To efficiently protect the body from infectiousorganisms, leukocytes circulate as nonadher-ent cells in the blood and lymph where, uponencountering an inflammatory stimulus, theyarrest and migrate into the affected tissues1–3.Once arrested from the bloodstream, theleukocyte faces a formidable barrier made ofendothelial cells linked to each other byinterendothelial junctions (Fig. 1)4. To initiatethe transmigration process through this vascu-lar barrier—which is also called diapedesis orextravasation—activated leukocytes mustsense the interendothelial junction and engage

The last molecular fortress in leukocyte trans-endothelial migrationMICHEL AURRAND-LIONS, CAROLINE JOHNSON-LEGER AND

BEAT A. IMHOF

Leukocyte arrest on inflamed endotheliumconstitutes only the first phase of theirrecruitment into the tissues. New datapoints to the roles played by JAM-1 andCD99 in leukocyte passage through thebarrier posed by the vascular endotheliumduring inflammatory responses.

in molecular interactions in order to crawlthrough the cleft between adjacent endothelialcells and gain access to the underlying base-ment membrane. In contrast to leukocyteadhesion and arrest, the molecular details ofleukocyte transmigration are not well under-stood. In this issue of Nature Immunology,Muller5 and Weber6 and colleagues explorethe molecular interactions that are crucial forleukocyte transmigration into the tissues. Inaddition, they identify candidate moleculesthat help propel leukocytes through the vascu-lar barrier and into sites of infection.

Leukocyte homing begins with a multi-step adhesion process that captures theleukocyte from circulating blood and adheresit to the vascular wall1–3. Adhesive selectinmolecules initiate the rolling of leukocytesalong inflamed endothelium. Chemokinesinduce the activation of integrins on rollingleukocytes that then bind to vascular ligands;this process leads to the tight adhesion ofleukocytes to the endothelium7. One of theseintegrins is the heterodimeric lymphocytefunction–associated antigen 1 (LFA-1),which interacts with intercellular adhesion

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These concerns aside, the study by Martinand Goodnow will undoubtedly stimulatefuture investigations into the functional dif-ferences between IgM and IgD BCRs andthose expressed by B cells after CSR, as wellas the molecular mechanisms responsible forsuch differences. Published data concerningthis area are limited, despite the fact that amajor fraction of memory B cells in mice andhumans expresses class-switched BCRs.Because all forms of BCRs are found associ-ated with the signal-transducing Igα and Igβcoreceptors11, differences in activity amongthe classes of BCR are likely accounted forby unique interactions between their mem-brane and cytoplasmic domains and yet-to-be-identified integral membrane and cyto-plasmic factors. Such specific interactionscould lead to differences in plasma mem-brane distribution and density, transmem-brane signaling and endocytic potential. Thelatter idea is supported by previous in vitrostudies in the case of mouse IgG1 and IgG2aisotypes12.

Taken together with previous studies of the“maturation” of IgV region structure andfunction during the development of the mem-ory B cell compartment, the study by Martinand Goodnow inspires a new working modelfor BCR function at different stages of B celldevelopment (Fig. 1). Primary B cells

express IgM and IgD BCRs, whose V regionshave not been altered by somatic hypermuta-tion. Such “affector” BCRs efficiently pro-mote foreign antigen–independent develop-ment in central lymphoid organs and mediatetolerance induction to the self-antigens towhich they bind with high avidity. Once Bcells enter the periphery, the low-avidity self-antigen reactivity of subpopulations of“affector” BCRs may further direct primarydevelopment towards particular mature B cellsubsets that are phenotypically and function-ally distinct8,9,13.

At the onset of an immune response,“affector” BCRs also mediate clonal selec-tion into the response. However, the develop-ment of protective immunity to mostpathogens requires the transformation of“affector” into “effector” BCRs. This takesplace during memory B cell development,most probably in the GC. The V regions of“affector” BCRs are subjected to iterativehypermutation and both positive selection bythe driving foreign antigen and negativeselection by self-antigens14. The resulting“effector” V regions possess increased affini-ty and specificity for the driving foreign anti-gen, properties that confer exquisite sensitiv-ity for this antigen to memory B cells. Manymemory B cell precursors also undergo CSR,which endows their progeny with the second

component of an “effector” BCR, an IgG,IgA or IgE C region. This results in the acqui-sition of unique functions that promote effi-cient secondary differentiation, such as rapidand sustained proliferation and generation ofAFCs in the case of IgG1. As alluded to byMartin and Goodnow3, this model predictsthat humoral immunity is accounted for notso much by quantitative differences in thefrequency of pathogen-specific B cells, butby qualitative differences in the function ofthe BCRs expressed by primary versus mem-ory B cells.

1. Berek, C. & Milstein, C. Immunol. Rev. 96, 23–41 (1987).2. Honjo,T. Immunol.Today 3, 214–217 (1982).3. Martin, S. & Goodnow, C. Nature Immunol. 3, 182–188 (2002).4. Kaisho,T., Schwenk, F. & Rajewsky, K. Science 276, 412–415

(1997).5. Achatz, G., Nitschke, L. & Lamers, M.C. Science 276, 409–411

(1997).6. Roth, P. E. et al. J. Exp. Med. 178, 2007–2021 (1993).7. Kenny, J. J. et al. J. Immunol. 154, 5694–5705 (1995).8. Haughton, G.,Arnold, L.W.,Whitmore,A. C. & Clarke, S. H.

Immunol.Today 14, 84–87 (1993).9. Martin, F. & Kearney, J. F. Immunity 12, 39–49 (2000).10. Pogue, S. L. & Goodnow, C. C. J. Exp. Med. 191, 1031–1043

(2000).11. Reth, M. Annu. Rev. Immunol. 10, 97–121 (1992).12. Tarlinton, D. Science 276, 374–375 (1997).13. Hayakawa, K. et al. Science 285, 113–116 (1999).14. Hande, S., Notidis, E. & Manser,T. Immunity 8, 189–198 (1998).

Kimmel Cancer Center and Department ofMicrobiology and Immunology, Jefferson MedicalCollege,Thomas Jefferson University, Philadelphia, PA19107, USA. (Manser@Springfield.jci.tju.edu)©

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molecule 1 (ICAM-1) expressed by vascularendothelium upon inflammation7. However,considerably less is known about the mech-anisms used by leukocytes to cross theendothelium. Until now, platelet-endothe-lial cell adhesion molecule 1 (PECAM-1,also known as CD31) was the only knownmolecule involved in diapedesis andexpressed on leukocytes andendothelial cells at the intercel-lular contacts8,9.

Muller and colleagues nowidentify a second molecule,CD99, that is essential formonocyte transmigration and isconcentrated at interendothelialcontacts5. They show that dia-pedesis occurs as a multistepcascade involving sequentialmolecular interactions betweentransmigrating leukocytes andendothelial cells. Using a panelof antibodies generated againstendothelial cells, they showthat transmigrating monocytesfirst use homophilic PECAM-1interactions to link leukocytesto the luminal surface ofendothelial cells and initiatediapedesis. Homophilic CD99interactions then allow theinvading leukocytes to transmi-grate through clefts in theendothelial wall. This mecha-nism probably occurs underinflammatory and noninflam-matory conditions because thedistribution of PECAM-1 andCD99 at endothelial cell-cellcontact regions is not affectedby inflammatory stimuli. Thesetwo molecules may thus formthe minimal basic housekeep-ing elements for leukocytetransmigration.

A role for inflammatorystimulation of endothelial cellsin the control of leukocytetransmigration is provided bythe study by Weber and col-leagues6. They identify the mol-ecule junctional adhesion mole-cule 1 (JAM-1) as a ligand forLFA-1. In contrast to PECAM-1 or CD99,JAM-1 is exclusively localized at junctionalcomplexes between quiescent endothelialcells. Previously, JAM-1 was shown to relo-calize to the apical surface of endothelialcells after inflammatory stimulation10.Weber and colleagues show that JAM-1-

and are formed by a network of transmem-brane proteins that are specific to each typeof junction. Gap junctions are clusters oftransmembrane, hydrophilic channels thatallow the direct exchange of ions and smallmolecules between adjacent cells. However,no evidence exists for the involvement ofgap junctions in transmigration of leuko-

cytes. Adherens junctions areformed by transmembrane pro-teins of the cadherin family,which exhibit homophilic inter-actions and are associated withintracellular catenins and theactin cytoskeleton. Numerousstudies have shown that thebinding of cadherins to thecatenin-actin cytoskeleton isessential for morphogenesisand maintenance of cadherinsat cell-cell contacts. In endo-thelial cells, adherens junctionsare formed by vascular-endo-thelial (VE)-cadherin, whichplays a central role in vasculo-genesis and in the regulation ofmacromolecular permeability.It has been proposed that VE-cadherin acts as a gatekeeperfor the passage of leukocytes,which, themselves, do notexpress VE-cadherin. Themigrating leukocyte inducesdelocalization of VE-cadherinaway from adherens junctions,resulting in a gap throughwhich the transmigrating leu-kocytes can pass12. Althoughthe mechanism by which delo-calization of VE-cadherinoccurs is still unknown, it isclear that the opening of theendothelial adherens junctionis restricted to a limited region.

The third type of intercellularadhesive complexes are the tightjunctions that form close con-tacts between endothelial cellsand are located at the most api-cal part of the junction betweenadjacent cells. Electron micro-scopy studies have shown thatthe number of tight junctions in

endothelial cells varies with the requirementfor permeability control11. For example, tightjunctions are well developed in the brain vas-cular bed that forms the blood-brain barrier,whereas tight junctions are barely detectablein the high endothelial venules of lymphoidorgans where constitutive extravasation of

–LFA-1 interaction is involved in tight adhe-sion or transmigration of leukocytes,depending on the apical or junctional local-ization of JAM-1 on endothelial cells. Theseresults support the hypothesis that endothe-lial cells actively control the efficiency ofleukocyte transmigration by regulating thestructure of intercellular junctions.

Vascular junctions are molecular complex-es that contribute to the barrier function ofthe blood vessel, hindering leukocyte dia-pedesis11. At least three types of intercellularadhesive complexes have been described: gapjunctions, adherens junctions and tight junc-tions5. These were defined morphologically

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Figure 1. New molecular mechanisms in leukocyte transendothelialmigration. (a) An inflammatory stimulus leads to expression of ICAM-1 on theluminal face of the vascular endothelium.The same signal affects the junctional local-ization of JAM-1 at interendothelial contacts while the lateral distribution of CD99and PECAM-1 is not affected. (b) After tethering, rolling, triggering and tight adhe-sion, leukocytes transmigrate through the vascular endothelium using JAM-1,PECAM-1 and CD99. Chemokines activate integrins on leukocytes (triggering),which leads to LFA-1 engagement with vascular ICAM-1 and JAM-1. Subsequently,sequential trans-homophilic interactions of PECAM-1 and CD99 contribute totrans-endothelial migration of leukocytes.

Inflammatorysignal

JAM-1

ICAM-1

PECAM-1

CD99

LFA-1

Leukocyte

Chemokine activation

Endothelial cell

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NEWS & VIEWS

lymphocytes occurs. Three types of transmem-brane proteins are found in tight junctions:occludin, claudins and the JAMs4. Tight junc-tions consist of strands of the apparently fusedplasma membranes of adjacent cells in whichthe tetraspan proteins occludin and claudinsare incorporated. JAM-1 is an immunoglobu-lin (Ig) superfamily molecule that is peripher-ally associated with tight junctions via cyto-plasmic adaptor proteins bridging JAM-1 andclaudins. Thus, inflammatory signals mayinduce the relocalization of JAM-1 onendothelial cells by regulating its associationwith adaptor proteins.

Monoclonal anti–JAM-1 blocked mono-cyte transmigration in a murine model13.However, because murine monocytes do notexpress JAM-1, it was postulated that theantibody interfered either with the remodel-ing of interendothelial junctions duringtransmigration or that additional ligands forJAM-1 exist on transmigrating cells. Weberand colleagues bring a valuable missingpiece to this puzzle by showing that LFA-1on leukocytes binds to JAM-1 expressed byendothelial cells6. They show that the mem-brane-proximal Ig domain of JAM-1 sup-ports its interaction with LFA-1, whereas themembrane-distal Ig domain of JAM-1 isresponsible for its homophilic dimerizationat interendothelial junctions14 (Fig. 1). Thisstructural duality opens the possibility thatJAM-1–JAM-1 interactions at interendothe-lial contacts occur simultaneously to LFA-1–JAM-1 interactions between the transmi-grating leukocyte and endothelial cells. Inaddition, when JAM-1 is localized on theluminal side of the blood vessel, it inducestight adhesion of leukocytes. Until now thisfunction was assigned to ICAM-1 interactionwith LFA-13. However, the exclusive expres-sion of ICAM-1 on the luminal surface ofinflammatory endothelium could not explainthe involvement of LFA-1 in transmigration.The dual involvement of LFA-1 in tightadhesion and transmigration of leukocyteshas now been identified6. The scenario out-lined above, however, does not provide a rolefor the expression of JAM-1 on human circu-lating cells nor does it provide an explana-tion for the signals that lead to relocalization

of JAM-1 in endothelial cells. Answeringthis question may be more complex than pre-viously thought because it seems that furthermolecules are involved in leukocyte dia-pedesis.

At present it is not clear whether transmi-gration starts with PECAM-1–PECAM-1 orLFA-1–JAM-1 interactions, whether thesemechanisms operate at the same time orwhether there are qualitative differencesbetween these adhesion pairs. PECAM-1 caninfluence the cellular actin cytoskeleton, themachinery for cell migration15. However, thedirect involvement of PECAM-1 in leuko-cyte migration has not been identified. Thiscontrasts with the integrin LFA-1, whichdirectly participates in leukocyte migrationby linking the extracellular environment tothe intracellular cytoskeleton. The currentmodel suggests that PECAM-1 ligationtransduces signals into cells through its asso-ciation with the phosphatases SHP-1 andSHP-2. This process occurs either in trans-migrating leukocytes or endothelial cells inwhich it may contribute to open the junction-al complexes. Both mechanisms contributeto facilitating leukocyte diapedesis throughthe vascular wall.

The final step in leukocyte transmigrationinvolves CD99. This molecule was identifiedin the early 1990s and is expressed by allleukocytes and red blood cells. Muller andcolleagues show that vascular endothelialcells constitutively express CD99 at lateralcontacts5. In contrast to anti–PECAM-1, anti-CD99 arrests leukocytes only after penetrat-ing deep into the interendothelial junctions,just before transmigration is completed. Thelate arrest of the leukocyte may be explainedby blockade of the uropod, the tail of themigrating cell and a membrane region thatconcentrates high numbers of adhesion mole-cules, such as ICAM-1 and possibly others.Unfortunately we do not know yet whetherCD99 also becomes enriched in this region.By analogy to PECAM-1, the question arisesof whether CD99 is directly or indirectlyinvolved in leukocyte migration. CD99 regu-lates LFA-1 integrin expression and affinityvia an unknown signal-transduction mecha-nism16. Thus, it is likely that CD99 interferes

indirectly with leukocyte transmigration, pos-sibly by regulating the function of integrinsin the uropod.

The reports by Muller5 and Weber6 andcolleagues provide the first solid evidencefor the existence of a multi-step molecularmechanism involved in leukocyte transmi-gration. The role of JAM-1 and CD99 inguiding leukocytes through the junctionalcomplexes as well as the refined dissectionof this multi-step cascade will be researchactivities for the immediate future; real-timeintravital microscopy will be instrumentalfor this12. The results should tell us why ablood vessel does not become leaky whileleukocytes proceed through the gap in theendothelium, and confirmation of the twomechanisms in vivo will also be of com-pelling interest. It took ten years for us tounderstand the three steps of leukocyte adhe-sion and many pharmaceutical companieshave now targeted these steps to identifycompounds that halt chronic inflammation.Thus, identification of the moleculesinvolved in leukocyte transmigration mayprovide important future targets for thera-peutic intervention. The current state ofaffairs suggests that this may be achievedrapidly.

1. Butcher, E. C.,Williams, M.,Youngman, K., Rott, L. & Briskin, M.Adv. Immunol. 72, 209–253 (1999).

2. Springer,T. A. Cell 76, 301–314 (1994).3. Imhof, B. A. & Dunon, D. Adv. Immunol. 58, 345–416 (1995).4. Johnson-Leger, C., Aurrand-Lions, M. & Imhof, B. A. J. Cell. Sci.

113, 921–933 (2000).5. Schenkel, A. R., Mamdough, Z., Chen, X., Liebman, R. M. &

Muller,W. A. Nature Immunol. 3, 143–150 (2002).6. Ostermann, G.,Weber, K. S. C., Zernecke, A., Schroder, A. &

Weber, C. Nature Immunol. 3, 151–158 (2002).7. von Andrian, U. H. & Mackay, C. R. N. Engl. J. Med. 343,

1020–1034 (2000).8. Albelda, S. M., Muller,W. A., Buck, C. A. & Newman, P. J. J. Cell.

Biol. 114, 1059–1068 (1991).9. Liao, F., Ali, J., Greene,T. & Muller,W. A. J. Exp. Med. 185,

1349–1357 (1997).10. Ozaki, H. et al. J. Immunol. 163, 553–557 (1999).11. Simionescu, M., Simionescu, N. & Palade, G. E. J. Cell. Biol. 67,

863–885 (1975).12. Shaw, S. K., Bamba, P. S., Perkins, B. N. & Luscinskas, F.W. J.

Immunol. 167, 2323–2330 (2001).13. Martin-Padura, I. et al. J. Cell. Biol. 142, 117–127 (1998).14. Kostrewa, D. et al. EMBO J. 20, 4391–4398 (2001).15. Poggi, A. Panzeri, M. C., Morretta, L. & Zocchi, M. R. Eur. J.

Immunol. 26, 817–824 (1996).16. Hahn, J.H. et al. J. Immunol. 159, 2250–2258 (1997).

Department of Pathology, Centre MédicalUniversitaire, Geneva 1204, Switzerland.(beat.imhof@medecine.unige.ch)

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