doi:10.1006/smim.2001.0345, available online at http://www.idealibrary.com onseminars in IMMUNOLOGY, Vol. 14, 2002: pp. 83–92

Chemokines in rapid leukocyte adhesion triggering andmigration

Brent Johnston∗ and Eugene C. Butcher

Leukocyte subsets are recruited from the blood to lymphoidand non-lymphoid tissues via a multi-step process thatinvolves distinct adhesive and activation steps. Chemokines,a family of chemotactic cytokines that signal throughG-protein-coupled receptors, play critical roles in regulatingthe leukocyte recruitment cascade. Chemokines can betransported and immobilized on the surface of vascularendothelial cells, where they activate leukocyte subsetsexpressing specific receptors. Activation signals inducefirm adhesion of rolling leukocytes by rapidly upregulatingintegrin affinity and/or avidity. Chemokines can also directmigration of adherent cells across the endothelium, andcontrol segregation of cells into specific microenvironmentswithin tissues. The regulated expression of chemokinesand their receptors is a critical determinant for homing ofspecialized lymphocyte subsets, and controls both tissue andinflammation-specific immune processes.

Key words: adhesion / chemokine / integrin / lymph node/ selectin

c© 2002 Elsevier Science Ltd. All rights reserved.

Introduction

It is well appreciated that lymphocytes continuallyrecirculate during immune surveillance, movingfrom the blood into organized lymphoid tissuesand subsequently returning to the blood via the

From the Laboratory of Immunology and Vascular Biology, Departmentof Pathology, Stanford University School of Medicine, Stanford,CA 94305-5324, USA and Center for Molecular Biology andMedicine, Veterans Affairs Medical Center, MC154B, Palo Alto,CA 94304, USA. *Corresponding address. Veterans Affairs MedicalCenter, MC154B, 3801 Miranda Avenue, Palo Alto, CA 94304, USA.E-mail: wbjohnst@stanford.edu

c©2002 Elsevier Science Ltd. All rights reserved.1044–5323/02/$ - see front matter

lymphatics.1 Similarly, lymphocytes and otherleukocyte populations are recruited from the bloodto sites of inflammation and tissue damage. Evidencefrom many groups supports a multi-step model ofadhesive interactions and activation signals duringthe recruitment process2–4 (Figure 1). Initially,marginated leukocytes tether to the endotheliumvia microvillous processes and engage in transientinteractions that manifest as rolling as due tohemodynamic shear forces. Leukocytes then undergoa rapid activation step that upregulates integrinfunction and allows them to arrest and adherefirmly to the endothelium. Adherent leukocytessubsequently migrate across the endothelium, andcan localize to distinct microenvironmental sites bysequential (step-by-step) integration of signals frommultiple chemoattractant sources.5

Tethering and rolling are usually mediated bythe selectin family of adhesion molecules (L-selectin expressed constitutively on leukocytes, andP-selectin and E-selectin upregulated by activatedendothelium), that bind to ligands modifiedwith specific carbohydrate epitopes.6,7 Leukocyteadhesion is mediated by interactions of the integrinfamily of adhesion molecules [LFA-1 (αLβ2), Mac-1(αMβ2), VLA-4 (α4β1), and LPAM-1 (α4β7)]with members of the immunoglobulin family ofadhesion molecules (ICAM-1, ICAM-2, VCAM-1, andMAdCAM-1).8,9 However, in vitro and in vivo evidenceshows that the α4-integrins (α4β1 and α4β7) can alsosupport leukocyte tethering and rolling.10–13 Onekey to the participation of selectins and α4-integrinsin tethering is their expression on microvillousprocesses on the surface of circulating cells.10,14

As integrins are normally expressed on leukocytesin a state that has low affinity for ligands,15–18

it is clear that they must become functionallyupregulated to mediate firm adhesion. Several invitro studies have demonstrated that cross-linkingL-selectin on leukocytes or binding to E-selectincan upregulate integrin function,19–21 suggesting

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Figure 1. The multistep model of leukocyte-endothelial interactions in the recognition and recruitment of leukocytes fromthe blood. The adhesive and activation steps that take place during the recruitment of naive lymphocytes to the peripherallymph nodes (PLN) and memory lymphocyte subsets to the skin and gut illustrate the sequential recruitment cascade. Eachstep in the cascade involves independent receptor–ligand interactions that can enhance specificity through combinatorialcontrol of leukocyte recruitment.

that this interaction could provide the activationsignal required for firm adhesion. However, thissignal is not sufficient to initiate adhesion underphysiological conditions: leukocytes have been shownto roll on P-selectin, E-selectin, and glycosylatedL-selectin ligands, without upregulating adhesionto ICAM-1.15,22 Additionally, neutrophils andlymphocytes both exhibit rolling behavior in highendothelial venules (HEV) of lymphoid tissues, butonly lymphocytes adhere,23 suggesting that a moreselective signal regulates adhesion. This review willfocus on the role that chemokines play in mediatingthe specificity of the leukocyte recruitment paradigmthrough adhesion triggering and directed migration.We will review some of the mechanisms knownto regulate these events, and highlight how thesepathways operate during lymphocyte homing in vivo.

Chemokines and their receptors

The chemokines are a superfamily of about 50low molecular weight chemotactic cytokines (8–

14 kD) that bind to and signal through seventransmembrane-spanning G-protein-linked receptorsexpressed on leukocytes and other cell types.24,25

Chemokines share homologous sequences and canbe subdivided into four main classes (C-C, C-X-C,C-X3-C and C) depending on the orientation ofconserved cysteine residues in their amino acidsequences. The chemokine receptors preferentiallysignal through heterotrimeric G-proteins of the Gαiclass as most responses are sensitive to inhibitionwith pertussis toxin (PTX).26–28 However, in somestudies PTX has not completely blocked chemokineresponses, suggesting that some chemokine receptorsmay also couple to Gαq or Gα16 in certain cell typesor under certain circumstances.29,30

The expression of chemokine receptors variesgreatly among different leukocyte subsets. Fur-thermore, the chemokines they recognize aredifferentially expressed in tissues and inflammatoryconditions.25,31 Therefore, chemokines acting incombination with different adhesion receptors,have the potential to increase the specificity ofleukocyte homing by providing an additional level

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of control through which cells home to tissue sites(Figure 1).

Chemokine-induced changes in affinity andavidity

Chemokines induce rapid but transient increasesin adhesion of leukocytes to immobilized integrinligands.17,18,32,33 This can occur via two differentmechanisms, upregulation of integrin affinitythrough conformational changes, or changes inintegrin avidity which occur through integrin cluster-ing.16,34–36 Chemokine-induced changes in integrinaffinity can be observed by measuring the binding ofsoluble ICAM-1 and VCAM-1 to activated cells,17,18

or the binding of antibodies that act as ligandmimetics.32,36 However, it should be noted thatchemokine-induced binding to soluble ligands variesin different cell types and with different integrins.For example, the chemokine SDF-1α induces rapidbinding of soluble ICAM-1 to lymphocytes,17 whilevery little binding to soluble VCAM-1 is observed.18

In contrast, SDF-1α induces significant increases inthe binding of soluble VCAM-1 to monocytes.18 It isnot clear what signaling pathways upregulate integrinaffinity after chemokine stimulation, but the srckinase p56lck has been implicated in the regulationof VLA-4 (α4β1) affinity.37

Avidity changes through clustering of low affinityreceptors can strengthen adhesion to immobilizedintegrin ligands.16 This process requires protease-dependent release of integrins from cytoskeletalrestraints in order to facilitate lateral mobility ofintegrins in the plasma membrane.34,35 Chemokinescan induce rapid clustering of integrins,17,32

suggesting that chemokines induce increased lateralmobility of adhesion molecules in addition toaffinity changes. Chemokine induced clusteringof LFA-1 (αLβ2) can be blocked by inhibitors ofphosphoinositide-3 kinase (PI3K) or the proteasecalpain, which have no affect on the ability ofchemokine-stimulated lymphocytes to bind solubleICAM-1.17 This suggests that chemokine-inducedchanges in integrin affinity and avidity are regulatedthrough different pathways.

The contribution of affinity and avidity changesto leukocyte adhesion appears to vary dependingon the site density of the adhesive substrate. Whilechemokine-induced adhesion to low density ICAM-1 (<1500 sites µm−2) is inhibited by blockingPI3K or protease activity, adhesion to high density

ICAM-1 (>3000 sites µm−2) is insensitive to theseinhibitors.17 This suggests that affinity changesare sufficient to mediate adhesion to high densityligands when the integrin does not need to move toencounter ligand, but chemokine-enhanced integrinmobility is required to facilitate adhesion to lowdensity sites where ligands are spaced further apart.In vivo, lymphocyte adhesion to Peyer’s patch HEVis insensitive to PI3K or protease inhibitors.17 At13 800 ± 1500 sites µm−2, the ICAM-1 site density inPeyer’s patch HEV is well in excess of the density atwhich lateral mobility becomes necessary to mediateadhesion.17 However, it is possible that lateralmobility of integrins could play a role in the homingof lymphocytes to sites with lower ICAM-1 density.

Rapid adhesion triggering under flow

In order to examine the ability of chemokines toinduce adhesion in the context of the multi-steprecruitment cascade, many groups have utilized invitro flow chamber assays that mimic blood flow. Incontrast to static adhesion assays, selectin-mediatedrolling interactions are important under flowconditions, as leukocytes will not interact on surfacescoated only with ICAM-1.15,22,26 Lymphocytes andmonocytes can also initiate selectin-independentlow affinity rolling interactions with VCAM-1 orMAdCAM-1.10,11 Chemoattractant upregulatedintegrin function can then induce arrest and firmadhesion of rolling cells. Many chemokines cantrigger adhesion of leukocyte subsets expressingthe appropriate chemokine receptors. For example,IL-8 will induce adhesion of neutrophils whichexpress CXCR1 and CXCR2;38,39 MCP-1 will triggerthe adhesion of monocytes through CCR2;40 andeotaxin can trigger CCR3-dependent adhesion ofeosinophils.41 Adhesion of naive lymphocytes canbe induced by the CCR7 ligands SLC and ELC,26,42

while different memory lymphocyte subsets willadhere in response to ligands for CXCR3 (IP-10 orMig),43 CCR4 (MDC or TARC),44 CCR6 (MIP-3α),26

or CCR1 and CCR5 (RANTES).45

In most in vitro adhesion triggering studies, leuko-cytes tether and adhere almost immediately,26,38

while in vivo they often engage in significantrolling interactions before arresting and adheringfirmly.12,23,46 DiVietro et al.39 addressed this issue bychanging the site density of immobilized chemokine.As the concentration of immobilized chemokine wasincreased, the distance that cells rolled and time of

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interaction before arrest decreased significantly. Thissuggests that the neutrophils were integrating thechemokine signal and had to reach a threshold ofactivation before they would trigger adhesion. Theobservation that the rolling velocity of cells decreasedas they rolled also suggests a gradual integration ofsignal.39 Therefore it appears that adhesion can betriggered more quickly if the chemoattractant densityis increased.

Chemokines on a stick

Most chemokines are produced as secretedmolecules. However, there are two chemokines,fractalkine and CXCL-16, that are known to beexpressed as transmembrane proteins attachedto a mucin stock.47,48 Fractalkine can mediaterapid adhesion of CX3CR1+ NK cells, CD8+ Tlymphocytes, and monocytes under conditions ofshear flow.49,50 However, unlike other chemokines,cells can tether and adhere directly to fractalkinein a PTX-independent manner.49,50 This effectrequires intact chemokine as no adhesion is seenif the chemokine domain and mucin stock areimmobilized separately or in trans. It is not knownwhether CXCL16 can act in the same manner, and itis unclear whether these chemokines can elicit directadhesion of leukocytes to endothelium in vivo.

Chemokine immobilization

In flow assays, chemokines can initiate leukocyteadhesion if they are coimmobilized on a surfacewith adhesion molecules,26,38 or if they are addedas soluble factors in the perfusion fluid.39,40

However, it is unlikely that soluble chemoattractantssecreted from localized areas in vivo would persistat the blood-endothelium interface due to thedilution factors induced by shear flow. Therefore,chemoattractants that bind to the endothelial surfacewould be most likely to promote leukocyte adhesionand migration.51 This point is illustrated by theobservations of Weber et al.:52 TNFα-stimulatedendothelial cells produce two chemokines, Groα

which binds to the endothelium, and MCP-1 whichis secreted into the supernatant without bindingthe endothelium. Under flow conditions, only Groα

mediates monocyte adhesion to the endothelium,while the MCP-1 is diluted and washed away byfluid flow. As MCP-1 has been shown to induce

monocyte adhesion under flow conditions when itis present homogeneously in the perfusion buffer,40

this confirms that chemokine gradients from localsources are most efficient when immobilized on theendothelium. Soluble chemokines exposed to shearflow are dispersed and unable to mediate adhesion.

Many chemokines have been shown to bindto the surface of endothelial cells.45,53 As mosthave highly charged amino acid residues that canmediate binding to heparan sulfate and otherglycosylaminoglycans (GAGs),54–56 and heparinasetreatments block binding of chemokines to endothe-lial cells,52,57 this interaction is likely to facilitate theimmobilization of chemokines on the endothelialsurface. Another molecule that may immobilizechemokines on the endothelial surface is the Duffyantigen receptor for chemokines (DARC). Thischemokine receptor is expressed on endotheliumand red blood cells, and binds promiscuously tomembers of the CC and CXC classes of chemokineswithout activating intracellular signaling pathways.58

However, it is not clear whether DARC facilitatespresentation of chemokines on the endothelium. Infact, responses in DARC deficient mice suggest thatthis molecule could act to sequester chemokines andprevent them from activating leukocytes.59

Activated endothelial cells can produce and binda number of chemokines that mediate leukocytearrest.38,45,52 In addition, Middleton et al.57 haveshown that endothelial cells transport chemokinesfrom the basolateral to luminal surface, indicatingthat chemokines produced in the tissues can alsoimpact on leukocyte recruitment. IL-8 or RANTESinjected into the skin accumulate at the basolateralsurface of the endothelium. Instead of diffusingthrough endothelial cell junctions, the chemokinesare transported through the endothelium in vesiclesand presented on the apical surface to intravascularleukocytes.57 A truncated IL-8 peptide that isunable to interact with heparan sulfate was nottransported through the endothelium to the apicalsurface, implicating heparan sulfate in this transportprocess.57 These authors also postulated that DARCcould play a role in transport across the endotheliumas the chemokines that could be transported to theapical surface of endothelial cells (IL-8 and RANTES)also bind to this promiscuous chemokine receptor.57

In contrast, MIP-1α does not bind to DARC and wasnot transported across the endothelium even thoughit binds to heparin.57

Chemokines, including SLC, ELC, IL-8, and MCP-1,can also be transferred from sites of injection or

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inflammation to the draining lymph nodes.60–63 Thisalso appears to require binding to heparan sulfate,as SLC with a truncated carboxyl terminus doesnot accumulate in draining lymph nodes.60 In thelymph node, transferred chemokines move from thesubscapular sinus through a network of reticularconduits,64 and can be transported across HEV to theluminal surface.60,61,63 MCP-1 transported from theinflamed skin triggers monocytes to adhere in HEVand gain access to the lymphoid tissues,63 where theycould influence immune responses.

Lymphocyte homing to secondary lymphoidtissues

Recruitment of lymphocytes from the blood tosecondary lymphoid tissues is directed by bindinginteractions at specialized HEV which express ICAM-1 and a number of glycosylated L-selectin ligandscollectively known as peripheral node addressins(pNAd) (Figure 1). Lymphocytes roll on HEV in vivo,with approximately 25% of rolling cells becomingfirmly adherent to the vessel wall.23 It is importantto note, that <1% of the cells adhere without rolling,demonstrating the importance of the multi-stepprocess. In peripheral lymph node HEV, lymphocytetethering and rolling are completely blocked by L-selectin antibodies, while the firm adhesion step canbe blocked by antibodies against LFA-1 (αLβ2).23 Inthe Peyer’s patch however, lymphocyte homing hasan additional adhesive component: α4β7 expressedon lymphocytes interacts with MAdCAM-1 to dramat-ically slow rolling (and can support some tetheringand rolling even in the presence of L-selectin anti-bodies), while antibodies against α4β7, MAdCAM-1,or LFA-1 each block adhesion completely.13 Thissuggests that α4β7/MAdCAM-1 interactions functionas an essential ‘bridge’ between the tethering andfirm adhesion steps in Peyer’s patch HEV.

A spontaneous deletion in the functional codingregion for two closely associated chemokines, SLCand ELC,65,66 results in a serious defect in the abilityof normal naive T lymphocytes to home to secondarylymphoid organs in DDD/1-plt (paucity of lymphnode T cells) mice.67,68 A targeted mutation inCCR7, the receptor for SLC and ELC, producesa similar phenotype.69 Consistent with this data,T lymphocytes adhere primarily in HEV that stainwith SLC (Figure 2). Furthermore, lymphocytesdesensitized to SLC or ELC do not adhere inHEV of PLN or Peyer’s patches.60,70 In contrast,

Figure 2. T lymphocytes localize primarily to Peyer’s patchHEV that express SLC. TRITC-labeled T lymphocytes (red)were allowed to accumulate in Peyer’s patch HEV for 10min after iv. injection into normal animals. An AlexaTM 488-conjugated anti-SLC antibody (green) was then injected toilluminate SLC-expressing HEV segments. T lymphocytespreferentially accumulate in SLC+ interfollicular areas butnot in the follicular (F) areas. B lymphocytes accumulatepreferentially in perifollicular areas that are often SLCnegative (not shown) (20X objective).

desensitization with SDF-1α, the ligand for CXCR4,does not affect lymphocyte rolling or arrest in HEV.

While DDD/1-plt and CCR7 deficient mice aredeficient in the number of naive lymphocytes insecondary lymphoid tissues, they have significantnumbers of memory T lymphocytes.67,69 It wasproposed that this resulted from the recruitment ofT lymphocytes from the tissues through the afferentlymphatics. However, it is also possible that otherchemokines can mediate the recruitment of memorylymphocytes from the blood. For example, followingsensitization to a contact allergen, MIP-1α andMIP-1β are produced in inflamed lymph nodes, andMIP-1β is transported to the surface of HEV.71 This isassociated with increased recruitment of lymphocytesto the lymph node, and can be partially blockedby treating animals with neutralizing antibodiesagainst these chemokines. Other chemokines thatattract memory lymphocytes, including MDC, TARC,RANTES, Mig and MCP-1, are also expressedin the lymph node during inflammation,72–74

and chemokines may also be transported from

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inflammatory tissue sites via the draining lymphaticsand specialized reticular conduits.64

Interestingly, B and T lymphocytes exhibitdifferences in lymphoid homing to secondarylymphoid organs even though they both respond toSLC and ELC in chemotaxis and adhesion assays.75

While adhesion of both subsets can be blocked in vivoby PTX treatment, desensitization with SLC or ELCfails to block the arrest of B lymphocytes in HEV.70

Furthermore, B and T lymphocytes accumulate indifferent areas of the Peyer’s patch. T lymphocytesarrest primarily in interfollicular HEV which stainwell with antibodies against SLC (Figure 2), whileB lymphocytes adhere primarily in perifollicularHEV which often lack SLC.70 These findings areconsistent with the observations that B lymphocyteshome normally to the secondary lymphoid tissuesof DDD/1-plt mice.67,68 B cells express CXCR5, thereceptor for a chemokine (BLC/BCA-1) expressedprimarily in B cell follicles.76 However, B lymphocytesfrom CXCR5-deficient mice home normally to lymphnodes and Peyer’s patches when transferred intowild type77 or DDD/1-plt mice (Warnock andECB, unpublished observation), suggesting thatBLC/BCA-1 does not provide the signal for B celladhesion in HEV of secondary lymphoid organs. Thechemokines or other chemoattractants mediatingCCR7-independent B cell recruitment to secondarylymphoid tissues remain to be determined.

Lymphocyte homing to skin versus intestinalsites

Memory lymphocyte populations exhibit selectivityin their homing to non-lymphoid tissues. Themost clearly defined of these populations are askin homing subset and a population that homespreferentially to intestinal sites (Figure 1). Thespecificity of these subsets for their target tissuesderives from selective expression of specific adhesionmolecules and chemokine receptors that recognizeligands that are differentially expressed at thesetissue sites. The use of a limited number of adhesionmolecules and chemokine receptors in variouscombinations can increase the potential diversity ofspecific homing responses.

Lymphocytes that home to inflamed skin expressthe cutaneous lymphocyte-associated antigen (CLA),a carbohydrate modification that recognizes E-selectin.78,79 CLAhi cells migrate in responseto the chemokines TARC (a CCR4 ligand) and

CTACK (a CCR10 ligand).44,80 As TARC is displayedon skin microvascular endothelium of patientswith psoriasis and lichen planus, and can triggeradhesion of CLA+ memory T lymphocytes toICAM-1 under flow conditions,44 it is likely tobe involved in lymphocyte trafficking to the skin.CTACK is produced constitutively by epidermalkeratinocytes,80 suggesting that it may also play arole in the recruitment or localization of CLA+

lymphocytes in the skin. Indeed, either CCR4 orCTACK support the homing of E-selectin-bindinglymphocytes to inflamed skin in mice, while blockingboth inhibits recruitment.81 However, a formaldemonstration of chemokine-induced adhesiontriggering within the skin microvasculature awaits insitu microscopic studies.

Memory lymphocytes that migrate preferentiallyto intestinal sites express high levels of the α4β7-integrin compared with naive lymphocytes orsystemic memory cells.82 A population of thesecells also express CCR9, the receptor for TECK,83

which is expressed in the epithelium of the smallintestine but not the colon.84 Almost all lymphocytesin the small intestine, but few in the colon or otherextra-lymphoid tissues express the TECK receptor,CCR9,83,84 suggesting a specialized role for CCR9and TECK in small intestinal immunity. Interestingly,CCR9 is expressed by many IgA antibody secretingcells (ASC), but not by IgM or IgG ASC, suggestinga mechanism for selective localization of IgA plasmacells to the gut.85 It remains to be determinedwhether TECK mediates lymphocyte adhesionand/or diapedesis within the microvasculature of thesmall intestine, and what other factors can mediatelymphocyte recruitment to other intestinal sites.

Chemokines in transendothelial migration

While chemoattractants bound to the endothelialsurface are most likely to promote leukocyteadhesion, it has been assumed that soluble orimmobilized chemoattractant gradients across theendothelium induce adherent cells to migratefrom the vascular surface into the tissues.51,52

Cinamon et al.86 have recently postulated thatsurface immobilized chemokines can facilitatetransendothelial migration in the absence ofchemoattractant gradients across the endothelium.Transendothelial migration required a higherconcentration of bound chemokine than needed toelicit firm adhesion, and also required the presence

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of continual shear forces. A shear dependentenhancement of monocyte migration has alsobeen reported,52 but it was attributed to therelease of soluble chemokine on both sides ofthe endothelium and the formation of a gradientonly when shear forces removed chemokine fromthe apical surface. However, transendothelialmigration in the absence of a chemotactic gradientas purported by Cinamon et al.86 would suggestpotential roles for additional factors includingshear-dependent mechanoreceptors in facilitatingthe migration process. This concept could haveimportant implications for the leukocyte recruitmentprocess, and clearly requires more investigation.

Concluding remarks

The homing of leukocytes from the blood to thetissues needs to be a well controlled process to ensurethe recruitment of the proper cells to the correctlocation at the right time. The use of chemokinesat critical control points in the adhesion processallows for complex combinations of interactionsto facilitate specific homing of specialized celltypes to tissues. Although we have come a longway in our understanding of how leukocytes arerecruited from the blood, there are many issues thatstill need to be resolved. The signaling pathwaysby which chemokines trigger leukocyte adhesionare poorly understood, as are the mechanismsregulating chemokine transport and presentationon endothelial surfaces. Current thinking aboutthe requirement for chemokine gradients acrossthe endothelium may need to be revised, and therole of shear forces in mediating migration in vivoneed to be elucidated. In complex inflammatoryconditions that involve multiple chemokines, it willbe important to distinguish whether they act totrigger cell adhesion within the vasculature, inducemigration across the endothelium, or act to localizecells to distinct locations within the tissues.

Acknowledgements

We thank E. J. Kunkel for critical review of themanuscript, and A. Warnock for providing confocalimages of Peyer’s patch HEV. This work is supportedin part by USPHS grants and an award from theDepartment of Veterans Affairs to ECB. BJ is therecipient of a fellowship from the Canadian Institutesof Health Research.

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