Pathophysiology 13 (2006) 1–14

Chemokines and leukocyte trafficking in rheumatoid arthritis�

Teresa K. Tarranta, Dhavalkumar D. Patelb,∗a Department of Medicine, Division of Rheumatology, Allergy, and Immunology, Thurston Arthritis Research Center, University of North Carolina,

3330 Thurston Bldg., CB#7280, Chapel Hill, NC 27599, USAb Departments of Medicine and Microbiology and Immunology, Division of Rheumatology, Allergy, and Immunology, Thurston Arthritis

Research Center, University of North Carolina, 3330 Thurston Bldg., CB#7280, Chapel Hill, NC 27599, USA

Abstract

Leukocyte infiltration into the joint space and tissues is an essential component of the pathogenesis of rheumatoid arthritis (RA). In thisreview, we will summarize the current understanding of the mechanisms of leukocyte trafficking into the synovium, focusing on the roleof adhesion molecules, chemokines, and chemokine receptors in synovial autoimmune inflammation. The process by which a circulatingleukocyte decides to migrate into the synovium is highly regulated and involves the capture, firm adhesion, and transmigration of cells acrossthe endothelial monolayer. Adhesion molecules and chemokine signals function in concert to mediate this process and to organize leukocytesinto distinct structures within the synovium. Chemokines play a key regulatory role in organ-specific leukocyte trafficking and activation bya wnstreas©

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ffecting integrin activation, chemotaxis, effector cell function, and cell survival. Consequently, chemokines, their receptors, and domignal transduction molecules are attractive therapeutic targets for RA.2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Rheumatoid arthritis; Chemotaxis; Chemokine; Synovial; Leukocyte

. Leukocyte migration

A key step in the development of an inflammatory process,ncluding autoimmune diseases like RA, is the recruitment ofeukocytes to the site of inflammation. This involves multipleegulatory steps initially proposed in the multi-step modelsf Springer[1] and Butcher and co-workers[2]. This process

nvolves: (1) leukocyte rolling; (2) rapid activation of leuko-yte integrins and subsequent adhesion to endothelial ligandsfirm adhesion); (3) transendothelial migration (diapedesis);nd (4) migration of inflammatory cells through tissues inesponse to chemokine gradients.

.1. Leukocyte capture and rolling

Migration of leukocytes to sites of inflammation beginsith cell capture and subsequent rolling along endothelium

Fig. 1). This initial capture of leukocytes by the endothe-ium is mediated primarily by cell surface proteins of the

This paper was part of the Rheumatoid Special Issue, See Pathophysiology2/3.

selectin family and their ligands during resting and inflamtory conditions[3]. However, other receptor-ligand interations including CD44-hyaluronan and CX3CR1-fractalk(CX3CL1) are effective at cell capture during specific tyof inflammation[4–6].

1.1.1. Selectins and their ligandsThere are three major classes of selectins that are

dominantly, albeit not exclusively, expressed on the cellwhich they are named. L (leukocyte)-, P (platelet)-, an(endothelial)-selectins are also named CD62L, CD62PCD62E, respectively[7,8]. The selectins recognize and bto similar carbohydrate moieties at the lectin domain duinflammation.

When a cell is in the resting state, P-selectin is preinternally within the�-granules of platelets or within WeibePalade bodies of endothelial cells. Once activated by inflmatory or thrombogenic mediators, the granules fuse witplasma membrane and P-selectin is expressed on the ceface where it functions in cell adhesion[9]. Within minutesof an inflammatory signal, cell surface P-selectin is remo

∗ Corresponding author. Tel.: +1 919 966 0552; fax: +1 919 966 0550.E-mail address: ddpatel@med.unc.edu (D.D. Patel).

by proteolytic cleavage thus limiting the effective window ofadhesion.

928-4680/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.

oi:10.1016/j.pathophys.2005.11.001

2 T.K. Tarrant, D.D. Patel / Pathophysiology 13 (2006) 1–14

Fig. 1. Schematic depicting the current understanding of leukocyte captureand adhesion that is critical to cellular migration to sites of inflamma-tion. Depicted are circumstances in which (A) selectins, (B) CD44 and (C)CX3CR1 participate in leukocyte capture by endothelial cells.

E-selectin is expressed on activated endothelial cells hoursafter exposure to inflammatory mediators such as IL-1�,TNF-�, interferon-�, substance P, and LPS[10]. E-selectinis specifically upregulated on endothelial cells in inflamedsynovium, and may be important in the recruitment of inflam-matory cells in RA[11–14].

L-selectin is expressed on all leukocytes with the excep-tion of a subset of memory T cells[15,16], and attachmentof leukocytes to the endothelium both in vivo[17,18] andin vitro [19,20] is largely L-selectin dependent. L-selectinregulation is particularly important in the organ-specific traf-ficking of leukocytes[21]. In L-selectin deficient animals, Tcells are unable to home to peripheral lymph nodes, support-ing a critical role of L-selectin in homeostatic lymphocytetrafficking [22].

L-selectin is rapidly cleaved from the cell surface after cel-lular activation, and may play an important role in receptorregulation and function. L-selectin shedding is not requiredfor diapedesis or for naı̈ve lymphocyte homing and recircula-tion to peripheral lymph nodes[23,24]. However, inhibitionof L-selectin shedding on antigen-specific lymphocytes andneutrophils alters their migratory pathways[25,24].

1.1.2. CD44 and hyaluronanAn additional capture molecule present on activated T

cells is CD44[26]. CD44 is present on most cell types, and is amember of the hyaldherin family with similar structure to theselectins[27,5]. There are several different isoforms identi-fied, which contribute to its multifunctionality, and many arepresent in the inflamed joint. Upregulation of CD44 and asubsequent increase in its ability to bind to hyaluronate islikely to be functionally important in inflammatory diseases

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tates such as RA[5].

.1.3. Fractalkine (CX3CL1) and CX3CR1Fractalkine is a unique molecule that functions a

hemokine in its secreted form, and as a selectin-like aion molecule when it is expressed on the cell surface[28,4].ike the selectins, fractalkine can mediate the rapid captuirculating leukocytes. Fractalkine has a mucin domainxtends from the cell surface and is structurally and funclly similar to the short consensus repeats of the endotxpressed selectins (E- and P-selectin)[29]. Tyrosine sulfaion, which enhances ligand binding between P-selectints ligand P-selectin glycoprotein ligand-1 (PSGL-1), is amportant in fractalkine-CX3CR1 interactions that medeukocyte capture[30].

.2. Leukocyte firm adhesion

The types of molecular interactions involved in cell cure are generally weak and lead to leukocyte rolling onndothelium. During the process of rolling, leukocytes

riggered by endothelial surface bound chemokines tond become firmly adherent to the endothelium throurocess termed activation-dependent stable arrest. Thdhesion is mediated by interactions between integrins

heir ligands.Integrins are usually in an inactive state on leukoc

nd become activated after the triggering of certairotein coupled receptors like chemokine receptors[31].heir ligands on the endothelium are members of

T.K. Tarrant, D.D. Patel / Pathophysiology 13 (2006) 1–14 3

immunoglobulin superfamily known as intercellular adhe-sion molecules (ICAMs) as well as vascular cell adhesionmolecule 1(VCAM-1), platelet-endothelial cell adhesionmolecule-1 (PECAM-1), and mucosal addressin cell adhe-sion molecule-1 (MAdCAM-1)[32,33]. Important integrin-ligand interactions include the leukocyte specific�2 inte-grins, leukocyte functional antigen-1 (LFA-1, CD11a/CD18),complement receptor type-3 (CR3, CD11b/CD18, Mac-1),and p150,95 (CD11c/CD18) with their ligands ICAM-1 and-2 [9]. The�1 integrins, and in particular very late antigen-4(VLA-4, CD49d/CD29), are particularly important in woundhealing and cell trafficking in embryogenesis[33]. VLA-4,which is expressed on monocytes, basophils, and eosinophilsin addition to T- and B-lymphoctes, interacts with its lig-and VCAM-1 to induce cell adhesion[33]. When integrin-ligand interactions are impaired, firm adhesion and subse-quent recruitment of leukocytes toward sites of inflammationis impaired. A clinical example that highlights this defectis Leukocyte Adhesion Deficiency syndrome type 1 (LAD-1) [34], where individuals who are deficient in the commonchain (CD18) of the�2 integrins have a syndrome of poorwound healing and recurrent bacterial infections.

Integrin ligand expression varies on vascular endotheliumand may regulate lymphocyte migration patterns. ICAM-2(CD102) is constitutively expressed, which suggests a role inmaintaining homeostatic leukocyte trafficking[35]. In con-t nset morei

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killer cells in a variety of immune responses such as vas-cular inflammation, allograft rejection and tumor clearance[39–44] and unpublished observations. Thus, by regulatingfirm adhesion in different leukocyte subsets, chemokines canfunction as control signals in cell-specific endothelial cellrecognition and recruitment that may be important in inflam-matory disease states.

1.3. Transendothelial migration (diapedesis)

Diapedesis is the process whereby the leukocyte migratesacross the endothelial monolayer and basement membraneinto the tissues. It is a complex process that is poorlyunderstood and likely involves several molecular interac-tions acting in concert. After transendothelial migration andextravasation into tissues occurs, the leukocyte must receivenew signals, often in the form of chemokine gradients, tocontinue the process of recruitment toward the site of inflam-mation.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is an immunoglobulin-related molecule involvedin a broad scope of biologic functions but it is felt to playa role, albeit not exclusively, in transendothelial migration.It is expressed on leukocytes as well as at the endothelialcell junctions of the monolayer. Blockage of PECAM-1 onthe endothelium, monocytes, or neutrophils, inhibits leuko-cb intoi ),t them lw py,c tis( o-c

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rast, ICAM-1 (CD54) is markedly upregulated in respoo proinflammatory cytokines and thus, appears to bemportant in inflammatory responses[36].

Integrins are not constitutively active on leukocynd integrin-ligand engagement requires a second s

o strengthen its interaction (i.e. activation-induced strrest). Signaling through the chemokine receptor in a Rependent mechanism leads to a conformational chan

eukocyte integrins[2,37]. As a result, ligand binding occuith high avidity and affinity, which leads to leukocyte arrnd firm adhesion[31]. Rapid integrin-dependent adhes

hen occurs through the pertussis toxin (PTX) sensitivei�ubunit of a heterotrimeric GTP-binding protein[31].

Chemokines facilitate integrin binding to establish fidhesion and differentially do so amongst different leukoopulations. Epstein-Barr virus-induced molecule-1 (EIP-3�, CCL19), secondary lymphoid tissue chemok

SLC, 6-Ckine, CCL21), and stromal cell derived factoSDF-1, CXCL12) trigger rapid adhesion through ICA

binding of most lymphocyte populations in static ahysiologic flow conditions in vitro[31], whereas liver anctivation-regulated chemokine (LARC, MIP-3�, CCL20)nd monocyte chemoattractant protein (MCP-3, CCpecifically activate integrins on subpopulations of T lyhocytes[31,27,5].

Fractalkine (CX3CL1) has been shown to preferentnduce adhesion of different leukocyte subsets both innd in vivo. Fractalkine leads to stable arrest of CDonocytes[38]. In vivo fractalkine plays an important ro

n the recruitment and adhesion of monocytes and na

ytes from transendothelial migration[45], and PECAM-1lockade in animal models reduces leukocyte infiltration

nflamed tissues[46]. In rat adjuvant-induced arthritis (AIAreatment with anti-PECAM-1 resulted in a decrease inean number of adherent leukocytes per 100�M sized vesseithin the synovium, as visualized by confocal microscoompared to controls[47]. In the collagen-induced arthriCIA) mouse model, inhibition of PECAM-1 with a monlonal antibody ameliorated but did not abolish disease[48].

In genetically engineered animals deficient in PECA, neutrophils display an abnormal migration throughasal lamina; however, there are normal numbers of leytes recovered from sites of inflammation[49]. Surprisinglyhese animals have exacerbated inflammatory arthritis iIA model [50]. In general, the PECAM-1 deficient aals develop spontaneous autoimmunity in that they devutoantibodies, proteinuria, and immune complex glom

onephritis over time[51]. The etiology of this apparearadox is not clear, but could be explained by a functiedundancy in PECAM-1 deficient animals or a roleECAM-1 in regulating tolerance.

. Chemokines and chemokine receptors

Chemokines are small, structurally related peptide–10 kD that function to upregulate integrin adhesess and promote leukocyte migration into tissues[5,9].he chemokine system is extensive with an estim0 chemokine ligands and 20 receptors. Chemokine

4 T.K. Tarrant, D.D. Patel / Pathophysiology 13 (2006) 1–14

secreted from a wide variety of cells, and are involved in bothinnate and adaptive immune responses. They are similar inamino acid sequence and bind to receptors containing seventransmembrane spanning helices. They are classified basedon the N-terminal structure of cysteine residues into eitherthe C, CC, CXC, or CX3C families. The classification doesnot imply function, but rather to which family of receptorsa chemokine binds. Consequently, most chemokines appearto have redundant specificity since they bind to numerousreceptors within the same family.

The two larger groups of chemokine families are theCC and CXC families. CC chemokines have adjacent cys-teine residues and bind to one or several of the nine CCRreceptors. Typically, these peptides are chemoattractants formonocytes, but have also been described to affect T cell, NKcell, basophil, and eosinophil migration[52].

The CXC family has one nonconserved amino acid locatedin between the two conserved cysteines. They serve as lig-ands for the six known CXCR receptors. In general, CXCchemokines with a Glu-Leu-Arg (ELR) motif located beforethe first cysteine promote the migration of neutrophils andangiogenesis[9,52].

Fractalkine (CX3CL1) is the only member of the CX3Cfamily described to date, and has several unique attributes.Fractalkine is expressed on endothelial cells, and its recep-tor, CX3CR1, is expressed in humans on T lymphocytes,N off CCc cidsbe oundp tede firm

adhesion of leukocytes under physiologic flow conditions thatis both integrin and G-protein signaling independent[4].

Chemokines signal through G protein coupled receptors(GPCRs). Signal transduction typically occurs through thePTX-sensitive Gi� subunit of the chemokine receptor, butother cases have been described where G protein coupling isPTX insensitive[54–56]. Receptor-ligand interaction inducesa conformational change in the GPCR leading to GTP hydrol-ysis, release of G� and G�� subunits that activate phospho-lipase C (PLC), PI3 kinase (PI3K), and receptor tyrosinekinases. Chemotaxis is dependent on activation of PI3K[57,58], whereas activation of the small GTP binding pro-tein RhoA increases integrin adhesiveness[2,37].

Recent evidence from our group and others has shownthat�-arrestin 2 signaling plays an important role in chemo-taxis [59]. �-Arrestins mediate G protein receptor desensi-tization and endocytosis via clathrin coated pits[60]. Theyalso serve as scaffolds involved in G-protein independent sig-naling pathways such as the ERK/MAPK pathway[61–63](Fig. 2). The downstream pathways of�-arrestin 2 activationthat result in chemotaxis are still unclear, but splenocytesisolated from animals deficient in either�-arrestin 2 or Gprotein coupled receptor kinase 6 (GRK6) have markedlyimpaired chemotactic responses to SDF-1[59]. In vivo, �-arrestin 2 deficient animals have diminished hyper-airwayresponsiveness in a murine model of allergic asthma, andh uet

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K cells, and monocytes[28]. The chemokine domainractalkine is highly homologous to members of thehemokine family; however, fractalkine has three amino aetween the N-terminal cysteine residues[53]. More inter-stingly, it exists both as a secreted and a membrane-brotein. In its membrane-tethered form on TNF-activandothelium, fractalkine participates in the capture and

ig. 2. Signal transduction pathways through chemokine receptors. Cnd lead to cellular responses via activation of PI3K dependent patheceptor tail, and binding of b-arrestin. b-arrestin binding not only leactivate new signaling pathways, possibly leading to different cellular

ave diminished CD4+ T cells within the airways, likely do a defect in trafficking[64].

Chemokines are either constitutively expressed or induring inflammation. In general, the constitutive chemokuch as ELC and SLC act on naı̈ve lymphocytes wherheir receptors, CXCR5 and CCR7, are expressed[65].his is in contrast to macrophage inflammatory prot

heterotrimeric G-protein dependent pathways are activated by chemoe agonistgonist activation also leads to GRK-mediated phosphorylation of theokineeceptor desensitization to further G-protein mediated signals, butalso serves tses.

T.K. Tarrant, D.D. Patel / Pathophysiology 13 (2006) 1–14 5

1� (MIP-1�, CCL4), regulated upon activation normal Tcell expressed and secreted (RANTES, CCL5), monokineinduced by interferon-� (Mig, CXCL9), eotaxin (CCL11),and IP-10 (CXCL10) chemokines, which are upregulated ininflammatory tissues and target specific leukocyte subsets[66]. Chemokines and their receptors differentially recruitTh1 versus Th2 subsets. In general, CXCR3 and CCR5are predominantly expressed on Th1 cells and monocytes,whereas CCR3 and CCR4 are differentially expressed onTh2 cells [67]. Antibody blockade of CXCR3, which isupregulated in rheumatoid arthritis and is the receptor forinflammatory chemokines such as IP-10, MIG, and IFN-inducible T cell� chemoattractant (I-TAC), inhibits recruit-ment of Th1 cells in an adjuvant-induced peritonitis model[68]. Thus, chemokines maintain normal homeostatic migra-tion of leukocytes in addition to participating in both innateand adaptive immune responses.

Chemokines have been appreciated as playing a major rolein organ-specific trafficking since the landmark finding that adefect in CXCR5 led to defective migration of B cells to thespleen and Peyer’s patches[69]. B cell attracting chemokine 1(BCA-1, CXCL13), SDF-1 (CXCL12), ELC (CCL19), andSLC (CCL21) have been identified as being constitutivelyexpressed in normal lymphoid tissues and are responsiblefor the homeostatic trafficking and eventual positioning ofB-, T-, and dendritic cells within these structures[69–73].L inalc viuma reg-u allyt t then

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dent. E-selectin expression is upregulated in the inflamedsynovium, and subsequently decreases in disease remissionafter TNF-� blocking therapy[14]. Serum levels of both L-and P-selectin are increased in RA patients[74], but only P-selectin levels correlate with disease activity[5,75]. In the ratadjuvant-induced arthritis (AIA) model, blocking antibodiesto P-selectin inhibited the accumulation of neutrophils andmonocytes[76]. However, P-selectin deficient mice develop amore severe form of collagen-induced arthritis (CIA), whichdemonstrates that inflammatory arthritis is not entirely P-selectin dependent[77].

CD44 is upregulated in RA patients[6,78], and may beimportant in disease pathogenesis. In humans, inflamma-tory arthritis disease activity correlates with the ability ofantigen-specific T cells to roll on hyaluronic acid, a ligand forCD44 [5]. Blocking antibodies to the CD44-ligand interac-tion inhibit leukocyte trafficking in mouse models of arthritis[79] and decrease in vitro migration of synovial-like fibrob-lasts[80]. The role of CD44 in synovitis is pleotropic sincecross-linking CD44 on RA synovial cells in vitro up regu-lates Fas (CD95)-expression, and both hyaluronan fragmentsand CD44 engagement stimulate early Fas-mediated apop-tosis of RA synovial cells in vitro[81]. Spontaneous growtharrest and remission are observed in RA synovial cells thatexpress functional Fas antigens in addition to frank apoptosis[81,82]. This imbalance between cell proliferation and celld o thep

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ymphoid aggregates with formations similar to germenters are seen within the inflamed rheumatoid synond are discussed in further detail below. Targeting theselation pathways within different tissues could potenti

reat autoimmune inflammatory disease states withoueed for global immunosuppressive therapy.

. Regulation of leukocyte trafficking to inflamedynovium

Organ-specific leukocyte trafficking can occur at mifferent points and is highly regulated. Increased cell recent occurs by upregulating selectin or integrin adheolecule expression on the cell surface or by incr

ng leukocyte migration and activation through differenhemokine production. The resulting infiltrate consistany leukocyte subsets including granulocytes, T cel

ells, monocytes, macrophages, dendritic cells and mastach contributing in unique ways to the inflammation. Sets of leukocytes may also be retained for sustained pef time within inflamed tissues. When one or several of thegulatory mechanisms become altered, the result can lehronic inflammation or autoimmunity such as in RA.Table 1ummarizes the roles in inflammatory arthritis of some oolecules involved in leukocyte trafficking.

.1. Adhesion molecules and rheumatoid arthritis

There is accumulating evidence that leukocyte traffico the inflamed synovium in RA is adhesion molecule de

eath, mediated by CD44 interactions, could contribute tathological process of synovitis in RA.

The role of integrins in organ-specific leukocyte hominhe RA synovium is less clear. Blocking the different clasf integrins does not inhibit leukocyte binding to the vas

ar endothelium in RA synovium in vitro[5], and anti-LFA-1ntibodies do not inhibit T lymphocyte or neutrophil mig

ion in AIA [83]. Although VCAM-1 is upregulated in Rynovium and soluble levels correlate with clinical severeviewed in[84]), VCAM-1 blockade does not dramaticaeduce inflammatory cell migration into arthritic jointsrevent disease in CIA[85]. However, combined anti-LFA-nd anti-VLA-4 blockade inhibits monocyte migration to

nflamed synovium[86], and ICAM-1 (CD54) deficient micave decreased disease expression in CIA[87].

One potential mechanism for integrin-mediated region in RA is at the level of the synoviocyte as oppoo the leukocyte. Interaction of�1 integrins with ICAM-1nd Fas antigen on RA synovial cells induces Fas-medpoptosis[82]. This pathway could potentially lead to tpontaneous growth arrest and dysregulation of synovioeen in inflammatory RA.

.2. Chemokines and chemokine receptors inheumatoid arthritis

Chemokines and their receptors are key players inecruitment of inflammatory cells to the synovium in Rumerous chemokines have been found to be expresoth the protein and mRNA level in the inflamed syno

6T.K

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(2006)1–14

Table 1Properties of selected molecules in RA that affect leukocyte trafficking and recruitment

Molecule Family Names Function Animal data in arthritis Human data in RA

SelectinsCD62E Selectin E-selectin, ELAM-1,

LECAM-2Leukocyte rolling. Upregulated in RA, levels decrease after

anti-TNF� treatment.CD62L Selectin L-selectin, LAM-1,

LECAM-1, Leu-8Leukocyte rolling. Levels are not consistent with RA flares.

CD62P Selectin P-selectin, PADGEM,GMP-140

Leukocyte rolling. (1) Blockade inhibitsneutrophil/monocyte accumulation inAIA.

Increased in RA, correlates with diseaseactivity.

(2) Blockade ameliorates CIA.(3) CD62P deficient animals haveincreased CIA.

Adhesion moleculesCD11a/CD18 Integrin LFA-1 Firm adhesion. Blockade does not alter leukocyte

migration.

CD31 Ig family PECAM-1, GpIIa, EndoCAM Diapedesis. (1) Blockade ameliorates CIA andAIA.

Immunoregulation? (2) CD31 deficient animals haveincreased CIA and enhancedautoimmnity.

CD44 Hyaldherin H-CAM, Pgp-1, Hermes,In(Lu)-related

Lymphocyte rolling, cell adhesion. (1) Blockade decreases leukocytetrafficking in rodent models of RA.

(1) Numerous isoforms present ininflamed joint.

(2) Increased Fas expression andapoptosis of synoviocytes aftercross-linking CD44 in vitro.

(2) Present in inflamed synovium.

CD54 Ig family ICAM-1 Firm adhesion of leukocytes. (1) CD54 deficient animals havereduced CIA.

(1) Randomized, placebo controlled trialwith inhibitor could not demonstrateefficacy.

(2) Fas mediated synoviocyteapoptosis in vitro.

CD106 Ig family VCAM-1, INCAM-110 Firm adhesion. (1) Blockade ameliorates CIA. (1) Increased in inflamed synovium.(2) Levels correlate with clinical diseaseseverity.

ChemokinesCCL3 CC MIP-1� Th1 chemoattractant. (1) Blockade ameliorates CIA. Elevated in RA synovial fluid.

Ligand for CCR1, 3, 5. (2) Polyclonal antibody blockadedoes not improve AIA.

CCL5 CC RANTES Chemoattractant for monocytes,Th memory cells, and eosinophils.

(1) Polyclonal antibody blockadeameliorates AIA.

(1) Produced by RA synoviocytes inresponse to proinflammatory cytokines.

Ligand for CCR1, 3, 4, 5. (2) MetRANTES ameliorates CIA. (2) Levels similar to patients with OA.

CXCL13 CXC BCA-1 Lymphocyte homing, germinalcenter formation.

May contribute to organizedlymphoid aggregates in RAsynovium.

CX3CL1 CX3C Fractalkine, neurotactin Integrin independent leukocytecapture and firm adhesion.

Blockade ameliorates CIA. Receptor CX3CR1 increased oncirculating and residing T cells in RA.

T.K.Tarrant,D

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1–147

Table 1 (Continued)

Molecule Family Names Function Animal data in arthritis Human data in RA

Chemokine receptorsCCR1 CCR Present on monocytes, immature

dendritic cells, T cells, basophils.Phase IIb study of CCR1 inhibitordemonstrated safety and trend towardimprovement.

CCR2 CCR Present on monocytes, immaturedendritic cells, basophils, T cells,particularly Th2.

CCR2 deficient animals haveincreased CIA.

(1) Ligand MCP-1 upregulated byactivated synoviocytes with TNF-�.

Blockade ameliorates CIA. (2) Tissue infiltrating monocytes haveincreased CCR2.

CCR5 CCR Present on monocytes, immaturedendritic cells, T cells, Th1 cells.

CCR5 deficient animals have similarCIA scores as controls.

(1) Upregulated in RA.

(2) Polymorphism�32 associated withmilder disease.(3) Ligands MIP-1�, MIP-1�, RANTESelevated in RA.

CXCR3 CXCR Present on activated T cells,particularly Th1.

(1) Upreguated in RA.

(2) Ligands IP-10, Mig elevated in RA.

CXCR4 CXCR Present on basophils, Th2 cells,and mature dendritic cells.

Blockade ameliorates CIA.

8 T.K. Tarrant, D.D. Patel / Pathophysiology 13 (2006) 1–14

fluid, which mirrors the diverse inflammatory cellular infil-trate histopathologically[88]. In particular, Th1 chemoat-tractants such as MIP-1�, MIP-1�, IP-10 and Mig and theirreceptors CCR5 and CXCR3 are upregulated within thesynovial microenvironment[89,88]. CCR2 and CCR5 areexpressed on T cells and macrophages within the inflamedjoints of RA patients[90–92] and in CIA in animals[93].RA patients also have increased CX3CR1 expression on cir-culating and synovial-residing T cells that produce Th1-typecytokines[94,95]. IL-8 (CXCL8) and ENA-78 (epithelial-neutrophil activating protein 78, CXCL5), which attract neu-trophils and monocytes, and MCP-1 (monocyte chemoattrac-tant protein 1, CCL2), which attracts monocytes, are secretedby activated synovial fibroblasts in response to proinflam-matory cytokines such as TNF-� [88,5,96]. The sources ofchemokines are both the synovial lining cells as well as infil-trating leukocytes[97].

Although macrophage-lymphocyte chemokines are ele-vated, the data do not entirely support a pathogenic role. MIP-1�, which is expressed by synovial macrophages, is elevatedin RA synovial fluid [98], but MIP-1� is variably elevatedin RA when compared to synovial fluid from osteoarthritis(OA) patients[99]. Although RANTES mRNA is inducedin response to proinflammatory cytokines by RA synovialfibroblasts, RANTES protein levels are similar between RAand OA patients[100].

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which suggests that it could be functioning in recruitment aswell as maintenance of these organized lymphoid structures[106,107].

Evaluation of chemokine receptor expression in RA hasyielded some intriguing observations. Epidemiologic stud-ies have suggested that the CCR5�32 polymorphism, whichresults in a defective receptor, may confer milder diseasein patients with RA[109], but this correlation has not beenconsistently confirmed[110]. The chemokine receptors mostabundantly expressed on activated leukocytes in RA patientsare CCR5 and CXCR3[88,89], and CCR2 on tissue infil-trating monocytes[111]. However, the genetic deletion ofCCR5 in the murine CIA and collagen-Ab-induced arthritis(CAIA) models showed no difference in disease expressioncompared to wild-type controls[111]. This result highlightsthe redundancy of the chemokine system, and how targetingone molecule may not be therapeutically efficacious.

Earlier animal work in either CCR2 deficient mice or inmice treated with a monoclonal antibody to CCR2 (MC-21) showed a pronounced defect in leukocyte migration[112,113]. However, CCR2 deficient animals immunizedto develop CIA had a more accelerated and severe formof inflammatory arthritis as well as increased autoantibodyproduction compared to controls[113]. Further mechanisticstudies elucidated that early (days 0–15) versus late (days21–36) blockade of CCR2 with MC-21 antibody could pro-d bateda b-p erea ss ingi oim-m dis-e

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The microenvironment within the chronically inflamoint may also retain and compartmentalize pathogenichrough chemokine interactions, and there is accumulvidence to suggest that the SDF-1 interaction with its reor CXCR4 may play a role in the inflammatory proc101]. TGF-� upregulates CXCR4 expression on syno

cells, which then functionally adhere to both ICAM-1 abronectin after exposure to SDF-1[102]. Cultured synoviabroblasts from RA patients express high levels of Sand VCAM-1, but only the SDF-1/CXCR4 interactio

ffect T cell migration in vitro[103]. T cell subsets displayepecific patterns and rates of migration in co-cultureynovial fibroblasts, suggesting that SDF-1/CXCR4 inteions may contribute to the compartmentalization of CDersus CD8+ inflammatory T lymphocytes in RA[103,101].XCR4/SDF-1 interaction also protects T cells from unoing activation-induced cell death, which may lead to aulation of inflammatory effector cells within the joint spa

104].Approximately 20% of RA patients have B and T ly

hocyte aggregates beneath the synovial lining with aar organization to germinal centers[105]. CXCL13, whosexpression is primarily confined to B cell follicles, has aeen identified in the RA synovium and may participat

he establishment of these structures[106–108]. Two groupsdentified that CXCL13 was primarily expressed in germenters within lymphoid aggregates, which was depenn the presence of follicular dendritic cells[107,108]. Inddition, the Takemura group found CXCL13 expresithin endothelial cells of small arterioles and capillar

uce different phenotypes of protected versus exacerrthritis in the CIA model[114]. This study identified a suopulation of CCR2+/CD25+ regulatory T cells, which wnergic to collagen-specific activation[114]. These resultuggest that the regulatory control of lymphocyte trafficks highly complex and could even lead to enhanced aut

unity when the regulatory balance is tipped in certainase states.

Given the observation that synovial macrophages exigh levels of CCR2 in inflammatory arthritis and tacrophages are still present in the inflamed joint deCR2 deletion[111], the role of the monocyte/macropha

n disease pathogenesis continues to be an actively exprea. A new paradigm has been suggested by the Litroup that describes monocyte/macrophage subsetsn CCR2 and CX3CR1 expression and their relative r

n inflammation [115]. In this model, circulating periphral blood monocytes that are CX3CR1lo/CCR2+/Gr1+ inice (CD14+/CD16− in humans) are actively recruited

nflammatory sites whereas CX3CR1hi/CCR2-/Gr1− resi-ent macrophages (CD14lo/CD16+ in humans) predomina

n non-inflamed tissues[115]. This polarization of monoyte/macrophage subsets and their role in RA needsurther studied.

. Therapeutic considerations

Inhibitors to adhesion molecules have had some sun animal models of inflammatory arthritis. A P-selec

T.K. Tarrant, D.D. Patel / Pathophysiology 13 (2006) 1–14 9

glycoprotein ligand (rPSGL-1Ig) fusion protein was ableto ameliorate established CIA in mice, partially throughdecreased production of TNF-� [116]. VCAM-1 blockadewith neutralizing antibodies increases circulating B cells andis able to reduce clinical severity but not incidence of CIA[85]. A recently generated monoether derivative of probucil,compound 4ad (AGIX-4207), which has been shown to havepotent inhibitory effects on VCAM-1, inhibited paw edemain a collagen II sensitized rat model and is currently in clinicaltrials for RA [117].

The �2 integrins and ICAM-1 (CD54) have been down-regulated both directly and indirectly by therapeutic interven-tions in patients with inflammatory arthritis. Methotrexateand leflunomide, which are disease modifying antirheumaticdrugs (DMARDs), downregulate the expression of ICAM-1and VCAM-1 in the synovial tissue of patients with activeRA [118,33]. Anti-ICAM-1 monoclonal antibodies have hadlimited success in patients with refractory RA, but did showalterations in T cell recruitment and responsiveness[119].Consequently, a follow-up phase I/II study was performedin early RA patients where a single course of therapy wasassociated with clinical improvement to a greater extent thanpreviously had been observed in patients with longstanding,aggressive RA[120]. However, a randomized, placebo con-trolled trial of an antisense oligodeoxynucleotide ICAM-1inhibitor could not demonstrate clinical efficacy beyond thato died[

t the� dids iasis[g ryb nicalb wnt atica .

herdc ma-t thes teda thes infil-t

ctionh f RA.A notp st elio-r rb -i els.I inma ody

treatment of CIA does not alter anti-type II collagen anti-body production or IFN-� production by stimulated effectorT cells in vitro [129]. Humoral and cell mediated immuneresponses were left intact, but the infiltration of adoptivelytransferred splenic macrophages to the inflamed synoviumwas impaired[129]. These data suggest that cell migrationcould be an important target in the pathogenesis and treatmentof RA.

Due to the considerable redundancy of chemokine func-tion and promiscuity within chemokine families using sharedreceptors, targeting one specific chemokine-receptor pairmay not be effective. Consequently, small molecule inhibitorsof the GPCR are being developed for possible therapeutic use.Already reported are compounds that inhibit CCR1, CCR2,CCR3, CCR5, CXCR2 and CXCR4[130–134], and clinicaltrials in human disease with these compounds are forthcom-ing.

Results from the inhibition of CCR1 in a double blind,placebo controlled, phase IIb clinical trial has recently beenreported in RA[135]. In 16 randomized patients with activeRA on day 15 after treatment, there was decreased cellularityof infiltrating macrophages and T cells within the synoviumof the patients treated with the CCR1 antagonist. No majoradverse effects were reported, and there was a trend towardclinical improvement in the patients treated with active drug[135]. Limitations of this study include small sample size ands

mallm nter-a firsti sholdf ownip lsh ialt eptori R2d iso-l o seeni efi-c ulei mayb rossr ni-m onsw tors.F e atb er-b

rac-t -i emo-t Rs,a ptorm smallm ibit

f placebo in the 43 patients with active RA who were stu121].

Efazulimab, a humanized monoclonal antibody againsL integrin, showed no therapeutic benefit in RA, buthow clinical efficacy in decreasing skin plaques in psor122]. Natalizumab, a monoclonal antibody against�4 inte-rins (CD49d), is in phase III clinical trials for inflammatoowel disease and multiple sclerosis, and may have clienefit in RA[122]. Alefacept, anti-(LFA)-2, has been sho

o be efficacious in a limited number of patients with psorirthritis[123], and also may prove to be of benefit in RA

Chemokine inhibition in RA can be achieved eitirectly or indirectly. Neutralizing TNF-� and IL-1 withurrently available biologic agents downregulates inflamory chemokine and chemokine receptor expression inynovial joint. Infliximab, a monoclonal antibody direcgainst TNF-�, decreases IL-8 and MCP-1 expression inynovium, which correlates with a decreased leukocyterate and clinical improvement[96].

Therapies targeted at the chemokine-receptor interaave had some pre-clinical success in animal models onti-MIP-1� and MIP-1� antibodies decreased but didrevent disease in CIA[124]. MetRANTES, which block

he CCR1, CCR3, CCR4, and CCR5 receptors, also amated disease in the CIA model[125]. Targeting MCP-1 eithey using an antagonist protein[126] or neutralizing antibod

es[127], decreases inflammatory arthritis in rodent modnhibition of the SDF-1/CXCR4 interaction improved CIAice[128] as does inhibition of fractalkine[129]. Providingn insight to potential mechanism, anti-fractalkine antib

hort-term follow-up.Although the data are encouraging to proceed with s

olecule inhibitors targeted at the chemokine-receptor iction, there are potential pitfalls to be considered. The

s the risk of immunosuppression and a decreased threor infection. CCR1 and CCR2 deficient mice have shncreased susceptibility toToxoplasma gondii infection com-ared to controls[136,137], and the CCR1 deficient animaad increased mortality[137]. There is also the potent

o exacerbate autoimmune disease with chemokine recnhibitors as illustrated by the worsening CIA in the CCeficient animals[111]. The result does not appear to be

ated to Th1-mediated diseases since exacerbation is alsn the OVA challenged allergic asthma model in CCR2 dient mice[138]. Finally, animal studies using small molec

nhibitors to further investigate mechanism and safetye limited secondary to species specificity and lack of ceactivity. In addition, studies with genetically deficient aals may not be as informative as inhibiting interactiith exogenous substances like small molecule inhibior example, murine CCR2 inhibitors are very effectivlocking CIA, while CCR2 deficient animals have exacated CIA.

Recent intriguing data suggest that specific ligand inteion with a GPCR can activate non-classical�-arrestin signalng pathways leading to downstream effects such as chaxis [139]. If these data generalize to chemokine GPCrguably different chemokines binding to the same receay activate separate signaling pathways. Therefore,olecule inhibitors of chemokine receptors could inh

10 T.K. Tarrant, D.D. Patel / Pathophysiology 13 (2006) 1–14

classical GPCR pathways but also activate non-classical path-ways leading to lack of efficacy or unexpected side effects.Further study will need to be conducted to better understandthe underlying mechanisms of these complex regulatory path-ways.

5. Conclusions

Although anti-TNF-� therapies have provided consider-able benefit to numerous patients with RA, there are stillthose who obtain only partial benefit or are unable to tol-erate these drugs. Consequently, there is still a need foradditional therapies that can be used either in conjunctionwith anti-TNF-� drugs or to act as solo agents. Leukocytetrafficking to sites of inflammation is a highly regulated pro-cess and may be a potential therapeutic target in chronicautoimmune diseases like RA. Adhesion molecules as well aschemokine-receptor interactions that regulate organ-specificmigration are ideal targets to downregulate the inflammatoryresponse without compromising immune surveillance againstinfection. Designing therapies aimed at multiple chemokinereceptors will likely be more effective than inhibition of a sin-gle GPCR due to the considerable redundancy and overlapof function within chemokine families.

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