Selectins, T-cell rolling and inflammation

Reviews

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Selectins, T-cell rolling and inflammation The selectins, a family of Ca2+-dependent lectins, are expressed on inflamed vascular endothelium and some leukocyte subsets, and mediate adhesive contacts between blood cells and vessel walls. These interactions are loose and reversible, operate under conditions of shear flow, and result in leukocyte rolling along the vessel wall. The structure of the selectins and their ligands makes them uniquely suited for supporting the type of bond formation and dissociation that must prevail in order for a cell to be able to roll under conditions of flow. Because rolling precedes (and appears to be essential for) the integrin-mediated firm arrest before extravasation in response to inflammatory or infectious stimuli, inhibition of selectin function has potential for anti-inflammatory therapy, but also presents some significant challenges because of the complexity of the processes involved. FOR memoryT cellsto participatein an immuneresponse in peripheraltissues,theymustfirst exit thebloodstream efficiently at the correct site.To achievethis end,an elegantsystemof lymphocytetrafficking hasevolvedthat ensures that naiveT cellsexit the vasculature mostefficiently in peripherallymph nodes(wherepotentialexposure to new antigensis maximal), while memoryT cells preferentially hometo peripheraltissuessuchasskin or lung, whereforeign antigensto which the hosthasbeenpreviouslyexposedarelikely to be re-encountered in thesettingof infectionor injury’. Here, we describethe extravasationparadigm,togetherwith the specializedadhesionmoleculesinvolved, andthen summarizesome experimentalmodelsthat probe the possibilityof inhibiting extravasationasan anti-inflammationtherapy.Although augmentation of extravasationcould alsobe clinically relevant,we will not discuss this aspecthere.

Thomas S. Kupper* MD, PhD Chief Division of Dermatology, Brigham and Women pital, ston, 20 Shattuck Stre MA02115, USA. Ronen Alon PhD Senior Investigator Dept of Immunology, Weizmann Institute of Science, Rehovot, 76100 Israel.

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Division of Dermatology, Brigham and Women’s Hospital, 20 Shattuck Street, Boston, MA 02115, USA. Tel: tl 617 278 0993 Fax: tl 617 278 0305 *e-mail: tskupper0bic.s. bwh.harvard.edu

The multi-step

extravasastion paradigm

Extravasationof T cells takesplace as a sequenceof overlapping steps,each of which can be independentlyregulated,providing multiple opportunitiesfor control and specificity. Although originally describedin detailfor neutrophils2 , this paradigm,with some C<>pynehtOlYY7

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Stimulus Integrin-activating

a

signals

d

Endothelium Key Endothelial

selectin ligand

Endothelial

selectin

Integrin-activating High-affinity

signal

l

integrin n

Low-affinity

integrin

lntegrin ligand II

Figure1. The extravasation paradigm shown schematically. (For simplicity, only one type of selectin-ligand interaction has been depicted for each cell). (a) Memory T cells travelling through the blood stream express selectin ligands and low-affinity integrin ligands. (b) An inflammatory stimulus causes venule dilation, reduction in blood flow velocity and expression of selectins on the endothelial cell surface. (c) Interaction of the selectins with the carbohydrate domains of their ligands on the T-cell surface results in (c+z) transient tethering of the cells and rolling along the vessel walls in response to forces exerted by blood flow. The cells still express low-affinity integrin ligands. (f) If the appropriate ‘activating signals’ are encountered, the leukocyte integrins increase the binding affinity for their specific ligands by an unknown mechanism; the cells become firmly attached via their integrin receptors and flatten, and can then (g) extravasate.

modifications,nowalsoappears to applyto the extravasationprocess of T cells in both secondarylymphoid organsandperipheralextralymphoidtissues(Fig. 1). Leukocytes.arriving at a point in a vessel wherethe diameteris suddenlyincreased,areforcedtowardsthe vessel wall by the erythrocytes.becauseof their concaveshapeand greaterflexibility. Thisdiameterincreasecanoccuracutely,mediated by vasoactivefactorsreleasedin the settingof inflammation,or can exist as a function of static anatomy,when a narrow capillary becomesa wider postcapillaryvenule. If the appropriateadhesionmolecule-ligandpair is displayedon both circulatingcellsandendothelium,the leukocytebecomesengagedin looseadhesions (tethers) with the vesselwall. The forcesexertedby the flow of blood on the leukocytecausebondsto breakon the upstreamsurfaceof the cell, as the whole cell is pushedforward, allowing the formation of new bondsat the downstreamsurface.This formation and releaseof bonds.while the cell is beingpushedforward,resultsin a rolling motion along the endothelialsurfacein the direction of blood flow.

Ultimately,if appropriatesignalsare encountered by the rolling cell: leukocyteintegrinsareactivatedandinducedto increasetheir affinity for their counterreceptors on endothelialcells. The initial tetheringandrolling stepsarelooseandreversible.but can operateefficiently undershear.Conversely,the integrin-mediated firm adhesion operates only at low shearor followingprolongedcontact with thevesselwall. Oncestabilized,thisintegrin-mediated adhesion is quitestrong,leadingto thecompletearrestof theleukocyte.In the final step of the extravasationcascade,the cells flatten and develop pseudopodia, which areinsinuatedbetweenthe endothelialcell junctionsby a mechanism involving adhesion moleculessuchasplateletendothelialcelladhesion molecule1 (PECAM-1).Eventually,the entire cell passesout of the bloodstream and into the perivasculartissue, whereit is subjectto chemotacticgradientsthat can direct it to the sourceof the inflammatoryor infectiousprocess.The T cell is now poisedto respondto antigen,appropriatelypresented in the contextof majorhistocompatibility (MHC) molecules on antigen-presenting cells. 215

The molecules that mediate adhesion to endothelium The complex interactions underlying the leukocyte extravasation process are mediated by distinct families of adhesion molecules’. Members of the immunoglobulin superfamily, represented by adhesion molecules such as intercellular adhesion molecules 1 and 2 (ICAM-1, ICAM-2), vascular cell adhesion molecule 1 (VCAM-I), and mucosal addressin cellular adhesion molecule 1 (MAdCAM-l), are expressed predominantly on cytokine-activated endothelium, and act as ligands for their respective counterreceptors. These latter molecules belong to the integrin family, and include lymphocyte functionassociated antigen 1 (LFA-I), very late antigen 4 (VLA4) and a$,. After appropriate activation, integrins can mediate firm adhesion to vascular endothelium. Integrins and integrin-ligand interactions will not be discussed extensively in this review (reviewed in Ref. 3). Two other families of adhesion molecules, the selectins and their ligands, play a central role in the initiation of the contact between leukocytes and endothelial cells that ultimately results in extravas-

ation: these molecules will be the subject of this review. The selectins comprise a family of three closely related Ca?+-dependent lectins, which primarily mediate both very early transient adhesions and rolling interactions between leukocytes and endothelial cells. Their ligands belong to the mucin family - long, rod-like glycoproteins containing multiple serinelthreonine-rich, heavily 0-glycosylated domains that are essential for binding to the selectins. The structure of the selectins (and that of their ligands) renders them uniquely suited for both tolerating shear forces and mediating the rapid bond formation and dissociation necessary for cells to roll along the luminal aspect of the vessel wall. Selectin-mediated rolling adhesion initiates the cascade of events that results in the successful arrest of leukocytes (including T cells) at specific sites of extravasation. Presumably, a cell that rolls slowly along the endothelial surface has ample opportunity to become exposed to chemotactic or activating stimuli originating at, or near, the vessel walls. Selectins can therefore be viewed as playing a pivotal role in determining whether a

Table 1. The selectins and their ligands” Selectin

Synonyms

Site of expression

Ligand

Selectin

L-selectin

CD62L, Peripheral lymph node homing receptor, LECAM-1, MEL-14

Constitutively, on nearly all circulating leukocytes; localized to the tips of microvilli.

GlyCAM-1 (sgp50); CDS4 (sgp90) in peripheral lymph nodes; MAdCAMon mesenterfc lymph nodes. Binding depends on appropriate sialylation, fucosylation and sulphation.

T cells: Directs the homing of T cells to peripheral lymph nodes; mediates rolling and extravasation at these sites.Contributes to T-cell

function

Ref(s)

4-a

homing to mucosal lymph nodes and gut Peyer’s patches.

Neutrophils: Mediates rolling at inflamed or injured sites. Rapidly shed from the cell surface after activation by proteolytic cleavage. Possible signalling function. E-selectin

CD62E, ELAM-1

Induced on the luminal aspect of endothelial cells, especially in skin, by inflammatory agents such as IL-l, TNF-cx or LPS. Requires de nova mRNA synthesis for expression.

CLA on skin-homing T cells; N-linked glycans, one of which shows homology to chicken fibroblast growth factor receptor.

Thought to represent a skin vascular addressin; specifically binds skin-homing T cells. Mediates rolling of leukocytes in viva, and cloned CD4+ T cells in vitro.

9-12

P-selectin

CD62P, PADGEM, GMP-140

Stored pre-formed in Weibel-Palade bodies of endothelial cells and a granules of platelets. Inflammatory mediators such as histamine and thrombin induce the release of stored P-selectin and cytokinesinduce synthesis of P-selactin mRNA.

PSGL-1 when appropriately decorated with sialic acid- and fucose-bearing carbohydrates. Sulphotyrosine linkages might also be important for binding.

Mediates

5-8

leukocyte

rolling in vivo and in vitro.

aAbbreviations: CIA, cutaneous lymphocyte-associated antigen ; IL-I, interleukin 1; LPS, lipopotysaccharide; MAdCAM-1, mucosal addressin cell adhesion molecule 1; PSGL-1, P-selectin glycoprotein ligand 1; TNFa, tumour necrosis factor (1.

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leukocyte returns to the circulation or commits to an inflammatory response.

The structure of the selectins and their ligands The nomenclature of the selectins has undergone a series of revisions since their first identification (Table 1). Each of the three se0 lectins consists of: a domain analogous to that CD34 % > .s+of the plant C-type lectins; an epidermal c-g v growth factor (EGF)-like domain; a variable number of consensus repeats homologous to complement-binding proteins; a transmembrane region; and an intracytoplasmic C-terminal domain (Fig. 2). Their striking structural similarity probably reflects the multiplication of a single common ancestral gene. Selectins can also bind a class of complex oligosaccharides: known as lactosaminoglycans, that contam the core tetrasaccharide structure sialyl Lewis x (SLe”) (Fig. 3). The SLe” tetrasaccharide can be supported on either cell-surface glycoprotein or glycolipid scaffold?; it is then said to be ‘decorating’ these structures. In addition, synthetic Le” derivatives that are sulphated, and branched oligosaccharides that Key bear additional fucose groups, have been @ C-type lectin domain Immunoglobulin-like domain shown to bind the selectins with even higher 15 affinities than the originally described tetrasac8 Epidermal growth factor domain s Mucin-like domain with O-linked chat-ides,at least in vitro (reviewed in Ref. 5). (o) and N-linked ( ) 0 0 Consensus repeat To what extent these different possible carboglycosylation domains and hydrate selectin ligands are relevant in vivo resulphotyrosine ( s) residues mains to be determined. Binding of the selectins to their ligands is Ca?+dependent and Figure 2. Schematic representation of the structure of the selectins and their ligands. Note that E- and Poccurs predominantly via the lectin domain, alselectins are expressed on different types of endothelium from that expressing appropriately glycosylated CD34 and mucosal addressin cellular adhesion molecule 1 (MAdCAM-1). GlyCAM-1, which is secreted, though the integrity of the EGF domain seems has been omitted from this diagram of endothelial selectin ligands. CD34 and MAdCAMare transmemto be important for interactions with E- and brane proteins. Adapted from Ref. 7. P-selectin as well (reviewed in Refs 6,7). The cDNA for the protein backbone of the ligands for T-cell L-selectin and P-selectin glycoproteinligand 1 (PSGL-1)havebeencloned(reviewedin Refs 6-8) andall arepredictedto havemucin-likestructures - characterized Sia Sia by serine-andthreonine-rich sequences potentiallyableto supporthigh a2,3 I a2,3 6’ I degreesof O-linked glycosylation(Fig. 2). Interestingly,the protein so4Gal Gal backbones of a numberof the selectinligands,suchasCD34,havea widetissuedistribution,but only supportselectinbindingin a restricted I 8114 al 3 G,J$14 Fuc a1y3 Fuc 1 numberof anatomical sites,suggesting that unique,tissue-specific postGlcNAc translational modifications at the levelof carbohydrate groupsarecentral to adhesive function(reviewedin Ref.6). Gal Gal While PSGL-1 is well establishedas the principal ligand for I I P-selectin,theidentityof the ligandfor E-selectinismorecontroversial. F@ue a Structure of sialyl Lewis x (slex). (a)sLex is the core tetrasaccharA ligandfor E-selectinhasbeenidentifiedfrom mousemyeloidcells, ide molif shered by all the selectin figands. (b) 6’-sulphated sle” is the major andcloningof its cDNA predictsa 94% ammoacid sequence hommbohydrate groupon GlyCAM-1 chains, and coufd be the defining modifiology with chickenfibroblastgrowth factorreceptorlO. It is surprising c&n for L-selectin recogrcitiwp. Fuc, fucwe; Gal, galactose; GlcNAc, thata moleculethathasessentially the structureof apeptidegrowthfacI%@@&EI&; Sit, sialic acid. tor receptorcanfunctionasa ligandfor E-selectin.In humans,E-selectinligandscan bedetectedby the monoclonalantibodies CSLEX-1 andHECA-452,which recognizeSLe%earingstructures. HECA-4.52 similarto SLe”on a skin-homingsubset of memoryT cells’.An E-sealsorecognizesa relatedbut poorly definedoligosaccharide structure lectin-immunoglobulin chimerahasbeenusedto isolateE-selectin-

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Figure 4. L-selectin on the tips of T-cell microvilli. (a) Low-magnification of a false-coloured scanning electron micrograph of a typical L-selectin-positive cell. Bar, 1 km. (b) Higher magnification of a sorted human lymphocyte stained with immunogold. The gold particles are clustered on the apical surfaces of microvilli (arrow). Bar, 200 nm. Photomicrograph kindly provided by Dr Stanley Erlandsen, Dept of Cell Biology, University of Minnesota Medical School, MN. USA, who modified the original in order to highlight the L-selectin molecules. The original photomicrograph was published in Ref. 5, and is reproduced here with permission of Chapman and Hall, International Thomson Publishing Services Ltd, and the authors.

binding glycoproteins from human neutrophils and the promyelocytic cell line HL60. In this setting, the ligand seemed to be identical to the P-selectin ligand PSGL-1, suggesting that PSGL-1 on these cells can also act as an E-selectin ligand’“. In accordance with these findings, preliminary data from our laboratory ‘I indicate that the cutaneous lymphocyte-associated antigen (CLA) on memory T cells has both important similarities to and differences from conventional T-cell PSGL-1. Recently, increased activity of the enzyme al ,3 fucosyl transferase VII (FucT-VII), which adds the fucose groupto the sialylatedtrisaccharideprecursorto form SLe’, hasbeenassociated with the elevatedexpression of CLA on peripheral-blood T cells’landculturedlymph-nodeT cells’.

Molecular

mechanisms of rolling

The cellular localizationof the selectinsand their ligandsmakes them uniquely suited for allowing a cell to roll. For example, L-selectin is displayed selectively on cell-surfacemicrovilli of humanlymphocytes(Fig. 4) andneutrophilsrh. andPSGL-1 (Ref. 17) hasalsobeenlocalizedto thesestructuresin neutrophils.The accessibility of the moleculesis further enhancedby their great length. The mucin-likeligandsfor L- andP-selectin,andthe P-selectinmoleculeitself, arevery long. eachprojecting-40 nm from the cell surface.Furthermore,the consensus repeatsegments of all the selectins extendthe actualbindingsiteson the selectinsfor their ligandsaway from the cell surface,and might contributeto the flexibility of the molecules(Fig. 2). Studieson the kinetics of bond formationbetweenneutrophilsandP-selectinr8 predictedan unusuallyhighrateof P-selectin-ligandbond associationand dissociation,implying that, althoughthesebondsform rapidly. they are transient.The dissociation constantfor the P-selectin-PSGL-1bond(1 s ’ in the absence of shear)wasrelatively insensitiveto increasingshearforce, indicating high tensilestrength.Calculationshadshownthat the fluid drag

operatingon rolling cells wasmuch greater thanthe forcesnecessary to pluck transmembraneproteinsout of the plasmamembrane. The ability of the selectin-ligandbondto resist suchdislocationis thus critical for the initial tetheringof a flowing leukocyteto the vesselwall. Failureof the first bondto tether the cell would makeit unlikely that further bonds could form. thereby precluding the morestablemultimericinteractionsthat permit rolling’“. L-selectin-mediated rolling of a transfectedmousepre-B cell line” andstress resistance of E-selectin expressed on cytokine-activated human umbilical vein endothelialcells (HUVECs)?O dependon an intact cytoplasmicdomain. suggestingthat anchoringto the cytoskeletonis necessary for shear-resistant interactionsmediatedby thesetwo molecules.

Selectins mediate T-cell rolling

Severalin I!~L’O studiesin mouseandrat modelshad suggested that lymphocyteextravasation occursas a seriesof steps,involving the sequentialinteractionof cells with selectins,chemo-attractants andintegrins,analogousto that foundfor neutrophils(reviewed in Ref. 1). The applicationof temperature-controlled flow chambers, in which flow parameters prevalentin post-capillaryvenulescan be simulatedand controlled, combinedwith video-microscopy”,has madepossiblethe dissectionof theseindividualstepsat the molecular andcellularlevel in vitro, andhasdisclosedsimilaritiesaswell as interestingdifferencesin the adhesionmoleculesusedby neutrophils andlymphocytes. One of the first studiesto demonstrate that cell-surfaceE- andPselectincouldsupportshear-dependent rolling of specificT-cell subsetsin vitro usedhumanCD4+T-cell clones.isolatedfrom the skinof patientswith atopic dermatitisor leprosy’:. While certain T-cell clonesfrom suchpatientsexpressed functionalligandsfor P-selectin only, other clonesexpressed ligandsfor P- andE-selectin.All clones expressedPSGL-1. while only the E-selectin-bindingclonesexpressedCLA. Ligandsfor both selectinscould mediaterolling on their respectiveselectins.The expressionof one, or more, selectin ligandson a singlecell couldreflect thedifferent homingcapabilities of T-cell subsets,but might also allow’for sequential(or simultaneous)interactionsthat could work synergistically.The clinical implicationsof suchmechanisms will be discussed in the next section, The in vitro confirmationthat T cellscanutilize selectin-mediated interactionsas the first in a seriesof sequentialstepsfor extravasationhascomefrom a numberof studies.PurifiedhumanT cells from peripheralbloodhavebeenvariouslyshownto attachto androll on peripheralnodeaddressin (PNAd)::. E-selectin22,2i and cytokinestimulatedhumanHUVECS?‘,~~. Activation of the T cellswith phorbol ester significantly increasedthe velocity of rolling on PNAd (compatiblewith activation-dependent sheddingof L-selectin,reviewedin Refs6,7), andreducedthe rolling velocity on PNAd plus ICAM- of Jurkat T lymphoblaststo almostzero??,demonstrating that T cellscanparticipatein the processof selectin-mediated rolling andactiv!ation-dependent arrest.

In contrast to circulating neutrophils. however, T cells can utilize a number of other adhesion-molecule-Ii&and pairs for tethering and rolling as well as for stable arrest. T cells can attach to and roll on VCAM-1 in an u,-integrin dependent fashion?? and (YJ$ integrinexpressing lymphoid cells can roll on the mucosal addressin MAdCAM- in the absence of carbohydrate ligands for L-selectin2h. However. if properly glycosylated on its mucin domain, MAdCAMcan also support rolling of L-selectin-expressing cells:?. In sharp contrast to E- or P-selectin-mediated rolling, which is never followed by firm adhesion to these selectins. the interaction of VCAM-1 and MAdCAM- with CY~integrins is often characterized by immediate a~rest on contact with the substrate, or rolling and subsequent arrest. The T cells experiencing immediate arrest probably either express very high levels of cyqintegrin (e.g. memory T cells) or are pre-activated by antigen contact or chemokine exposure, for example, and exhibit high avidity for their respective ligands. Finally, the recently described vascular adhesion protein 1 (VAP-I), a 170 kDa sialoglycoprotein expressed normally on high endothelial venules (HEVs) of peripheral lymph nodes, and upregulated in inflammatory diseases such as arthritis, gut inflammation and allergic lesions, can support binding of activated lymphocytes under conditions of shear stress, in the absenceof L-selectit?“. while the proteoglycan CD44 interacting with its ligand hyaluronate. mediates rolling of activated mouse lymphocytes and T-cell lines?’ on cultured mouse endothelial cells. The activity of one or both of these two pathways might explain discrepancies observed between rolling mechanisms on selectin-transfected cells as opposed to cytokinestimulated endothelial cell layers”.

Significance of selectin-mediated

T-cell rolling in disease

L-selectin Most immunologically mediated inflammatory diseases involve extravasation of memory T cells at peripheral sites. Whereas L-selectin clearly mediates T-cell homing to secondary lymphoid tissues and interactions with HEVs there”‘, it does not seem to be sufficient for lymphocyte rolling at peripheral sites, perhaps because there are fewer and/or inefficient L-selectin ligands there. This view is supported by studies on the double E-selectin-P-selectin-deficient mouse strain”, in which in viva rolling, observed by video-microscopy of tumour necrosis factor cx (TNF-cr)-stimulated exteriorized mesentery is virtually absent. If Lselectin ligands were present in inflammatory sites, it should have been possible to demonstrate significant leukocyte rolling in these mice. Furthermore. L-selectin is absent from some memory T cells, especially the gut-homing fraction that expresses high levels of a& (the integrin ligand for the mucosal adhesion molecule. MAdCAM 1). The 01~integrins have been shown to mediate tethering and arrest without vesselassociated integrin activationZ5,and perhaps this mechanism is particularly well developed in the gut. Such cells might be activated at some distance from the site of actual adhesion. Furthermore. site-specific adhesion mechanisms would allow the selective recruitment of T-cell subsets, thereby increasing the efficiency of tissue-specific homing. E-selectin CLA- memory T cells are enriched in the skin, a site in which Eselectin is readily and persistently expressed during inflammation. CLA is selectively expressed on T cells that have undergone the naive-tomemory transition in lymph nodes draining the skin, while its ligand, E-selectin, is predominantly upregulated in skin by pro-inflammatory (also known as primary) cytokines. such as interleukin la (IL-la) or TNF-a. These cytokines are among the first to be released in cutaneous

tissues in response to injury or infection. The combination of sitespecific adhesion-molecule induction to retain skin-homing T cells. and the potential for antigen presentation in situ to the extravasated cell, provide an elegant mechanism whereby a T cell can ‘remember’ both the antigen and the anatomical location in which it was most recently encountered. However, not all sites expressing Eselectin and integrin ligands will recruit CLA+ T cells, suggesting that E-selectin expression alone might be necessary but insufficient for T-cell emigration into skin. Furthermore, although E-selectin appears to contribute to cutaneous T-cell-mediated delayed-type hypersensitivity (DTH) responses, there seems to be some functional redundancy with P-selectin, because in E-selectin-null mice, P-selectin can compensate in such a response’?. and P-selectin itself has been shown to be important in T-cell-dependent DTH in normal animals”. E-selectin overexpression and/or CLA+ T-cell infiltrates have been associated with a number of chronic skin diseases31. Atopic dermatitis is characterized by infiltration of antigen-specific T cells of the Th2 phenotype, producing a typical set of cytokines. namely IL-4, IL-5. IL-10 and IL-13 (Ref. 35). T-cell clones specific for the house dust mite antigen Dpt and expressing high levels of CLA have been isolated from the skin of patients suffering from this disease. The cells adhered to and rolled on E-selectin”. demonstrating that T cells directly involved in skin disease could potentially participate in an extravasation process. Although the hallmark of psoriatic lesions is epidermal keratinocyte hyper-proliferation, they also exhibit increased E-selectin expression on the microvasculatun?, and a CLA’ T-cell infiltrate. A subset of patients with psoriasis also develops psoriatic arthritis, and a recent study has shown that T cells from cutaneous psoriatic lesions were CLA+. while T cells in the same patient derived from inflamed joints were CLA (Ref. 36). Finally, cutaneous T-cell lymphoma seems to be a malignancy of skin-homing T cells, because the relevant transformed cell is CLA+, CD45RO’. Cutaneous T cells in each of the above diseases, as well as allergic contact dermatitis and many other diseases, bear high levels of CLA. P-selrctin P-selectin expression occurs very rapidly on endothelial cells at inflammatory sites, and P-selectin can also be expressed at very high levels on platelets, which can be deposited at sites of injury. It is known that neutrophils can bind platelets via a P-selectin-dependent, L-selectin-independent mechanism’?, providing an additional adhesion pathway that could potentiate an immune response. Platelets that have been activated in vitro can also support rolling of lymphocytes’s, but the significance of this pathway irz uivo remains to be demonstrated. However. because the protein backbone of PSGL-I is expressed on all leukocytes, there are potential interactions between all leukocytes and P-selectin. P-selectin is also induced in some chronic inflammatory sites, such as arthritic jointsj9, and might help to localize T cells to these areas at later stages of inflammation. Pselectin might also serve as an accessory rolling receptor for CLA+ T cells, because all CLA’ T cells thus far identified also express functional PSGL-1, and P-selectin can be co-expressed with E-selectin on vessels in inflamed skin. The in viva functional redundancy of these two selectins with respect to rolling and/or extravasation has already been noted above. Interestingly, mice deficient in FucT-VII exhibit a defect in rolling and extravasation”O, similar to that of E- and P-selectin-deficient mice, suggesting a role for this enzyme in the generation of functional E- and P-selectin ligands. 219

Reviews

Glossary Chemotactic stimuli; chemo-attractants - Soluble molecules, often proteins such as chemokines, that form a concentration gradient along which responsive cells move from low to increasingly higher concentrations. C-type lectin - Protein originally identified in plants, bearing an amino acid sequence that can bind carbohydrate structures in a C&+-dependent manner. Domain - Defined area on a large molecule, such as a protein. Extravasation - The process by which a cell leaves the blood vessel, via the intercellular junctions, and enters the surrounding tissues. Glycosylation - Covalent addition of oligosaccharides to specific hydroxyl groups of serine or threonine (O-linkages), or amino groups of asparagine (N-linkages) of proteins. Glycoprotein; glycolipid - Proteins or lipids bearing covalently bound carbohydrate groups. lntegrin - Cell-surface heterodimeric cell-adhesion receptor that links extracellular ligand with the actin cytoskeleton via cytoplasmic domains. lntegrins are expressed on almost all cell types; on leukocytes they exist in multiple activation states. In response to appropriate stimuli, leukocyte-integrin adhesive function is increased, resulting in enhanced adhesion to ligand. Knockout mouse - A genetically engineered mouse strain that has a specific gene functionally deleted from its genome.

Therapeutic

potential of inhibition

Ligand -A molecule that can specifically bind to another molecule via a noncovalent bond. Memory T cell -A T cell that has encountered its specific antigen and, as a result, has undergone differentiation to the most mature form of T cells. They are distinguishable from naive T cells by the expression of the cell-surface marker CD45RO. Memory T cells can express selectin ligands such as P-selectin glycoprotein ligand 1 (PSGL-i), cutaneous lymphocyte-associated antigen (CLA), (Y$, or a$, and thus exhibit tissue-specific homing behaviour. A subset of memory T cells do not express L-selectin on their cell surface. Mucin - A heavily glycosylated glycoprotein, usually with acidic sugar moieties, O-linked to highly repetitious serinelthreonine residues. Mucins have highly extended rod-shaped structures. Naive T ceil - A T cell that has not encountered its specific antigen. Naive T cells express the cell-surface antigen CD45RA, and L-selectin, and recirculate throughout the body, extravasating from the vessels into the tissues through the high endothelial venules in the lymph nodes. Selectin - A protein, expressed on several subclasses of leukocytes, able to mediate rolling interactions of blood cells with vascular endothelium via interaction of a lectin domain with carbohydrate structures on its ligands. Tethering - Weak and reversible adhesive contact of leukocytes with vascular endothelium, usually taking place under conditions that simulate blood flow.

of selectin function

Considerable effort hasnow beendevotedto the searchfor inhibitors Studieson the selectin-deficientmice(reviewedin Refs6-8) have of selectinfunction. Studiedmostextensivelyin animalmodelsdeshownmoreclearly thananyin vitro experimentthat the selectinsare pendent on neutrophil-mediated inflammation (reviewed in Refs essentialboth for protectionagainstpathogenicmicroorganisms and 5,6,8), selectininhibitors have traditionally targetedthe physical for homing.They also appearto be associated with physiological interaction between selectinsand their ligands. They include leukocyte homeostasi9’,by a mechanismthat is not completely antibodiesagainstselectins,selectinoligopeptides.solubleselectinunderstood. Furthermore, selectins might play a role in immunoglobulin chimeras,smallmolecularweightcarbohydrateanahaemopoiesis.becausethis process was perturbed in the E- loguesandanionicpolymerssuchasfucoidin. selectin-P-selectin-deficient animals,perhapsowingto anincreasein Far feweranimalmodelsexist in which the inhibitionof adhesion haemopoieticcytokinesin the serum31. A wide variety of disorders, moleculesspecificallyin the context of T-cell-mediated diseasehas rangingfrom chronicsyndromes (suchasasthma,inflammatoryskin beeninvestigated. Onestudydemonstrated that anantibodyagainstPdisease,arthritis andcancer)to moreacute processes (suchasadult selectinwaseffectivein ameliorating symptomsin arodentglomerulorespiratorydistresssyndrome,sepsis,traumaor ischaemia-reperfu- nephritis/DTHmodeP3;and in another.antibodiesagainstL-selectin sion injury) are associatedwith selectinoverexpressionin the in- inhibitedspontaneous insulitisin youngnon-obese diabeticmicel’.An flamedtissuel?,suggesting that aberrantselectinexpression is intrin- antibodyagainstL-selectin,togetherwith SLe” andrelatedoligosacsically associatedwith the inflammatorydiseaseprocess.Chronic charides,inhibitedlymphocytesfrom bindingto the endotheliumof overexpression of selectinsin immunologicallymediatedinflamma- cardiactransplants undergoingrejectionin [email protected] humans,infusion tory diseaseis probably due, at leastin part, to increasedcytokine of rheumatoidarthritis patientswith an antibody againstTNF-a productionby extravasatedT cellsthat have encounteredantigenin significantlyreduces serumselectinlevels,T-cell infiltration andextissueandbecomeactivated.As long asantigenpersists,the T-cell pressionof E-selectinandVCAM-1 in rheumatoidsynovialtissues4s,4h activationresultsin a positivefeedbackloop of selectinexpression, - presumably by inhibitingTNF-a-dependent selectinexpression. leukocyte extravasationand attendantinflammation.Inhibition of extravasationby blockingselectinscould preventthe initial accumulationof T cellsin the tissues,therebypreventingthe subsequent pro- There are,however,a numberof factorsthat mustbe consideredin duction of cytokines by thesecells, and interrupting the cycle. relation to anti-selectintherapy, in both neutrophil- and T-cellBlocking selectinscould thus play an important role in treating mediateddisease settings.First, solubleselectinscanbe foundin the chronicinflammatorydisorders. bloodin many inflammatorydisease9’.aswell asin healthyadults.

Srlrctins

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imrltlr

trnrl

disrrrsc

Reviews

Any effective anti-selectin compound would theoretically have to saturate these, as well as cell-surface-borne molecules. This would require high systemic concentrations of the agent, or some mechanism of local delivery to achieve high local concentrations, Furthermore, because T cells can attach and roll using the VLA-4VCAM-1 receptor pair, inhibition of selectin-mediated rolling alone might not be sufficient to prevent attachment and extravasation. Indeed, one study@ showed that even when rolling was inhibited by 90% (by the L-selectin-binding polymer fucoidin), there were still significant numbers of adherent cells, possibly rolling via their integrin receptors, suggesting that a combination of inhibitors might often be necessary. Although small molecular weight carbohydrate analogues of the selectin ligand-binding sites are being actively pursued as potential inhibitors of selectin function (reviewed in Refs 6,8), compounds investigated to date display low affinity, as well as little specificity for a particular selectin. To overcome these drawbacks, a promising new PCR-based technique, termed systematic evolution of ligands by exponential enrichment (SELEX), has been applied to generate DNA fragments that bind specifically, and with high affinity, to the lectin domain of L-selectin in a Cal+-dependent manner’“. These fragments do not cross-react significantly with the other selectins; they inhibit the binding of L-selectin to SLe”; and they prevent homing of human lymphocytes to the lymph nodes of severe combined immunodeficient mice, suggesting that they might find application as therapeutic agents. Theoretically, analogous DNA fragments that bind specifically to the other selectins could also be generated, permitting therapy directed at E- or P-selectin individually.

The outstanding questions Will inhibitionof selectin-dependent T-cell rolling sufficeto control chronic disease,or will other rolling pathwaysneedto beinhibitedaswell? l Do high levelsof solubleselectinsin somediseases preclude the feasibility of usinganti-selectincompoundssystemicallyto blockdisease-related leukocyteextravasation? l What is the biochemicalnature of cutaneouslymphocyteassociatedantigen and of the endothelialligands(peripheral nodeaddressin) for L-selectin?Doesthe hgand-siteincludeprotein structuresaswell asoligosaccharides and,if so,will blocking the latterbeeffective enoughto preventadhesion of T cells? l Canglycosylationpathwaysof the biosynthesis of P-selectin ligandsbe inhibited,while retainingeffective E-selectinligand biosynthesis? 0 What is the natureof the specificactivatingsignal(s)that operateon rolling T cellsto modulateadhesiveness of their integrinsfor the respectiveendothelialligands? l Do restingandactivatedT cells differ in their requirements for selectin-mediated rolling beforefirm adhesion? l What is the natureof the differentiationand activationsignalsthat regulatethe c11,3fucosyltransferases implicatedin selectin ligandcarbohydratebiosynthesis? l

Concluding l&e jirture:

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remarks

Like otherleukocytes,T cellshavethecapacityto roll on endothelial Another approachto controllingthe courseof an inappropriatein- surfacesunderphysiologicalflow usingselectin-ligandinteractions. flammatoryresponsecould be the inhibition of selectinligandsyn- both in vitro and in viva. CertainT-cell-endothelialselectininteracthesis.The cul,3-fucosyltransferasegroupof enzymescatalysesthe tions might be especiallyimportantin diseases involving particular final fucosylationstepin the synthesisof the carbohydratemoieties tissues.For example,CLA is a T-cellligandfor E-selectin,andCLA’ of the selectinligands.Theseenzymes,althoughpreferentiallyex- memoryT cells are abundantin inflamedskin (which is characterpressedby different leukocyte subclasses. exhibit large degreesof ized by prolongedE-selectinexpression)but not in inflamedsynoverlapwith respectto function andcellularexpression(reviewedin ovium.While T cellscanalsotetherandroll on P-selectin.no tissueRef. 8). One of them,FucT-VII, hasbeenshownto be the principal specificpathwayhasbeenimplicatedin this regard.However,it has regulatorof E-selectinligand synthesisin humanT cells” but ap- alsobecomeclearthat T cellsarequiteheterogeneous with respectto pearsto be essentialalsofor P- and L-selectinligand biosynthesis, their potentialfor adhesionand rolling, aswell asin the activation because FucT-VII-null micelack theseligands.Rollingand lympho- state of their constitutively expressedligands.Much more work cyte homing are also severelycompromisedin thesemice, again needsto be donebeforethe relativecontributionof selectinsto T-cell demonstratingthe global importanceof the selectinsand their li- extravasationand, more importantly.T-cell-mediatedinflammatory gandsfor theseprocesses”O. In a diseasesettingsuchasatopic der- diseasebecomesclear,Nevertheless,it is certainthat the processes matitis,which is characterizedby E-selectinoverexpressionin the by which T cellsarrestand roll along a vesselwall are an integral skin andconcomitantT-cell infiltration, selectiveinhibition of FucT- part of (anda prerequisitefor) extravasation,andtheir understanding VII is attractive,becauseno functionalE- or P-selectinligandscould will shed light on the selectivity and specificity of an immune be synthesized,and the courseof the diseasecould be mitigated. response. However,to achievedisease-specific enzymeinhibition, an idealinhibitory compoundwould be requiredto distinguishbetweenother Acknowledgements. We thank Dr Robert Fuhlbrigge for helpful discussions and Dr Dieter members of the enzymefamily in the samecell. aswell asbeingspe- Armerding for invaluable help in the preparation of the diagrams. This work was supported cific for cellsinvolved in the disease; otherleukocytesubclasses that by NIH Grant AR42689/Harvard Skin Disease Research Center (H.R.. T.S.K.) and R.A. is alsoexpressFucT-VII (suchas‘patrolling’T cells)would haveto be the recipient of the Alon Scholarshlp, awarded by the Israel1 Mmistry c&Education. sparedto prevent appropriateanti-inflammatoryresponsesfrom being compromised. Whethersucha degreeof specificity is attain- References able is purely speculative.Clearly, the therapeuticpotentialof se- 1 Springer, T.A. (1995) Traffic signals on endothelium for lymphocyte recirculectin function inhibitionfor controlof T-cell-mediated inflammation lation and leukocyte emigration, Arznu. Rev Phwol. 57. 827-872 is only beginningto be explored,andmanyquestionsare still to be 2 Butcher. E.C. (1991) Leukocyte-endothelial cell recognition: three (or more) answered. steps to specificity and diversity, G/l 67, IO33- IO36

Reviews

3 Hynes, R.O. ( 1992) Integrins: versatility, modulation, and signaling in cell adhesion, CeU 69. I I-25 4 H&en. S.R. el u/. ( 1995) Spatial distribution of L-selectin (CD62L) on human lymphocytes and transfected murine Ll-2 cells, Histo&w. J. 27.547-554 5 Rosen, S.D. and Bertorzi, C.R. (1994) The selectius and their ligands. Curr: Opin. Cdl Bd. 6,663-673 6 Tedder, T.F. er al. ( 1995) The selectins: vascular adhesion molecules, FASEB J. 9, x66-873 7 McEver, R., Moore, K. and Cummings, R.D. (I 995) Leukocyte trafficking mediated by selectinxarbohydrate interactions, J. Biol. Check. 270, 11025-l 1028 8 Lasky, L. ( I995 ) Selectinxarhohydrate interactions and the initiation of the inflammatory response, Annu. Ret>. Biochem. 64, 113-139 9 Berg, E.L. Edul. ( 1991) The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule-l, J. E,xp. Med. 174. 1461-1466 10 Steegmaier, M. et u/. (1995) The E-selectin ligand ESL-1 is a variant of a receptor for Iibroblast growth factor, Nclture 373,615420 11 Olofsson, A.M. ef al. I 1994) E-selectin mediates leukocyte rolling in interleukinl-treated rabbit mesentery venules. Blood 84, 2749-2758 12 Alon, R. et ul. (I 994) Distinct cell surface ligands mediate T lymphocyte attachment and rolling on P- and E-selectin under physiological flow, J. Cell Viol. 127, 1485-1495 13 Asa, D. ef cri. ( 1995) The P-selectin glycoprotein ligand functions as a common human leukocyte ligand for P- and E-selectins, J. Bioi. Chern. 270, 11662-I 1670 14 Fuhlbrigge. R.C. ef u/. (lYY7) Molecular identification of cutaneous lymphocyte associated antigen (CLA): CLA is an isoform of P-selectin glycoprotein ligand 1 (PSGL-I), J Invest. Dermarol. 108. 53X (Abstract) 15 Knibbs, R.N. rr cri. (1996) The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells. J. Ceil Bioi. 133, Y I l-920 16 Picker, L.J. ef al. 11991) The neutrophil selectin LECAM-1 presents carbohydrate ligands to the vascular selectins ELAM-1 and GMP-140, Cell 66, 921-Y33 17 Moore, K.L. er ul. ( 1995) P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin. L Cell Biol. 128.66 I-67 I 18 Alon, R.. Hammer, D.A. and Springer, T.A. (1995) Lifetime of the Pselectin-carbohydrate bond and its response to tensile force in hydrodynamic flow. Nutm

374.539-542

19 Kansar, G.S. et rrl. ( 1093) Regulation of leukocyte rolling and adhesion to high endothelial venules through the cytoplasmic domain of L-selectin, ./. E.xp.Med. 177,833-83X 20 Yoshlda. M. ef ul. (I 996) Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton, J. Cell fM. I33,445;155 21 Lawrence, MB. and Springer, T.A. (1991 I Leukocytes roll on P-selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins, Cell 65,859%873 22 Lawrence, M.B. d ul. (1995) Rolling of lymphocytes and neutrophils on peripheral node addressin and subsequent arrest on ICAMin shear flow, Eur J. Zmrnunol. 25. 1025s1031 23 Jones, D.A.. M&tire, L.V.. Smith. C.W. and Picker. L.J. 119941 A two-step adhesion cascade for T cell/endothelial cell interactions under flow conditions, J. C/in. Invert.

94, 2443-2450

24 Luscinskas, F.W., Ding. H. and Lichtman, A.H. (1995) P-selectin and vascular cell adhesion molecule 1 mediate rolling and arrest, respectively. of CD4+ T lymphocytes on tumor necrosis factor u-activated vascular endothelium under flow, J. E.xp. Med. 18 1. 1 I79m I I86 25 Alon, R. etcrl. ( 1995) The integrin VLA-4 supports tethering and rolling in flow onVCAM-I. J. Ce/li?io/ 128. 1243-1253 26 Berlin, C. ef u/. (1995) cxI integrins mediate lymphocyte attachment and rolling under physiologic flow, Cell 80, 4 13422 222

27 Bargatre, R.F., Jutila, M.A. and Butcher. E.C. (1995) Distinct roles of Lselectin and integrins (Y$, and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep model confirmed and refined, Immunity 3, 99-108 28 Salmi, M. and Jalkanen, S. (1996) Human vascular adhesion protein-l (VAP-1) is a unique sialoglycoprotein that mediates carbohydrate-dependent binding of lymphocytes to endothelial cells. J. Exp. Med. 1X3,569-579 29 de Grendele, H.C. et ul. ( 1996) CD44 and its ligand hyaluronate mediate rolling under physiologic flow: a novel lymphocyte+mdothelial cell primary adhesion pathway, J. Exp. Med. 183. I 119-I 130 30 Arbones. M.L. et al. (1994) Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin-deficient mice, /WWIU~IQ I, 247-260 31 Frenette, P.S. rf ul. (1996) Susceptibility to infection and altered hematopoiesis in mice deficient in both P- and E-selectins. Cell 84,563-574 32 Labor. M.A. et crl. (1994) Characterization of E-selectin-deficient mice: demonstration of overlapping function of the endothelial selectins, Znzmunif~ I, 709-720 33 Tipping, P.G. er cl/. (I 996) P-selectin directs T lymphocyte-mediated injury in delayed-type hypersensitivity responses: studies in glomerular nephritis and cutaneous delayed-type hypersensitivity, Eur J. Immunoi. 26,454460 34 Grover, R.W. ef rrl. (I 991) Endothelial leukocyte adhesion molecule-l (ELAM. 1) expression in cutaneous inflammation. Llr J. Uertizato/. 124, 117-l 23 35 Romagnani, S. ( 1995) Biology of human T,l and T,2 cells, J. C/in. Immurwl. 15, 121-129 36 Pitzaliq, C. et ul. (1996) Cutaneous lymphocyte antigen positive T lymphocytes preferentially migrate to skin hut not to the joint in psoriatic arthritis, Arthrir~s Rheum. 39, 137-145 37 Buttrum, SM., Hatton, R. and Nath, G.B. I 1993) Selectin mediated rolling of neutrophils on immobilized platelets, Blood X2, I 165-l 174 38 Diacovo. T.G. er ui. I 1996) Platelet mediated delivery of lymphocytes to high endothelial venules, Science 273,252~255 39 Damle. N.K. ef u/. (1992) GMP-140 (P-selectinKD62) binds to chronically stimulated but not resting CD4’ T lymphocytes and regulates their production of proinflammatory cytokines, ENK J. frnmmol. 22. 1789-1793 40 Maly. P. et ul. (1996) The cY(1.3)fucosyltransferase Fuc-TV11 controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell 86,643-663 41 Butcher, E. and Picker, L.J. (1996) Lymphocyte homing and homeostasis. Science 272,60-66 42 Bevilacqua, M.P. er ol. ( 1994) Endothelial-leukocyte adhesion molecules in human disease, Amu. Rev Med. 45, 361-378 43 Yang. X. et al. (I 996) The role of cell adhesion molecules in the development of IDDM. Diuhrres 45, 705-7 10 44 Turunen. J.P. et ul. ( 1996) Ue IWO expression of endothelial sialyl Lewis (a) and sialyl Lewis (x) during cardiac transplant rejection: superior capacity of a tetravalent sialyl Lewis (x) oligosaccharide in inhibiting L-selectindependent lymphocyte adhesion, J. Exp. Med. 182, I 133-l 142 45 Paleolog, E.M. et ai. (1996) Deactivation of vascular endothelium by monoclonal anti-tumor necrosis factor Q antibody in rheumatoid arthritis, Arthritis Rheum. 39, 10X2-109 I 46 Tak, P.P. ef al. (I 996) Decrease in cellularity and expression of adhesion molecules by anti-tumor necrosis factor (Y monoclonal antibody treatment in patients with rheumatoid arthritis, Arrhrltir Rheum. 39, 1077-IO81 47 Gearing, A.J.H. and Newman, W. (1993) Circulating adhesion molecules in disease, Immunol. 7ixiu~ 14, 5066512 48 Kubes, P.. Jutlla, M. and Payne, D. (1995) Therapeutic potential of inhibiting leukocyte rolling in ischemia/reperfusion. J. Urn. Invest.95, 25 I O-25 19 49 Hicke, B.J. et ul. (1996) DNA aptamers block L-selectin function in viva, J. C&I. Invrsr.

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