CC Chemokine Receptors and Chronic Inflammation--Therapeutic Opportunities and Pharmacological Challenges

1521-0081/65/1/47–89$25.00 http://dx.doi.org/10.1124/pr.111.005074PHARMACOLOGICAL REVIEWS Pharmacol Rev 65:47–89, January 2013Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: CHRISTOPHER J. GARLAND

CC Chemokine Receptors and ChronicInflammation—Therapeutic Opportunities

and Pharmacological ChallengesGemma E. White, Asif J. Iqbal, and David R. Greaves

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

A. The Chemokine Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48B. Chemokine Structure and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49C. Receptor Structure and Signaling Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50D. Chronic Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

II. CC Chemokine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52A. CCR1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52B. CCR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53C. CCR3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57D. CCR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58E. CCR5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60F. CCR6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62G. CCR7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63H. CCR8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65I. CCR9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66J. CCR10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67K. Atypical CC Chemokine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

III. Role of Chemokines in Chronic Inflammatory Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69A. Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

1. Summary of Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692. Current Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693. Evidence Supporting a Role for CC Chemokines in Development of Pathology . . . . . . . . . . 704. Key Chemokine Receptors as Drug Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

B. Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721. Summary of Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722. Current Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733. Evidence Supporting a Role for CC Chemokines in Development of Pathology . . . . . . . . . . 734. Key Chemokine Receptors as Drug Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

C. Obesity, Metabolic Syndrome, and Type 2 Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751. Summary of Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752. Current Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773. Evidence Supporting a Role for CC Chemokines in Development of Pathology . . . . . . . . . . 774. Key Chemokine Receptors as Drug Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

IV. Chemokine Receptor Drugs in Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79V. Critical Assessment of Chemokine Receptors as Anti-inflammatory Drug Targets . . . . . . . . . . . . . . 79

A. CCR1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Address correspondence to: David R. Greaves, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford,OX1 3RE, UK. E-mail: [email protected]

The work in the Greaves laboratory is funded by the British Heart Foundation, Programme Grant Number RG/10/15/28578. G.E.W. isfunded by British Heart Foundation project Grant Number PG/10/60/28496.

dx.doi.org/10.1124/pr.111.005074.

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B. CCR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79C. CCR3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80D. CCR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80E. CCR5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80F. CCR6 and CCR7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80G. CCR8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80H. CCR9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80I. CCR10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Abstract——Chemokines are a family of low molec-ular weight proteins with an essential role in leukocytetrafficking during both homeostasis and inflammation.The CC class of chemokines consists of at least 28members (CCL1-28) that signal through 10 knownchemokine receptors (CCR1-10). CC chemokine receptorsare expressed predominantly by T cells and monocyte-macrophages, cell types associated predominantly withchronic inflammation occurring over weeks or years.Chronic inflammatory diseases including rheumatoidarthritis, atherosclerosis, and metabolic syndrome arecharacterized by continued leukocyte infiltration intothe inflammatory site, driven in large part by excessivechemokine production. Over years or decades, persistentinflammation may lead to loss of tissue architectureand function, causing severe disability or, in the caseof atherosclerosis, fatal outcomes such as myocardial

infarction or stroke. Despite the existence of severalclinical strategies for targeting chronic inflammation,these diseases remain significant causes ofmorbidity andmortality globally, with a concomitant economic impact.Thus, the development of novel therapeutic agents forthe treatment of chronic inflammatory disease continuesto be a priority. In this review we introduce CCchemokine receptors as critical mediators of chronicinflammatory responses and explore their potentialrole as pharmacological targets. We discuss functionsof individual CC chemokine receptors based on in vitropharmacological data as well as transgenic animalstudies. Focusing on three key forms of chronicinflammation—rheumatoid arthritis, atherosclerosis,and metabolic syndrome—we describe the pathologicfunction of CC chemokine receptors and their possiblerelevance as therapeutic targets.

I. Introduction

A. The Chemokine Family

Chemokines are a family of around 50 low molecularweight polypeptides with a conserved tertiary struc-ture. They are divided into four classes, C, CC, CXC,and CX3C, on the basis of the location of key cysteine

residues that participate in disulfide bonding and areeither juxtaposed (CC) or separated by 1 or 3 aminoacids (CXC and CX3C, respectively). Virtually all chemo-kines are secreted from the cell after synthesis, withtwo exceptions, CX3CL1 (fractalkine) and CXCL16(SR-PSOX), which can remain tethered to the cellsurface by a transmembrane mucin-like stalk (Bazan

ABBREVIATIONS: AAD, asthmatic airway disease; ABN912, high-affinity human anti-human CCL2/MCP-1 monoclonal antibody of theIgG4/k isotype (MW 145 kd); ACAT, acyl-CoA cholesterol acyl transferase; AHR, airway hyperresponsiveness; AIDS, acquired immunedeficiency syndrome; AMD3100, plerixafor; AMPK, AMP-activated protein kinase; Apo, apolipoprotein; ASC, antibody secreting cell;AZD4818, (1R,9S,12S,13S,14S,17R,20S,21R,23S,24R,25S,27R)-17-Ethyl-1,14,20-trihydroxy-12-{(1E)-1-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-propen-2-yl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-a zatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetrone; AZD5672,3-[1-[(4-chlorophenyl)methyl]-3-(3,3-dimethylbutanoyl)-5-(quinolin-2-ylmethoxy)indol-2-yl]-2,2-dimethylpropanoic acid; BAL, bronchoal-veolar lavage; CCX354, 1-[4-(4-chloro-3-methoxyphenyl)piperazin-1-yl]-2-[3-(1H-imidazol-2-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl]ethan-1-one; BX471,(R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloric salt); CFA, complete Freund’s adju-vant; CIA, collagen-induced arthritis; CNS, central nervous system; con A, concanavalin A; COPD, chronic obstructive pulmonary disease;CP481,715, 2-Quinoxalinecarboxamide, N-((1S,2S,4R)-4-(aminocarbonyl)-1-((3-fluorophenyl)methyl)-2,7-dihydroxy-7-methyloctyl)-, 212790-31-3;DAG, diacylglycerol; DARC, Duffy antigen receptor for chemokine; DC, dendritic cell; DMARD, disease-modifying antirheumatic drug; DSS,dextran sulfate sodium; DTH, delayed type hypersensitivity; EAE, experimental autoimmune encephalomyelitis; EBV, Epstein Barr virus; EGFR,epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular regulated kinase; GAG, glycosaminoglycan; GM-CSF, granulocyte macrophage colony-stimulating factor; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; GVHD, graftversus host disease; GWAS, genome-wide association study; HEK, human embryonic kidney; HHV, human herpes virus; HIV, humanimmunodeficiency virus; HSP, heat shock protein; IAP, inhibitor of apoptosis protein; ICAM-1, intercellular adhesion molecule 1; INCB3284, 1-hydroxy-4-[[(1R,3S)-3-propan-2-yl-3-[3-(trifluoromethyl)-7,8-dihydro-5H-1,6-naphthyridine-6-carbonyl]cyclopentyl]amino]cyclohexane-1-carbonitrile;INCB-3344, N-[2-[[(3S,4S)-1-[4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxypyrrolidin-3-yl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide;INCB8761, N-[2-[(3S)-3-[[4-hydroxy-4-(5-pyrimidin-2-ylpyridin-2-yl)cyclohexyl]amino]pyrrolidin-1-yl]-2-oxoethyl]-3-(trifluoromethyl)ben-zamide; IFN, interferon; iNOS, inducible nitric oxide synthase; IP3, inositol trisphosphate; JIA, juvenile idiopathic arthritis; JNK, Jun N-terminalkinase; LDL, low-density lipoprotein; LN, lymph node; LPS, lipopolysaccharide; mAb, monoclonal antibody; MALT, mucosal-associated lymphoidtissue; MAPK, mitogen-activated protein kinase; Maraviroc, 4,4-difluoro-N-[(1S)-3-[(1S,5R)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxamide; MEK, MAPK/ERK kinase; MI, myocardial infarction; MK 0812,1,5-Anhydro-2,3-dideoxy-3-{[(1S,3R)-3-isopropyl-3-{[3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl]carbonyl}cyclopentyl]amino}-4-O-methyl-D-erythro-pentitol; MK-0812, 1,5-Anhydro-2,3-dideoxy-3-[[(1R,3S)-3-[[7,8-dihydro-3-(trifluoromethyl)-1,6-naphthyridin-6(5H)-yl]carbonyl]-3-(1-methylethyl)cyclopentyl]amino]-4-O-methyl-D-erythro-pentitol; MLN1202, Humanised mAb against CCR2; MMP, matrix metalloproteinase

48 White et al.

et al., 1997; Matloubian et al., 2000). Chemokines can bebroadly classified as homeostatic or inflammatorydepending on whether they have a role in physiologiccell trafficking [e.g., CCL19 (ELC) and CCL21 (SLC)] orare synthesized on demand in response to an inflamma-tory stimulus [e.g., CCL2 (MCP-1)].Chemokines have a systematic nomenclature based

on the class and a numerical designation e.g., CCL3,CXCL10 (Murphy et al., 2000; Zlotnik and Yoshie,2000). This greatly simplifies the previous systemwhereby chemokines were named predominantly byfunction and could therefore have several differentnames, e.g., CCL2 was originally named monocytechemoattractant 1 (MCP-1), small inducible cytokineA2 (SCYA2), and monocyte chemotactic and activatingfactor (MCAF) (Furutani et al., 1989; Yoshimura et al.,1989b; Mehrabian et al., 1991). Pairings betweenindividual chemokine receptors and their chemokineligands are shown in Fig. 1.

B. Chemokine Structure and Function

The first chemokine, IL-8 (CXCL8), was originallydescribed for its ability to chemoattract neutrophils,and many other chemokines have since been identifiedby their role in mediating chemotaxis of specificleukocyte subsets (Yoshimura et al., 1987). All chemo-kines have a similar tertiary structure comprisinga disordered N terminus of 6–10 amino acids followedby a long loop (known as the N loop), a 310 helix,a three-stranded beta-sheet, and finally a C-terminalalpha helix (Allen et al., 2007).The N terminus has a critical role in receptor

activation, and N-terminal truncations can renderchemokines inactive or even able to act as antagonists.Deletion of residues 2–8 in the N terminus of CCL2, forexample, generates a chemokine that still binds to thereceptor but fails to inhibit cAMP synthesis and acts asan antagonist in calcium flux and chemotaxis assays(Jarnagin et al., 1999). In contrast, several of theligands for the chemokine receptor CCR1 undergoproteolytic processing during inflammation to generate

full agonists (Proost et al., 2000; Berahovich et al.,2005). Chemokine processing may also alter receptorusage—CCL4 (MIP-1b) can be processed by CD26(dipeptidyl peptidase IV) to leave residues 3–69, whichgenerates a protein capable of activating CCR1 andCCR2b in addition to CCR5, which is activated by thewild-type chemokine (Guan et al., 2002). Similarly, N-terminal truncation of the CCR4 ligand CCL22 (MDC)generates a chemokine that loses chemoattractantactivity for a T-cell line but retains chemotactic formonocytes, suggesting the presence of an alternativereceptor that is differentially activated by the twospecies (Struyf et al., 1998b). N-terminal processing

Fig. 1. CC chemokine receptor and ligand pairings. The known chemo-kine receptors belonging to each of the chemokine families, (C, CC, CXC,and CX3C) are represented around the outer ring of the wheel, with theirchemokine ligands shown along the wheel spokes. Receptors with a singleknown ligand are shown in the area of the circle shaded yellow. A receptornamed CCR12 has been described (Miao et al., 2007), although this is notcurrently listed in the IUPHAR database of recognized chemokinereceptors.

MOG, myelin oligodendrocyte glycoprotein; MPC, myeloid progenitor cell; MR, mannose receptor; NK, natural killer; NO, nitric oxide; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; NSAID, nonsteroidal anti-inflammatory drug; OVA, ovalbumin; PBL, peripheralblood leukocyte; PBMC, peripheral blood mononuclear cell; PD980549, 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; pDC,plasmacytoid dendritic cell; PI3K, phosphoinositode 3-kinase; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; PKB, protein kinase B;PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; PPAR, peroxisome proliferator-activated receptor; PPD, purifiedprotein derivative; PPD, purified protein derivative; PTX, pertussis toxin; RA, rheumatoid arthritis; RAG, recombinase activating gene;RANTES, regulated on activation normal T cell expressed and secreted; ROCK, Rho-associated coiled coil-forming protein kinase; SB-328437,methyl (2S)-2-(naphthalene-1-carbonylamino)-3-(4-nitrophenyl)propanoate; SCH351125, [4-[4-[(Z)-C-(4-bromophenyl)-N-ethoxycarbonimi-doyl]piperidin-1-yl]-4-methylpiperidin-1-yl]-(2,4-dimethyl-1-oxidopyridin-1-ium-3-yl)methanone; SEA, Schistosoma mansoni egg antigen;SHP, Src homology region 2 domain-containing phosphatase; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index;SMC, smooth muscle cell; SNP, single nucleotide polymorphism; SRE, sterol regulatory element; SR-PSOX, CXCL16; TAK-779, dimethyl-[[4-[[3-(4-methylphenyl)-8,9-dihydro-7H-benzo[7]annulene-6-carbonyl]amino]phenyl]methyl]-(oxan-4-yl)azanium chloride; T-ALL, T-cell acutelymphoblastic leukemia; T-CLL, T-cell chronic lymphoblastic leukemia; TCR, T-cell receptor; TGF, transforming growth factor; TGF-b,transforming growth factor-b; TLR, Toll-like receptor; TNBS, trinitrobenzene sulfonic acid; TNF, tumor necrosis factor; Treg, regulatoryT cell; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; UTR, untranslated region; WT, wild type; ZK 756326, 2-{2-[4-(3-phenoxybenzyl)piperalzin-1]ethoxy}ethanol.

CC Chemokine Receptors in Inflammation 49

thus exists as a mechanism to finely control theactivity of chemokines, facilitating a wider range ofcellular responses to a given spectrum of chemokines.Of note, the ability of chemokines to undergo proteolyticprocessing means that quantification of absolute chemo-kine levels by enzyme-linked immunosorbent assay(ELISA) may not accurately reflect the amount of activechemokine present in a given sample—more sensitivebioassays may need to be developed to measure chemo-kine activity.Structural studies have demonstrated that many

chemokines form dimers or tetramers in solution, andsome, e.g., CCL5 (RANTES), may form higher orderaggregates of several hundred kilodaltons (reviewed inAllen et al., 2007). CCL2 has been crystallized in twoforms: a dimeric form and a tetrameric form of twoassociated dimers with different structures (Lubkow-ski et al., 1997). The residues required for aggregationin the chemokines CCL3 (MIP-1a), CCL4, and CCL5have been identified via mutagenesis studies, and, inthe case of CCL3, two acidic residues at positions 26and 66 are required (Czaplewski et al., 1999). Chemo-kines can also heteroligomerize with other chemokinesof the same or different classes (Allen et al., 2007). Forexample, CXCL4 (platelet factor 4) and CCL5 are bothstored in platelet a granules and were found toheteroligomerize via a specific region of CCL5 re-quired for aggregation (von Hundelshausen et al.,2005). This interaction has a functional consequence:amplifying the effect of CCL5 on monocyte adhesion toactivated endothelial cells. However, it is known thatchemokines bind their receptors as monomers, becausemutants that are unable to oligomerize retain the samereceptor binding affinity and in vitro chemotacticactivity as wild-type chemokines (Czaplewski et al.,1999; Proudfoot et al., 2003; Allen et al., 2007).In vivo, most chemokines are thought to bind to

glycosaminoglycans (GAGs), proteoglycans such as hep-arin sulfate and chondroitin sulfate that are expressedon the surface of most cells (Allen et al., 2007). Proudfootet al. (2003) determined the GAG binding regions ofCCL2, CCL3 (MIP-1a), and CCL5 and generatedmutants of these chemokines that show reducedheparin-binding activity. In vitro, these chemokinesretain equivalent chemoattractant activity to wild-type proteins. However, intraperitoneal administra-tion of the GAG-binding mutants in mice fails torecruit leukocytes, demonstrating the importance ofGAG binding for establishment of chemokine gra-dients in vivo.

C. Receptor Structure and Signaling Mechanisms

All chemokine receptors are class A G protein-coupledseven-transmembrane receptors that induce signal trans-duction via Gi and occasionally other G proteins. Thus,most chemokine responses can be inhibited by pertussistoxin (PTX) (Wu et al., 1993). By using cells transfected

with a variety of G protein-coupled receptors (GPCRs)that couple to Gi, Gs, or Gq heterotrimeric G proteins,Neptune and Bourne (1997) demonstrated that Gi acti-vation is necessary for chemotaxis. In addition, thecotransfection of these cells with Gatransducin, which se-questers Gbg subunits, completely prevented cell migra-tion. indicating that Gbg subunits activate pathwaysrequired for chemotaxis (Neptune and Bourne, 1997).

The network of signaling pathways activated bychemokines is complex, allowing multiple functionaloutcomes including chemotaxis, adhesion, proliferation,and control of gene expression. There is little consen-sus about canonical pathways for chemokine receptorsignaling, because the pathways activated depend onthe specific receptor and cell types involved. Many ofthe early studies focusing on chemokine receptorsignaling used transfected cells overexpressing a givenreceptor, which makes it hard to extrapolate thesefindings to signaling in primary cells. What is clear isthat the majority of chemokines induces calcium flux incells expressing their cognate receptor, either viacalcium influx or release from intracellular stores.This is often induced via direct activation of phospholi-pase C by Gbg subunits, causing an increase in in-tracellular inositol trisphosphate (IP3) and diacylglycerol(DAG) and leading to calcium release from intracellu-lar stores. Numerous other studies have shown animportant role for phosphoinositide 3-kinase (PI3K)in coordinating chemokine responses via coupling todownstream effectors, including extracellular regulatedkinase (ERK) and Akt (Curnock et al., 2002). It is clearthat chemotaxis requires the integration of multiplesignaling pathways, culminating in actin polymerizationto facilitate movement toward any given chemokinestimulus.

The structural regions required for ligand bindingand receptor activation have been defined for severalchemokine receptors. For many chemokine receptors,such as CCR2, the N terminus is a critical determinantof ligand binding and is involved in, but not sufficientfor, efficient signal transduction (Monteclaro andCharo, 1996; Samson et al., 1997). The N terminus isoften glycosylated or tyrosine sulfated, and this may berequired for high-affinity chemokine binding, as is thecase for CCR5 (Bannert et al., 2001). Other regions ofCCR5 required for ligand selectivity and intracellularsignaling have been determined, with the secondextracellular loop of the receptor having a key role(Samson et al., 1997). In contrast, for another chemo-kine receptor, CX3CR1, residues in the N terminus andthird extracellular loop are required for ligand bindingand receptor signaling (Chen et al., 2006). A two-stepmodel for chemokine receptor binding and activationhas been postulated, whereby the N terminus andextracellular loops of the receptor are involved inbinding the core domain of the chemokine ligandwhereas the N terminus of the chemokine penetrates

50 White et al.

directly into the helical bundle of the receptor (Guptaet al., 2001). Chemokine receptors, in common withother rhodopsin-like GPCRs have a DRY (asparticacid–arginine–tyrosine) conserved motif at the cyto-plasmic end of the third transmembrane segment. Thisregion has been shown to be critical to signaling,because chemokine-like receptors that lack the DRYmotif, such as the receptor D6, act as molecular sinksor decoys for chemokines, showing binding but nointracellular signaling in response to chemokines(Bonini et al., 1997; Nibbs et al., 1997). The C terminusof the receptor, as for many GPCRs, contains keyserine and threonine residues which can be phosphor-ylated by G protein-coupled receptor kinases (GRKs) toinduce recruitment of arrestin proteins leading toreceptor internalization and signal termination (Vroonet al., 2006). It is now known that aside from its role inreceptor internalization, beta arrestin recruitment mayalso enable the formation of a G protein-independentsignaling complex (reviewed extensively in Rajagopalet al., 2010). Because many chemokine receptors arepromiscuous in their ligand binding, differentialactivation of G protein- versus b-arrestin-dependentsignaling pathways may facilitate functional selectiv-ity of cellular responses. This may also be regulated atthe level of phosphorylation, whereby different GRKrecruitment may modulate signaling complex forma-tion, as is the case for CCR7, which binds both CCL19and CCL21 with different outcomes (Zidar et al.,2009). The generalized structural regions of

chemokine receptors required for function are sum-marized in Fig. 2.

To date, only a single chemokine receptor, CXCR4,has been crystallized and the structure determined bygeneration of a stabilized receptor with a T4 lysozymefusion inserted between helices 5 and 6 and one or morethermostabilizing point mutations (Wu et al., 2010). Thereceptor was cocrystallized with small molecule andcyclic peptide antagonists, aiding in identification of theligand binding site, which seems to differ significantlyfrom other GPCRs. The small molecule isothioureaderivative (IT1t) was cocrystallized and found to contacthelices 1, 2, 3, and 7. The authors suggest that thebinding of this molecule mimics the binding of thechemokine N terminus, which is believed to protrudeinto the helical bundle of the receptor in the two-stepmodel of chemokine receptor activation. Further-more, CXCR4 was found to consistently exist as ahomodimer, with the key regions for homodimeriza-tion identified as transmembrane helices 5 and 6.These data support multiple studies in transfectedcell systems showing that chemokine receptors canhomo and heterodimerize with various consequences(Rodriguez-Frade et al., 1999; Mellado et al., 2001;El-Asmar et al., 2005).

D. Chronic Inflammation

Acute inflammation is the normal response ofvascularized tissues to injury, irritation, and infection.Tissue injury or the activation of tissue residentmacrophages and mast cells causes the release ofvasoactive substances that activate endothelial cellsin nearby venules, leading to the elaboration of aninflammatory exudate comprised of cells (initiallyneutrophils) and plasma proteins (including comple-ment, antibodies, and serum albumin). These localchanges in the properties of vascular endothelium leadto the cardinal features of inflammation: heat, redness,swelling, and pain. Acute inflammation is an essentialphysiologic response to injury and infection thatrapidly recruits the cells and molecules of the innateimmune response to potential sites of microbial in-fection (Majno and Joris, 1996). The initial wave ofneutrophil recruitment is followed by recruitment ofmonocytes, which differentiate into macrophagesthat orchestrate the process of tissue repair. Chronicinflammation is a harmful process that can occurthrough failure to resolve acute inflammation orthrough persistence of an inflammatory stimulus(Nathan and Ding, 2010). However, many prevalentinflammatory diseases, for example, atherosclerosis,show no progression from acute to chronic but insteadhave the hallmarks of chronic inflammation (suchas monocyte influx and macrophage differentiation)from the outset. Other chronic inflammatory diseases,such as rheumatoid arthritis (RA), show perpetual

Fig. 2. Schematic representation of the key functional regions ofchemokine receptors. Mutagenesis and chimeric receptor studies haveidentified regions of chemokine receptors important for ligand binding,receptor activation, and internalization, although specific sequencesinvolved in signaling differ between different CC chemokine receptors.


recruitment of cells characterizing both acute andchronic inflammatory responses.In this review we have chosen to focus specifically on

the function of CC chemokines in chronic inflamma-tion. CC chemokines are known to have a key role inthe recruitment of monocytes and macrophages, celltypes crucial to the development of atherosclerosis, RA,and adipose inflammation. Although the CXC family ofchemokines have essential roles in neutrophil, B-, andT-cell recruitment, they are outside the scope of thisreview, and we refer the reader to some excellentreviews on the biology of CXC chemokines and theirrole in various pathologies (Romagnani et al., 2004;Weathington et al., 2005; Bizzarri et al., 2006; Strieteret al., 2007).

II. CC Chemokine Receptors

A. CCR1

CCR1 was cloned in 1993, several years after theidentification of two of its ligands, CCL5 and CCL3(Schall et al., 1988; Sherry et al., 1988; Gao et al.,1993). Both CCL5 and CCL3, but not CCL2, werefound to induce calcium flux in human neutrophilswith an IC50 of 5 and 50 nM, respectively, suggestingthe expression of a CC chemokine receptor in neutro-phils (Gao et al., 1993). During cloning of the firstchemokine receptor, IL-8R B (CXCR2), four relatedcDNAs were identified in the HL-60 promyelocyticleukemia cell line (Murphy and Tiffany, 1991). Com-plementary RNA corresponding to the longest of thesecDNAs was injected into Xenopus oocytes, which werethen challenged in a calcium flux assay with variouschemokines (Gao et al., 1993). The oocytes were foundto respond to CCL3 and CCL5, but not CCL2 or any ofthe CXC chemokines tested. A second group used adegenerate PCR-based approach to clone the receptor,which was then expressed in human embryonic kidney(HEK) 293 cells and found to bind CCL1 (I-309) andCCL5 with high affinity and respond in a calcium fluxassay to these chemokines (Neote et al., 1993b). Furtherstudy demonstrated that receptor mRNA was expressedin human B lymphocytes and in the promyelocytic celllines THP-1 and U937 and in differentiated HL-60 cells,which display a more neutrophil-like phenotype. Fi-nally, the receptor was mapped to chromosomal location3p21. Once a specific antibody against CCR1 had beenraised, a more detailed expression analysis was per-formed that showed that T cells, NK cells, monocytes,and CD341 bone marrow progenitor cells expressedCCR1 on the cell surface, whereas no expression couldbe detected on B cells or granulocytes (Su et al., 1996).CCR1 was found to be expressed preferentially onCD45RO1 memory T cells. The murine CCR1 homologwas cloned a few years later by two independent groupson the basis of homology with the human receptor (Gaoand Murphy, 1995; Post et al., 1995).

By using GTPgS exchange, calcium flux and chemo-taxis assays in HL-60 cells the CCR1 ligands wereplaced in the following rank order of potency CCL3 .CCL23 (MPIF-1) . CCL5 $ CCL4 (Chou et al., 2002).Furthermore, CCL4 was found to antagonize responsesto the other ligands, suggesting it could be an en-dogenous inhibitor of CCR1 activity. Other chemo-kines, e.g., CCL15 (HCC2), have also been shown to beweak CCR1 ligands. CCL15 is an unusual chemokinewith an extended N terminus of 16–20 amino acids,similar to CCL23 and two murine chemokines withno direct human homolog, CCL6 (C10) and CCL9(MIP-1g) (described in Berahovich et al., 2005). An im-portant finding was that these ligands can be pro-teolytically cleaved to truncate the N terminus andgenerate agonists with increased binding to CCR1 andapproximately 1000-fold higher potency comparedwith intact forms (Berahovich et al., 2005). This couldbe mediated by inflammatory proteases, supernatantsfrom human cells, and physiologic fluids. Furthermore,CCL15 and CCL23 truncated at the N terminus butnot CCL3 or CCL5 were detected in synovial fluid fromRA patients. In contrast, truncation of two amino acidsfrom the N terminus of CCL5 makes the chemokineinactive at CCR1 (Struyf et al., 1998a).

A characterization of CCR1 signaling in humanmonocytes demonstrated that receptor activation byCCL23 led to phospholipase C (PLC) activation andintracellular calcium release as well as some calciumentry from the extracellular medium (Nardelli et al.,1999). CCL23 was not found to alter cAMP levels inthe cell, but did activate phospholipase A2 (PLA2) inmonocytes, leading to arachidonic acid release. In-hibition of PLA2 and 5-, 12-, and 15-lipoxygenasecompletely blocked F-actin polymerization induced byCCL23. In the monocytic THP-1 cell line, CCL15,CCL23, and CCL24 (eotaxin 2) were found to inducephosphorylation of NFkB (Lee and Wong, 2009). Thisactivation was PTX insensitive, suggesting the involve-ment of G proteins other than Gai in receptor coupling.In transfected cells, CCR1 was found to couple to Ga14/16

to activate this pathway. A more detailed dissection ofthis pathway in THP-1 cells using pharmacologicalinhibitors suggested the involvement of several down-stream pathways including PLCb, protein kinase C,Ca21/calmodulin dependent protein kinase II, Raf-1,MAPK/ERK kinase (MEK1/2), and c-Src.

Mice lacking CCR1 developed normally, showing nohistologic differences in lymphoid organs, peripheralblood counts or any altered susceptibility to spontane-ous infections (Gao et al., 1997). However, spleensfrom knockout mice contained fewer granulocyte-macrophage and multipotential progenitor cells, sug-gesting defective mobilization of these cells from thebone marrow. Upon lipopolysaccharide (LPS) chal-lenge, Ccr1 knockout mice showed normal egress ofprogenitor cells from the bone marrow but these cells

52 White et al.

did not migrate normally to the spleen. Cells found inthe spleen were also in a slow- or noncycling state inCcr1 knockout mice, as opposed to the rapid pro-liferation of these cells in response to LPS seen in wild-type mice. CCR1 was also found to be essential for theresponse of murine neutrophils to CCL3, both inmigration and calcium flux assays. By using anAspergillus fumigatus infection model (intravenousadministration), where neutrophil function is critical,CCR1 knockout mice showed only 30% survival after30 days compared with 60% survival in controlanimals. Mortality was also accelerated, with deathoccurring within 10 days of infection. In a model ofgranuloma formation (Schistosoma mansoni egg in-jection), Ccr1-deficient mice showed reduced granu-loma formation in the lung, suggesting defectivemigration of multiple leukocytes required to formgranulomatous tissue. Further analysis demonstratedthat knockout mice showed a Th1 skewed cytokineprofile in the lung, with enhanced interferon-g (IFN-g)production that inhibits granuloma formation.This excessive Th1 response in Ccr1 knockout mice

was highlighted in another study using a nephrotoxicnephritis model (Topham et al., 1999). CCR1-deficientmice showed increased renal injury, as measured withhistologic and functional parameters, compared withwild-type animals. Ccr1 knockout mice showed en-hanced recruitment of macrophages and T cells intothe kidney, suggesting that CCR1 is not essential forthe migration of these cells. Further analysis showeda Th1 skewed response in these mice with high IgG2atiters, increased IFN-g production by splenocytes andenhanced tumor necrosis factor a (TNFa) and TNFbproduction by mononuclear cells. This suggests thatCCR1 has a role in regulating cell-mediated immuneresponses in mice.CCR1 deficiency was also detrimental in a model of

Leishmania major infection, a pathogen that differen-tially affects various inbred mouse strains: C57Bl6/Jmice (and others) usually develop a Th1 response andcontrol the infection, whereas Th2 polarized Balb/cmice are highly susceptible to infection and producelarge amounts of IL-4. In Ccr1-deficient C57Bl6/J mice,the response to Leishmania was enhanced comparedwith wild-type (WT), smaller lesions were formed, andthe infection was cleared more rapidly (Rodriguez-Sosaet al., 2003). In these experiments, both wild-type andCcr1 knockout mice produced similar levels of Th1cytokines, but Ccr1-deficient mice generated signifi-cantly less IL-4 and IL-10, cytokines that inhibitparasite clearance in this model.Deletion of CCR1 may have other beneficial effects;

for example it reduces airway remodeling in a pulmo-nary A. fumigatus model (Blease et al., 2000), attenu-ates the damaging pathologic response to respiratorysyncitial virus infection (Miller et al., 2006), protectsagainst enteritis induced by Clostridium difficile toxin

in mice (Morteau et al., 2002) and suppresses cardiacallograft rejection (Gao et al., 2000).

Interestingly, CCR1 has recently been shown to havea critical role in bone formation via effects on thedifferentiation and function of osteoblasts and osteo-clasts (Hoshino et al., 2010). Ccr1 knockout animalswere found to have fewer and thinner trabecular bones,and osteoblasts showed defective differentiation. Cul-tured Ccr12/2 bone marrow cells generated fewerosteoclasts due to reduced cell fusion, and these cellsshowed no osteolytic activity.

Thus, CCR1 has differential effects depending on thepathologic context: it has a critical role in controllingcell-mediated immunity to enable pathogen clearance,but may also generate detrimental pathophysiologicalresponses, e.g., in airway remodeling. Studies analyz-ing the phenotype of mice deficient for CCR1 and theother CC chemokine receptors discussed in this revieware presented in Table 1.

A recently published abstract has identified anassociation between a single nucleotide polymorphism(SNP) ;38 kb from the 39-untranslated region (UTR) ofthe CCR1 gene and risk of Behcet’s disease, a complexform of systemic vasculitis characterized by recurrentinflammatory attacks throughout the body (Kirinoet al., 2011). This SNP was reported to be in a potentialregulatory region of the gene, and the protective minorallele was found to be associated with increased CCR1expression.

B. CCR2

The high-affinity ligand for CCR2-CCL2 had beendescribed several years earlier as a potent monocytechemoattractant that was purified from the culturesupernatant of a glioma cell line (Yoshimura et al.,1989a) and cloned from a HL-60 cDNA library(Furutani et al., 1989). In 1994, CCR2 was cloned fromthe Monomac 6 monocytic cell line using a PCR-basedstrategy with degenerate primers based on conservedregions of other chemokine receptors (Charo et al.,1994). This identified a novel PCR product that wasthen used to screen a cDNA library and identify twoclones with predicted transmembrane segments. Thesetwo clones differed only in the C terminus and 39-UTRof the receptor and were designated as splice variantsof the CCR2 receptor and named CCR2-A and -B.cRNA of both variants was microinjected into Xenopusoocytes and generated a robust calcium flux in responseto CCL2. The murine CCR2 receptor was initiallyidentified by screening cell lines for a response to themurine CCL2 homolog [also known as JE (Boring et al.,1996)]. WEHI 274.1 cells were found to respond to CCL2by calcium flux assay; a cDNA library was constructedand a full length CCR2 receptor was cloned.

CCR2 is expressed by multiple cell types includingmonocytes, dendritic cells (DCs), and endothelial cells(Charo et al., 1994; Sozzani et al., 1997; Weber et al.,


TABLE 1Summary of chemokine receptor knockout phenotypes

Gene Mouse Strain NormalGrowth? In Vivo Model Effect of Receptor Deletion Reference

CCR1 C57Bl/6 Yes Analysis of BM and spleen myeloidprogenitor cell development

.

Defective migration of progenitorsto spleen, reduced cell cycling.

.

.

Gao et al., 1997

C57Bl/6 Yes A. fumigatus i.v. infection Reduced survival, acceleratedmortality

C57Bl/6 Yes Granuloma formation (S. mansoniegg injection)

Reduced granuloma formation,excessive Th1 response

Sv129 x C57Bl/6 Yes Nephrotoxic nephritis model Increased renal injury,excessive Th1 response.

Topham et al., 1999

Sv129 x C57Bl/6 Yes Leishmania infection Less Th2 cytokine production,failure of parasite clearance.

Rodriguez-Sosaet al., 2003

Balb/c (10 gen) Yes Pulmonary A. fumigatus infection Reduced airway remodeling. Blease et al., 2000Sv129 x C57Bl/6 Yes RSV infection Reduced pathologic response

and tissue damage.Miller et al., 2006

Sv129 x C57Bl/6 Yes Clostridium difficile toxin injection Protective against enteritis. Morteau et al., 2002Sv129 x C57Bl/6 Yes Cardiac allograft Reduced graft rejection. Gao et al., 2000;

Hoshino et al., 2010C57Bl/6 Yes Analysis of bone formation Fewer trabecular bones, impaired.

osteoblast differentiation,defective osteoclastogenesis.

CCR2 Sv129 x C57Bl/6 Yes Thioglycollate peritonitis Reduced macrophage recruitment; Boring et al., 1997Sv129 x C57Bl/6 Yes Granulomatous disease PPD

of M. bovisReduced DTH and Th1 responses

Sv129 x C57Bl/6 Yes Analysis of BM and spleen myeloidprogenitor cell development

Enhanced progenitor cell cyclingand increased apoptosis.

Reid et al., 1999

Sv129 x C57Bl/6 Yes MOG-induced EAE Pathology abrogated. Fife et al., 2000Sv129 x ICR Yes L. monocytogenes infection Failed bacterial clearance Kurihara et al., 1997C57Bl/6 DSS colitis Protective. Andres et al., 2000Sv129 x C57Bl/6 Yes Inflammatory pain model

(formalin and CFA)Reduction of inflammatory

pain responseAbbadie et al., 2003

Sv129 x C57Bl/6 Yes Neuropathic pain (sciaticnerve ligation)

Abrogation of neuropathicpain response

C57Bl/6 West Nile Virus infection . Absence of monocytosis,increased lethality of virus.

Lim et al., 2011

CCR3 Balb/c Yes Analysis of eosinophil trafficking Defective trafficking tointestinal mucosa

Humbles et al., 2002

Balb/c Yes OVA-induced skin inflammation Defective eosinophil recruitment. Ma et al., 2002Sv129 x C57Bl/6 Yes OVA-induced asthma model Defective eosinophil recruitment. Pope et al., 2005

CCR4 C57Bl/6 (4 gen) Yes OVA-induced airwayinflammation;lethal endotoxemia

No effect; survival Chvatchko et al., 2000

C57Bl/6 Yes Intraperitoneal Escherichiacoli infection

Reduced bacterial numbers,enhanced PMN andmonocyte recruitment

Ness et al., 2006

C57Bl/6 Yes Polymicrobial sepsis model Decreased mortality, enhancedbacterial clearance from organs.

Traeger et al., 2008

C57Bl/6 Yes Dengue virus infection Reduced lethalityand tissue damage.

Guabiraba et al., 2010

C57Bl/6 Yes A. fumigatus-inducedinflammation

Reduced eosinophil recruitmentand airway hyperresponsiveness.

Schuh et al., 2002b

CCR5 Sv129 x ICR Yes L. monocytogenes infection Reduced bacterial clearance Zhou et al., 1998Sv129 x ICR Yes Lethal endotoxemia SurvivalSv129 x ICR Yes DTH (FITC) model Increased DTH reactionSv129 x ICR Yes T-cell-dependent antigen

challengeIncreased humoral response

C57Bl/6 (8 gen) Yes MOG-induced EAE. No effect Tran et al., 2000Sv129 x C57Bl/6 Yes DSS colitis Protective Andres et al., 2000Sv129 x C57Bl/6 Yes Chronic fungal asthma. Protective—disease transient Schuh et al., 2002aSv129 x C57Bl/6 Yes Lymphocytic choriomeningitis

virus infection model.No effect Nansen et al., 2002

Sv129 x C57Bl/6 Yes Cryptococcus neoformans infection Reduced survival, defectiveleukocyte recruitment to brain

Huffnagle et al., 1999

Sv129 x C57Bl/6 Yes Graft versus host disease Reduced survival, defectiveTreg recruitment

Wysocki et al., 2005

C57Bl/6 Yes Paracoccidioides brasiliensisinfection.

Smaller granulomas, better controlof fungal growth, reducedTreg recruitment

Moreira et al., 2008

CCR6 Sv129 x C57Bl/6 Yes Cellular composition of MALT Reduced DC numbers, alteredT-cell populations in mucosa

Cook et al., 2000

Sv129 x C57Bl/6 Yes Oral antigen administration (KLH) Reduced humoral response(fewer antibody-producingcells in mucosa)

(continued )

54 White et al.

1999). Indeed, CCR2 is now known to be differentiallyexpressed on the known subsets of both mouse andhuman monocytes (Geissmann et al., 2003). Aside fromCCL2, CCR2 has several other high-affinity ligandsincluding MCP-2 (CCL7), MCP-3 (CCL8), MCP-4(CCL13), and MCP-5 (CCL12)—a murine chemokinewith close homology to human CCL2 (Combadiereet al., 1995; Berkhout et al., 1997; Gong et al., 1997b;Sarafi et al., 1997). However, CCL7, CCL8, and CCL13all bind other chemokine receptors, whereas CCL2 andCCL12 signal exclusively through CCR2. The rankorder of potency of CCR2 ligands at the human receptoris reported to be CCL2 . .CCL13 5 CCL8 . CCL7.Numerous studies have sought to determine the

intracellular signaling induced by CCL2 acting onCCR2. In stably transfected HEK-293 cells, CCR2activation induced calcium release from intracellularstores and inhibited adenylate cyclase with highpotency (90 pM), and both responses were blocked by

PTX, suggesting coupling to Gai (Myers et al., 1995).CCL2 was also shown to induce ERK 1/2 (p42/p44MAPK) activation via a PTX-sensitive mechanism ina T-cell hybridoma (Dubois et al., 1996). Followingreceptor activation in transfected HEK-293 cells it wasshown that the receptor is rapidly phosphorylated(within 1 minute) and internalized (Franci et al., 1996).When the receptor was coexpressed in Xenopus oocyteswith various members of the b-adrenergic receptorkinase (bark) family, expression of the bark2 isoformcompletely blocked CCR2 activation, suggesting thatphosphorylation by this kinase induces receptor de-activation, a mechanism now known to be common tomany GPCRs. By using site-directed mutagenesis,several serine and threonine residues in the C terminusof the receptor were shown to be essential to receptorinternalization and deactivation. A crucial discoverycame when it was shown that Gbg heterodimers releasedafter activation of CCR2 were essential for chemotaxis of

TABLE 1—Continued

Gene Mouse Strain NormalGrowth? In Vivo Model Effect of Receptor Deletion Reference

Sv129 x C57Bl/6 Yes Rotavirus infection Reduced IgA response andviral clearance

Sv129 x C57Bl/6 Yes Contact hypersensitivity. More severe and persistentinflammation

Varona et al., 2001

Sv129 x C57Bl/6 Yes DTH No inflammatory responseC57Bl/6 (8 gen) Yes DSS colitis Protective Varona et al., 2003C57Bl/6 (8 gen) Yes TNBS colitis in C57BL/6J mice Confers susceptibility—- mice

usually resistant.C57Bl/6 (8 gen) Yes Cockroach antigen pulmonary

inflammationReduced airway resistance,

eosinophil. recruitment and IL-5production

Lukacs et al., 2001

C57Bl/6 (8 gen) Yes MOG-induced EAE Reduced disease score Liston et al., 2009CCR7 CD1 x Balb/c LNs

smallerAnalysis of cellular composition ofblood and lymphoid organs

Increased Th cells in blood, bonemarrow, and spleen; ReducedTh cells in LN and morphologicalterations in LN

Forster et al., 1999

CD1 x Balb/c Spleensenlarged

Contact and delayed-typehypersensitivity

No response to antigen challenge

C57Bl/6 and Balb/c Analysis of lymphocytedevelopment

Th2 polarization, increasedIL-4 in LN, B cell activation

Moschovakiset al., 2012

CCR8 Sv129 x C57Bl/6 Yes Granuloma formation (S. mansoniegg injection).

Impaired Th2 response, reducedeosinophil recruitment

Chensue et al., 2001

Sv129 x C57Bl/6 Yes Cockroach antigen pulmonaryinflammation

Impaired Th2 response, reducedeosinophil recruitment

Sv129 x C57Bl/6 Yes PPD of M. bovis injection No effect, Th1 response normalC57Bl/6 (10 gen) Yes Analysis of monocyte trafficking

(latex bead labeling)Defective monocyte-derived

DC trafficking to LNQu et al., 2004

C57Bl/6 Yes Chronic fungal asthma Protective, enhanced clearanceof fungi

Buckland et al., 2007

C57Bl/6 Yes Atopic dermatitis Less eosinophilic inflammation,defective recruitment of Th2cells to inflamed skin

Islam et al., 2011

CCR9 C57Bl/6 Yes Analysis of lymphocytedevelopment

No major effect on T orB cell development

Wurbel et al., 2001

C57Bl/6 Yes Analysis of plasma cell localization Defective recruitment ofIgA-secreting cells intosmall intestine

Pabst et al., 2004

C57Bl/6 Yes Oral antigen administration Defective IgA responseSv129 x C57Bl/6 Yes Rotavirus infection Normal recruitment of IgA1

plasmablasts to small intestineFeng et al., 2006

Sv129 x C57Bl/6 Yes ConA-induced hepatitis Protected from hepatitis unlessCCR91 macrophages present

Nakamoto et al., 2012

CCR10 Balb/c (7 gen) Yes Analysis of IgA-secretingcells to mucosa

Defective recruitment ofIgA-secreting cells tomammary gland

Morteau et al., 2008

C57Bl/6 (8 gen) Yes Analysis of gd T-cell development. Defective migration of skinintraepithelial Vd31 lymphocytes.

Jin et al., 2010b


a transfected lymphocyte cell line [300.19 (Arai et al.,1997)]. This was achieved by coexpression of theGatransducin (Gat) protein that binds free Gbg hetero-dimers with high affinity, preventing their interactionwith downstream substrates. In contrast, Gat expressiondid not affect ERK phosphorylation in response to CCL2and only had a small effect on calcium flux, suggestingthat this is not mediated via the Gbg subunit.CCR2-deficient mice were generated in 1997 and

found to be developmentally normal (Boring et al.,1997). The absence of CCR2 impaired recruitment ofleukocytes (mainly macrophages) into the peritonealcavity of mice injected with the inflammatory stimulusthioglycollate. To determine the role of CCR2 inimmunity, CCR2 knockout mice were injected withbeads coated with purified protein derivative (PPD)antigen from Mycobacterium bovis, which inducesgranuloma formation in the lung and is associatedwith a Th1 cytokine profile in the draining lymph node.Ccr2 knockout mice had significantly smaller granulo-mas containing fewer macrophages in response to PPDchallenge. Associated with this, Ccr2-deficient cellsisolated from draining lymph nodes produced less IFN-g and virtually no detectable IL-12 in response to PPD.In other experiments, splenocytes from Ccr2 knockoutmice were stimulated with concanavalin A (con A) andfound to produce significantly less IFN-g than wild-type cells, suggesting defective antigen-induced cyto-kine responses in these animals. Ccr2-deficient micewere generated in a second laboratory, and theseanimals failed to clear an infection by the intracellularbacteria Listeria monocytogenes, suggesting a directrole for CCR2 in responses to bacterial pathogens(Kurihara et al., 1997).CCR2 also has a role in myeloid progenitor cell

cycling in mice. Mice deficient for Ccr2 were found tohave a dramatic increase in the number of myeloidprogenitor cells (MPCs) in S phase, indicating increasedproliferation of these cells (Reid et al., 1999). However,the absolute number of MPCs in bone marrow or spleenwas unaffected in knockout mice. This discrepancy wasshown to be due to increased apoptosis of c-kit1 lin2

immature cells in the bone marrow, suggesting animportant role for this receptor in MPC survival.CCR2 has been shown to have an essential role in

the induction of experimental autoimmune encephalo-myelitis (EAE), a mouse model for multiple sclerosis(Fife et al., 2000). The absence of CCR2 completelyprevented the development of EAE pathology, andknockout mice showed reduced infiltration of bothmonocytes and T cells into the central nervous system.T cells from these mice showed similar antigen-inducedcytokine production to wild-type controls, and adoptivetransfer of wild-type or Ccr22/2 T cells into knockoutrecipients failed to induce the onset of EAE, indicatingthat CCR2 expression on host cells is required fordisease induction.

CCR2 also has an important role in the developmentof inflammatory and neuropathic pain (Abbadie et al.,2003). Knockout mice showed a 70% reduction in painbehavior in an inflammatory pain model (intraplantarformalin injection). Intraplantar administration of theligand CCL2 into wild-type mice also induced a greaterdegree of mechanical allodynia, a pain response toa stimulus that is not usually painful. By using a modelof chronic pain, sciatic nerve injury, Ccr2 knockoutmice did not develop the mechanical allodynia seen inwild-type animals. This was associated with reducedinfiltration of monocytes/macrophages into the sciaticnerve and dorsal root ganglion and less activation ofmicroglia in the spinal cord.

In 2007, CCR2 was shown to play a critical role in bothbone marrow egress of classic monocytes and in mono-cyte trafficking from the blood to sites of inflammation(Tsou et al., 2007). The key murine ligands required forthis process were identified as CCL2 and CCL7.

CCR2 is also important for survival in a murinemodel of West Nile Virus (Lim et al., 2011). West NileVirus induces a large degree of monocytosis, and thesecells are recruited into the brain, where they limit viralreplication. In Ccr22/2 animals, this monocytosis isabolished and infected animals show reduced survivalassociated with diminished accumulation of classicmonocytes in the brain. This is not due to defectiverecruitment to the brain because transfer of WT andCcr22/2 monocytes leads to similar accumulation inthe brain of infected animals. The authors did notspecifically analyze whether failed monocytosis in Ccr2-deficient animals was due to reduced bone marrowegress (as described above) or altered survival in theperiphery or possibly defective mobilization froma splenic reservoir, a process that does not requireCCR2 after myocardial infarction but that has not beenextensively tested in other models (Swirski et al., 2009).

Taken together, these data suggest that CCR2 hasa key function in cellular homeostasis, particularly formonocytes, while also playing a critical role in thedevelopment of inflammatory responses in response toa wide range of insults.

A nonsynonymous single nucleotide polymorphism(V64I) in the first transmembrane domain of CCR2 hasbeen identified and found to be associated with a fasterprogression to acquired immunodeficiency syndrome(AIDS) in human immunodeficiency virus (HIV)-positiveindividuals, although it has no relationship with in-cidence of HIV infection (Smith et al., 1997). Thismutation has not been studied extensively in inflam-matory disease, but a few publications have assessedthe association of this SNP with pathology. No associ-ation was found with the incidence or severity of RA(Bayley et al., 2003) or with systemic lupus erythema-tosus [SLE (Aguilar et al., 2003)], and similarly themutation did not correlate with the incidence of lupusnephritis, although the V allele was associated with

56 White et al.

a less severe disease phenotype as measured by theSLEDAI (SLE disease activity) index (Malafronte et al.,2010). Interestingly, carriers of the I allele withpsoriasis were found to be more likely to progress topsoriatic arthritis once the disease was established, butCCR2 genotype did not correlate with the incidence ofpsoriasis per se (Soto-Sanchez et al., 2010). Finally, the Iallele was associated with abdominal aortic aneurysm(Katrancioglu et al., 2011). In summary, these datasuggest that the CCR2 V64I mutation may lead tofaster disease progression once pathology is establishedbut does not seem to be a strong risk factor for theincidence of inflammatory disease.

C. CCR3

CCR3 was first described by two independent groups(Daugherty et al., 1996; Ponath et al., 1996) andinitially referred to as CC CKR3. The receptor, a pro-tein of 355 amino acids in length was isolated andcloned from human eosinophils on the basis ofsimilarity to other chemokine receptors and found toshare 63% sequence homology with CCR1 and 51%with CCR2. In contrast to other members, CCR3 wasshown to lack sites for N-linked glycosylation (Ponathet al., 1996). The N-terminal region of CCR3 was alsoshown to contain a leucine residue instead of proline inthe proline-cysteine motif conserved in the N terminusof other chemokine receptors.CCR3 is expressed upon a variety of immune cells,

including eosinophils (Fujisawa et al., 2000; Badewaet al., 2002), Th2 lymphocytes (Gerber et al., 1997),mast cells (Brightling et al., 2005; Uguccioni et al.,1997), and basophils (Daugherty et al., 1996). Withrespect to eosinophils, basophils, and Th2 lymphocytes,CCR3 expression is restricted to the cell surface(Uguccioni et al., 1997). In stark contrast, CCR3 inmast cells is stored in intracellular granules that aremobilized and recruited to the cell surface following Fcreceptor engagement/cross-linking with IgE (Priceet al., 2003). The murine homolog of CCR3 was alsocloned in 1996 and found to be expressed on eosino-phils and mediate chemotaxis toward eotaxin (CCL11)(Gao et al., 1996).CCR3 binds to a wide range of ligands, which can all

lead to the activation of the receptor. Examples includeCCL5, CCL2, CCL7, CCL11, CCL13, CCL15, CCL24(eotaxin-2), and CCL26 (eotaxin-3) (Daugherty et al.,1996). Evidence from competitive binding studies hasshown that CCR3 displays maximal affinity for CCL11with a Kd of 0.1 nM. CCL5 and CCL7 were shown tobind at slightly lower affinities with Kd values between2.7 and 3 nM (Daugherty et al., 1996). Similar trendswere observed in functional assays, including intracellu-lar calcium mobilization, with ED50 in the subnanomolarrange for all three ligands aforementioned. Chemotaxisassays performed with both primary eosinophils andcells stably transfected with CCR3 revealed CCL11 to

possess the most potent migratory effect followed byCCL5 and CCL7, respectively (Daugherty et al., 1996;Ponath et al., 1996).

A study performed by Kitaura et al. (1999) identifiedCCL26 as a novel functional ligand for CCR3. L1.2 cells(a murine pre B cell line) stably transfected with CCR3were shown to generate a calcium flux in response toCCL26 with an EC50 of 3 nM. The authors also reportedcrossdesensitization between CCL26 and other knownCCR3 ligands with the rank order of potency reportedas: CCL11, CCL13 . CCL26 . CCL24, CCL5. Fur-thermore, in chemotaxis assays CCL11 induced a typ-ical bell-shaped dose-dependent response with maximaleosinophil migration observed at 100 nM in contrast toCCL26, which only had an effect on migration at 1 mM.This trend was also observed with basophils, withCCL11 inducing migration between 30 and 300 nM, incontrast to CCL26 where chemotaxis was only observedat 300 nM (Kitaura et al., 1999).

A non-CC chemokine ligand, the HIV Tat protein,has also been shown to bind and activate CCR3on monocytes and macrophages. Albini et al. (1998)generated a synthetic Tat protein and peptide(CysL24-51) that contained the “chemokine-like” regionof Tat. Both protein and peptide were shown to inducePTX-sensitive calcium fluxes in monocytes. Receptordesensitization and direct/displacement binding assayswere performed with CCR2 and CCR3 transfectedCHO-K1 cells. The authors reported both Tat and theCysL24-51 peptide could partially desensitize the re-sponse to CCL2, CCL7 and completely block theresponse to CCL11 in human monocytes. Tat wasshown to specifically displace CCL7 from membranesof CCR3 transfected cells. The peptide CysL24-51 wasalso shown to specifically bind both CCR2 and CCR3transfected CHO-K1 cells with Kd of 66.4 6 8.4 nM forCCR3 binding. The results from this study suggest thatthe HIV Tat protein can mimic the effects of b-chemo-kines, which may have a role in the recruitment ofmonocytes/macrophages to sites of HIV producing cells,enhancing the spread of infection within the host.

CCL11 binding to CCR3 has been shown to activatesignaling via the mitogen activated protein kinase(MAPK) signaling pathway in eosinophils. Treatmentof human eosinophils with CCL11 (10–100 nM) acrossa time course was shown to induce a rapid phosphor-ylation and activation of ERK1/2. Pretreatment ofeosinophils with a MEK inhibitor (PD980549) reducedkinase activity following stimulation with 10 nMCCL11, highlighting that the phosphorylation andactivation of ERK 1/2 occur via a MEK-dependentsignaling pathway. Furthermore pretreatment withthe MEK inhibitor resulted in a substantial reductionof both CCL11-induced eosinophil rolling in the mousemesenteric circulation and on CCL11-induced chemo-taxis of eosinophils (Boehme et al., 1999). Similarobservations were made by another independent group


who reported dose-dependent activation of ERK2 andp38 following stimulation with CCL11. In the presenceof specific inhibitors against both ERK2 and p38,CCL11-induced eosinophil cationic protein releaseand chemotaxis were inhibited (Kampen et al., 2000).Collectively these studies highlight the role of MAPKactivation in regulating CCL11-induced eosinophilrolling, degranulation and migration via CCR3.Given that CCR3 is highly expressed upon eosino-

phils and basophils it is not surprising that thereceptor has been highlighted as a leading therapeutictarget in diseases that have a strong allergic in-flammatory component. The generation of mice lackingCCR3 was first described in 2002 (Humbles et al.,2002). Ccr32/2 mice display normal development withno impairment during and after gestation (Humbleset al., 2002). Under basal conditions Ccr32/2 micedisplay impaired eosinophil trafficking to the intestinalmucosa with reduced cell numbers observed comparedwith littermate controls, highlighting the role of thereceptor in regulating eosinophil trafficking underhomeostatic conditions. In a model of airway hyper-responsiveness, Ccr32/2 mice showed defective eosin-ophil recruitment, with the majority of cells constrictedwithin the subendothelial space unable to migrate outinto the lung parenchyma. Interestingly the authorsalso reported accumulation of mast cells in the airwaysof Ccr32/2 mice, unveiling a novel role for the receptorin mast cell homing. Similar observations have beenreported by several other groups of defective eosinophilrecruitment in ovalbumin (OVA)-induced skin inflam-mation (Ma et al., 2002) and an OVA-induced exper-imental asthma model (Pope et al., 2005). Morerecently, an in vivo study examined the role ofepithelial CCR3 in an LPS-induced model of lunginflammation (Li et al., 2011). A specific CCR3 in-hibitor [SB-328437, methyl (2S)-2-(naphthalene-1-car-bonylamino)-3-(4-nitrophenyl)propanoate] at 5 mg/kgwas given via intratracheal instillation to mice withLPS-induced acute lung injury. The inhibitor wasshown to reduce neutrophil recruitment into thealveolar space and attenuate IL-8 production inbronchoalveolar lavage (BAL) fluid of these mice whencompared with LPS treatment alone. Improved histo-logic scores were also observed in mice treated withSB-328437 inhibitor. Collectively these findingsrevealed for the first time a potential role for epithelialexpressed CCR3 in promoting LPS-induced lung in-flammation through mediating the release of IL-8 (Liet al., 2011).Interestingly, a nonsynonymous SNP of CCR3

(L324P) in the C terminus of the receptor was foundto ablate transport of the receptor to the cell surfacewhen expressed in transfected cells (Wise et al., 2010).The receptor was found to be expressed intracellularly,and these cells were unable to migrate toward CCL11.However, this polymorphism is found at very low

frequency in the population, and thus the significanceof this finding remains unclear.

D. CCR4

CCR4 was cloned from a basophilic cell line in 1995using an RT-PCR strategy with degenerate primersbased on conserved regions in the CXCR2 and CCR1receptors (Power et al., 1995). The murine homologwas subsequently cloned the following year (Hoogewerfet al., 1996). The human receptor mRNA was highlyexpressed in thymus and peripheral blood leukocytesand at a low level in spleen (Power et al., 1995). CCR4cRNA was transiently expressed in Xenopus oocytes,and various chemokines were assayed for their abilityto induce calcium flux and opening of a voltage-gatedcalcium channel using a patch clamp assay. CCL3,CCL2, and CCL5 were found to induce a signal inCCR4-transfected cells but very high doses were used(1 mM).

Subsequently, TARC (CCL17) and CCL22 havebeen identified as the high-affinity ligands of CCR4,which were not displaced by other CC chemokines ina competitive binding assay (Imai et al., 1997, 1998).Imai et al. (1997) confirmed that CCR4 was expressedin T cells (mainly CD41), but was undetectable in anyother leukocytes. CCR4 was subsequently shown tobe expressed by Th2-polarized lymphocytes (Bonecchiet al., 1998a).

Although intracellular signaling pathways down-stream of CCR4 have not been as extensively studiedas for other chemokine receptors, two papers haveexplored CCR4 signaling in a T-cell line and in vitrodifferentiated Th2 cells. In the human leukemic cellline CEM, CCL22 was found to induce a rapid andtransient accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) as well as a slower phosphoryla-tion of protein kinase B (PKB/Akt) (Cronshaw et al.,2004). This effect was blocked by PI3K inhibitors,although these had no effect on chemotaxis in responseto CCL22, indicating that PI3K activation is notrequired for CCR4-dependent migration of these cells.In contrast, a ROCK (Rho-associated coiled coil-forming protein kinase) inhibitor did block CCL22-induced chemotaxis in CEM cells. The induction ofPKB phosphorylation by CCL17 and CCL22 wasreplicated in Th2 cells differentiated in vitro fromprimary human peripheral blood leukocytes (PBLs), aswas the inability of PI3K inhibitors to block CCR4-mediated chemotaxis.

In a subsequent study using CEM cells, both CCL17and CCL22 were found to induce calcium flux ina PLC-dependent manner (Cronshaw et al., 2006).Calcium release was found to be dependent on inositoltrisphosphate (IP3)-sensitive store operation. However,CCL22-induced chemotaxis required PLC activationbut not IP3-mediated calcium release. An inhibitoragainst the novel protein kinase C (PKC) isoforms that

58 White et al.

are activated by diacylglycerol (DAG) blocked chemo-taxis induced by CCL22. Furthermore, CCL22 wasfound to induce phosphorylation of PKC d. Inhibitors ofPKC and PLC were also confirmed to block Th2 cellmigration toward CCR4 ligands.Ccr42/2 mice were developmentally normal, and

splenocytes from these mice were shown to be un-responsive to the high-affinity ligands CCL17 andCCL22 and also to CCL3 (Chvatchko et al., 2000).Thus, although CCL3 is thought to be a low affinityligand at CCR4, loss of the CCR4 receptor abrogatesthe functional response of splenocytes to CCL3. In vivo,deletion of CCR4 was found to have no effect ongeneration of a Th2 immune response in an OVA-induced airway inflammation model (Chvatchko et al.,2000). However, loss of CCR4 was found to protect micein a lethal endotoxemia model, with a concomitantdecrease in peritoneal macrophage numbers andplasma TNFa and IL-1b levels in response to LPS(Chvatchko et al., 2000). This initial observation wasconfirmed and extended by Ness et al. (2006) whoanalyzed the responses of CCR4-deficient mice back-crossed for 10 generations to C57BL/6J challengedwith a range of bacterial Toll-like receptor (TLR)ligands and defense against intraperitoneal infectionwith Escherichia coli. The authors showed thatdecreased numbers of viable bacteria in Ccr42/2

animals were associated with enhanced recruitmentof CD11b1 neutrophils and monocytes into theperitoneal cavity at early time points following in-oculation (Ness et al., 2006). Intriguingly, detailedcharacterization of macrophages from Ccr42/2 ani-mals showed an alteration in gene expression patternskewed toward that seen in alternatively activated(M2) macrophages.Traeger et al. (2008) further confirmed the impor-

tance of CCR4 in host defense against bacterialinfection by studying the effect of CCR4 gene deletionin a model of polymicrobial sepsis following abdominalsurgery. Ccr42/2 mice showed decreased mortality andgreatly enhanced clearance of bacteria from majororgans (lung, liver, kidney, spleen) but no change inviable bacteria numbers in the peritoneal cavity orblood (Traeger et al., 2008). The authors speculatedthat CCR4 deficiency protects mice from lethal se-quelae of systemic bacterial infection by reducing therecruitment of anti-inflammatory regulatory T (Treg)cells into sites of bacterial infection.Recently Guabiraba and colleagues (2010) compared

the effects of dengue virus infection in three differentstrains of chemokine receptor deficient mice (Ccr12/2,Ccr22/2, and Ccr42/2 animals). The mouse model ofdengue used shares many features of severe dengueinfection in humans, including liver damage, thrombo-cytopenia, cytokine storm, systemic inflammation, anddeath. Dengue infection of Ccr42/2 animals wascharacterized by reduced lethality and reduced tissue

damage despite a similar viral load to that observed inwild-type and CCR1- and CCR2-deficient animals(Guabiraba et al., 2011). Taken together with thedecreased lethality observed in Ccr42/2 mice inendotoxemia and sepsis models, a picture emerges ofCCR4 as an important chemokine receptor in thesystemic response to overwhelming innate immuneactivation.

The role of CCR4 in allergic inflammation has beenthe subject of extensive investigation. This stems fromoriginal observations that expression of CCL17 andCCL22 is potently upregulated by Th2 cytokines andthe preferential expression of CCR4 on Th2 CD41

T lymphocytes (Bonecchi et al., 1998b; Imai et al.,1999; Greaves et al., 2001). The association betweenCCR4 and allergic inflammation was strengthened byobservations made with clinical material. For instance,Panina-Bordignon et al. (2001) reported upregulationof CCL17 and CCL22 expression in airway epithelialcells of lung biopsy samples from atopic asthmaticpatients immediately following allergen challenge andNakatani et al. (2001) reported elevated numbers ofCCR41 T cells in chronic skin lesions of patients withchronic dermatitis. These studies suggesting the in-volvement of CCR4 in human atopic allergy werereinforced by studies undertaken using animal modelsof allergic inflammation. By using a murine T-celltransfer model of asthmatic airway disease (AAD)Lloyd et al. (2000) demonstrated that T cells polarizedin vitro to Th2 differentiation, but not cells polarized toTh1, could induce airway hyper-responsiveness (AHR)following allergen inhalation in sensitized recipientmice. Transferred Th2 lymphocytes preferentiallyexpressed the CCR3 and CCR4 chemokine receptorsin vitro and in the lung after transfer. By usingvalidated anti-CCL11 and anti-CCL22 polyclonal anti-bodies, the authors showed decreased recruitment ofTh2 donor lymphocytes in the early stages of lungdisease by blocking the activity of the CCR3 ligandCCL11, whereas antibodies against the CCR4 ligandCCL22 blocked the later stages of AAD. Lung in-flammation has been studied in Ccr42/2 mice usinga range of different experimental models. In theoriginal report describing the generation of Ccr42/2

mice Chvatchko et al. (2000) showed no differencebetween wild-type and CCR4-deficient mice in thedevelopment of AHR following OVA sensitization.However, a subsequent report by Schuh et al. (2002b)using the same Ccr42/2 mice in a different model oflung inflammation that used sensitization and sub-sequent challenge with Aspergillus conidia showedreduced eosinophil recruitment and reduced AHR inCCR42/2 mice compared with wild-type animals.

Various explanations for the conflicting results indifferent animal models of allergic inflammation havebeen proposed, but optimism about targeting of CCR4for therapeutic benefit in allergic asthma has come


from more recent studies in a humanized model ofasthma. NOD/SCID (nonobese diabetic/severe com-bined immunodeficient) mice reconstituted with pe-ripheral blood mononuclear cells (PBMCs) from normaland atopic human volunteers underwent bronchialchallenge with house dust mite antigen. Allergicinflammation, goblet cell hyperplasia, and AHR wereonly seen in mice reconstituted with PBMCs fromatopic donors, and this inflammatory response wasabolished by administration of a CCR4 blockingmonoclonal antibody (Perros et al., 2009). Furthervalidation of CCR4 as a useful therapeutic target inhuman asthma has come from Vijayanand et al. (2010)who showed that CCR41 but not CCR4- T cells fromasthmatics are the major source of Th2 cytokines,including CCL17. By using explanted bronchial tissuefrom asthmatic donors the authors went on to showthat a small molecule CCR4 antagonist markedlyreduced Th2 cell chemoattractant activity in culturemedium of such explants (Vijayanand et al., 2010).Most of the foregoing discussion of CCR4 has focused

on the proinflammatory role of CCR41 cell recruitmentin response to upregulation of CCL17 and CCL22expression. However, in common with several other CCchemokine receptors, CCR4 also plays a homeostaticrole in leukocyte trafficking in addition to its welldocumented effects in innate immune cell activationand Th2 immunopathology. In 1999, Campbell et al.(1999) showed that many CCR41 memory T cells inblood coexpressed CCR4 and cutaneous lymphocyteantigen. The authors went on to demonstrate thatthese cells underwent CCL17-triggered tight adhesionto intercellular adhesion molecule 1 (ICAM-1) underconditions of physiologic flow (Campbell et al., 1999).Later experiments confirmed the importance of theCCR4 receptor rather than the CCR10 receptor for skinhoming and recruitment of T cells to sites of skininflammation (Soler et al., 2003). Campbell et al.(2007) elegantly demonstrated the importance ofCCR4 for the development of appropriate skin-homing T lymphocyte populations in competitiverepopulation experiments using equal numbers ofCcr42/2 and Ccr41/1 cells in Rag2/2 (recombinaseactivating gene) mice and in experiments using T-cellreceptor (TCR) transgenic models (Baekkevold et al.,2005).A deeper appreciation of the central role of CCR4 in

the biology of skin-homing T lymphocyte biology shouldnot prevent the exploration and hopefully the exploi-tation of CCR4 as a therapeutic target in diseases suchas asthma where there is clear evidence linking CCL17and CCL22 as major players in disease pathology.One study has examined the potential association

of a CCR4 SNP (C1014T) with atopic dermatitis(Tsunemi et al., 2004). There was no associationbetween CCR4 genotype and disease incidence, bloodIgE levels, or eosinophil counts.

E. CCR5

The CCR5 receptor was described by three indepen-dent groups at around the same time (Combadiereet al., 1996; Raport et al., 1996; Samson et al., 1996a).The receptor was found to be expressed in monocytesand macrophages, the THP-1 monocyte cell line, CD41

and CD81 T cells, but not in eosinophils or neutrophils(Combadiere et al., 1996; Raport et al., 1996). In HEK-293 cells transfected with CCR5, several chemokineswere found to induce calcium flux with the rank orderof potency CCL3 . CCL5 . CCL4 (Combadiere et al.,1996). In CCR5 transfected COS-7 cells, inositolphosphate production was elicited by low nanomolarconcentrations of chemokine and was found to havea different rank order: CCL5 . CCL4 . CCL3 (Raportet al., 1996). By using competitive radioligand bindingassays in transfected HEK-293, all three ligands werefound to have relatively low affinity for CCR5 (IC50

;100 nM); instead both CCL5 and CCL3 were found tobind with higher affinity at CCR1 (Combadiere et al.,1996). In CCR5 transfected COS-7 cells, all threechemokines bound with higher affinity, with competi-tion binding IC50 values all around 7 nM (Raport et al.,1996). The different values reported by different groupsare likely to represent different levels of expression and/or receptor coupling in the various transfected cell linesused. The receptor was mapped to chromosomal location3p21, very close to the CCR2 gene (Raport et al., 1996).The murine homolog of CCR5 was described a fewmonths prior to the identification of the human receptorand was also found to bind CCL3, CCL4, and CCL5(Boring et al., 1996).

Subsequent studies have shown that CCR5 can alsobind other CC chemokines, notably CCL8 and CCL13,although both are less potent than CCL5 in inducingmigration of transfected cells—doses of 100 nM CCL13are required to elicit migration (Ruffing et al., 1998).These data were confirmed by another group who alsoshowed that CCR5 could bind the chemokines CCL7and HCC-1 (CCL14), although the biologic activity ofthe chemokines was not assessed (Napier et al., 2005).CCL11 also has some biologic activity at CCR5,although very high concentrations (1 mM) were neededto induce a response (Ogilvie et al., 2001).

Just before the identification of CCR5, its ligandsCCL5, CCL3, and CCL4 were found to be produced bycytotoxic CD81 T cells and were capable of suppressingHIV infection (Cocchi et al., 1995). Subsequently,CCR5 was found to be the major coreceptor for entryof macrophage-tropic strains of the virus (Alkhatibet al., 1996; Deng et al., 1996) . Indeed, individualshomozygous for a 32-bp deletion in the coding region ofCCR5 (CCR5 D32) are resistant to HIV infection(Samson et al., 1996b).

The regions of the CCR5 receptor important forligand binding and signaling have been extensively

60 White et al.

studied. Samson et al. (1997) generated chimericreceptors with different combinations of transmem-brane and extracellular domains of the CCR2 andCCR5 receptors. By using competitive binding assaysand a microphysiometer to assess biologic responses(via a change in extracellular pH following signaling),the second extracellular loop of the receptor was foundto be the key region determining ligand specificity.However, using a similar approach with chimericreceptors, it was the N-terminal domain and the firstextracellular loop of CCR5 that were found to beimportant in infection with HIV (Rucker et al., 1996).Furthermore, it was shown that binding of the HIVenvelope protein to CCR5 induces a signaling responsethat is not required for entry in vivo but instead mayinduce chemotaxis of T cells or enhance viral replica-tion (Weissman et al., 1997).CCR5, like all chemokine receptors, is Gai coupled,

leading to a reduction in intracellular cAMP levels(Aramori et al., 1997). Following activation, the receptorwas found to be desensitized via phosphorylation byGRKs 2, 3, 5, and 6, leading to b-arrestin recruitmentand receptor internalization (Aramori et al., 1997).Binding of CCL4 to CCR5 was also shown to induceactivation of RAFTK/Pyk2 (a member of the focaladhesion kinase family), leading to activation of theJun N-terminal kinase (JNK) and p38 MAPK path-ways and the cytoskeletal protein paxillin (Ganjuet al., 1998). This provided the first evidence of howchemokine receptors might link ligand binding tocytoskeletal rearrangement and migration. CCL4 wassubsequently also shown to activate Syk via RAFTK andrecruit the phosphatases SHP1 (Src homology region 2domain-containing phosphatase-1) and SHP2 associatedwith the adapter protein Grb2 (Ganju et al., 2000).The generation and phenotyping of a mouse lacking

CCR5 was first described in 1998 (Zhou et al., 1998).Macrophages isolated from Ccr52/2 mice generatedreduced amounts of inflammatory cytokines (includingIL-6, IL-1b ,and TNFa) when classically activated withLPS and IFN-g ex vivo. By using a Listeria mono-cytogenes infection model, Ccr52/2 mice showed de-fective macrophage-dependent clearance, with highertiters of bacteria found in the liver. CCR5 deficiencyalso offered protection in a lethal endotoxemia model inwhich CCR5 ligands are known to play an importantrole in macrophage recruitment, contributing to pa-thology. In wild-type mice, expression of CCR5 inT cells was shown to be dramatically upregulatedfollowing in vitro activation with anti-CD3 and anti-CD28 antibodies. Activated Ccr52/2 T cells producedmore IFN-g, GM-CSF (granulocyte macrophage colonystimulating factor) and IL-4 compared with wild-typecells. This enhanced cytokine production was mirroredin an enhanced delayed-type hypersensitivity (DTH)reaction and increased antibody production followingantigen challenge in CCR5 deficient mice.

Ccr52/2 mice were also found to have drasticallyreduced survival in a model of intratracheal delivery ofCryptococcus neoformans, an AIDS-associated patho-gen (Huffnagle et al., 1999). Knockout mice showednormal recruitment of leukocytes to the lung but defec-tive accumulation of leukocytes in the brain, associatedwith increased accumulation of capsule polysaccharideand brain edema. Thus, CCR5 appears to play a criticalrole in the generation of both innate and adaptive im-mune responses to control a variety of pathogens.

Other studies have shown that CCR5 deficiency hasno effect in an EAE model or in antiviral immunity butprotects mice from a dextran sodium sulfate (DSS)-induced colitis model and from chronic fungal asthma(Andres et al., 2000; Tran et al., 2000; Nansen et al.,2002; Schuh et al., 2002a).

Several studies have analyzed the importance ofCCR5 expression Treg cells. In murine models of graftversus host disease (GVHD), Tregs are required toinhibit rejection but suppress effector responses.Wysocki et al. (2005) demonstrated a requirement forCCR5 in this process since irradiated mice giventransplants supplemented with CCR5-deficient Tregsshowed reduced survival compared with mice receivingWT Tregs. Ccr52/2 Tregs showed a normal initialaccumulation in lymphoid tissues, but a later defect inrecruitment to both lymphoid tissues and GVHD targetorgans. A second study by Moreira et al. (2008) usedthe fungal pathogen Paracoccidiodes brasiliensis,which induces the formation of granulomas containingviable yeast particles, allowing disease reactivation.This process is driven by recruitment of Treg cells tothe infected side, leading to inhibition of effectorresponses and fungal persistence. CCR5 was shownto have a critical role in the process because CCR5knockout Tregs showed a reduced accumulation ingranulomas, leading to better control of fungal growthand dissemination. Finally, CCR5 expression on Tregshas been shown to be important for evasion of theimmune response by tumors. By using a CCR5 antago-nist, Tan et al. (2009) demonstrated that CCR5 blockadereduced Treg migration to tumors, thus leading todiminished tumor growth in a pancreatic cancer model.

Thus CCR5 expressed on leukocytes has disparateroles that both facilitate effective immune responsesagainst pathogens, but may also promote immunetolerance. This may be beneficial in certain scenarios,such as transplantation, but may allow tumors toevade the host immune system.

In terms of epidemiologic studies, the CCR5 D32polymorphism mentioned above has also been exten-sively studied for potential association with inflamma-tory disease, in particular RA. A meta-analysis of fivecase-control studies with European participants dem-onstrated a mean allele frequency of 6% for D32 in RApatients and 10% in controls (Prahalad, 2006). Asignificant negative association of the D32 allele with


RA was identified, indicating that this polymorphismis protective. Similarly, a case-control study from theUK replicated the negative association of the D32 allelewith juvenile idiopathic arthritis [JIA (Hinks et al.,2010)]. These authors also performed a meta-analysisof three studies including .2000 patients and con-firmed the protective effect of the polymorphism. Oneof the studies included in this meta-analysis, however,failed to independently show an association of D32 withJIA (Lindner et al., 2007). Another recent small studyshowed no correlation of the D32 polymorphism witheither the incidence of RA or SLE or disease severity inthese patients (Martens et al., 2010). These studiessuggest that the influence of CCR5 polymorphism onarthritis is relatively subtle, and large-scale studiesare needed to demonstrate an involvement of thisreceptor with disease. The recent failure of the licensedCCR5 antagonist maraviroc (Selzentry; Pfizer, Sand-wich, UK) to reduce arthritis severity beyond estab-lished therapies (see IV. Chemokine Receptor Drugs inClinical Trials), also suggests that CCR5 may not bea viable therapeutic target in RA.

F. CCR6

Several groups independently identified an openreading frame located on chromosome 6q27 as a poten-tial GPCR with homology to known chemoattractantreceptors (Zaballos et al., 1996; Baba et al., 1997;Greaves et al., 1997; Liao et al., 1997a, b; Power et al.,1997). The formal demonstration that these cDNAclones encoded the CCR6 chemokine receptor onlybecame possible with the availability of recombinantprotein for the CCL20 chemokine, variously known asMIP-3a, Exodus, and LARC (Hieshima et al., 1997;Hromas et al., 1997; Rossi et al., 1997). CCR6 trans-fected cells were shown to generate a calcium flux onlywhen exposed to recombinant CCL20 protein andlabeled CCL20 bound to CCR6 transfected cells withhigh affinity (;0.1 nM Kd) (Greaves et al., 1997).CCL20 remains the sole high-affinity chemokine ligandof the CCR6 receptor but low affinity binding of humanbeta defensin-1 and -2 to the CCR6 receptor has beenreported (Hoover et al., 2002; Yang et al., 1999a).Initial mRNA expression data across panels of

human tissues and primary cells suggested thatCCR6 was expressed predominantly by DCs andT cells. This observation was confirmed and extendedusing CCR6-specific antibodies that showed surfaceexpression of CCR6 on immature DCs that underwentdose-dependent chemotaxis in response to CCL20(Carramolino et al., 1999; Dieu-Nosjean et al., 2000).Maturation of DCs derived from CD341 cord bloodprogenitors in vitro following treatment with IL-4 orTNFa led to a marked decrease in CCR6 expressionand a corresponding decrease in DC responsivenessto CCL20 (Carramolino et al., 1999). ImmatureDCs generated by culturing human monocytes with

GM-CSF, IL-4 and TGF-b1 (transforming growthfactor b1) showed good levels of CCR6 expression andchemotaxis to CCL20 (Yang et al., 1999b). Furtherevidence that CCL20 is a potent chemoattractant ofimmature DCs came from experiments using CD1a1

Langerhans cell precursors generated from humanskin (Dieu-Nosjean et al., 2000). In keeping with theinitial reports of CCR6 mRNA expression patterns, useof CCR6-specific antibodies in flow cytometry showedthat resting memory T cells in human blood both ex-pressed CCR6 and were responsive to CCL20 (Liaoet al., 1999). More recently CCR6 was shown to beselectively expressed by Th17 CD41 T cells, a subset ofeffector cells associated with protection against extra-cellular microbes. Moreover, these cells showed func-tional responses to CCL20 in vitro (Annunziato et al.,2007; Singh et al., 2008). CCR6 expression on humanB lymphocytes and activated neutrophils has beenreported, but CCR6 expression is not always associatedwith chemoattractant effects of CCL20 in these celltypes (Yamashiro et al., 2000; Liao et al., 2002).

Relatively few papers have addressed the signalingpathways downstream of the CCR6 receptor in pri-mary cells. Keates et al. (2007) demonstrated CCR6expression by the epithelial cell lines Caco-2 and HT-29 and then demonstrated that CCL20 addition tothese cells leads to epidermal growth factor receptor(EGFR) transactivation via shedding of the cell-associated EGFR ligand amphiregulin by metallopro-teinase activation. More recently Lin et al. (2010)undertook a detailed proteomics profiling of proteinsmobilized to lipid rafts in CCR6 transfected Jurkatcells soon after addition of CCL20. The authorsidentified 85 proteins that are rapidly mobilized tolipid rafts containing CCR6 and siRNA knockdown ofone of these proteins that is associated with actincytoskeletal rearrangement (L-Plastin) decreasedCCL20-mediated chemotaxis but did not affect cal-cium mobilization in response to CCL20. The authorsadditionally demonstrated a role for the chaperoneprotein heat shock protein 90 (HSP90) in CCR6signaling in transfected Jurkat cells.

Cloning of the murine homolog of CCR6, whichshares 74% homology with the human CCR6 receptor,was reported in 1998 along with the demonstrationthat murine CCL20 caused calcium mobilization inmCCR6 transfected cells (Varona et al., 1998). Soonafterward two independent groups reported the gener-ation of Ccr62/2 mice. Consistent with the reportedexpression pattern of CCL20 in the intestinal epithe-lium of mice and humans (Tanaka et al., 1999) anda specific role for CCR6 in intestinal T-cell homeostasisand DC recruitment, Ccr62/2 mice displayed altered T-cell populations in mucosal associated lymphoid tissue(MALT) and impaired IgA responses to orally admin-istered antigens and viral pathogens (Cook et al., 2000).Ccr62/2 mice displayed no defect in humoral immune

62 White et al.

responses to subcutaneously delivered antigens. Var-ona et al. (2001) independently generated a second lineof Ccr6-deficient mice and confirmed the previousobservation of altered T-cell numbers and T-cell subsetswithin the intestinal mucosa. Additionally, theseauthors showed that Ccr62/2 mice displayed alteredimmune responses in DTH and contact hypersensitiv-ity. The involvement of CCR6 expressing T cells incontact hypersensitivity to hapten sensitization andchallenge was confirmed in a subsequent study(Paradis et al., 2008). Further evidence for the in-volvement of CCR61 cells in intestinal immunityand inflammation came from the demonstration thatCcr62/2 mice showed less severe intestinal pathologyin the DSS model of colitis, whereas the absence ofCCR6 conferred susceptibility to the trinitrobenzenesulfonic acid-induced (TNBS) model of colitis inC57BL/6J mice, a usually resistant strain (Varonaet al., 2003). These results were complemented by laterstudies showing that a CCL20 blocking antibodyreduced intestinal pathology, decreased PMN recruit-ment, and reduced the number of CCR61 T cells in theTNBS model of colitis (Katchar et al., 2007).Ccr6-deficient mouse strains have been used to study

the role of CCR6 expression in a range of animaldisease models in which DC mobilization and T-cellresponses are known to be important in pathogenesis.Lukacs et al. (2001) reported that expression of theCCR6 receptor is required for the allergic immuneresponse that drives allergic pulmonary inflammationin mice sensitized to cockroach antigen. Comparedwith sensitized wild-type mice, sensitized Ccr62/2 micedisplayed reduced airway resistance, fewer eosino-phils, and less IL-5 in lung tissue following antigenchallenge. Taken together with the initial descriptionof Ccr62/2 mouse IgA responses to rotavirus infection,this study suggests a key role for CCR6-expressingcells in the generation of adaptive immune responsesto mucosal antigens. Further investigation of the roleof CCR6 expressing cells in a cigarette smoke-inducedlung injury model was reported by Bracke et al. (2006),who showed reduced numbers of DCs, activated CD81

T cells, and neutrophils in Ccr6-deficient mice leadingto decreased emphysema in this model of chronicobstructive pulmonary disease (COPD).Liston et al. (2009) examined the role of CCR6-

expressing cells in both the induction and effectorphase of the EAE model of demyelination and centralnervous system (CNS) inflammation. Ccr62/2 mice hadreduced disease scores compared with wild-type micefollowing immunization with myelin oligodendrocyteglycoprotein (MOG) peptides (Liston et al., 2009). Theauthors extended this observation by showing thatpeak EAE disease scores and spinal cord CD41 T-cellnumbers in wild-type mice could be significantlyreduced by use of a CCR6 antagonist [a truncatedform of the CCR6 ligand, CCL20(6-70)] and a rabbit

anti-mouse CCR6 antiserum. In contrast Elhofy et al.(2009) demonstratedmore severe CNS inflammation inCcr62/2 mice compared with wild-type animals usinga similar, but not identical, EAE induction protocol. Byusing adoptive transfer of different cell populations,the authors mapped the increased severity of EAE inCcr6-deficient animals to an absence of Ccr61 DCsrather than Ccr61 T cells. Another explanation for thecontrasting results obtained with CCR6-deficient micein the EAE model of CNS inflammation comes from thework of Yamazaki et al. who demonstrated that CCR6is expressed on both Th17 effector cells and Tregpopulations. Taken together these results suggesta complex role for CCR6 mediated T cell and DCrecruitment in autoimmune disease.

Genome-wide association studies (GWAS) have id-entified single nucleotide polymorphisms near theCCR6 gene on chromosome 6q27 as novel risk loci forautoimmune Graves’ disease, generalized vitiligo, andRA (Chu et al., 2011; Jin et al., 2010a; Quan et al.,2010; Stahl et al., 2010). Of note Kochi et al. (2010)identified a dinucleotide polymorphism in the CCR6gene that was associated with RA susceptibility in twoindependent cohort studies and was correlated withthe level of CCR6 expression and the presence of IL-17in patient sera. The demonstration that a functionalpolymorphism within the human CCR6 gene isassociated with an increased risk of RA and increasedIL-17 cytokine levels in the sera of RA patients hasfueled increased interest in Th17 cell biology in a rangeof autoimmune diseases (Lubberts, 2010).

G. CCR7

CCR7 was first identified in 1993 by Birkenbach andcolleagues (1993) and was soon after assigned itsnomenclature in a study by Yoshida et al. (1997), whocarried out molecular cloning and site-specific mappingof a novel ligand for the receptor that would later bedesignated CCL19. CCR7 was initially referred to asEBI1 (Epstein Barr virus-induced gene 1) because thegene that encoded the receptor was induced by EBVinfection (Birkenbach et al., 1993; Schweickart et al.,1994). The CCR7 gene is located on human chromo-some 17q12-q21.2. Expression of CCR7 has beendetected on T lymphocytes (Bardi et al., 2001; Kimet al., 2005), B lymphocytes (Birkenbach et al., 1993),DC (Walker et al., 2005), and natural killer (NK) cells(Inngjerdingen et al., 2001). In activated B cells, CCR7is highly upregulated following EBV infection (Birken-bach et al., 1993); similar observations were made withCD41 T cells following infection with human herpesvirus 6 (HHV6) and HHV7 (Hasegawa et al., 1994). Themouse homolog of CCR7 was cloned in 1994 and foundto display 86% homology with the human receptor(Schweickart et al., 1994).

CCL19 (also known as EBI-1-ligand chemokine;ELC, MIP-3b, CK b-11) and CCL21 (also known as


secondary lymphoid-tissue chemokine; SLC, TCA-4,6Ckine) are the primary ligands for CCR7. Studiesfrom the group led by Osamu Yoshie were the first todescribe the isolation, cloning, and characterization ofboth these ligands (Nagira et al., 1997, 1998; Yoshidaet al., 1997, 1998). CCL19 was shown to share closehomology with other CC chemokines, including CCL3,CCL7, and CCL5. Binding studies utilizing K562 cellsstably transfected with a variety of CC chemokinereceptors revealed only specific and high-affinitybinding of CCL19 to CCR7 (Yoshida et al., 1997). Thisbinding was also shown to induce calcium mobilizationwith an EC50 of 0.9 nM (Yoshida et al., 1997).Furthermore, 293/EBNA-1 cells stably transfected withCCR7 and HUT78 cells (human T-cell line) expressingendogenous CCR7 were shown to undergo chemotaxistoward CCL19, with maximal effects observed in bothcell types at 300 ng/ml (Yoshida et al., 1997).CCL21, a polypeptide of 134 amino acids in length,

shares approximately 21–33% homology with otherhuman CC chemokines. High levels of CCL21 mRNAexpression have been reported in human lymph nodesand appendix, with intermediate levels detected inspleen and low levels in the thymus. Similar to CCL19,CCL21 expression is virtually absent on peripheralblood leukocytes (Nagira et al., 1997). However, morerecent evidence has shown CCL19 to be expressed bymature DCs and CCL21 upon endothelial cells ofafferent lymphatics (Ansel et al., 2000; Baekkevoldet al., 2001) and high endothelial venules (Gunn et al.,1998; Stein et al., 2000; Warnock et al., 2000). Nagiraet al. (1997) demonstrated the ability of CCL21 toinduce PTX-sensitive chemotaxis of T-cell lines Hut78and Hut102 at doses of 10–100 nM. A similarchemotactic effect was observed with freshly isolatedperipheral blood lymphocytes (PBL) with maximalchemotaxis at 0.1 nM. In contrast, chemotaxis wasabsent when testing CCL21 with neutrophils, mono-cytes, or monocytic cell lines. Interestingly, CCL21 wasnot found to induce calcium flux in PBL; however,calcium mobilization was observed with culturedT cells with a reported EC50 of 1 nM (Nagira et al.,1997). These initial observations were confirmed andexpanded upon in a subsequent study by Sullivan et al.(1999), whereby the authors used stably transfectedCCR7 HEK-293 clones and demonstrated high-affinity(pM) binding of both CCL19 and CCL21. Furthermore,the authors also reported an enhanced chemotacticresponse to both ligands in the subnanomolar range(0.1–1 nM) in addition to rapid calcium mobilizationand MAPK activation (Sullivan et al., 1999).During the course of DC maturation, CCR7 cell

surface expression is upregulated (Dieu et al., 1998;Sallusto et al., 1998). Both CCL19 and CCL21 areconstitutively expressed in lymph nodes and playa central role in the trafficking of CCR7 expressingDCs toward these highly organized lymphoid

structures where antigen presentation to naiveT cells takes place (Ngo et al., 1998). CCR7 has beenshown to activate two independent signaling modulesin human DCs. The first involves Gai-dependent MAPKactivation of ERK1/2, JNK, and p38 that regulatechemotaxis of DCs. The second involves activation ofthe GTPase Rho, the tyrosine kinase Pyk2, and theinactivation of cofilin, with all three components shownto have a role in DC migratory speed (Riol-Blancoet al., 2005). CCR7 has also been shown to play animportant role in the survival of mature DCs via anti-apoptotic signaling (Sanchez-Sanchez et al., 2004).Stimulating DCs with either CCL21 or CCL19inhibited apoptosis by 50–60% with the maximal effectobserved at 200 ng/ml. When pretreating DCs witha neutralizing antibody against CCR7, the protectiveeffects of CCL19 and CCL21 were abolished. Theseeffects observed with CCL19 and CCL21 were partlyattributed to the rapid activation of PI3K and Akt,which are both key regulators of survival in a variety ofcell types. NFkB was also shown to be involved inpromoting the antiapoptotic effects of both chemokines,because inhibition of NFkB resulted in attenuatedeffects of CCL19 and CCL21 on DC survival (Sanchez-Sanchez et al., 2004).

The generation and phenotyping of CCR7-deficientmice (Ccr72/2) was first described by Forster et al.(1999). In the absence of CCR7, elevated levels of CD41

T cells were found in the peripheral blood, spleen, andbone marrow of knockout mice. In contrast, lowerlevels of CD41 T cells were detected in the mesentericlymph nodes (LN), peripheral LNs, and Peyer’spatches. Specific immunostaining of LNs from Ccr72/2

mice revealed an impaired distribution and in somecases reduced numbers of both B and T cells within theouter cortex, paracortex, and marginal sinus areas ofLNs when compared with WT mice. Another keyobservation in the Ccr72/2 mice was that increasednumbers of activated B cells were recovered from LNs,whereas migration of activated skin DCs into drainingLNs was impaired. One of the most significantobservations reported in this study was the impairedability of Ccr72/2 mice to mount T-cell responses inboth models of contact hypersensitivity and DTH withno ear swelling observed following rechallenge withOVA at 24 or 48 hours. Impaired humoral responseswere also apparent in the Ccr72/2 mice followingapplication of the T-cell-dependent antigen DNP-KLH(Forster et al., 1999). Other in vivo evidence indicatesa critical association between CCR7 and CXCR5 in thedevelopment and organization of secondary lymphoidorgans. Mice deficient in both receptors (Ccr72/2

Cxcr52/2) were shown to lack all peripheral LNs (Ohlet al., 2003). Furthermore, CD3-CD41IL-7Rahi cellswhich play a critical role during the early phase ofsecondary lymphoid organ development were shown toco-express both receptors (Ohl et al., 2003). More

64 White et al.

recently, CCR7 deficiency was shown to promote Th2polarization and B cell activation, characterized byelevated IL-4 levels in lymph nodes (Moschovakiset al., 2012). Collectively these studies highlight a dualfunctionality for CCR7, first in mounting effectiveprimary immune responses and, second, in homeo-static mechanisms that include developing andmaintaining the architecture and activity of localmicroenvironments in secondary lymphoid tissues.Several SNPs in the CCR7 gene have been identified

but are found at very low frequency and were shown tohave no association with several diseases includingSLE (Kahlmann et al., 2007).

H. CCR8

Molecular cloning of the CCR8 receptor (formerlyknown as TER1, ChemR1, CY6, CKR-L1) was firstdescribed by three independent groups at around thesame time (Napolitano et al., 1996; Samson et al.,1996c; Zaballos et al., 1996). The gene that encodes thereceptor was mapped onto chromosome 3p21 and shownto share close homology with other known chemokinereceptors (Napolitano et al., 1996; Samson et al., 1996c;Zaballos et al., 1996). The human and murine CCR8genes have been shown to share approximately 71%homology, with both receptors shown to be activatedwith either murine or human CCL1 (I-308) (Roos et al.,1997; Goya et al., 1998).CCR8 mRNA expression appears to be restricted to

lymphoid tissue and several cell lines. Zaballos et al.(1996) reported mRNA expression of CCR8 in PBMCs,more specifically monocytes/macrophages, CD41 andCD81 T cells, and to a lesser extent in CD191 B cells.CCR8 was also shown to be selectively expressed uponTh2 polarized cells and clones (Zingoni et al., 1998).Human NK cells have been reported to express CCR8upon activation with IL-2 (Inngjerdingen et al., 2000).However, several other independent studies have beenunable to show detectable CCR8 mRNA levels inhuman PBMC subsets (Napolitano et al., 1996; Rooset al., 1997), therefore data relating to the expressionprofile of CCR8 in primary human immune cells shouldbe interpreted with caution.CCL1 is the principal functional ligand for CCR8.

CCL1 binds with high affinity to CCR8 (;1.2 nM Kd)and induces transient intracellular calcium mobili-zation (EC50 2 nM) (Roos et al., 1997; Tiffany et al.,1997). Cells transfected with the CCR8 receptorhave also been shown to migrate toward CCL1 ina dose-dependent manner, with maximal activityobserved at 10 nM (Tiffany et al., 1997). Otherligands, including CCL17 and CCL4, have beenreported to induce chemotaxis in Jurkat cells stablyexpressing CCR8 (Bernardini et al., 1998). However,a recent study tested all previously describedligands for CCR8 and could only confirm CCL1 andvMIP-1 (viral macrophage inflammatory protein-1:

a viral chemokine mimic from Kaposi’s sarcoma-associated herpes virus) as potent activators of thereceptor in chemotaxis and calcium flux assays (Foxet al., 2006).

CCL1 protects murine thymic lymphoma cell linesfrom dexamethasone-induced apoptosis (Van Snicket al., 1996). This initial observation was confirmedand extended by Spinetti et al. (2003) who demon-strated that both CCL1 and vMIP-1 induce CCR8-mediated rescue from dexamethasone-induced apopto-sis via an ERK dependent pathway. The use ofa specific antagonist (MC148/MCC-I) of CCR8 wasshown to inhibit the rescue effect of CCL1 and vMIP-1,highlighting the involvement of CCR8 in cell survival(Spinetti et al., 2003).

A study performed by Haskell et al. (2006) was thefirst to describe a novel nonpeptide chemokine receptoragonist for CCR8. The agonist 2-{2-[4-(3-phenoxybenzyl)piperalzin-1]ethoxy}ethanol (ZK 756326) was shownto inhibit binding of CCL1 to U87 cells expressingCCR8 with an IC50 of 1.8 mM. ZK 756326 inducedPTX-sensitive calcium mobilization in CCR8-expressingcells in a dose-dependent manner. ZK 756326 wasalso shown to activate murine cells expressingCCR8, promoting their chemotaxis and inducingERK1/2 phosphorylation. Akin to CCL1, ZK 756326at 10 mM was shown to inhibit HIV fusion of cellsexpressing CD4 and CD8 (Haskell et al., 2006).Collectively these observations highlighted the useof this CCR8 agonist as a potential tool to studyCCR8 biology.

CCR8-deficient mice have been instrumental indeciphering the role the receptor plays in the re-cruitment of specific leukocyte subsets, in regulatingimmune responses, and as a potential therapeutictarget. Phenotypically, Ccr82/2 mice were shown todevelop normally, to be fertile, and not to developspontaneous disease (Chensue et al., 2001). CCR8deletion was shown to specifically impair local Th2cytokine responses in models of S. mansoni soluble eggantigen (SEA)-induced granuloma formation and OVAand cockroach antigen-induced allergic airway inflam-mation. This impaired Th2 response was associatedwith a significant reduction in eosinophil levels in thelung (50–80%) and Th2 cytokine (IL-5 and IL-13)production in the lungs and draining LNs of Ccr82/2

mice compared with WT mice. Interestingly, Th1specific immune responses elicited with M. bovis PPDremained unaffected in Ccr82/2 mice, emphasizing thespecificity of CCR8 in Th2 functional responses(Chensue et al., 2001). In a follow-up study from thesame group, CCR8 expression was shown to be re-stricted to SEA elicited (specific type 2 response) IL-101

CD41 T cells (Freeman et al., 2005). These cells werefurther characterized and shown to express CD251 andCD441 but lacked Foxp3 expression, indicating thatthey were not Treg cells. Collectively these observations


highlight the importance of a defined subset of IL-10-producing CCR81 CD41 CD251 CD441 T cells inmediating Th2 type responses to parasitic antigens.The importance of CCR8 in Th2 responses was also

highlighted in an atopic dermatitis model in whichCcr8-deficient animals showed less eosinophilic in-flammation, and adoptive transfer studies showedCCR8 was critical for homing of Th2 cells to inflamedskin (Islam et al., 2011). This process was shown todepend on murine CCL8, which was highly expressedin inflamed skin.Other interesting observations made with CCR8-

deficient mice include leukocyte trafficking studiesthat implicate a role for the receptor in the migration ofmonocyte-derived dendritic cells to draining lymphnodes (Qu et al., 2004). In a model of chronic fungalasthma, the absence of CCR8 receptor was shown topromote the clearance of fungal material in the lungs ofthese animals, highlighting the receptor as a potentialtherapeutic target in fungal associated pulmonaryconditions (Buckland et al., 2007).

I. CCR9

In 1997, a novel CC chemokine, thymus-expressedchemokine (TECK, CCL25), was identified from anal-ysis of a RAG-1 deficient mouse thymus cDNA libraryand found to be expressed at the mRNA level inthymus and the small intestine (Vicari et al., 1997).Recombinant murine CCL25 was shown to be chemo-tactic for murine macrophages, DCs, and thymocytes.Chemotaxis could be blocked by pretreatment withPTX, implicating a Gai-coupled receptor as mediatingthe chemoattractant effects of CCL25 (Vicari et al.,1997). In 1999, Zabellos et al. (1999) demonstratedthat a previously identified orphan GPCR, GPR-9-6,encoded the CCL25 receptor (designated CCR9) asHEK293 cells transfected with full length versions ofthe GPR-9-6 open reading frame showed CCL25mediated calcium flux and dose-dependent chemotaxisto CCL25. Other groups confirmed the assignmentof GPR-9-6 as the receptor for CCL25 and furtherdemonstrated that human thymocytes expressingCCR9 displayed chemotaxis to CCL25 (Youn et al.,1999; Norment et al., 2000). Additionally, Yu et al.(2000) demonstrated that alternative splicing of theCCR9 gene generated two different CCR9 mRNAsencoding two different versions of CCR9. CCR9Acontains 12 additional amino acids at its N terminuscompared with CCR9B and had a lower EC50 forCCL25-mediated calcium flux compared with CCR9Bin transfected cells (Yu et al., 2000). CCL25 remainsthe only identified high-affinity ligand for CCR9. Themurine homolog of CCR9 was identified in 2000(Norment et al., 2000).Generation of an anti-CCR9 monoclonal antibody

allowed a detailed examination of the cells expressingCCR9 in different anatomic sites (Kunkel et al., 2000).

Butcher and co-workers demonstrated that CD41 andCD81 T cells expressed the highest level of CCR9in human peripheral blood and that CCR91 T cellscoexpressed high levels of the gut-homing a4b7integrin. Consistent with this observation, the authorsused flow cytometry to demonstrate expression ofCCR9 by all intraepithelial T cells and lamina proprialymphocytes in the small intestine (Staton et al.,2006). These findings were confirmed in a complemen-tary study using a CCR9-specific polyclonal antibody,which also showed that a significant fraction ofperipheral gd T cells express CCR9 (Uehara et al.,2002).

Possibly because of the limited availability of CCR91

primary cells, relatively little information is availableon the signaling pathways downstream of CCR9. Thefew published studies of CCR9 signaling have comefrom CCR91 T-cell lineage acute lymphocytic leukemia(T-ALL) cells. In a survey of 38 T-cell leukemia cases,Qiuping et al. (2003) showed that CCR9 was highlyexpressed on peripheral blood CD41 T-ALL cells butwas less well expressed and expressed on fewer CD41

cells from patients with T-cell lineage chronic lympho-blastic leukemia (T-CLL). CCR9 expression was asso-ciated with chemotaxis of T-ALL cells but not T-CLLcells to CCL25 (Qiuping et al., 2003). The same groupsubsequently reported that CCL25 treatment ofCCR91 T-ALL cells enhanced resistance to TNFa-induced apoptosis, and this protection was associatedwith CCL25-mediated upregulation of the inhibitor ofapoptosis protein (IAP) family member Livin (Qiupinget al., 2004). The authors demonstrated that CCL25-mediated Livin induction was via a JNK1 pathway andthat shRNA inhibition of Livin specifically blockedCCL25-mediated resistance to TNFa-induced apopto-sis. Other CCR9 signaling studies undertaken usingthe MOLT4 T-ALL cell line revealed a role for Rho-ROCK and Ezrin in mediating cytoskeletal rearrange-ment following CCL25 treatment (Zhou et al., 2010;Zhang et al., 2011).

The first description of Ccr92/2 mice reported nomajor developmental or immunologic abnormalities,and the fact that Ccr92/2 thymocytes showed nochemotaxis to CCL25 strongly suggested that CCR9is the sole functional receptor for CCL25 (Wurbel et al.,2001). Parenthetically, it should be noted that a non-signaling orphan chemokine receptor (CCX CKR) with;30% sequence homology with CCR9 was identified byGosling et al. (2000) and shown to bind the CCR7ligands CCL19 and CCL21 and the CCR9 ligandCCL25 with high affinity (Comerford et al., 2006).Further detailed analysis of lymphocyte homing inwild-type and CCR9-deficient mice revealed CCR9expression on IgA1 plasma cells in mesenteric lymphnodes and Peyer’s patches that was downregulated onmigration to the small intestine (Pabst et al., 2004).Ccr92/2 animals failed to mount a normal IgA response

66 White et al.

to orally administered antigens (Pabst et al., 2004),although Ccr92/2 animals showed no gross defect inrotavirus-specific plasmablast recruitment to the in-testine during rotavirus infection (Feng et al., 2006).Given the expression of the CCR9 receptor on gut-

homing lymphocytes in the blood and lymphocytes inthe gut, a number of laboratories looked at the effectsof CCR9 ablation and CCL25 blockade in mousemodels of intestinal inflammation. Apostolaki et al.(2008) generated Ccr9- and Ccl25-deficient mice on theTNFDARE/1 mouse background. TNFDARE/1 mice haveelevated levels of TNFa and develop a Crohn’s disease-like inflammation of the ileum, which is characterizedby increased CD81 T-cell recruitment. Ablation ofeither the Ccr9 gene or the Ccl25 gene did not alter thedisease pathogenesis in this study (Apostolaki et al.,2008). A similar study using TNFDARE/1 Ccr9-deficientmice generated by a second group showed increaseddisease severity in Ccr92/2 mice compared with Ccr91/1

mice at 4, 8, and 20 weeks of age that was accompaniedby an increased number of CD81 effector cells anda decreased number of Treg cells in the lamina propriaof Ccr9-deficient animals (Wermers et al., 2011). In thissecond study, the authors also treated TNFDARE/1

animals with a CCR9 blocking monoclonal antibodyand observed a similar increase in intestinal inflam-mation to that seen in their TNFDARE/1 Ccr9-deficientmice. Given that both genetic deletion of CCR9 andantibody blockade of CCR6 exacerbated ileitis in thismodel, the authors concluded that CCR9 has beneficialeffects in this spontaneous model of intestinal in-flammation. In a subsequent study, Walters et al.(2010) reported that treatment of TNFDARE/1 micewith subcutaneous injections of the CCR9 antagonistCCX282-B reduced disease scores judged by histology.This study seems difficult to reconcile with the twoprevious reports on the role of CCR9 in the TNFDARE/1

model. Further clarification of the role of CCR9 in thedevelopment and chronicity of ileitis could come fromtesting CCX282-B in Ccr92/2, TNFDARE/1 mice, andfrom testing CCX282-B in other models of intestinalinflammation.Wurbel et al. (2011) studied the role of CCR9 in

inflammation of the colon by testing the effects ofCCR9 and CCL25 deficiency in the DSS model ofcolitis. The authors showed that Ccr92/2 and Ccl252/2

mice are more susceptible to DSS colitis than wild-typelittermate controls and that these animals take longerto recover from colitis upon withdrawal of DSS fromthe drinking water. Taken together, the publishedwork using the TNFDARE/1 and DSS models areconsistent with a homeostatic rather than a proinflam-matory role for CCR91 cells in murine models ofintestinal inflammation.Interestingly, a recent report implicated activated

macrophages expressing CCR9 in the pathogenesis ofmurine acute liver injury following con A injection.

Ccr92/2 mice did not develop hepatitis following con Ainjection unless they also received CCR91-activatedmacrophages from a con A-injected wild-type donor.Moreover, con A-induced hepatitis could be amelioratedby injection of CCL25 blocking antibodies (Nakamotoet al., 2012). Another interesting CCR91 myeloid cellpopulation was identified by Zeyda et al. (2010) in theiranalysis of adipose tissue macrophages obtained fromhigh fat diet-induced obese mice. Using F4/80, MannoseReceptor (MR), and CD11c, the authors showed differ-ential expression of CCR9 in F4/801 MR- CD11c2 cells.The relationship of this myeloid cell population to theCCR91 tolerogenic immature plasmacytoid DCs (pDCs)described by the group of Eugene Butcher remains to bedefined (Hadeiba et al., 2008).

Given the phenotype of CCR9-deficient mice inmodels of intestinal inflammation it is a little sur-prising that no genetic association between CCR9or CCL25 has been seen in GWAS studies and case-control cohorts for inflammatory bowel disease.However, Inamoto et al. (2010) described an SNP(rs12721497, G926A) within the human CCR9 gene,which alters the amino acid sequence within the thirdextracellular domain loop from valine to methionine(V272M). Stably transfected Jurkat cells expressingboth variants of CCR9 at similar levels were generatedand compared for their chemotactic response to CCL25response. Jurkat cells expressing the CCR9-926Gvariant were more responsive to CCL25 than Jurkatcells expressing the CCR9-926A variant (Inamotoet al., 2010).

J. CCR10

The first citation of a receptor named CCR10 was in1997 (Bonini et al., 1997; Bonini and Steiner, 1997).The receptor was found to be highly expressed inplacenta and fetal liver and bound several CC chemo-kines, including CCL2, CCL7, CCL13, and CCL5.Sequence analysis showed the receptor did not havea DRY box motif and was unable to induce calcium fluxin transfected cells. In the same year, this receptor wasalso identified by a second group who named thereceptor D6, which is now known to be a decoy receptorfor chemokines without signaling capacity (Nibbs et al.,1997).

The true CCR10 receptor was identified in 2000when the orphan receptor GPR2 was cloned in humansand mice and found to bind a chemokine, ESkine/CTACK, now named CCL27 (Homey et al., 2000;Jarmin et al., 2000). The receptor had been previouslylocalized to chromosomal locus 17q21 in a cluster withseveral CC chemokines (Marchese et al., 1994). Highlevels of human CCR10 mRNA were found in testis andsmall intestine and foetal lung and kidney, whereasmany other tissues including spleen, thymus, lymphnode, and colon showed low level CCR10 expression(Jarmin et al., 2000). In the mouse, CCR10 mRNA was


not found in the testis, but high levels were detectedin small intestine, colon, lymph node, and Peyer’spatches, with lower levels found in spleen and thymus.This expression pattern suggested a potential role forthe receptor in leukocyte trafficking. Functionally,CCR10-transfected L1.2 cells were found to releaseintracellular calcium and migrate in response toCCL27 but not to 17 other chemokines tested (Jarminet al., 2000). A second group also demonstrated thatCCR10 mediated migration and calcium flux of trans-fected Baf/3 cells toward CCL27 (Homey et al., 2000).These authors further demonstrated that the receptoris expressed in T cells and Langerhans cells but notmonocytes or DCs. Finally, CCR10 was found to beexpressed by several cell types in normal skin and wasupregulated by the inflammatory cytokines IL-1b andTNFa in melanocytes (Homey et al., 2000).A second chemokine ligand for CCR10, CCL28

(MEC), was subsequently identified in humans andmice and found to chemoattract both CD41 and CD81

T cells (Wang et al., 2000). Another group demon-strated that this chemokine could also induce migra-tion of eosinophils via the CCR3 receptor (Pan et al.,2000).CCL27 has been shown to have a key role in T-cell

migration during cutaneous inflammation, and CCR10is expressed on most skin-homing T cells in patientswith psoriasis and atopic and allergic dermatitis (Reisset al., 2001; Homey et al., 2002).The CCL28-CCR10 axis is believed to be important

in migration of IgA antibody-secreting cells (ASCs) tomucosal surfaces (Wilson and Butcher, 2004). By usingCCR10 knockout mice (which are developmentallynormal), this was shown to be crucial to migration ofIgA ASCs to the lactating mammary gland but not tointestinal surfaces (Morteau et al., 2008).A very recent study suggests that CCL28-mediated

Treg recruitment plays a role in tumor development(Facciabene et al., 2011). Hypoxia occurs in manytumors and contributes to angiogenesis. However, thisprocess releases damage-associated molecular patternsand could therefore drive tumor rejection without theinduction of tolerance via Treg cell recruitment.Facciabene et al. (2011) demonstrated that hypoxiainduces CCL28 expression, leading to Treg cell re-cruitment, tolerance induction, and angiogenesis,hence promoting tumor survival.

K. Atypical CC Chemokine Receptors

In addition to the receptors described above, anumber of nonsignaling receptors are expressed bymammalian cells that bind CC chemokines with highaffinity without generating a functional response.There are three nonsignaling or "atypical" receptorsknown to be CC chemokine receptors: DARC, D6, andCCX-CKR.

DARC (the Duffy antigen receptor for chemokines) isan antigenic determinant expressed on the surface ofred blood cells, where it is also the entry receptor forthe malarial parasite Plasmodium vivax (Cutbush andMollison, 1950; Miller et al., 1976). DARC binds a widerange of both CXC and CC chemokines, includingCCL2 and CCL5 (Neote et al., 1993a; Neote et al.,1994). DARC is also expressed on the surface ofvenular endothelial cells, where it is known to mediatetranscytosis of chemokines from the basolateral toapical surface of the cell (Pruenster et al., 2009).Rather than being degraded, transported chemokineremains intact and promotes migration of leukocytesacross the endothelial monolayer. On red blood cells,DARC may function as a sink for chemokines in blood,which may prevent excessive inflammatory reactionsas has been shown to occur in DARC-knockout mice inan endotoxemia model (Dawson et al., 2000).

D6 was cloned in 1997 and initially thought to bea functional chemokine receptor designated as CCR10(Bonini et al., 1997). D6 was subsequently shown to bean atypical receptor with a mutated DRY motif and theability to bind a broad range of inflammatory CCchemokines (Nibbs et al., 1997). D6 is expressed ontissues, including placenta, skin, gut, and lung, whereit is primarily found on lymphatic endothelial cells. Onlymphatic endothelium, D6 has been shown to mediatechemokine scavenging of chemokines, leading to in-ternalization and degradation (Fra et al., 2003).Interestingly, D6 can selectively recognize only theactive forms of some chemokines, e.g., CCL14 withoutbinding inactive molecules that have been truncated byCD26 (Savino et al., 2009). Numerous studies havedemonstrated that D6 knockout mice display excessiveinflammatory responses in several experimental mod-els, supporting a chemokine scavenging role for D6 invivo (Jamieson et al., 2005).

CCX-CKR was originally identified as a signalingreceptor and named CCR11, although subsequentstudies failed to replicate this finding, and it is nowknown to be a nonsignaling receptor (Schweickartet al., 2000). CCX-CKR binds the homeostatic CCchemokines CCL19, CCL21, and CCL25 (Gosling et al.,2000). Like D6, CCX-CKR is believed to have a scaven-ger role, binding chemokines for internalization anddegradation. The function of CCX-CKR in vivo has notbeen as extensively explored as for the other decoyreceptors, but two studies have suggested a role in DCtrafficking and in the regulation of immune responses,because CCX-CKR knockout mice have exaggeratedresponses in the EAE model (Heinzel et al., 2007;Comerford et al., 2010).

Clearly the atypical chemokine receptors have animportant role in the regulation of inflammatoryresponses in vivo. Further discussion of these receptorsis outside the scope of this review, and we refer thereader to an excellent recent review exploring the

68 White et al.

biology of these receptors in more detail (Graham et al.,2012).

III. Role of Chemokines in ChronicInflammatory Diseases

In this section, we review the role of chemokines andtheir possible utility as therapeutic targets in three keydisease areas, namely, atherosclerosis, rheumatoidarthritis, and metabolic syndrome. Although disparatein their clinical manifestations, these three forms ofchronic inflammation share several common features:they often persist for several decades, have similarunderlying pathologic mechanisms (notably monocyterecruitment and macrophage activation), and allconstitute a significant healthcare burden. Further-more, these diseases often exist as comorbid patholo-gies in the same individual: patients with metabolicsyndrome are at increased risk of atherosclerosis, asare individuals with RA.

A. Atherosclerosis

1. Summary of Pathology. Atherosclerosis is achronic inflammatory disease of the major arteries.Atherosclerotic plaques composed largely of modifiedlipids, recruited macrophages, T cells, and smoothmuscle cells (SMCs) accumulate in the arterial wallover many decades. Eventually these plaques mayocclude the lumen of the artery, leading to angina (ifsited in the coronary arteries) or transient ischemicattacks (when localized in the carotid or cerebralarteries). Acute plaque rupture can lead to potentiallyfatal outcomes, including myocardial infarction (MI)and stroke. In the UK and the US, cardiovasculardisease accounts for one in three of all deaths, with halfof these occurring as a result of coronary heart disease.Cardiovascular disease also has a significant economicimpact, with direct costs of £14.4 billion to the UKeconomy in 2006, mainly for hospital-based treatment.Indirect costs, which are more difficult to measure,include production losses as a result of mortality andmorbidity, particularly for stroke.The histologic features of atherosclerosis have been

well described for over a century, and multiple modelsto explain pathogenesis have been proposed (Williamset al., 2012). The key initiating event in atherogenesisis believed to be endothelial dysfunction, which canoccur as a result of hypertension, diabetes, smoking, orelevated plasma low-density lipoprotein (LDL) levels.Endothelial dysfunction leads to decreased nitric oxide(NO) production and expression of adhesion moleculescapable of mediating monocyte adherence, productionof inflammatory cytokines, and enhanced permeabilityof the endothelium. Apolipoprotein B (Apo B)-containinglipoproteins enter the vessel wall by diffusion,where they can become modified to become oxidizedLDL (oxLDL) or minimally modified LDL (mmLDL),

pro-inflammatory species that are likely detected byresident macrophages in the vessel wall. Monocytesare recruited from the blood into the intima in responseto chemokines such as CCL2 expressed on the en-dothelium. In the subendothelial space of the tunicaintima, monocytes differentiate into macrophages thatthen phagocytose the oxLDL in the vessel wall viascavenger receptors. This nonsaturable pathway ofmodified LDL uptake leads to accumulation of choles-terol droplets in the cytoplasm of the macrophage,generating the canonical "foam cells" that are typical ofearly atherosclerotic lesions. T cells, particularly CD41

Th1 cells, are also recruited into early atheroscleroticlesions and have been found to recognize self antigens,including oxLDL and HSP60. Th1 cells produce largequantities of IFN-g that activate macrophages, leadingto further cytokine and chemokine production.

Continued recruitment of inflammatory cells andaccumulation of modified lipid leads to the generationof a necrotic core to the plaque composed of dead anddying cells as well as extracellular cholesterol. As theplaque continues to develop, SMCs are recruited fromthe tunica media into the tunica intima where theyproliferate and secrete extracellular matrix, forminga "fibrous cap" that covers the inflammatory necroticcore of the plaque. This process may continue for manydecades, leading to arterial stenosis and gradual loss ofthe vessel lumen. Furthermore, apoptosis of SMCs ordegradation of extracellular matrix proteins in the capweakens this structure, leading to plaque rupture andrelease of thrombogenic material into the bloodstream.An arterial thrombus can form rapidly, leading tocessation of blood flow and ischemia manifested as MIor stroke.

2. Current Treatments. Primary and secondaryprevention strategies for atherosclerotic disease cur-rently target modifiable risk factors. Lifestyle anddietary advice are offered to help patients stopsmoking, reduce plasma cholesterol, exercise more,and lose weight. Pharmacological interventions includethe use of antihypertensive medications, lipid lower-ing, and antiplatelet drugs to reduce the likelihood ofthrombus formation and MI or ischemic stroke. Inpatients at high risk of an acute cardiovascular event,or immediately following such an event, anticoagulantsor antiplatelet drugs are used routinely to limit arepeat event.

Several agents exist to modify plasma cholesterol,including bile acid sequestrants, niacin and ezetimibe(Zetia), but statins are by far the most widely pre-scribed, accounting for approximately 1 million pre-scriptions per week in the UK. Statins competitivelyinhibit the rate-limiting enzyme in cholesterol bio-synthesis, HMG-CoA-reductase, which converts HMG-CoA to mevalonic acid. A fall in cholesterol synthesisleads to an upregulation of hepatocyte LDL-R expres-sion, increasing clearance of LDL from the plasma. It is


now known that statins have numerous actions thatare independent of their cholesterol-lowering activity,some of which may be mediated by products of themevalonate pathway that prenylate or farnesylateseveral membrane-bound enzymes.3. Evidence Supporting a Role for CC Chemokines in

Development of Pathology. Evidence supporting a rolefor CC chemokines in atherogenesis is derived from threemain sources: hypercholesterolemic mouse models, epi-demiology of human chemokine or chemokine receptorpolymorphisms, and histologic evidence for chemokineexpression within human atherosclerotic lesions.The majority of atherosclerosis research in animals

has used two well characterized models of acceleratedatherogenesis: the Apoe2/2 and Ldlr2/2 mouse modelsthat develop severe hypercholesterolemia and plaqueformation when fed a high-fat diet [reviewed in(McNeill et al., 2010)]. The development of atheroscle-rosis in these animals may be further accelerated bythe use of mechanical stimuli, such as arterial wireinjury, which causes significant endothelial damage.The first evidence for a causal role for chemokines in

atherogenesis in this model came from the generationof Ccr22/2 Apoe2/2 mice (Boring et al., 1998). Quanti-tative histologic analysis of lesions in the aorta of theseanimals revealed that the absence of CCR2 inhibitedplaque formation and reduced macrophage infiltrationinto the vessel wall. Subsequent data generated inCcl22/2 Ldlr2/2 mice confirmed that absence of theCCR2 ligand (JE) also significantly reduced lesion

formation (by 83%) in mice fed a high-fat diet (Gu et al.,1998). Since these seminal studies, numerous reportsutilizing chemokine receptor knockout animals crossedonto Apoe- or Ldlr-deficient mouse strains have beenpublished. The results of these studies are presented inTable 2.

Studies utilizing CCR1 knockout animals have pro-vided conflicting data. Deletion of CCR1 in the bonemarrow of LDLR knockout animals led to a dramatic70% increase in plaque area in the thoracic aorta after12 weeks of fat feeding (Potteaux et al., 2005). Incommon with studies described above (see II: CCChemokine Receptor, A: CCR1), the absence of CCR1 inbone marrow led to an enhanced Th1 response to con Aand to increased levels of IFN-g in the spleen,specifically localized to areas of oxidized lipid accumula-tion, suggesting an enhanced adaptive immune responsein these animals. Other studies have shown no effect ofCCR1 deficiency on neointima formation following wireinjury (Zernecke et al., 2006) and increased plaqueformation and T-cell content in Ccr12/2 Apoe2/2 mice feda high-fat diet (Braunersreuther et al., 2007b).

Studies aimed at demonstrating a nonredundantrole for CCR5 in mouse models of atherogenesis havemet with limited success. Initial studies reported nosignificant effect of deleting CCR5 on lesion size inApoe2/2 mice on chow diet (Kuziel et al., 2003) andsmaller lesions in Ccr52/2 Apoe2/2 animals fed a high-fat diet (Braunersreuther et al., 2007b). Bone marrowtransfer of Ccr52/2 stem cells was reported to have no

TABLE 2Effects of individual chemokine receptor knockout in atherosclerosis mouse models

Gene KO Mouse Model Effect Reference

CCR1 Knockout bone marrow transfer intoLDLR deficient mice fed a high-fat diet

No effect at 8 weeks. 70% increase inlesion size in thoracic aorta at 12 weeks.

Potteaux et al., 2005

Apoe2/2 fed high-fat diet Increased plaque size, T cell and IFN-g content. Zernecke et al., 2006Wire injury in Apoe2/2 mice No effect Braunersreuther

et al., 2007bCCR2 Apoe2/2 fed a high-fat diet Smaller lesions with fewer macrophages Boring et al., 1998

Ccr22/2 bone marrow transplant intoApoE3-Leiden mice fed high-fat diet

86% reduction in lesion size Guo et al., 2003

Transfer of Ccr22/2 bone marrow into Apoe2/2

mice with established lesionsNo inhibition of lesion progression comparedwith WT bone marrow transfer

Guo et al., 2005

CCR5 Apoe2/2 fed a chow diet No effect on lesion size at 16 weeks Kuziel et al., 2003Apoe2/2 fed high-fat diet Smaller lesions with fewer macrophages and T cells Potteaux et al., 2006Wire injury in Apoe2/2 mice Reduced neointima formation associated

with increased IL-10 productionZernecke et al., 2006

Knockout bone marrow transfer intoLDLR-deficient mice fed a high-fat diet

No effect on lesion size at 8 weeks, 30% fewermacrophages. Small reduction in lesion sizeat 12 weeks. Increased collagen content

Braunersreutheret al., 2007b

CCR6 Apoe2/2 fed high-fat diet. 40% smaller lesions with fewer macrophagesat 16 weeks, circulating inflammatorymonocyte numbers reduced

Wan et al., 2011

CCR7 Apoe2/2 fed a high-fat diet 16 weeks 1 2week treatment with LXR agonistto promote regression

LXR agonist induced emigration of CD681 cellsfrom WT plaques but no effect inCCR7 KO, i.e., regression failed.

Feig et al., 2010

Ldlr2/2 fed a high-fat diet 50% smaller plaques in aorta after 12 weeks’ fatfeeding, decrease in plaque macrophage numbers,increased DC and T-cell numbers, probably dueto failed migration to 2° lymphoid organs.Reduced plasma IFN-g.

Luchtefeld et al., 2010

Apoe2/2 fed a high-fat diet for 16weeks followed by 4 week statin treatment

Statins-induced emigration of CD681 cells fromWT plaques but not in CCR7 KO.

Feig et al., 2011b

70 White et al.

effect on lesion size but was associated with a reductionin plaque macrophage content (Potteaux et al., 2006).Taken together, the published data on CCR5 in mousemodels of atherosclerosis is not as impressive as theresults obtained by ablation or targeting of CCR2 (orCX3CR1), and currently CCR5 is not seen as a prom-ising target for therapeutic intervention in cardiovas-cular disease.The role of CCR7 and its ligands CCL19 and CCL21

in lymphocyte trafficking, DC migration, and lympho-cyte motility in the context of mounting adaptiveimmune responses is well established (Gunn et al.,1999; Robbiani et al., 2000; Worbs et al., 2007). Therole of CCR7 in atherogenesis is, however, morecontroversial due to the demonstration that CCR7 isimportant for macrophage emigration out of estab-lished atherosclerotic plaques in an aortic transplan-tation model of atherosclerosis (Trogan et al., 2006;Feig et al., 2010, 2011a). Ablation of the CCR7 gene inLdlr2/2 mice followed by 12 weeks of a high-fat dietwas associated with reduced plaque size and reducednumbers of both CD681 cells (macrophages) andCD11c1 cells [macrophages and DCs (Luchtefeldet al., 2010)]. An intriguing recent publication fromthe Fisher and Garabedian laboratories demonstratedthat Apoe2/2 mice a high-fat diet and treated witheither atorvastatin or rosuvastatin demonstrated nochange in atherosclerotic plaque area but did showsignificant reductions in CD681 macrophage stainingarea (Feig et al., 2011b). Plaques from statin-treatedanimals had reduced CCL2 expression compared withcontrol animals but showed increased CCR7 mRNAexpression. The statin-induced decrease in CD681

macrophages was not seen in Ccr72/2 Apoe2/2 micetreated with atorvastatin or rosuvastatin. The authorswent on to show that statin treatment increasedmacrophage CCR7 expression via a sterol regulatoryelement (SRE) within the CCR7 promoter.Somewhat surprisingly given the focus on CCR9

expressing cells in mucosal immunity, Abd Alla et al.(2010) demonstrated that RNA-interference mediatedknockdown of CCR9 in hematopoietic progenitor cellsof Apoe-deficient mice retarded atherosclerotic lesiondevelopment. Supporting evidence for a role for theCCL25/CCR9 axis in atherogenesis came from demon-stration of CCR91 and CCL251 cells in human athero-sclerotic lesions. It will be interesting to determine moreprecisely which cell types in atherosclerotic plaquesexpress CCR9 and whether genetic deletion or antibodyblockade of CCL25 and CCR9 similarly affects athero-genesis in Apoe2/2 and Ldlr2/2 mouse models.The functions of key CC chemokine receptors that

have been well defined in atherogenesis are summa-rized in Fig. 3.4. Key Chemokine Receptors as Drug Targets.

Many of the CC chemokine receptors discussed pre-viously as promising candidates for therapeutic

intervention in atherogenesis on the basis of knockoutmouse studies have been targeted in animal models ofatherosclerosis using small molecule antagonists, N-terminal deleted or modified chemokines, monoclonalantibodies, and viral CC chemokine binding proteins.CC chemokine receptors targeted in animal models ofatherosclerosis using small molecules include targetingof CCR2 using GSK 134486B in Apoe2/2 mice feda high-fat diet and implanted with angiotensin II-containing osmotic mini pumps (Olzinski et al., 2010),oral delivery of the CCR2 antagonist INCB-3344 inApoe2/2 mice (Aiello et al., 2010), targeting of bothCCR2 and CCR5 with a nonpeptide small molecule inApoe2/2 mice with an angiotensin II mini pump (Majoret al., 2009), and TAK-779 targeting of both CCR5 andCXCR3 in Ldlr2/2 mice (van Wanrooij et al., 2005).Peptides used to antagonize CC chemokine activity inanimal models of atherosclerosis include a peptidethat disrupts CXCL4-CCL5 heterodimerization(Koenen et al., 2009). Mutated chemokines tested fortheir antiatherogenic properties in mouse models ofatherosclerosis include the CCL5 variant [44AANA47](Braunersreuther et al., 2008), met-RANTES (Veillardet al., 2004), and N-terminally deleted CCL2 (Inoueet al., 2002). Blocking monoclonal antibodies (mAbs)tested in animal models of atherogenesis includea reagent that blocks murine chemokines CCL2 andMCP-5 (Lutgens et al., 2005). Broad spectrum CCchemokine blockade in Apoe2/2 mice has been testedusing in vivo delivery of a viral CC chemokine bindingprotein derived from poxvirus (known as 35K or vCCI)(Bursill et al., 2004; Bursill et al., 2009).

Despite the wealth of intervention studies in pre-clinical models so far there is only one reportedexample of CC chemokine receptor blockade con-ducted in patients at increased risk of cardiovasculardisease using the anti CCR2 blocking monoclonalantibody MLN1202 generated by Millennium (Gilbertet al., 2011). The double-blind placebo trial ofMLN1202 recruited 112 patients with two or morerisk factors for coronary artery disease who hadelevated levels of C-reactive protein. Patients receivinga single dose of the anti CCR2 humanized monoclonalantibody displayed significantly decreased levels ofplasma CRP between 4 and 12 weeks post treatmentand a transient 70% decrease in circulating monocytenumbers. This trial showed that a single dose ofMLN1202 is generally well tolerated but did not allowconclusions to be drawn about the efficacy of thisreagent to reduce monocyte recruitment into athero-sclerotic lesions or decrease the incidence of cardiovas-cular disease.

Thus, although preclinical animal models of athero-sclerosis have been critical in determining pathologicmechanisms and in identifying the key chemokinesinvolved, this is yet to translate into clinical benefit.It remains to be seen whether chemokine blockade,


in a disease spanning several decades, could be aviable therapeutic strategy. Any future therapywould need to add value to existing treatments, inparticular the use of statins, which may prove challeng-ing. Short-term targeting of chemokines, for example toprevent the accelerated atherogenic processes oftenoccurring following angioplasty, stenting, or organtransplant, may be a more realistic aim.

B. Rheumatoid Arthritis

1. Summary of Pathology. RA is a chronic in-flammatory autoimmune condition characterized bysynovitis, pannus formation, joint tenderness, andstructural damage/destruction of bone, cartilage, andligaments. The inflammation of the synovial tissue

lining the joints leads to the development of symmet-rical polyarthritis, which tends to manifest in the wristand small joints of the hands. RA affects 0.2–1% of theglobal population, which illustrates the need for thedevelopment of effective therapeutic interventions(Imboden, 2009). The severity of RA can vary amongpatients with some displaying a mild self-limitingdisease while in others a more chronic progressivedisease may persist, which can eventually lead to totalloss of joint function and permanent disability (Choet al., 2007). In addition to joint inflammation/destruction, other systems, including the cardiovascu-lar and respiratory, may also be affected, which, takentogether, can account for the increased morbidity/mortality associated with RA (van Vollenhoven, 2009).

Fig. 3. Diagram emphasizing the role of critical CC chemokine receptors in key aspects of atherogenesis. The normal endothelium in a healthy arteryprovides a smooth surface for blood flow and has an essential role in maintaining vascular tone. Numerous atherogenic risk factors can induceendothelial dysfunction, particularly at sites of turbulent blood flow. This leads to activation of endothelial cells, inducing expression of adhesionmolecules and presentation of chemokines on GAGs, in particular CCL2. Monocytes expressing CCR2 and Th cells enter the blood vessel wall and areretained in the subendothelial space. Monocytes differentiate into macrophages and begin to take up oxidized LDL accumulated in the vessel wallbecoming foam cells. As the lesion progresses, continued monocyte (via CCR2 and CCR5) and Th cell recruitment (via CCR5) occurs, and smoothmuscle cells are recruited from the tunica media into the tunica intima to form a fibrous cap overlying the plaque. In the regressing lesion, foam cellsupregulate CCR7, facilitating emigration either via lymphatic vessels or reverse transmigration across the endothelium. Although other chemokinereceptors have important roles in atherogenesis (e.g., CXCR3 for Th cell recruitment, CX3CR1 for monocyte recruitment and survival, and CXCR2 formonocyte adhesion) these are outside the scope of this review. We refer the reader to a more detailed review of individual chemokines in atherogenesis(Braunersreuther et al., 2007a).

72 White et al.

Chronic inflammation of the synovium drives theprocess of cartilage and bone destruction through therelease of a variety of mediators, including chemo-kines, matrix metalloproteinases (MMPs), cytokines,and growth factors. This persistent inflammationcontinues as a result of dysregulation in mechanismsinvolved in the resolution of inflammation, thusleading to continual activation of both innate andadaptive immune systems.Animal models of autoimmune arthritis have been

an invaluable tool in furthering our understanding ofsome of the key mechanisms involved in the patho-genesis of human RA, such as the involvement ofspecific cellular subsets and pro inflammatory media-tors. Furthermore, animal models have proven to befundamental in testing and developing novel thera-peutics used in the treatment of RA, for example, thedevelopment of biologics such as those directedagainst TNFa (van den Berg, 2001, 2009). A varietyof experimental arthritis models are currently usedwith some better characterized then others. Examplesinclude those that require immunization with antigen/proteoglycan such as collagen-induced arthritis (CIA)(Courtenay et al., 1980), aggrecan-induced arthritis(Finnegan et al., 1999), and streptococcal cell wallarthritis (Koga et al., 1985) and those that are inducedwith adjuvant such as complete Freund’s adjuvant(CFA) and pristane (Hopkins et al., 1984) and thoseinduced by serum transfer such as the K/BxN model(Kouskoff et al., 1996).2. Current Treatments. RA along with other in-

flammatory autoimmune conditions has been tradi-tionally treated with a combination of glucocorticoids(e.g., prednisolone), gold, and nonsteroidal anti-inflammatory drugs [NSAIDs (Malemud, 2009)]. How-ever, treatment with NSAIDs often results in gastricand cardiac side effects and provides only symptomaticrelief rather than targeting disease progression (Scottet al., 2010). The introduction of disease-modifyingantirheumatic drugs (DMARDs) overhauled existingdrug therapies, and to date these remain the leadingchoice for the treatment of RA. They have been shownto reduce joint swelling and pain and limit jointdamage, thereby improving overall function (Malemud,2009). When first diagnosed with active RA, approxi-mately 50% of patients are started on methotrexate,which still remains the most widely used DMARD(Suarez-Almazor et al., 2000). In some patients,combinational therapy with other DMARDs includinghydroxychloraquine, sulfasalazine, and leflunomidehas proven to be highly effective (Strand et al., 1999).However, many patients who have been on a singleDMARD or a combination over a long period of timecan develop adverse side effects and/or becomeunresponsive.More recently, the advent of biologic therapies (bio-

logics), which target proinflammatory cytokines such as

TNFa have revolutionized the current treatments avail-able for RA. In the case of RA, biologics are prescribedonly when treatment with NSAIDs or DMARDs hasfailed. There are three anti-TNF therapies currentlyapproved in the UK for treatment of RA: etanercept,a fusion of soluble TNFR with the Fc domain of humanIgG1; infliximab, a chimeric monoclonal antibody(mAb) against TNFa; and adalimumab, a fully humanmAb [reviewed in (Khraishi, 2009]. Two more recentlydeveloped anti-TNF drugs—certolizumab, a PEGylatedFab9 fragment of a humanized mAb, and golimumab,a humanized mAb administered monthly—can also beused if other anti-TNFs have failed. Several studieshave shown that patients treated with an anti-TNFaconcomitantly with MTX display more effective diseasesuppression compared with patients treated with MTXalone (Maini et al., 1999; Weinblatt et al., 1999;Keystone et al., 2004). Moreover, treatment with theanti-TNFa was also shown to inhibit the progression ofjoint bone erosion in RA patients (Keystone et al.,2004).

Other cytokines targeted by biologics include IL-1and IL-6, which are inhibited by the soluble antago-nist anakinra and the mAb toculizumab, respectively,which both block receptors for these cytokines(Bresnihan, 1999; Salliot et al., 2011). Other biologicsapproved for use in the UK include abatacept, an Fcfusion protein of CTLA-4 that binds CD80-CD86 toprevent T-cell costimulation (Ostor, 2008) and rituximab,a chimeric monoclonal antibody that binds CD20 onB cells to induce depletion of these cells via antibody-dependent cellular cytotoxicity and complement depen-dent cytotoxicity (Dass et al., 2006; Nakou et al., 2009).Severe side effects of biologics can include susceptibilityto serious infections such as tuberculosis and rare allergicreactions.

3. Evidence Supporting a Role for CC Chemokinesin Development of Pathology. Several CC chemokines(summarized in Fig. 4) can readily be detected in RAsynovial fluid and tissue biopsies. CCL2, a potentchemoattractant for monocytes/macrophages, is highlyelevated in synovial fluid and sera of RA patients (Kochet al., 1992; Akahoshi et al., 1993). Ex vivo culture ofsynovial fibroblasts stimulated with IL-1, TNF-a, orIFN-g results in the production of CCL2 (Koch et al.,1992; Villiger et al., 1992; Hachicha et al., 1993). CIAcarried out in rats was shown to generate increasedlevels of CCL2 in lavage fluid collected from the joints,in addition to increased CCL2 mRNA expression insynovial tissue. Administration of a neutralizing mAbtargeting CCL2 was shown to reduce ankle swelling by30% when compared with controls. This reduction inswelling was associated with a decrease in the numberof monocyte/macrophages recruited to the joints (Ogataet al., 1997). In the MRL-lpr mouse model of arthritis,a strain of mice that spontaneously develop arthritisresembling human RA, daily administration of a CCL2


antagonist, MCP-1(9-76), prevented the onset of dis-ease in these animals. When administered therapeu-tically, a reduction in clinical scores was also observedboth macroscopically and histologically (Gong et al.,1997a).CCL3, a potent chemoattractant of lymphocytes,

monocytes, and eosinophils, can be readily detected insynovial fluid and tissue of RA patients (Matsui et al.,2001; Ruth et al., 2003). RA fibroblasts stimulated withIL-1, TNF-a, and IL-18 induce CCL3 production, withpeak levels observed after 48 hours. Neutrophilsisolated from synovial fluid have also been reportedto contain increased levels of CCL3 protein and mRNAcompared with peripheral blood neutrophils (Hatanoet al., 1999). Infusion of CCL3 antibodies in mice with

CIA was shown to delay the onset and reduce theseverity of disease (Kasama et al., 1995).

CCL20, a chemoattractant of lymphocytes andmonocytes, is detected in large quantities in both RAsynovial fluid and tissue (Matsui et al., 2001; Ruthet al., 2003). Synovial tissue fibroblasts produce CCL20in response to a range of cytokines, including TNF-aand IL-17 (Chabaud et al., 2001; Matsui et al., 2001;Ruth et al., 2003).

CCL5 mRNA expression in synovial fibroblasts isincreased following stimulation with IL-1 or TNF-a(Rathanaswami et al., 1993; Hosaka et al., 1994).These effects can be augmented with INF-g or sup-pressed by IL-4 (Rathanaswami et al., 1993). Immu-nohistochemical analysis also revealed elevated CCL5

Fig. 4. An overview of CC chemokine-mediated leukocyte recruitment and retention in RA. This figure depicts the plethora of cells involved in theinflammatory processes that take place in the RA synovial compartment. The normal joint consists of a thin synovial membrane that spans the ends ofthe joints and is the innermost layer of the joint capsule. During RA, CC chemokines generated locally by activated cells, including endothelial cells,resident macrophages, and synovial fibroblasts, lead to recruitment of innate (e.g., monocytes) and adaptive immune cells (e.g., T and B cells) thatexpress a repertoire of CC chemokine receptors. The synovial membrane undergoes a dramatic structural change with proliferation of resident synovialfibroblasts in response to chemokines and other mediators leading to synovial hyperplasia. Chemokines induce synovial fibroblasts to produce furtherinflammatory cytokines and release tissue-degrading MMPs leading to pannus formation and cartilage destruction. Osteoclast accumulation withinthe joint leads to bone resorption, often causing irreversible tissue damage and disability. The continued production of CC chemokines and otherinflammatory cytokines and the failure of endogenous repair and resolution mechanisms drive the continual activation of both innate and adaptiveimmune systems.

74 White et al.

expression by macrophages present within RA synovialtissue (Volin et al., 1998). Treatment with an anti-CCL5 antibody ameliorates rat adjuvant- inducedarthritis and was shown to be as effective as the NSAIDindomethacin in this model (Barnes et al., 1998).In human RA a range of CC chemokine receptors

including CCR1-7 have been reported as expressed byleukocytes found in synovial fluid, tissue, and periph-eral blood (Loetscher et al., 1998; Qin et al., 1998;Katschke et al., 2001; Ruth et al., 2001; Haringmanet al., 2006b; Szekanecz et al., 2006, 2010). The use ofantibodies against CCR2 and CCR5 was recentlyreported to have failed in inhibiting synovial fluid-induced monocyte chemotaxis. Interestingly the use ofeither an anti-CCR1 Ab or a CCR1 antagonist (BX471)inhibited synovial fluid-induced monocyte chemotaxis,highlighting this receptor as a potential target inreducing monocyte recruitment to RA synovium (Lebreet al., 2011). In terms of animal models, conflictingevidence surrounding CC chemokine receptors hasbeen reported. Treatment with Met-RANTES, a CCR1/CCR5 antagonist, was reported to reduce the incidenceof murine CIA in a dose-dependent manner. Animalsthat did go on to develop disease were shown to havereduced clinical scores following treatment (Plater-Zyberk et al., 1997). A nonpeptide antagonist for CCR5and CXCR3, TAK-779, administered before the onset ofclinical signs of CIA, was shown to reduce diseaseincidence and severity in addition to a reduction inhistologic scores (Yang et al., 2002). CCR2 blockadewith a neutralizing Ab during the initiation of CIA wasreported to improve clinical scores; however, whenadministered therapeutically exacerbation in clinicaland histologic scores was observed (Bruhl et al., 2004).CIA performed in Ccr2-deficient mice resulted ina more severe disease profile when compared withWT controls (Quinones et al., 2004). In another CIAstudy, treatment with a small molecule inhibitor ofCCR2 (MK-0812) was shown not to have any effect onthe disease severity (Min et al., 2010). A recent studyfrom Jacobs et al. (2010) systematically revieweda range of chemokine receptors and their involvement,if any, in the K/BxN serum transfer model. The resultsreported showed that mice deficient in a range of CCchemokine receptors (CCR1-7) displayed no differencein disease profile when compared with littermatecontrols (Jacobs et al., 2010). Collectively these studieshighlight the need for caution when interpreting datarelating to CC chemokine receptor blockade in animalmodels of arthritis.4. Key Chemokine Receptors as Drug Targets.

To date, a limited number of studies have investigatedthe effects of chemokine and chemokine receptor block-ade in human RA. A CCR1 antagonist, CP481,715, thatshowed promising preclinical efficacy through itsability to inhibit synovial fluid-induced monocytechemotaxis, was tested in a small phase Ib trial

(Gladue et al., 2003; Clucas et al., 2007). Patients withactive RA who were administered every 8 hours withCP481,715 over a course of 2 weeks showed a reductionin the total number of macrophages within the synovialintimal lining and an overall reduction in CCR11 cellswithin the synovium. Furthermore, one-third of allpatients showed improved clinical scores by reachingACR20 criteria following treatment (Gladue et al.,2003). A human CCR2 blocking antibody (MLN1202)tested in phase IIa clinical trial was shown to reducecirculating levels of CD141 monocytes; however, noclinical improvements were observed in active RApatients on the basis of ACR criteria (Vergunst et al.,2008). Similarly, in another randomized controlledtrial, no clinical benefit was observed in RA patientsfollowing monoclonal antibody treatment (ABN912)against the CCR2 ligand CCL2 (Haringman et al.,2006a). Maraviroc, a CCR5 antagonist recently usedin a phase IIa randomized, double-blind placebo-controlled trial, showed no efficacy in patients withactive RA on background methotrexate (Fleishakeret al., 2012). Similar negative outcomes in smallerclinical trials have also been previously reported withother CCR5 antagonists, AZD5672 and SCH351125, inRA (Gerlag et al., 2010; van Kuijk et al., 2010).Ongoing or completed clinical trials targeting chemo-kine receptors in RA and other chronic inflammatorydiseases are summarized in Table 3.

C. Obesity, Metabolic Syndrome, andType 2 Diabetes

1. Summary of Pathology. Over the last fewdecades, the incidence of obesity worldwide has in-creased dramatically, with almost 1 in 4 adults in theUK classed as obese in 2009 (http://www.dh.gov.uk/en/Publichealth/Obesity/DH_078098). Direct costs to theUK healthcare system are estimated at £4.2 billion peryear when costs for treatment of obesity-relateddiseases, including type 2 diabetes, cardiovasculardisease, and some cancers, are included. Metabolicsyndrome describes the coincidence of multiple cardio-vascular risk factors, including obesity, hyperglycemia,insulin resistance, hypertension, and hyperlipidemia.

It is now recognized that persistent, low-gradeinflammation provides a causal link between obesityand metabolic disorders such as insulin resistance andtype 2 diabetes. In 2003, two studies demonstratedthat obesity due to genetic factors or high-fat feeding inmice is associated with macrophage infiltration intoadipose tissue (Weisberg et al., 2003; Xu et al., 2003).Furthermore, these macrophages produce large amountsof TNFa, IL-6, and inducible nitric oxide synthase [iNOS(Weisberg et al., 2003)]. Further profiling has demon-strated that macrophages in lean adipose express genesassociated with alternative "M2" activation and producethe anti-inflammatory cytokine IL-10 to protect adipo-cytes from TNFa-induced insulin resistance (Lumeng


http://www.dh.gov.uk/en/Publichealth/Obesity/DH_078098

http://www.dh.gov.uk/en/Publichealth/Obesity/DH_078098

TABLE

3Summaryof

compo

unds

targetingch

emok

inereceptorsthat

have

enteredclinical

trials

Targe

tDru

gOther

Nam

esMan

ufacturer

Typ

eof

Molecule

Indication

HighestClinical

Phas

eCurren

tStatus

Pub

lish

edData

CCR1

CCX35

4Chem

ocen

tryx

andGSK

Smallmolecule

RA

Phas

eII

Ongo

ing

deve

lopm

ent

Dairagh

iet

al.,20

11SH

T04

268H

BAY86

-504

7,ZK81

1752

Bay

erSch

ering

Smallmolecule

End

ometriosis

associated

pelvic

pain

Phas

eII

Unkn

own

CP48

1,71

5Pfizer

Smallmolecule

RA

Pha

seII

Failed

Gladu

eet

al.,20

03;

Clucas

etal.,20

07;

MLN38

97Millennium

Smallmolecule

RA

Pha

seII

Failed

Vergu

nst

etal.,20

09BX47

1Berlex/Sch

ering

Smallmolecule

MS

Pha

seII

Failed

AZD48

18AstraZen

eca

Smallmolecule

COPD

Pha

seII

Failed

Kerstjens

etal.,20

10CCR2

CCX14

0Chem

ocen

tryx

Smallmolecule

Typ

e2diab

etes

and

diab

etic

neph

ropa

thy

Phas

eII

Ongo

ing

deve

lopm

ent

Sulliva

net

al.,20

12MLN12

02Millennium

andTak

eda

Human

ized

mon

oclonal

antibo

dyMS,b

onemetas

tases,

athe

rosclerosis

RA

Phas

eII

Ongo

ing

deve

lopm

ent,

failed

inRA

Vergu

nst

etal.,20

08;

Gilbe

rtet

al.,20

11PF-413

6309

INCB87

61Pfizeran

dIn

cyte

Smallmolecule

Chr

onic

pain,live

rfibrosis

(pha

seI)

Phas

eII

Ongo

ing

deve

lopm

ent

Xue etal.,20

12b

BMS-741

672

Bristol-M

yers

Squ

ibb

Smallmolecule

Insu

linresistan

ce,d

iabe

tic

neur

opathicpa

inPhas

eII

Unkn

own

INCB86

96In

cyte

Smallmolecule

MS

Phas

eI

Unkn

own

INCB32

84In

cyte

Smallmolecule

MS,R

A,Ins

ulin

resistan

cePha

seII

Unk

nown

Xue et

al.,20

12a

MK-081

2Merck

Smallmolecule

MS,R

APha

seII

Failed

Xia

and

Sui,20

09CCR3

GW76

6944

GSK

Smallmolecule

Asthma

Phas

eII

Ongo

ing

deve

lopm

ent

TPIASM8

Top

igen

andPharmax

isAntisense

against

CCR3

andIL

-3,I

L-5

and

GM-C

SF

receptors

Asthma

Phas

eII

Ongo

ing

deve

lopm

ent

Imao

kaet

al.,20

11

DPC16

8Bristol-M

yers

Squ

ibb

Smallmolecule

Asthma

Phas

eI

Unkn

own

DeLucca

etal.,20

05CCR4

Mog

amulizu

mab

KW-076

1Kyo

waHak

koKirin

centerCoan

dAmge

nHum

anized

defucosylated

mAbwithADCC

activity

Adu

ltT-cell

leuk

emia/ly

mph

oma

Phas

eII

Ongo

ing

deve

lopm

ent

Anton

iu,2

010;

Yam

amoto

etal.,20

10CCR5

Marav

iroc

Pfizer

Smallmolecule

RA

Pha

seII

Failed

Fleisha

ker

etal.,20

12GSK70

6769

GSK

Smallmolecule

RA

Pha

seI

Terminated

BMS-813

160

Bristol-M

yers

Squ

ibb

Smallmolecule

?Pha

seI

Ong

oing

deve

lopm

ent

Norman

,20

11

AZD56

72AstraZen

eca

Smallmolecule

RA

Pha

seII

Failed

Gerlag

etal.,20

10CCR9

Vercirn

onTraficet-EN,

CCX28

2,GSK16

0578

6

Che

mocen

tryx

andGSK

Smallmolecule

Crohn

’sdiseas

ePha

seIII

Ong

oing

deve

lopm

ent

Walters

etal.,20

10

76 White et al.

et al., 2007). Diet-induced obesity causes these cells toundergo a phenotypic switch to classically activated"M1" macrophages capable of producing high levels ofproinflammatory cytokines that induce insulin resis-tance in adipocytes (Lumeng et al., 2007).Insulin resistance describes the inability of meta-

bolic tissues to respond to the actions of insulin and isseen as impaired glucose uptake into muscle andenhanced lipolysis in adipose tissue. This results inhyperglycemia and hyperlipidemia, with multiplepathologic consequences. Subsequently, peripheral in-sulin resistance results in increased insulin secretionby b cells of the pancreas, causing hyperinsulinemia.Eventually, b-cell exhaustion may result in sustainedhyperglycemia and type 2 diabetes.2. Current Treatments. Therapy for metabolic syn-

drome generally involves lifestyle and dietary advice toreduce obesity as well as drug treatment to reduce riskfactors, such as hypertension, hypercoagulation, andhigh plasma cholesterol. More drastic measures toreduce weight include gastric surgery or use of orlistat(GlaxoSmithKline), an oral drug that irreversiblyinhibits gastric and pancreatic lipases, thereby re-ducing absorption of dietary fat. Unfortunately, un-pleasant side effects are common, potentially reducingpatient compliance. The only other pharmacologicaltreatments licensed for obesity treatment, sibutr-amine, a serotonin and noradrenaline reuptake in-hibitor, and rimonabant, a CB1 antagonist that actscentrally to reduce appetite, were withdrawn from themarket due to an increased rate of adverse cardiovas-cular events and psychiatric side effects, respectively.In patients who have progressed to type 2 diabetes,

there are several drug treatments available. Metfor-min lowers blood glucose via numerous mechanisms,including increasing glucose uptake and utilization inmuscle as well as reducing hepatic glucose productionvia a mechanism involving activation of AMP activatedprotein kinase (AMPK) (Zhou et al., 2001). It is usuallywell tolerated and is the first drug of choice in themajority of obese type 2 diabetic patients who fail tocontrol dietary intake and lose weight. Sulfonylureassuch as glibenclamide act directly on b cells in thepancreas to stimulate insulin release and therefore areonly useful if residual b cell activity is present. Theyare generally well tolerated but can lead to prolongedand severe hypoglycemia, particularly when long-actingsulfonylureas are prescribed. Thiazolidinediones such aspioglitazone reduce peripheral insulin resistance viatheir action on the peroxisome proliferator-activatedreceptor g (PPARg), a nuclear receptor that regulatesglucose and lipid metabolism. In adipocytes, activationof PPARg by endogenous agonists or pioglitazonecauses adipocyte differentiation, increased lipogene-sis, and uptake of fatty acids and glucose. Impor-tantly, PPARg also controls alternative activation ofmacrophages, thus reducing adipose inflammation

and improving insulin resistance (Odegaard et al.,2007). Another widely prescribed thiazolidinedione,rosiglitazone, was withdrawn in 2010 due to an asso-ciation with increased cardiovascular events. If oralmedications fail to sufficiently control blood glucoselevels, insulin may be prescribed.

Despite the existence of several therapies to treatdiabetes, there remains a significant unmet clinicalneed. As diabetes is a chronic, progressive condition,most of the established therapies eventually becomeless efficacious over time and new medications orincreased doses must be prescribed. Also, apart frommetformin, all the antidiabetic medications causeweight gain, meaning that the underlying inflamma-tory pathology occurring in obese adipose tissue willcontinue to worsen.

3. Evidence Supporting a Role for CC Chemokines inDevelopment of Pathology. There is strong evidencesuggesting a role for the CCL2-CCR2 axis in adiposeinflammation and obesity. CCL2 is constitutivelyexpressed by primary human preadipocytes and isupregulated by TNFa treatment in mature adipocytes(Gerhardt et al., 2001). Levels of CCL2, both systemi-cally and in white adipose tissue, are increased in fat-fedmice and plasma CCL2 levels correlate with body weight(Takahashi et al., 2003). In diabetic and nondiabeticobese patients, treatment with the thiazolidinedionerosiglitazone leads to a reduction in plasma CCL2 levels,suggesting a mechanistic basis for the efficacy of thesedrugs (Mohanty et al., 2004). CCL2 may have a directrole in insulin resistance because CCL2 added to matureadipocytes inhibits insulin-stimulated glucose uptakeand expression of several genes required for adipo-genesis (Sartipy and Loskutoff, 2003).

To confirm a role for CCR2 in obesity and adiposeinflammation, Ccr22/2 mice were fat fed for 24 weeks,and their metabolic phenotype was studied (Weisberget al., 2006). Ccr22/2 mice had a 15% lower body massthan wild-type animals after 24 weeks fat feeding andwere observed to consume fewer calories than Ccr21/1

animals. Furthermore, obese Ccr22/2 mice had lowerfasting blood glucose and insulin levels and were moreinsulin sensitive than control mice. CCR2 also seemedto have a direct role in macrophage recruitment, be-cause knockout animals had significantly fewer adi-pose tissue macrophages than wild-type mice. Finally,14-day treatment of wild-type obese mice with a CCR2small molecule antagonist (INCB-3344) reduced fast-ing glucose and insulin concentrations and increasedinsulin sensitivity. Adipose macrophage numbers werealso significantly reduced by CCR2 antagonism, sug-gesting that relatively short treatment could alteradipose content.

Another chemokine with a known role in obesity,adipose inflammation, and diabetes is CCL5. BothCCL5 and one of its receptors CCR5 were found to beupregulated at the mRNA level in adipose tissue of


male fat-fed mice (Wu et al., 2007). This was associatedwith increased numbers of T cells in obese adiposetissue, and the authors demonstrated that conditionedmedium from explanted adipose tissue of fat-fed micecould chemoattract T cells and this activity wasblocked by a neutralizing CCL5 antibody. In vitro,both murine and human adipocytes stimulated withTNFa produced abundant CCL5 mRNA. Finally, sub-cutaneous adipose tissue from obese individuals withmetabolic syndrome had significantly higher levels ofCCL5 mRNA compared with lean subjects, and thelevel of CCL5 was positively correlated with body massindex. CCL5 expression was increased further invisceral adipose tissue of morbidly obese patients andcorrelated with expression of the T cell marker CD3and the macrophage marker CD11b.Serum levels of CCL5 were also found to be elevated

in cohorts of patients with either impaired glucosetolerance or type 2 diabetes compared with controlsubjects (Herder et al., 2005). To assess whether CCL5has a causative role in diabetes, the associationbetween CCL5 serum levels, CCL5 polymorphisms,and the incidence of type 2 diabetes was investigated(Herder et al., 2008). Six SNPs in noncoding regions ofthe CCL5 gene (promoter, intronic, and 39 flankingregion) were analyzed for any correlation with serumCCL5 levels. The rarer alleles of four SNPs wereassociated with significantly lower serum CCL5 levels,but after adjustment for confounding factors, neitherthe CCL5 serum level or genotype were correlated with

the incidence of type 2 diabetes. The authors concludedthat CCL5 was elevated as a consequence of hypergly-cemia, but could not exclude a role for CCL5 in thepathogenesis of adipose inflammation.

A potential pathogenic role for CCL5 in humanadipose tissue was investigated by Keophiphath et al.(2010). The authors hypothesized that CCL5 may havea role in mononuclear cell recruitment and in pro-moting the survival of adipose tissue macrophagesfrom apoptosis induced by free cholesterol. CCL5 wasfound to induce both adhesion and transmigration ofmonocytes through an endothelial cell monolayer. Fur-thermore, macrophages in obese adipose tissue werefound in crown-like structures around adipocytes andwere shown to be apoptotic by terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) staining.In vitro induction of apoptosis in macrophages by freecholesterol loading [involving incubation of cells withfree cholesterol and an acyl-CoA cholesterol acyl trans-ferase (ACAT) inhibitor to block cholesterol esterifica-tion] could be reduced by treatment with CCL5. Takentogether, this suggests that a potential mechanism forthe action of CCL5 in obese adipose tissue is to bothrecruit and promote the survival of macrophages, thuscontributing to ongoing inflammation. The function ofCC chemokine receptors in adipose inflammation issummarized in Fig. 5.

4. Key Chemokine Receptors as Drug Targets.There is strong evidence from humans and animal mod-els that CCR2 may be a potential therapeutic target in

Fig. 5. CC chemokine functions in adipose inflammation. Classically activated macrophages in obese adipose tissue produce large amounts ofinflammatory cytokines, including IL-1b, TNFa, and IL-6, that induce insulin resistance in adipocytes leading to lipolysis and chemokine release.CCL2 released by adipocytes leads to continued monocyte recruitment from the blood, whereas CCL5 recruits T cells and promotes macrophagesurvival within adipose tissue. Accumulation and survival of monocyte/macrophages within inflamed adipose tissue leads to amplification of thisdamaging inflammatory cycle, eventually leading to systemic insulin resistance, metabolic syndrome, and diabetes.

78 White et al.

metabolic syndrome and diabetes. Several small mole-cule antagonists of CCR2 have been developed andtested in preclinical animal models (Shin et al., 2009;Kang et al., 2010; Sayyed et al., 2011). One of these,CCX140-B, developed by ChemoCentryx has completedphase II clinical trials (clinicaltrials.gov study identifierNCT01028963). The data are not currently publishedin a peer-reviewed journal, but the manufacturers citea dose-dependent decrease in plasma glucose and a signi-ficant decrease in HbA1c levels in patients receivingCCX140-B. This compound is now entering phase IItrials for diabetic nephropathy (clinical trials.gov studyidentifiers NCT01447147 and NCT01440257). The sta-tus of this and other CCR2 antagonists in clinical trialsis summarized in Table 3.Data from humans and mice suggest that CCR5 may

have a pathogenic role in metabolic syndrome and type2 diabetes, and licensed medications targeting CCR5already exist. Indeed, it has also been shown that theCCR5-D32 mutation is associated with better survivalin type 2 diabetic patients, suggesting that targeting ofCCR5 may be beneficial (Muntinghe et al., 2009). Toour knowledge, there are currently no studies assess-ing CCR5 antagonists in type 2 diabetes.

IV. Chemokine Receptor Drugs in Clinical Trials

A number of CC chemokine receptors have beentargeted for potential clinical benefit in inflammatorydisease. A summary of compounds currently in clinicaltrials is presented in Table 3.

V. Critical Assessment of Chemokine Receptorsas Anti-inflammatory Drug Targets

In 1998, the publication of a report showing thatdeletion of the chemokine receptor CCR2 in a mousemodel of atherosclerosis could dramatically reduce theinitiation of disease provided the first unequivocalevidence that chemokine receptors could be key targetsof new anti-inflammatory drugs (Boring et al., 1997).Since then, a plethora of studies, some of which wepresented above, have shown that chemokines playa nonredundant role in the development of multiplechronic inflammatory diseases. In this section, wereview the successes and failures of chemokine re-ceptor drug development to date and consider futureprospects for the field.Since the discovery that HIV cellular entry requires

a chemokine coreceptor (either CCR5 or CXCR4), it isno surprise that a large proportion of basic researchand clinical drug development has focused on these twotargets. Drugs targeting CCR5 (maraviroc) and CXCR4[plerixafor (Mozobil)/AMD3100] have been approvedfor use in HIV infection and for mobilization of he-matopoietic stem cells for transplantation, respec-tively, thus proving that drugs targeting chemokine

receptors can be well tolerated and demonstrate ther-apeutic efficacy. Furthermore, the development ofother orally administered drugs targeting inflamma-tory GPCRs, e.g., the CysLt1 leukotriene receptorantagonist montelukast orally used to treat asthma,demonstrates that in principle, targeting single in-flammatory GPCRs is a viable therapeutic strategy.However, 20 years since the description of receptorsfor the first known chemokine (IL-8, CXCL8), thereare still no marketed drugs targeting chemokine recep-tors for their presumed primary indication—as anti-inflammatories (Murphy and Tiffany, 1991). Indeed,a recently reported phase II trial utilizing maraviroc inpatients with RA already receiving methotrexate failed todemonstrate any improvement in clinical score comparedwith placebo (Fleishaker et al., 2012). This is perhaps notsurprising, because different regions of the CCR5 receptorare known to be involved in HIV binding and intracellularsignaling, and indeed maraviroc is reported to have noeffect on CCR5 signaling (Dorr et al., 2005).

Despite strong evidence from preclinical models de-monstrating a nonredundant role for CC chemokinereceptors in disease pathogenesis as well as the de-velopment of many potent receptor antagonists, whyhave drugs that target CC chemokine receptors so farfailed to yield any viable anti-inflammatory drugs?

A. CCR1

A number of clinical trials have targeted CCR1 in RA,but to date no compound has shown enhanced benefitcompared with established treatments (Szekaneczet al., 2011). The biology of CCR1 is complex, not leastbecause the most potent ligands for the receptor requireproteolytic activation in vivo at sites of inflammation.Several studies have shown that CCR1 knockoutanimals show an enhanced inflammatory response witha Th1 biased cytokine profile that often exacerbatesdisease progression—as is the case for the Apoe2/2

model of diet-induced atherosclerosis (Potteaux et al.,2005). CCR1 also seems to have a critical role inneutrophil recruitment, and blockade of the receptormay inhibit pathogen clearance. Although CCR1-mediated leukocyte recruitment seems to be associatedwith chronic inflammation rather than leukocytehomeostasis, it remains to be seen if CCR1 can betargeted for therapeutic benefit without skewing Th1/Th2 immune responses.

B. CCR2

Antagonism of CCR2 in type 2 diabetes and relatedpathologies seems to have shown promise in early clinicaltrials, but several compounds have failed in trials tar-geting RA or MS. In mice, CCR2 is known to have acritical role in regulating monocyte egress from the bonemarrow, which could make it a less attractive targetfor therapy. However, at least one CCR2 antagonist thathas shown clinical benefit in type 2 diabetes (CCX140-B)


http://clinicaltrials.gov

http://trials.gov

had no effect on blood monocyte levels in phase I trials.Thus, CCR2 remains a key target in metabolic in-flammation although anti-CCR2 trials in the settingof RA have been disappointing (Szekanecz et al., 2011).

C. CCR3

A large number of small molecule antagonistsagainst CCR3 have been developed and tested primar-ily in patients with asthma. However, no encouragingclinical data have been published so far. We do notknow whether this stems from a failure of animalmodels of allergic airway to predict good targets inhuman asthma, whether CCR3 blockade will only beefficacious in a subgroup of patients with asthma, or ifCCR3 blockade needs to be applied much earlier in thehuman disease process.

D. CCR4

CCR4 remains an interesting molecular target. Arecent study has suggested that CCR4 antagonists mayprove useful as vaccine adjuvants as a means ofbreaking peripheral tolerance induced by Treg cellsthat can hamper the efficacy of anticancer vaccines(Bayry et al., 2008; Pere et al., 2011). A monoclonalantibody against CCR4 (mogamulizumab) also exists,and trials are ongoing in patients with adult T-cellleukemia/lymphoma. This drug may also be developedin future by Amgen for the treatment of asthma, but asyet no clinical trials have been initiated (Antoniu,2010). However, in common with several other CCchemokine receptors, e.g., CCR9, the homeostatic roleof this chemokine receptor in leukocyte homing to theskin may limit long-term targeting of this receptoroutside of oncology.

E. CCR5

As discussed previously, CCR5 has been blocked asa coreceptor for HIV entry by maraviroc, showing thatit is possible to target CC chemokines for therapeuticbenefit in the infectious disease arena if not in thecontext of chronic inflammation. It should be notedthat at least two other anti-CCR5 drugs failed inclinical trials for HIV due to lack of efficacy (vicriviroc)or liver toxicity (apliviroc).

F. CCR6 and CCR7

Evidence from animal models suggests that the CCR6and CCR7 receptors have critical roles in immune ho-meostasis, particularly in intestinal immunity andlymphoid organization, respectively. Evidence thatthese receptors are important in inflammation isscarce, and it seems unlikely that they will proveuseful therapeutic targets. However, it is theoreticallypossible that agonism of the CCR7 receptor could provebeneficial in certain pathologies, e.g., in atherosclerosiswhere CCR7 has a key role in plaque regression (Troganet al., 2006; Feig et al., 2011b).

G. CCR8

The CCR8 receptor has a single known ligand, givinga simple one ligand–one receptor interaction, unlikemost chemokines. However, evidence for the role ofCCR8 in inflammation is weak, and this receptorremains relatively understudied. To our knowledge, noCCR8 antagonists have been developed for potentialtherapeutic application.

H. CCR9

A small molecule orally dosed antagonist targetingCCR9 [Traficet-EN (vercirnon)] has been developed byChemoCentryx (Walters et al., 2010). This compound iscurrently in phase III clinical trials for Crohn’s diseaseafter promising data were obtained from a phase II trial(although these data have not been published in a peer-reviewed journal). As detailed in II. CC ChemokineReceptors, I. CCR9, results obtained with CCR9 block-ade in animal models of colitis have provided conflictingresults and debate as to the suitability of CCR9 asa target for treatment of inflammatory bowel disease(IBD). At the heart of the debate is whether CCR9ligands are solely proinflammatory in colitis or whethertherapeutic blockade of CCR9 modifies the behavior ofgut-homing T lymphocyte populations to effectivelyreduce the recruitment of effector T-cell populationswhile sparing gut-homing Treg cells. One great ad-vantage of oral dosing CCR9 antagonists over usingtherapeutic antibodies to inflammatory cytokines orchemokines is that the dosage of the CCR9 blockadecan be modified and curtailed with "washout" of the anti-CCR9 drug within 48 hours. Given that the design ofreported phase II clinical trials was aimed at patientswith moderate to severe Crohn’s disease and that oneof the readouts was maintenance of clinical remission,CCR9 blockade may represent an important thera-peutic paradigm shift, i.e., long-term modification ofleukocyte homeostasis with an antichemokine drugrather than antibody-mediated anti-inflammatorytargeting of TNF or integrins. Future developmentsin this area are eagerly anticipated by chemokinebiologists, immunologists, and gasteroenterologists.

I. CCR10

As yet, no evidence suggests an inflammatory rolefor this receptor.

Despite the ongoing development of multiple chemo-kine receptor drugs and ongoing clinical trials, notablywith CCR2 and CCR9 antagonists, it is important toconsider why there has been a general failure totranslate promising data from in vitro and animalstudies into useful clinical therapies. One possiblereason is that we have overlooked the importance ofchemokine receptors in regulation and resolution inthe immune response. As seen with CCR4, Treg cells—not just effector cells—require chemokine receptors for

80 White et al.

efficient migration and hence maintenance of immunetolerance. In atherosclerosis, the CCR7 receptor hasbeen shown to be critical for exit of inflammatory cellsfrom sites of inflammation and for efficient dendriticcell homing and antigen presentation. We perhapshave underestimated the importance of other chemo-kine receptors in these processes, meaning thatantagonism of these receptors could hamper inflam-mation resolution.Another potential reason for clinical failures of

chemokine receptor drugs is the reliance on animalmodels, particularly mice, for identification of keytargets. Species differences in CC chemokine receptorbinding specificities have led to development of humanCC chemokine knock-in mice to test therapeuticefficacy and toxicity of small molecules active athuman but not mouse or rat CC chemokine receptors.Additionally, the use of transfected cells overexpress-ing chemokine receptors for initial stages of drugscreening may make it hard to translate findingsobtained in these cells to primary cell assays orpreclinical models. The fact that many chemokinereceptors may function on primary cells as homo- orheterodimers may mean that transfected cells lack keysignaling partners. It seems sensible to ensure thatearly drug screens should incorporate human primarycell assays to increase the likelihood of later successand assays should be conducted in the presence ofhuman plasma to better predict in vivo potency (Schalland Proudfoot, 2011).As discussed above another reason why CC chemo-

kine receptor blockade may have provided no newanti-inflammatory drugs may reside in the underap-preciated role of chemokine receptors in immunehomeostasis. It is clear that CCR4, CCR6, and CCR9mediate homeostatic leukocyte recruitment, and itcould be argued that CCR2 plays an important rolein homeostatic regulation of monocytes numbers inperipheral blood by mobilization of monocytes frombone marrow (Tsou et al., 2007). It is perhaps germaneto reflect on the fact that one of the few successfuldrugs that targets human chemokine receptors isplerixafor/AMD3100, which disrupts CXCR4 signalingthat is required for hematopoietic stem cell sequestra-tion in bone marrow niches.Another key consideration is that chemokine re-

ceptor knockout animals have been shown to haveraised circulating levels of their respective chemokineligands (Cardona et al., 2008), suggesting somescavenging roles of these receptors under homeostaticconditions. Thus, it is possible that antagonism ofchemokine receptors that prevents ligand binding mayincrease chemokine ligand levels. Because chemokinesgenerally bind to multiple receptors, these ligands maythen activate alternative receptors, abrogating theeffect of the antagonist. Targeting of multiple chemo-kine receptors may be required to overcome this

potential compensatory mechanism. To date, however,no strategies targeting multiple chemokine receptorshave approached clinical use, despite positive data inanimal models (Bursill et al., 2004; Shahrara et al.,2005; Millward et al., 2010).

Finally another important issue that needs to beconsidered when thinking about the paucity of novelanti-inflammatory drugs arising from research into CCchemokine receptors is the important issue of achiev-ing an effective therapeutic dose of chemokine blockadein randomized clinical trials. This issue was recentlyreviewed by Schall and Proudfoot through consider-ation of three different clinical trials of three differentanti CCR1 drugs (Schall and Proudfoot, 2011). Theauthors’ conclusion that anti-inflammatory effects ofCCR1 blockade will only be seen when antagonistoccupancy of the target receptor is greater than 98%over a 24-hour period sets the bar very high in terms ofreceptor "coverage" and, if true, will have importantconsequences for the development of drugs against thisclass of receptors.

Whatever the reason for a lack of therapeutic successto date, there remains a clear unmet clinical need fordevelopment of novel anti-inflammatory strategies. Itseems likely that with a wealth of available chemokinereceptor antagonists, useful drugs will be developed,but the examples of CCR5 and CXCR4 suggest thatthese may not be for the indications initially predictedfrom studies performed in animal models. A moremature understanding of the role of chemokines inboth homeostasis and inflammation resolution isneeded to inform future drug development and clinicaltrial design.

Authorship Contributions

Wrote or contributed to writing of the manuscript: White, Iqbal,Greaves.

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CC Chemokine Receptors and Chronic Inflammation--Therapeutic Opportunities and Pharmacological Challenges