yednock et al., 1992; Baron et al., 1993;.Kuchroo et al., 1993 . MS lesions contain a large popula-

tion of VLA-4-positive leukocytes. These VLA-4-positivecells are lymphocytes and monocyte in origin. Receptorson endothelial cells that potentially bind VLA-4 includeVCAM and the CS1-containing isoform of fibronectin.VCAM has received considerable attention, because it isinducible on endothelial cells in vitro by cytokines. Ourstudies failed to detect VCAM on endothelial cells in MS

Ž .lesions Bo et al., unpublished observations . Absence ofVCAM-positive vessels has also been reported in otherhuman inflammatory diseases, including rheumatoid arthri-

Ž .tis Elices et al., 1994 . Instead, we found that a subpopu-lation of microgliarmonocytes expressed VCAM-1 in ourMS autopsy material; these cells were localized predomi-nantly at edges of chronic-active and active foci of de-myelination. Many VCAM-1-positive microglia sur-rounded oligodendroglial perikarya; the results suggested

Žfunctional and phenotypic subpopulations of microglia Bo.et al., unpublished observations . Furthermore, although

VLA-4 appeared likely to be implicated in lymphocytermonocyte extravasation in MS lesions, the endothelialligand for this integrin remains to be unequivocally deter-mined.

6. Chemokines

Chemokines are a superfamily of small cytokines thatare divided into two major subfamilies, and two minor

Žbased on structural, functional and genetic criteria Lindley.et al., 1993 . The major subfamilies are the C–X–C, or

a-family, in which two N-terminal positionally conservedcysteine residues are separated by one amino acid, and theC–C or b-family, where the two corresponding cysteinesare adjacent. In humans, C–X–C polypeptides are encodedon chromosome 4, while C–C chemokines are found onchromosome 17. There is clear functional specialization ofthe two subfamilies: C–X–C family members are specific


towards neutrophils, activated T cells and varied tissue-specific cellular elements, while the C–C chemokines actupon monocytes, eosinophils, basophils and T-cells.Chemokine genes and products have been described in

Ž .humans, rats, rabbits and mice Rollins, 1997 .Many cell-types produce chemokines in response to

stimuli including bacterial cell wall products, lectin mito-gens, growth factors and inflammatory cytokines. Mono-cytesrmacrophages, lymphocytes, neutrophils, fibroblasts,endothelial cells, keratinocytes and astrocytes produce awide array of chemokines. Neoplastic cells of diverseorigins also express chemokine genes. Melanoma cells, for

Žexample, produce GRO-a initially characterized biochem-ically as an autocrine growth factor termed Melanocyte

� 4.Growth Stimulating Activity MGSA . Human tumors ofCNS origin secrete chemokines including IL-8, IP-10 and

Ž .MCP-1, -2 and -3 Yoshimura et al., 1989a . Chemokineexpression has been detected extensively in both sponta-

Žneous and experimental inflammatory states Furie and.Randolph, 1995; Luster, 1998 .

Most chemokines are highly basic and interact physi-cally with acidic extracellular matrix components such asthe glycocalyx that decorates the luminal aspect of theendothelium, or with cell-surface molecules such as the

Ž .Duffy antigen receptor for chemokines DARC . Theseinteractions may ‘present’ chemokines to leukocytes. Intissue, this immobilization may limit diffusion and provide

Ža relatively stable chemoattractant stimulus Tanaka et al.,.1993 . More importantly, it seems axiomatic that some

mechanism for preventing chemokine diffusion in theflowing bloodstream must be required for chemokine ac-

Žtion towards intravascular leukocytes Middleton et al.,.1997 .

The best-characterized biological activity of chemokinesis chemotaxis, defined as stimulation of directional migra-tion by target cells. The prototype a-chemokine is human

Ž .interleukin IL -8, a potent chemoattractant for neutrophilsŽ .Yoshimura et al., 1987; Larsen et al., 1989 . IL-8 lacks arodent homolog. However, KCrN51rCINC, rodent a-chemokines that are homologous to human GRO-a , shareneutrophil specificity. Several a-chemokines exhibit dif-ferent receptor-binding specificity from the neutrophil-directed chemokines, all of which possess a conserved

Ž .glutamate–leucine–arginine ELR motif in the N-terminalreceptor-binding domain. Non-ELR a-chemokines, includ-

Ž .ing IP-10 g-interferon inducible protein, 10 kDa and migŽ .monokine induced by gamma-interferon are chemotactic

Žfor activated T-cells and monocytes Luster and Leder,.1993; Taub et al., 1993b .

Chronic inflammation appears in general to be mediatedby the b-chemokines. The b-chemokine monocyte chemo-

Ž .attractant protein-1 MCP-1 is chemoattractant for mono-Žcytes and T-cells Rollins et al., 1989, 1991; Yoshimura et

. Žal., 1989a,b; Carr et al., 1994 . RANTES Regulated Upon.Activation, Normal T-cells, Expressed and Secreted , an-

other b-chemokine, is chemotactic for monocytes and

Žmemory T-cells but not for neutrophils Schall et al.,.1990 . MIP-1a attracts monocytermacrophages and CD8

q T-cells, whereas the closely related MIP-1b acts onŽCD4q T-cells Ming Wang et al., 1993; Taub et al.,

. Ž .1993a . MIP-1b but not MIP-1a increases endothelialŽ .adherence by T-cells Tanaka et al., 1993 .

Ž .Two newer chemokine families g and d have beenŽ .described Rollins, 1997 . These each have divergent struc-

tures from the ‘traditional’ members but exhibit sufficientconserved features and functional attributes to merit inclu-

Ž .sion in the chemokine field of study Rollins, 1997 . Theindex g-chemokine is lymphotactin. The prototype d-chemokine is termed either ‘neurotactin’ or ‘fractalkine’.

Studies of chemokine function convey a somewhatbewildering impression of redundancy. For example, thereare at least six human chemokines with strong specificitytowards neutrophils and it is uncertain what role theindividual products play in specific processes. One elegantmeans to address the biological roles of individualchemokines entails the deletion of selected chemokinegenes from the murine genome through gene-targeting,resulting in chemokine knock-out mice. Several such micehave been reported; to date, all but one are viable andsome exhibit informative, selective defects in host de-fenses. For example, mice that lacked MIP-1a were un-able to mount appropriate inflammatory reactions to viral

Žinfection of the pulmonary and cardiac systems Cook et.al., 1995 . Gene targeting results in loss of a functionally

unique product that is transcribed under defined circum-stances, governed by a genomic cis-regulatory apparatus.A further level of analysis will require ‘knock-in’ experi-ments, in which functionally similar chemokines are ex-changed in targeted loci, to determine whether informativephenotypes result from lack of a non-redundant functionalproduct or from absence of expression at a critical time inthe evolution of a response to physiologic challenge.

6.1. The chemokine hypothesis: chemokines as target cell-specific chemoattractants

Chemokines have been associated with the migration oflymphocytes, monocytes, eosinophils, basophils and neu-

Žtrophils Oppenheim et al., 1991; Rollins, 1997; Luster,.1998 . In vitro and in vivo, chemokines attract leukocytes

to migrate along concentration gradients. Chemokines alsoreversibly activate leukointegrins, consistent with a role in

Žmodulating leukocyte–endothelial interactions Detmers et.al., 1990 . Importantly, chemokine effects are selective for

target subpopulations of leukocytes, both in vitro and invivo. Thus, chemokines are proposed to function criticallyto define the cellular composition of inflammatory infil-

Žtrates Wolpe and Cerami, 1989; Oppenheim et al., 1991;Oppenheim, 1993; Springer, 1994; Taub, 1996; Butcherand Picker, 1996; Baggiolini et al., 1997; Rollins, 1997;

.Baggiolini, 1998; Luster, 1998 .


6.2. Chemokine receptors

Chemokines act through specific high-affinity receptorsthat belong to the superfamily of serpentine G-protein-cou-pled receptors. Signaling pathways elicited by chemokine–

Žreceptor ligation have recently been reviewed Kelvin etal., 1993; Horuk, 1994a,b; Murphy, 1994a; Ben-Baruch et

.al., 1995 . The receptors that respond to a-chemokines andb-chemokines differ, and are typically encoded by intron-less genes. There are at present nine cloned human recep-tors that respond to defined b-chemokines and five clonedreceptors that respond to a-chemokines. A simplifyingnomenclature has been proposed. The interest in chemokinereceptors was recently augmented by numerous observa-tions leading to the conclusion that chemokine receptorsŽincluding CCR3, CCR5; CXCR4; CCR2b among several

. Ž .others are obligate invasion co-receptors with CD4 forŽHIV-1 virus Choe et al., 1996; Doranz et al., 1996; Liu et

.al., 1996 .Relations between chemokines and their receptors are

complex: multiple chemokines can bind productively to asingle receptor and multiple receptors bind individual

Žchemokines Kelvin et al., 1993; Horuk, 1994a,b; Murphy,.1994a . Thankfully, individual chemokine receptors are

expressed selectively by target cells, providing for speci-ficity in biological responses, as shown by analysis of

Ž .chemokine-receptor-deficient mice Boring et al., 1997 .As with other G-protein coupled responses, chemokinesignaling is linked to phospholipase-dependent pathwaysand to the generation of transient elevations of cytosolicCa2q. Chemokine receptor signaling is frequently but notinvariably pertussis-toxin sensitive, indicating heterogene-ity in the system. Selected members of the G class of Gaq

components reconstitute IL-8 receptor signaling, uponŽ .overexpression in COS cells Wu et al., 1993 . It seems

apparent that there will be significant specificity in thevaried signaling pathways that can be evoked bychemokine-receptor interactions in individual cells. Thisspecificity may suggest opportunities for selective phar-macological intervention.

As indicated above, there is a non-selective chemokinereceptor that is identical to the human Duffy blood group

Žantigen, and is designated DARC Chaudhuri et al., 1993,.1994; Horuk et al., 1993b . The erythrocyte chemokine

receptor may serve in part as a chemokine ‘sink,’ limitingcirculating levels and functional activity of multiplechemokines. Reduction of circulating chemokine levelsmight maximize sensitivity of circulating leukocytes to

Žlocally elevated chemokine concentrations Neote et al.,.1993; Horuk et al., 1993a; Chaudhuri et al., 1994 . DARC

is the invasion receptor for Plasmodium ÕiÕax, and itsexpression on erythrocytes has been lost in several en-demic areas, presumably as a consequence of selectivepressure. DARC is also present universally on post-

Ž .capillary venules even in Duffy-negative individuals , po-tentially indicating an important function at that site. A

Žsurprising number of pathogens HerpesÕiridae in particu-.lar encode chemokine receptor homologs that are compe-

tent for ligand binding, indicating that these organismsmay gain a competitive advantage against host defenses by

Žblocking chemokine function Ahuja and Murphy, 1993;.Ahuja et al., 1994; Murphy, 1994b .

Chemokines evoke profound cellular responses, that arelimited partly by receptor downregulation. G-protein linkedreceptor responses can be attenuated by varied mecha-nisms that uncouple the receptor from the downstreamsignaling apparatus. At high chemokine concentrations,cells become transiently unresponsive to the individual

Ž .desensitizing agent homologous desensitization . Heterol-ogous desensitization involves unresponsiveness to multi-ple chemokines, after exposure to a single product. Recep-tor phosphorylation is involved in both homologous andheterologous desensitization. The G protein-coupled ki-nases, protein kinase A and protein kinase C have all been

Ž .implicated Murphy, 1994a; Ben-Baruch et al., 1995 .

6.3. Leukocyte recruitment to the CNS in EAE

EAE is a well-studied animal model of human demyeli-nating disease, that can be induced in susceptible mice,rats, rabbits, guinea pigs and non-human primates by

Žimmunization with myelin, myelin components myelinbasic protein: MBP; myelin proteolipid protein: PLP;

.myelin oligodendroglial protein: MOG or their immun-Žodominant domains Miller and Karpus, 1994; Tuohy et

. qal., 1994 . EAE can also be transferred with CD4 anti-Ž .gen-specific T lymphocytes Ben-Nun et al., 1981 . There

are different clinical variants of EAE, including chronicŽrelapsing forms Miller and Karpus, 1994; Tuohy et al.,

.1994 . The clinical and histopathological features of MSand EAE are regarded as similar in most essential respects,including the occurrence of multifocal lesions throughoutthe CNS, demyelinating plaques and perivascular inflam-

Ž .mation Lassmann, 1983; Raine, 1984 . It has been along-studied issue how inflammatory cells gain access tothe CNS during the course of these immune-mediatedinflammatory processes.

One important principle is that the great majority ofleukocytes within parenchymal CNS inflammatory lesionsare not T-cells that recognize myelin antigens, but areinstead nonspecific inflammatory cells. These nonspecificinflammatory cells, principally macrophages, are the proxi-mate mediators of tissue injury. In one series of studies,Cross et al. used radiolabeled antigen-specific cells totransfer EAE and showed that less than 4% of inflamma-

Žtory cells in perivascular cuffs were antigen-specific Cross.et al., 1990, 1993 . Labeled cells remained in perivascular

spaces despite massive leukocytic infiltration of the CNSparenchyma. Significantly, onset of clinical symptoms ofEAE coincided temporally with the influx of nonspecific

Ž .inflammatory cells into the CNS Cross et al., 1990, 1993 .This observation has been interpreted to indicate that


myelin-specific encephalitogenic T-cells exert their patho-genic potential by recruiting nonspecific inflammatorycells. A corollary is that encephalitogenic T-cells cross theBBB, but do not enter CNS parenchyma in substantialnumbers. This concept was also supported by Ohmori andcolleagues, who demonstrated that infiltrating antigen-specific T-cells do not exhibit proliferative clonal expan-

Ž .sion within the CNS target organ Ohmori et al., 1992 .Conclusions from these studies can be summarized as

follows:Ž .1 Immune-mediated inflammation within the CNS

Ž .begins when activated antigen-specific T-helper 1 Th1andror Th2 cells cross the BBB and recognize cognateantigen presented by perivascular cells. This encounterresults in retention of activated T-cells in the perivascularspace and local elaboration of pro-inflammatory cytokinesby both Th cells and CNS-resident perivascular mononu-

Žclear phagocytes Hickey and Kimura, 1988; Hickey, 1991;.Hickey et al., 1991 .

Ž .2 The T-cell-directed pathological process progressesvia endothelial activation, BBB disturbance and accumula-tion of antigen-nonspecific inflammatory cells. Tissue in-jury proceeds through myelin destruction by endogenousor infiltrating mononuclear phagocytes in a delayed-type

Ž . Žhypersensitivity DTH -like reaction Raine, 1984; Crosset al., 1990; Fabry et al., 1994; Skundric et al., 1994;

.Brosnan and Raine, 1996 . The DTH-mediated tissue de-struction may be augmented by anti-myelin antibodies, ofwhich the best-characterized is directed against MOGŽ .Lassman et al., 1988 . Mechanisms of antibody-dependentmyelin injury remain to be determined.

The influx of inflammatory cells is required for tissueŽ .injury in EAE and by analogy in MS but is also essential

for host defense against pathogens and for tissue repairafter injurious processes including ischemia and trauma.We consider it, therefore, important to characterize mecha-nistically the events that mediate both pathological andphysiological inflammatory cell recruitment to the CNS.The properties of the chemokines and their associationwith inflammatory states were consistent with a role inautoimmune demyelination, and several relevant studieswill be described below.

6.4. CNS chemokines in acute EAE

During recent years, endothelial and leukocyte adhesionligands within active MS lesions have been studied in

Žsome detail Bo et al., 1994, 1996; Cannella and Raine,.1995 . The investigation of chemokines has proceeded

more rapidly in the EAE system, primarily due to technicalconsiderations. Berman, Brosnan and their colleagues re-ported that MCP-1 mRNA and its gene product wereexpressed transiently in the CNS of rats with acute EAEŽ .Hulkower et al., 1993 . Kuchroo, Dorf and coworkersdemonstrated a consistent relationship between expression

of chemokine TCA-3 and encephalitogenic potential forŽ .antigen-specific T-cells Kuchroo et al., 1993 . In their

subsequent studies, however, TCA-3 antibodies did notaffect the course of EAE. We monitored the expression of

Ž .two chemokines IP-10 and MCP-1 within the CNS as afunction of the clinical course of acute PLP-peptide-in-duced EAE in SJLrJ mice, finding a brief and strikingburst of chemokine expression coincident with EAE clini-cal onset. In our studies, astrocytes near inflammatory fociwere the cellular source of IP-10 and MCP-1 transcriptsŽ .Ransohoff et al., 1993 .

Gray and colleagues demonstrated high-level expressionŽof multiple b-chemokines RANTES, MARCrMCP-3,

.TCA-3rI-309 by murine encephalitogenic PLP-peptide-specific T-cells in vitro, with vigorous expression in vivoin EAE lesions after T-cell transfer or active immunizationŽ .Godiska et al., 1995 . This study also showed accumula-tion of IP-10 and MCP-1 mRNAs, possibly as a result of

Žinduced synthesis by CNS parenchymal cells Godiska et.al., 1995 .

6.5. Chemokine expression follows, rather than precedes,the earliest infiltration of leukocytes at the onset of acuteEAE

We wondered if intrathecal parenchymal-cell chemokineexpression in acute EAE was initiated by circulating pro-inflammatory cytokines secreted after the peripheral sensi-tization to autoantigen. According to this hypothesis,parenchymal-cell chemokine expression could constitutethe initial stimulus for leukocyte entry into the CNS.

An alternative possibility was that a first wave ofantigen-specific inflammatory cells could cross the BBBwithout requirement for intrathecal chemokine production.These cells would then persist within the CNS compart-ment and produce inflammatory cytokines locally, stimu-lating chemokine expression. In this view, the productionof chemokines would exert an amplifying rather thaninitiating role in generating CNS inflammatory lesionsŽ .Tani and Ransohoff, 1994 . Alternatively, if chemokineexpression in the CNS could be elicited by systemicstimuli, parenchymal-cell chemokine production shouldprecede histological inflammation and chemokines couldbe viewed as initiators of CNS inflammation.

We studied the earliest detectable stages of histologicalEAE to differentiate these possibilities. The results were

Žunambiguous: CNS-specific chemokine expression de-termined by sensitive and specific RTrPCR dot-blot hy-

.bridization assay never preceded histological signs ofŽ .EAE Glabinski et al., 1996a .

Chemokine mRNAs were readily detectable in liversand spleens of these EAE mice at time points two to sevendays before the onset of EAE, indicating that circulatingpro-inflammatory cytokines were elevated well beforechemokine genes were expressed in the CNS compartmentŽ .Glabinski et al., 1996a . These results suggested that


activated, antigen-specific T-cells entered the CNS beforechemokine expression, consistent with the model of initia-tion of CNS inflammation, as proposed by Hickey and

Ž .coworkers Hickey, 1991; Hickey et al., 1991 . It remainsto be determined how these activated T-cells inducechemokine expression. Our results suggested that che-mokines are major amplifiers of CNS lesion formation, bypromoting recruitment of nonspecific inflammatory cells.

6.6. CNS chemokine expression is eleÕated during relapsesof C-R EAE

It was therefore of considerable interest to evaluatechemokine expression in mice with chronic-relapsing EAEŽ . ŽC-R EAE , an informative animal model of MS Yu et al.,

.1996a,b . We had documented that chemokine mRNAlevels decayed rapidly with resolution of acute EAE. Itwas pertinent to determine whether recrudescence of neu-rological symptoms in this disease was accompanied byrecurrence of chemokine gene expression in CNS tissuesand in systemic lymphoid organs.

To address CNS chemokine expression during relap-ses of C-R EAE, mice were immunized and monitoreddaily through resolution of the initial attack of neurologi-cal symptoms, during subsequent relapse and recoveryŽ .Glabinski et al., 1997 . Four cohorts of mice were ana-lyzed and compared. One group was sacrificed duringremission, after resolution of the first attack of EAE. Onecohort of mice was sacrificed after appearance of signs ofrelapse, defined as the occurrence of new neurologicalsymptoms accompanied by weight loss. A separate cohortof mice was sacrificed after appearance of signs of remis-

Ž .sion recovery from relapse , as determined by the im-provement of neurological signs accompanied by weightgain. Control mice were immunized with bovine serum

Ž .albumin BSA , which elicits potent systemic immuneactivation without CNS involvement; tissues from thesemice were harvested on the same days post-immunizationas EAE animals.

Ž .CNS brain and spinal cord tissues were aliquoted forcorrelative RTrPCR and ISH analysis for chemokine ex-

Žpression. We studied three b-chemokines MCP-1, MIP-. Ž .1a , RANTES and two a-chemokines GRO-a and IP-10 .

Liver and spleen were analyzed, to monitor chemokineexpression in the peripheral lymphoid compartment. Mus-cle served as a negative tissue control. Separate samples,obtained in parallel experiments, were subjected tochemokine ELISA.

Five chemokines were monitored in brain and liver,using both RTrPCR and ELISA. These five chemokinesŽ .MCP-1, IP-10, GRO-a , MIP-1a , RANTES exhibitedremarkably tight regulation during the evolution of clinicalrelapse. Brain chemokine transcripts and protein werepresent at baseline levels during the remission after thefirst EAE attack, rose abruptly at the onset of relapse, only

Žto decay again as the attack subsided Glabinski et al.,.1997 . ISH analysis and colocalization studies with lineage

markers showed that MCP-1, IP-10 and GRO-a wereexpressed by astrocytes bordering inflammatory infiltrates.By contrast, MIP-1a and RANTES were produced exclu-sively by leukocytes in parenchymal inflammatory fociŽ .Glabinski et al., 1997 . This pattern of expression of

Ž . Ž .MCP-1 by astrocytes and MIP-1a by leukocytes iscompatible with recent reports of chemokine expression by

Ž .cultured murine CNS-derived cells Hayashi et al., 1995 .RANTES expression by inflammatory cells in vivo inmurine EAE was also previously reported by Godiska et

Ž .al. 1994 . The co-ordinate expression of these five genesby cells of diverse lineage indicates stringent regulation invivo.

Control muscle tissue from EAE mice showed nochemokine expression; control CNS tissues from BSA-im-munized mice contained no detectable chemokine mRNA.We evaluated whether additional leukocyte infiltrates werepresent in CNS tissues of mice undergoing EAE relapses,but the presence of chronic inflammation precluded thedetection of newly-entering cells in these tissues. Addi-tional studies with labeled cells will be needed to addressthis important issue.

Chemokine expression during EAE relapse was con-fined to the CNS, with no evident elevation of levels in the

Ž .liver or spleen Glabinski et al., 1997 . This result wasstrikingly different from our previous findings during acuteEAE and argues that the inflammatory process was gener-ated within the CNS. Relapse in this model of C-R EAEwas recently shown to be contingent upon epitope spread-ing, defined as the process of antigenic diversification

Žduring a chronic tissue-specific autoimmune reaction Yu.et al., 1996a . Thus, mice immunized with a peptide

determinant of PLP develop immune reactivity to addi-tional determinants on PLP and autoreactivity spreads toother myelin antigens, such as MBP. We propose thatchemokine expression couples the new T-cell antigenrecognition with effector mechanisms that produce clinicalsymptoms.

6.7. CNS chemokine expression does not occur in EAEÕariants that lack macrophage recruitment to the brainparenchyma

Linington and colleagues reported variant forms ofEAE, in rodents that were immunized with CNS antigensincluding the astrocyte-specific calcium-binding protein,

ŽS100b Linington et al., 1993; Kojima et al., 1994; Bradl.and Linington, 1996 . These EAE models were character-

ized as variant in several ways: first, symptoms were mild,in the face of overwhelming inflammatory lesion forma-tion; second, the inflammatory lesions were predominantlyperivascular and contained a relative excess of T-cellsŽ .typically 90% , as compared with EAE lesions in MBP-immunized animals, that were consistently 10–20% T-cells.


It was of interest to determine if variable macrophageinvasion of the CNS compartment in these different EAEmodels was correlated with the abundance of macro-phagermonocyte-directed chemokines such as MCP-1 andMIP-1a . CNS RNA was analyzed for chemokine expres-sion by quantitative RTrPCR, at time points two, four,five and six days after T-cell transfer. Rats that receivedMBP-specific T-cells were compared with those receiving

ŽMOG- or either of two S100b-specific two different cell.lines T-cells. Abundance of MCP-1 and MIP-1a mRNA

was ten-fold higher in brains of rats that received theMBP-specific cells than in samples any of the variant EAE

Ž .animals S. Lassmann et al., unpublished observations .This result documented a striking correlation between theexpression of MCP-1 or MIP-1a and the entry of thetarget hematogenous monocytermacrophage cells into theCNS compartment. Of further interest, the T-cell-directedchemokine RANTES was equally expressed in the CNS ofrats with ‘typical’ EAE mediated by anti-MBP T-cells and

Ž .‘variant’ EAE panencephalomyelitis , caused by anti-S100b T-cells. This result delineated a striking correlationbetween CNS chemokine expression and the constituentsand distribution of a CNS inflammatory infiltrate.

6.8. EAE can be suppressed with chemokine antibodies

As described above, results from several laboratoriesincluding ours suggested a role for chemokines in attract-ing cells from the bloodstream into the CNS, but did notdirectly address the relevance of chemokine expression for

Ž .disease pathogenesis Karpus and Ransohoff, 1998 . Re-cently, Karpus et al. reported that injection of anti-MIP-1a

antibodies precluded development of passive transfer EAEand reduced inflammation in the CNS. The protectiveeffect was potent and could be used to treat ongoing EAE.Exposure of T-cells in vitro to the MIP-1a antisera failedto affect cytokine production or antigen-dependent prolif-eration. This result indicated that the chemokine antibodiesrequired components present in vivo to exert therapeuticeffects, and suggested blockade of the process of leukocyte

Ž .recruitment Karpus et al., 1995 . Alternatively, it is possi-ble that anti-MIP-1a caused inhibition of T-cell productionof Th1 cytokines, as it has been demonstrated in vitro thatMIP-1a treatment can polarize Th0 clones to Th1 differen-

Ž .tiation commitment Karpus et al., 1997 . MIP-1a produc-tion in the spinal cords of these mice far exceeded MCP-1,as monitored by ELISA, and MCP-1 antibodies did not

Ž .protect mice from acute EAE Karpus et al., 1995 . Impor-tantly, anti-MCP-1 antibodies blocked relapses of EAE inthe same model, demonstrating a temporal specialization

Žof chemokine function during the disease process Karpus.and Ransohoff, 1998 . These observations complemented

the expression studies described above.ŽIn our studies of active-immunization EAE described

.above , MCP-1 mRNA and protein accumulation predomi-nated slightly over MIP-1a , contrasting with findings in

the passive-transfer studies. This difference is not ex-plained by technical factors relating to assay sensitivity orspecificity, as chemokine ELISA in both studies was per-

Žformed in the laboratory of Dr. R.M. Strieter University. Žof Michigan School of Medicine, Ann Arbor, MI Karpus

.et al., 1995; Glabinski et al., 1997 .

6.9. In mechanical trauma to neural tissues, chemokineexpression precedes leukocytic inflammation and is rela-tiÕely restricted to MCP-1

Vigorous, diverse chemokine expression occurs in im-mune-inflammatory lesions of neural tissue, as describedabove. By contrast, in various models of mechanical traumato the rodent CNS and PNS, we found much more re-stricted chemokine expression. In each trauma model, theselective upregulation of chemokines preceded influx ofhematogenous monocytes. These results are briefly sum-marized below.

6.10. MCP-1 expression by astrocytes following penetrat-ing trauma or cortical cryolesion of the cerebral cortexand percussion trauma to the spinal cord

Traumatic injury to the brain initiates a complex tissue-specific response program that involves parenchymal andvascular elements, as well as infiltrating inflammatorycells. Approximately 24 h after penetrating mechanical

Ž .trauma to the adult rodent central nervous system CNS ,reactive astrogliosis develops and injury sites are infil-trated by mononuclear phagocytes derived from blood-borne monocytes and endogenous microglia. There is littleinformation regarding cellular interactions between astro-cytes and leukocytes during this process. To addresswhether chemokines were produced in the setting of post-traumatic inflammation, we analyzed chemokine expres-sion by ELISA and RTrPCR in lesional tissues of mousebrain after penetrating mechanical injury. We focused inparticular on early time points before histological detectionof infiltrating mononuclear phagocytes. Steady-state MCP-1 mRNA rose within 3 h after nitrocellulose membranestab or implant injury to the adult mouse brain, and MCP-1protein elevations were documented at 12 h post-injuryŽ . ŽGlabinski et al., 1996b . Other chemokines GRO-a ,

.MIP-1a , RANTES, IP-10 remained at baseline levelsŽthroughout the time period surveyed 48 h post-membrane

.insertion . ISH analysis for MCP-1 indicated that hy-bridization signal co-localized with immunohistochemistry

Ž .for the glial fibrillary acidic protein GFAP astrocytemarker, showing that astrocytes were the cellular source ofMCP-1 mRNA at these early time points after mechanical

Ž .brain injury Glabinski et al., 1996b . These results demon-strated that chemokine gene expression comprises onecomponent of the astrocyte activation program. The datawere consistent with a role for MCP-1 in the CNS inflam-matory response to trauma.


Compatible results were obtained in studies of non-penetrating traumatic injury to the cortex. In particular,following a cryogenic lesion of the cerebral cortex in adultmice, MCP-1 mRNA accumulated to high levels at 6 h onthe lesion side, remained elevated for 24 h and decayed to

Ž .baseline levels at 48 h Grzybicki et al., 1996 . On theunlesioned side, MCP-1 mRNA was elevated to a muchlesser extent and declined by 24 h. Upregulation of MCP-1mRNA was relatively specific, as mRNA encoding IP-10was not detectably increased.

MCP-1 mRNA was also elevated at the lesion epicenterfollowing percussive mechanical trauma to the rat spinalcord. Increased MCP-1 mRNA was readily detected byRTrPCR assay at 24 h post-injury, preceding mono-

Žcytermacrophage accumulation in lesions McTigue et al.,.1998 . This expression was selective as MIP-1a and

RANTES were not found to be produced in significantamounts, as compared to laminectomy controls.

Cumulative results from these studies suggested a rolefor chemokines in regulating post-traumatic inflammation

Ž .of the nervous system Ransohoff and Tani, 1998 .

7. Altered chemokine levels in CSF of patients withattacks of demyelination

There are limited data concerning chemokine levels inCSF of patients with neurological disease. Patients withasepticrviral meningitis have mononuclear pleocytosis.These patients have been shown to have increased levelsof chemokines, such as MCP-1, MIP-1a , and IP-10. Fur-ther, CSF cell counts correlate well with expression ofchemokines and neutralization studies indicate that CSFchemotactic activity is accounted for in large part bychemokines. Patients with bacterial meningitis and neu-trophilic pleocytosis exhibit high levels of neutrophilchemokines such as IL-8 and GRO-a. MCP-1 is alsoelevated, reflecting a minority population of monocytes inthe CSF. However, the chemotactic activity of CSF is notentirely abrogated by anti-chemokine antibodies, indicating

Žthat additional factors such as complement and bacterial.products are involved in attracting cells into the CSF

Žspace Saukkonen et al., 1990; Sprenger et al., 1996;Lahrtz et al., 1997; Spanaus et al., 1997; Vrethem et al.,

.1998 .We analyzed chemokine expression in sixty-nine

samples of CSF from patients with neurological diseaseŽ .Sørensen et al., 1999 . Thirty-eight patients had episodesof demyelination and were studied within two weeks ofsymptom onset. Symptoms were readily identifiable asabout three-fourths of patients had optic neuritis and theremainder had acute sensory or motor symptoms. Ofthirty-one neurological controls, twenty-one had either ten-sion headache or lumbar spondylosis and were consideredhealthy with respect to the central nervous system. Ten

Ž .patients were true other neurologic disease OND con-trols, with the majority having non-inflammatory condi-

tions such as cervical spondylotic myelopathy. Levels ofseven chemokines were evaluated by ELISA in the labora-

Žtory of Dr. Robert Strieter University of Michigan Medi-.cal Center, Ann Arbor, MI . The results were striking: MS

patients exhibited increased levels of two chemokines:IP-10 and RANTES, as compared with controls. IP-10 wasdetected at physiologically significant levels in all CSFs,but was approximately tripled in patients with acute at-tacks of demyelination. RANTES was undetectable inmore than 90% of CSFs from controls, but was present athigh levels in more than 50% of individuals with MSattacks. This upregulation of RANTES was relatively spe-cific, as no prior reports have indicated elevated CSF

ŽRANTES in conditions such as viral or bacterial meningi-.tis . Surprisingly, levels of MCP-1 were significantly de-

creased in patients with acute MS symptoms. Levels ofŽ .four other chemokines MIP-1a , MIG, GRO-a , IL-8

were either unaffected or only slightly altered. Regressionanalysis indicated a significant linear relationship betweenCSF cell count and IP-10 concentration. Although theresults do not address whether elevated CSF chemokinesare related to MS disease activity as compared with MSdisease state, it is striking that products which signal toTh1 T-cells are elevated during acute attacks.

8. Novel chemokine functions suggest additional physio-logic and pathogenic implications

A variety of previous and recent findings challenge alimited view of chemokine function. Initially described asleukocyte chemoattractants, chemokines are now known toelicit diverse cellular responses in non-hematopoietic cells,that influence developmental organogenesis, wound heal-

Ž .ing, angiogenesis and neoplasia Kunkel et al., 1995 . Onechemokine with mitogenic activity, GRO-a , was isolated

Žas a potent growth factor for melanoma cells melanoma.growth stimulatory activity: MGSA and independently

identified as an oncogene responsible for uncontrolledproliferation in transformed Chinese hamster fibroblastsand transformed chick fibroblasts. We recently identified anovel, major role for chemokines in the CNS. GRO-adramatically enhances proliferative responses of oligoden-drocyte progenitor cells to platelet derived growth factorŽ . Ž .PDGF , their major mitogen Robinson et al., 1998 . Thisfinding may have relevance for successful remyelination inMS as well as broader ramifications for the regulation ofoligodendrocyte development.

Further, CNS neurons possess binding-sites for anti-chemokine-receptor antibodies and CNS membranes bind

Ž .chemokines with receptor-like affinity Horuk et al., 1997 .Additional reports indicate that cultured neuronal cellsmanifest biological responses to chemokines such as

Ž .RANTES Bolin et al., 1998 These observations raise thepossibility of unanticipated CNS consequences of chronic

Ž .chemokine production during inflammation or blockadeŽ .as by therapeutic intervention .


Acknowledgements

The author’s research is supported by the NationalŽ .Multiple Sclerosis Society RG2362 and the National

Ž .Institutes of Health 1RO1-NS32151 . Dr. Ransohoff’slaboratory receives additional support from the WilliamsFamily Fund for Multiple Sclerosis Research.

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mechanisms of inflammation in ms tissue: adhesion molecules and chemokines

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