doi:10.1006/smim.2001.0350, available online at http://www.idealibrary.com onseminars in IMMUNOLOGY, Vol. 14, 2002: pp. 133–140

Clinical overview of leukocyte adhesion and migration:where are we now?

Diane Marshall a and Dorian O. Haskard ∗

This article discusses the potential for clinical translationof the large amount of information on the molecularbasis of leukocyte–endothelial cell interactions that has beencollected over the last twenty years. Areas of current interestinclude the identification of adhesion molecule expressionin inflammation by diagnostic imaging, understandingvariability in inflammatory responsiveness and diseasesusceptibility through identification of adhesion moleculeand chemokine polymorphisms and the application to thetreatment of inflammatory diseases of monoclonal antibodiesand conventional drugs with specific actions on leukocyteadhesion and migration.

Key words: human / adhesion molecule / chemokine /polymorphism / treatment

c© 2002 Published by Elsevier Science Ltd.

Introduction

The last twenty years has seen enormous advancesin understanding of the cellular and molecularmechanisms of leukocyte trafficking and it is nowtime to take stock to ask how this information can betranslated and applied to combating human diseases.While this article focuses primarily on leukocyte–endothelial cell interactions in inflammation,general principles also have bearing on theinteractions of tumour cells with endotheliumin cancer metastasis.1,2

From the BHF Cardiovascular Medicine Unit, Faculty of Medicine,Imperial College, Hammersmith Hospital, Du Cane Road, London W12ONN, UK. *Corresponding author. E-mail: d.haskard@ic.ac.uk.aPresent address: Pharmacology Department, Celltech R & D Ltd, 208 BathRoad, Slough, Berkshire, SL1 4EN, UK.

c©2002 Published by Elsevier Science Ltd.1044–5323/02/$ - see front matter

Studying leukocyte–endothelial interactions inhumans is limited by obvious ethical constraints.Much of what we believe to happen is extrapolatedfrom animal models, or from in vitro experimentswith human tissues, and both sources of informationhave their limitations. First, it is clear thatthere are species differences in the control ofadhesion molecule and chemokine expression,with endothelial P-selectin being a good example.3

Second, in vitro models often fail to incorporatethe complexity of the in vivo situation. Third, whatdirect knowledge we have from human in vivo studieshas been obtained from those microvascular bedsthat are accessible, such as skin and retina. Directinformation on the specific events that take-placein other tissues in human is largely limited tohistological studies of pathological specimens, oftenobtained relatively late in the course of disease.

Soluble adhesion molecules

Since cytokines and other mediators regulatethe expression of many adhesion molecules,quantification of adhesion molecule expressionprovides indirect information on inflammatoryactivity. Adhesion molecules can readily be measuredin soluble form in blood and other body fluidsusing commercially available ELISA kits that arenow available for each of the selectins, ICAM-1 andVCAM-1. Soluble adhesion molecules are thoughtto reach the blood mostly because of enzymaticcleavage from cell surfaces, although P-selectin canbe directly synthesised in human as the product ofan alternatively-spliced transcript. Although solubleE-selectin and L-selectin can be assumed to derivefrom activated endothelial cells and leukocytesrespectively, soluble P-selectin may come from bothplatelets and endothelial cells, and soluble ICAM-1and VCAM-1 may derive from many different cells.4

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A large literature now exists on the levels theseadhesion molecules achieve in inflammatory diseases,both in relation to clinical disease activity and inrelation to conventional markers of the acute phaseresponse (e.g. C-reactive protein (CRP), erythrocytesedimentation rate). In many clinical situations levelsof soluble adhesion molecules correlate with theacute phase response, and may not provide muchextra information to the clinician. For example,a recent study confirmed that soluble E-selectin,P-selectin, ICAM-1 and VCAM-1 act as prognosticmarkers for the development of coronary arterydisease, but ceased to have much prognostic valueonce other risk factors had been corrected for.5 Itremains to be determined whether the measurementof soluble adhesion molecules will prove clinicallyuseful in other fields.

Imaging

Autologous neutrophils, radiolabeled with indium-111 or technetium-99, have been used clinically forsome years to image inflammation, for examplein sepsis or inflammatory bowel disease.6 Thisapproach can be used to assess the effects oftherapeutics on leukocyte trafficking, for example inrheumatoid arthritis.7 The improved understandingof leukocyte–endothelial cell interactions now offersthe opportunity of extending the repertoire ofnuclear imaging to a more molecular level, andto probe localised endothelial activation. Thusmonoclonal antibodies or specific peptides havepotential for non-invasively monitoring expressionof individual molecules within inflamed tissues,and thereby reporting tissue activation. As yet,this approach has been confined to conventionalgamma-emitting isotopes, but it might be extended tomagnetic resonance imaging and positron emissiontomography.

E-selectin

E-selectin is an excellent target for imaging activatedendothelium, since it is expressed on the lumenalsurface of vascular endothelium and therefore fullyaccessible. Radiolabelled anti-E-selectin antibody hasbeen shown to image inflamed joints in rheumatoidarthritis8 and inflamed bowel in inflammatory boweldisease.9 An alternative approach is the use ofradiolabelled E-selectin-binding peptide.10,11

P-selectin

Radiolabelled anti-P-selectin antibodies have beenused to image platelet localisation followingpertcutaneous transluminal angioplasty.12 AlthoughP-selectin does not appear to have been used as atarget for imaging chronic inflammation, it maybe useful in this regard in view of its differentialexpression on endothelium in response to cytokines,when compared with E-selectin.13

ICAM-1 and VCAM-1Animal studies have shown the potential of ICAM-1

and VCAM-1 for imaging but as yet no clinical studiesappear to have been reported.14,15

Genetic influences

Although rare, the leukocyte adhesion deficiency(LAD) diseases provide important insights tomolecular mechanisms of leukocyte traffickingand function in human. The description in theearly-1980s of LAD Type I provided a powerfulinsight into the physiological importance andpharmacological possibilities of leukocyte adhesionin inflammation.16,17 The clinical course of β2integrin deficiency is characterised by recurrentpyogenic infections without pus formation, oftenleading to death from septicaemia in the first orsecond decade. A characteristic feature is a high whitecell count (sometimes as high as 150× 103 mm−3),in part due to the inability of leukocytes to emigratefrom the vascular compartment. In spite of theabnormalities of leukocyte migration, problematicviral infections are rare and patients are able tomount satisfactory humoral and cell-mediatedimmune responses, probably because β2 integrinsare less critical for mononuclear cell traffickingand function. Recently, patients with LAD I variantsyndromes have been described with adequate β2integrin expression but evidence of β2 integrindysfunction.18–20

The importance of selectins in humans is demon-strated by LAD Type II. In this genetic disease, there isdefective fucosylation of glycoproteins, caused by anabnormal GDP-fucose transporter.21 Affected individ-uals show developmental abnormalities, and also havea spectrum of abnormalities of leukocyte trafficking,overlapping with those of LAD Type I.

There is now an emerging literature dealing withgenetic polymorphisms of adhesion molecules,

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Table 1. Polymorphisms associated with inflammatory disease

Polymorphism Possible disease associations References

Adhesion moleculesE-selectin G98T substitution in 5′UTR T allele with atherosclerosis 24,25E-selectin S128R in EGF domain R128 with atherosclerosis and SLE 24–26E-selectin L554F in TM domain F554 with atherosclerosis 24P-selectin T715P in CR9 domain P715 with reduced incidence of

myocardial infarction27–29

ICAM-1 K29M in Ig domain 1 M29 with cerebral malaria 30ICAM-1 G241R in Ig domain 3 R241 more frequent in BD and

PMR/GCA31,32

ICAM-1 K469E in Ig domain 5 K469 more frequent in MS 33E469 more frequent in BD and ageof onset of type I DM

34,35

ChemokinesRANTES G/A at nucleotide position-403 in

5′UTRA allele with atopy, asthma,RA, PMR

36–38

Chemokine receptorsCCR2 V64I in 1st TM domain I64 with protection from sarcoidosis 39CCR3 T/C at nucleotide position 51

(silent mutation)C allele with asthma 40

CCR5 deletion of nucleotides 794–825 (132) 135 with less severe RA, MS, CAD 41–44CXCR2 C/T and C/T at nucleotide positions

785 and 1208785CC and 1208TT homozygosityand SSc

45

CX3CR1 V249I in 6th TM domain I249 and protection from CAD 46,47

BD Behcet’s disease DM Diabetes mellitusCAD Coronary artery disease GCA Giant cell arteritisMS Multiple sclerosis PMR Polymyalgia rheumaticaRA Rheumatoid arthritis SLE Systemic lupus erythematosusSSc Systemic sclerosis

chemokines and their receptors. These may underliemore subtle inter-individual differences which mayinfluence inflammatory responsiveness and diseaseexpression, and which may also provide insight on thepotential of individual molecules as pharmacologicaltargets. In the context of transplantation, poly-morphisms of adhesion molecules and chemokinereceptors may also influence graft survival throughacting as minor histocompatibility antigens.22,23

Table 1 provides a selection of polymorphisms thathave been linked to altered inflammatory diseaseexpression. More work is required to establish thereproducibility of these associations in different dis-eases and in different populations, and to determinewhich polymorphisms are associated with alteredprotein function and which are merely in linkage dis-equilibrium with other more critical polymorphisms.Polymorphisms which have so far been shown toalter expression and/or function include E-selectin

R128 E-selectin (gain in adhesive function),48,49

and M29 ICAM-1 (ICAM-1kilifi ) (reduced adhesivefunction),50,51 I249 CX3CR1 (reduced expression)46

and 135 CCR5 (reduced expression).52 Further-more, substitution of A for G at position -403 of theRANTES gene 5′ untranslated region leads to anaugmentation of promoter activity.38

Therapeutic targeting

There is now a considerable literature on the effectsin animal models of targeting adhesion moleculeswith antibodies, and on the effects of individualand combined gene deletions. These have yieldedresults that have been sufficiently encouraging tojustify a significant investment by the pharmaceuticalindustry in developing antibodies and drugs forclinical use. Products with exciting clinical potential

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are emerging, although there have also beendisappointments. As a generalization, attempts toalleviate chronic relapsing inflammatory diseases(e.g. inflammatory bowel disease, psoriasis, multiplesclerosis) are proving to be more successful thaninterventions for severe acute inflammation. Sofar, few, if any, of the trials aimed at amelioratinghypoxia-reperfusion in its various clinical guises (e.g.acute coronary syndromes, stroke, hypovolaemicshock) have delivered positive results. Possibly,redundancy of inflammatory mechanisms in theseacute situations acts against the benefits of blockingindividual pathways or molecules.

Selectins

The main effect of targeting selectins should beinhibition of the interactions between leukocytes,platelets and endothelial cells within the vascularcompartment, leaving tissue leukocyte functionintact. Studies using selectin-antagonists in animalmodels have been encouraging, particularly forischaemia-reperfusion injury.53 However the limitedexperience that exists in humans has not beenpromising. First, a carbohydrate inhibitor based onsialyl-Lewis X developed by Cytel had no demon-strable effect in patients undergoing angioplastyfor acute myocardial infarction. Second, a trial of ahumanized anti-E-selectin mAb, SPLAT-1, failed tolead to any significant improvement in psoriasis. Itremains possible however that inhibitors of selectinfunction may emerge that have higher affinity forselectins than sialyl-Lewis X analogues, and thatother clinical situations may be more amenable totherapeutic manipulation. The Genetics Instituteis developing a recombinant soluble antagonist ofPSGL-1, rPSGL-Ig, and by January 2001 was in phaseII trials. The agent is under consideration for acutemyocardial infarction and restenosis.54–56

Integrins and immunoglobulin-superfamily ligands

Support for the concept of targeting of leukocyteintegrins comes not just from the effects of LADType I (see above), but also from the ‘proof-of-principle’ in another field—that of targeting plateletIIb/IIIa. Thus monoclonal antibodies, peptidesand RGD-mimetic small molecules all exist fortargeting IIb/IIIa and have all been found to beclinically effective in the setting of acute coronarysyndromes.57

Unlike selectins, leukocyte integrins are essentialfor many leukocyte functions outside the vessel as

well as for leukocyte–endothelial cell interactions.Since treatment decisions in clinical practice usuallyfollow rather than precede unwanted leukocytetrafficking into tissues, inhibition of post-traffickingevents is likely to be a major advantage of agentsthat target leukocyte integrins. In the case of Tlymphocytes, inhibiting integrin function has thepotential not just to suppress on-going activationbut to contribute to long-term tolerance to allo- orautoantigen in transplantation and autoimmunediseases respectively (e.g. Reference 58).

β2 integrin subunitAgents that target the β2 integrin subunit (CD18)

have major effects on leukocyte function throughinhibiting all four associated integrins (i.e. αMβ2(Mac-1, CR3), αLβ2 (LFA-1), αXβ2 (p150, 95)and αDβ2). Trials using mAb to the common β2chain have been conducted in patients with acutestroke, myocardial infarction and acute traumatichypovolaemic shock, and all appear to have beenunsuccessful.59 Inhibiting the β2 integrin subunit asa strategy for treating chronic inflammatory diseaseswould mimic the effects of LAD Type I and probablycarries an unacceptable risk of infection.

αL integrin subunitTargeting the αL integrin subunit (i.e. LFA-1) is

likely to lead to a more restricted anti-inflammatoryeffect than inhibition of the β2 integrin subunit, sinceother β2 integrins that are important for host-defence(in particular αMβ2 on granulocytes and monocytes)remain intact. Recently completed phase III trials ofan anti-LFA-1 (Xanelim) in psoriasis have shown thepotential of this approach (Genentech/Xoma). Atleast four pharmaceutical companies have discoveredsmall molecular weight inhibitors of LFA-1, thecommon mode of action being allosteric inhibitionof the αL I domain. Given the apparent sensitivityof the αL I domain to inhibition, it remains to bedetermined if selective inhibitors can also be made toother α subunits with I domains (e.g. αM , αX , α1, α2).

ICAM-1Since ICAM-1 is one of several possible β2 integrin

ligands, it represents another potential target for therestricted inhibition of β2 integrin function. Patientswith rheumatoid arthritis have been treated with ananti-ICAM-1 mAb developed by Boeringer-Ingelheimwith beneficial results,60–62 although perhaps lessdramatic than those following the use of anti-TNFα.63

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Subsequently Boehringer-Ingelheim discontinueddevelopment of the antibody, following disappointingresults from a phase III study in acute stroke.

a4 integrin subunitThe α4 integrin subunit can associate with either

the β1 subunit to form very late Antigen-4 (VLA-4) orwith the β7 subunit to form lymphocyte-Peyer’spatch adhesion molecule-1 (LPAM-1). Theseα4 integrins are expressed at high density bylymphocytes, monocytes, eosinophils, basophils andnatural killer cells. In contrast, human neutrophilα4 integrin expression is low-absent. Consistentwith this distribution, α4 integrins are particularlycritical for lymphocyte, monocyte and eosinophilmigration and function and play important rolesin the generation of immunologically-mediatedinflammatory reactions. Inhibition of α4 integrinstherefore is an attractive strategy for modulatingthe upsteam immunological drive to chronicinflammation, whilst leaving neutrophil functionand innate host defence mechanisms relativelypreserved.64

Antibody inhibition studies have been verypromising in models of experimental allergicencephalomyelitis (EAE), colitis and asthma.However. since gene-targeting of α4 integrin leadsto embryonic lethality, we have less guidance fromanimal models on the in vivo effects of blockingα4 integrins in chronic disease. There are nowclinical trial data suggesting significant benefitsfor targeting the α4 integrin subunit with thehumanised monoclonal antibody natalizumab(Antegran) in multiple sclerosis65–67 and also inCrohn’s disease.68,69 It is not clear which α4 integrinis the more important target in inflammatory boweldisease, since lymphocyte trafficking into the gutinvolves α4β7 as well as α4β1. A humanized antibodyagainst α4β7 (LDP-02) is being developed for thetreatment of Crohn’s disease by Millenium andGenentech.70 Several pharmaceutical companiesnow have low molecular weight compounds thatselectively inhibit α4 integrins and which are enteringclinical trials.

VCAM-1AtheroGenics have discovered a series of

nitrobenzene compounds that selectively inhibitendothelial cell VCAM-1 (and not ICAM-1) geneexpression.71 In collaboration with Schering-Plough,they are developing an inhibitor (AGI-1067) for

potential prevention of atherosclerosis, and post-angioplasty restenosis. Preliminary results indicatedthat 6 months after angioplasty, patients whoreceived AGI-1067 had greater luminal diameters oftheir coronary arteries.

Chemokines and their receptors

In view of their potential tissue specificity and greatdiversity, chemokines and their receptors provideimportant alternatives to adhesion molecules astherapeutic targets. Although the chemokine fieldis relatively young, there are already promisingagents and the strategies put in place for establishingproof-of-principle for anti-adhesion therapy shouldspeed up their evaluation. A novel non-peptide CCR1antagonist (BX 471) has found to effectively reduceallograft rejection and rat EAE,72–74 and is now inphase I trials for the treatment of multiple sclerosis(Berlex Biosciences). Millennium and Kyowa Hakkaare also investigating orally available small-moleculeantagonists and monoclonal antibodies to twochemokine receptors, CCR1 and CXCR3, for thetreatment of rheumatoid arthritis, psoriasis, resteno-sis and multiple sclerosis. In March 2000, thesewere undergoing late-stage preclinical investigation.Several pharmaceutical companies currently havediscovery programmes for CCR2 and MCP-1 antag-onists, based upon the reduction in atherosclerosisand EAE in CCR2 and/or MCP-1 knock-out mice.

Abgenix Inc is developing the humanized anti-IL-8antibody, ABX-IL8 as a potential treatment of psori-asis and rheumatoid arthritis. In April 2000, a phaseIIa clinical trial of ABX-IL8 was initiated to includepatients with moderate-to-severe psoriasis. Improve-ment was demonstrated after four infusions of anti-body, with the number of CD3+ T cells and activatedkeratinocytes in plaques being reduced. The phaseIIb study has now been initiated. A phase IIa study forrheumatoid arthritis is also currently underway. Sev-eral companies are currently undertaking discoveryprogrammes of small molecule inhibitors of IL-8.

Since Th1 and Th2 lymphocytes express distinctpatterns of CC chemokine receptors, blockingselectively expressed receptors should enableimmune responses to be modified. Both CCR3 andCCR4 are preferentially expressed by Th2 cells75,76

and are particularly evident in allergic disease suchas asthma. Antagonists against CCR3 and its ligands(which include eotaxin, eotaxin-2 and eotaxin-3)are currently in development. Cambridge AntibodyTechnology is developing CAT-213, an anti-eotaxin

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monoclonal antibody, for potential treatment ofeczema, allergy and asthma and in September 2001received authorisation to begin a phase I/IIa study inpatients with allergic rhinitis.

The chemokine receptor CCR5 is preferentiallyexpressed by Th1 cells and macrophages, and actsas a coreceptor for HIV. Many companies havebeen pursuing CCR5 small peptide antagonistsand monoclonal antibodies, with clinical trialsexpected to start in 2001 for HIV infection. Thesecompounds may also be useful in Th1-driven chronicinflammatory diseases such as rheumatoid arthritisand multiple sclerosis.77,78

Conclusion

The field of leukocyte–endothelial cell interactionshas come along way in the twenty years since themolecules involved started to be discovered. It isclear that the information we now have providesa number of opportunities for clinical translation,not least in the treatment of chronic inflammatorydiseases. Much work is now needed to establish whichinflammatory clinical situations can be modified bytargeting adhesion molecules or chemokines, andto determine which molecular targets offer mostspecificity and the least general reduction in hostresistance. In turn, the merits of these strategies willhave to be compared with other established anddeveloping approaches, not least the inhibition ofupstream cytokine cascades.

Acknowledgement

The authors thank Dr Justin Mason for criticallyreviewing the manuscript.

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