Current Respiratory Medicine Reviews, 2005, 1, 15-32 15

Chemokines and their Receptors as Targets for the Treatment of COPD

Suzanne L. Traves and Louise E. Donnelly*

Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College London, DovehouseStreet, London, SW3 6LY, UK

Abstract: Chronic obstructive pulmonary disease (COPD) is a debilitating respiratory condition, characterisedby progressive, irreversible airflow obstruction. The major risk factor for development of COPD is cigarettesmoking, and the disease is predicted to become the 3rd leading cause of death by 2020. Currently, there are nopharmacological interventions that halt the progression of COPD; however one strategy is to reduce thechronic lung inflammation associated with this disease. An increased inflammatory infiltrate comprisingmacrophages, neutrophils and T-lymphocytes is a major hallmark of COPD. Furthermore, both macrophagesand neutrophils have the ability to cause all the pathological changes associated with COPD. Chemokines thatare elevated in sputum from COPD patients have the capacity to recruit neutrophils, the macrophage precursorcells, monocytes, and T-lymphocytes. Chemokines are considered predominantly chemotactic cytokineshowever; there is a growing body of evidence demonstrating that chemokines can also act as functionalantagonists thus leading to selective recruitment of inflammatory cells. Whilst inhibition of chemokinedependent recruitment of inflammatory cells via small molecule antagonists gives rise to potential treatmentsfor COPD, the discovery that chemokines are also natural antagonists could also be exploited in the ongoingsearch for treatment of this currently fatal disease.

Keywords: COPD, Chemokines, Receptors, Therapeutics, CXCR2, CXCR3.

INTRODUCTION In the UK, COPD causes over 30,000 deaths a year [3],in Europe, clinically relevant COPD affects 4-6% of adults[3], and in the USA, in 2000, an estimated 10 millionadults were diagnosed with COPD, yet 24 million peopleexhibited airflow obstruction thus highlighting the fact thatCOPD is under-diagnosed [1]. A model developed from theThird National and Examination Survey estimates that 3million people suffer from COPD in the UK [6]. It is notsurprising then, that in the UK, COPD is a frequent cause ofhospital admissions, costing more than £1 billion in directhealthcare costs each year [3]. The indirect cost of the diseaseamounts to more than £1.5 billion in disability, workabsence and reduced productivity in the UK [1], with 20million lost working days every year attributed to COPD[3]. This vast expenditure, coupled with the knowledge thatCOPD is on the rise in developed countries due to anincrease in population age and cigarette smoking [3], as wellas data which implicates that COPD starts at a much earlierage than previously thought [1], makes COPD a primedisease for research.

Chronic obstructive pulmonary disease (COPD) is theforemost cause of chronic morbidity and mortalityworldwide [1]. It is one of the most widespread diseases,increasing globally in prevalence [2] and is predicted tobecome the third most common cause of death [2, 3], andthe fifth most common cause of disability in the world by2020 [2, 3]. COPD is a disease of the lung and the generallyaccepted definition of this condition is that ‘COPD is adisease state characterised by airflow limitation that is notfully reversible. The airflow limitation is usually bothprogressive and associated with an abnormal inflammatoryresponse of the lungs to noxious particles and gases’ [4].The term COPD encompasses chronic obstructive bronchitis,with obstruction of the small airways, and emphysema, withenlargement of the airspaces and destruction of lungparenchyma (the part of the lungs beyond the airways atwhich gas exchange take place), loss of lung elasticity, andclosure of small airways [5]. Most patients with COPD haveall three pathophysiological features (chronic obstructivebronchitis, emphysema and mucus plugging), but therelative extent of each within individual patients may vary[5]. These factors result in COPD being frequentlyunderdiagnosed and undertreated [1]. The substantialmorbidity of COPD is currently underestimated byhealthcare professionals [1] and this coupled with itsconsideration as a self-inflicted (by smoking) disease,together with an irreversible disease process has resulted inCOPD being neglected by clinicians, researchers, and thepharmaceutical industry [3].

An increased inflammatory infiltrate in the lungs ofpatients with COPD is a key hallmark of the disease [7].This inflammatory infiltrate consists of macrophages, whichmature and differentiate from immigrating blood monocytes[8], and neutrophils, both of which are increased in thebronchoalveolar lavage (BAL) fluid and induced sputum ofpatients with COPD [9, 10]. T-lymphocytes are alsoincreased in the lung parenchyma and airways of thesepatients, with CD8+ T-lymphocytes being the mostprominent [11]. Both macrophages and neutrophils have thecapacity to cause all the pathological changes associated withCOPD [5, 12], with chemokines being responsible for theirrecruitment into the airways. Cigarette smoking is the majorrisk factor for developing COPD [5] and, at the present time,there are no pharmacological interventions which can halt theprogression of this disease [13].

*Address correspondence to this author at the Department of ThoracicMedicine, National Heart and Lung Institute, Imperial College London,Dovehouse Street, London, SW3 6LY, UK; Tel: +44 (020) 7352 8121 Ext.3061; Fax: +44 (020) 7351 8126; E-mail: l.donnelly@imperial.ac.uk

1573-398X/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

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The under-diagnosis of COPD coupled with poor in vivomodels makes COPD a difficult disease to investigate.Interspecies differences in chemokines and their receptorsmean that in vivo models are not necessarily the mostappropriate tool for investigating the pathogenesis of COPD.Often patients with COPD do not present to a clinician untilthe disease is quite advanced making disease initiationdifficult to study. A better understanding of the pathogenesisand subsequent pathophysiology of this disease will resultin new, more effective treatments for COPD. This reviewwill consider the hypothesis that the increased inflammatoryinfiltrate in COPD contributes to the lung destruction andirreversible nature of the disease. Therefore, we willconcentrate on the regulation of the recruitment andactivation of inflammatory cells in COPD by chemokinesand whether these mechanisms would provide targets for thetreatment of COPD.

CXC, CC and CX3C chemokines have four conservedcysteine residues, whereas C chemokines have two conservedcysteine residues. CXC and CX3C chemokines aredistinguished by the presence of one (CXC) or three (CX3C)amino acids between the first and second cysteine residues[18], whereas the first two cysteine residues of CCchemokines are juxtaposed (Fig. 1) [17]. CXC chemokinescan be further divided into ELR+ and ELR- chemokinesbased on the presence or absence of the tripeptide motifglutamic acid-leucine-arginine (ELR) between the amino(NH2)-terminus and the first cysteine residue [19, 20] (Fig.1). Chemokines have a low level of sequence identity;however their three-dimensional structure shows aremarkable homology in that they all possess the samemonomeric fold [21]. This fold, a carboxy (COOH)-terminalhelix and a flexible NH2-terminal region, is conferred onthese proteins by a four-cysteine motif that forms twodisulphide bridges. The flexible NH2-terminal region isbelieved to be important in receptor activation, asmodification of this region has been shown to affectchemotactic activity of these molecules [19, 20, 22].

CHEMOKINES

Recruitment of inflammatory cells into the airways is acritical event that triggers and sustains the clinicalmanifestations of inflammation [14]. The discovery of afamily of chemotactic cytokines, known as chemokines,which regulate cell trafficking within the immune system,has led to these molecules taking centre stage in the field ofinflammation [14]. Chemokines, like cytokines, aresecretory proteins produced by leukocytes and tissue cellseither constitutively or following induction, and exert theireffects locally in paracrine or autocrine fashion [15]. They aresmaller proteins than cytokines and exert their effects viaheptahelical G-protein-coupled receptors (GPCR) [15].Chemokines, typically consist of 70-130 amino acids [15]with a molecular weight of 8-10 kDa. They contain fourconserved cysteine residues linked by disulphide bonds [16].Chemokines can be subclassified into four groups, accordingto their structure and spacing of these conserved cysteineresidues, namely CXC, CC, C and CX3C (Fig. 1) [17].

To date, approximately 50 human chemokines but only19 receptors have been identified (Fig. 2) [15]. As there arenumerous chemokines, for the purpose of this review, thosethat might be or have been shown to be relevant in COPDwill be reviewed in depth. In humans, the majority of CXCand CC chemokines have been mapped to chromosomes 4and 17 respectively. Under normal conditions chemokinesare responsible for controlling leukocyte function,physiology and trafficking [23], however a greaterunderstanding of chemokines and their receptors have led tothem being implicated in a number of diseases includinginflammatory diseases such as COPD and asthma. The roleof the CC chemokines, including eotaxin-1, eotaxin-2,eotaxin-3, regulated on activation normal T-cell expressedand secreted (RANTES), macrophage inhibitory protein(MIP)-1α and monocyte chemoattractant protein (MCP)-2,-3, and -4 are of particular interest in asthma as they are

Fig. (1). Overview of the structure of the chemokines.

CXC chemokines consist of an NH2-terminus (N), four cysteines (C), the first two of which are separated by an amino acid (X), and aCOOH-terminus (CH). A subset of CXC chemokines has an ELR motif adjacent to the first cysteine. CC chemokines consist of an NH2-terminus (N), four cysteines (C), the first two of which are juxtaposed (C-C), and a COOH-terminus (CH). CX3C chemokines consist ofan NH2-terminus (N), four cysteines (C), the first two of which are separated by three amino acids (X3), and a COOH-terminus (CH). CChemokines consist of an NH2-terminus (N), two cysteines (C), and a COOH-terminus (CH).

Chemokines and their Receptors as Targets for the Treatment of COPD Current Respiratory Medicine Reviews, 2005, Vol. 1, No. 1 17

potent chemoattractants for eosinophils, basophils,monocytes and T-lymphocytes [24]. The role of CXC andCC chemokines have also been implicated in COPD, withinterleukin (IL)-8 [10, 25] growth-related oncogene (GRO)αand MCP-1 [26] being elevated in the sputum from thesepatients. These chemokines are important as they can recruitinflammatory cells, and IL-8 has been shown to causeelevated migration of neutrophils from patients with COPDwhen compared to control subjects [27]. Although aproinflammatory role of chemokines is mainly linked to

their ability to promote leukocyte migration, there isincreasing evidence that suggests an important role forchemokines in the control of cellular proliferation,differentiation and survival [14].

Inflammatory cells undergo regulated, multi-step,transendothelial migration from the blood stream to sites ofinflammation, a process which is facilitated by chemokines[28]. Up-regulated expression of adhesion molecules e.g. P-and E-selectin occurs on an inflamed endothelium [28].Leukocytes adhere to the vascular endothelium [29] then roll

Fig. (2). An overview of the chemokines and their receptors to date [21, 57].

Chemokines are divided into CXC, CC, C and CX3C subfamilies based on the spacing of the NH2-terminal cysteines residues. Theabbreviations for the chemokines are as follows: BCA-1, B-cell-attracting chemokine 1; CTACK, cutaneous T-cell attractingchemokine; DC-CK1. DC-derived chemokine; ELC, Epstein-Barr-virus-induced gene 1 ligand chemokine; ENA-78, epithelial-cell-derived neutrophil-activating peptide 78; GCP-2, granulocyte chemotactic protein; GRO; growth-related oncogene; IL-8, interleukin8; IP-10, interferon-inducible protein 10; I-TAC, interferon-inducible T cell α chemoattractant; MCP, monocyte chemoattractantprotein; MDC, macrophage-derived chemokine; MEC, mucosae-associated epithelial chemokine; MIG, monokine induced byinterferon γ; MIP, macrophage inflammatory protein; NAP-2, neutrophil-activating peptide 2, RANTES, regulated on activation,normal T-cell expressed and secreted; SDF-1, stromal-cell-derived factor 1; SLC, secondary lymphoid-tissue chemokine; TARC,thymus and activation-regulated chemokine; TECK, thymus-expressed chemokine.

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Fig. (3). Chemokine regulation of leukocyte movement [31].

Chemokines are secreted at sites of inflammation and infection by resident tissue cells, resident and recruited leukocytes, andcytokine activated endothelial cells. Chemokines are locally retained on the matrix and cell-surface heparan sulphate proteoglycans,establishing a chemokine concentration gradient surrounding the inflammatory stimulus, as well as on the surface of the overlyingendothelium. Leukocytes rolling on the endothelium in a selectin-mediated process are brought into contact with chemokinesretained on the cell-surface heparan sulphate proteoglycans. Chemokine signalling activates leukocyte integrins, leading to firmadherence and extravasation. The recruited leukocytes are activated by local pro-inflammatory cytokines and may becomedesensitised to further chemokine signalling because of high local concentrations of chemokines. The Duffy antigen receptor forchemokines (DARC), a non-signalling erythrocyte chemokine receptor, functions as a sink, removing chemokines from thecirculation and thus helping to maintain a tissue-bloodstream chemokine gradient.

along it under the shear flow of the blood stream [28].Chemokines which are bound to glycosaminoglycans e.g.heparin sulphate on the inflamed endothelium [28] can aidleukocyte adhesion by enhancing integrin-ligand-bindingaffinity [28, 29]. The leukocytes then move between orthrough endothelial cells [30], a process which is mediatedby chemokines [28]. Chemokines aid the diapedesis ofattached leukocytes through the endothelium by increasingpseudopod formation [28]. After traversing the endothelium,

the cells migrate towards the inflamed site under theguidance of chemotactic stimuli [28, 31]. This process iscontrolled in part by cell-surface molecules (e.g. receptorsand adhesion molecules), that allow migrating cells tointeract with the endothelium, tissue cells or ECM, and inpart by chemokines and other chemotactic molecules [31](Fig. 3).

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The typical response of leukocytes to a chemoattractantin vitro is to exhibit a bell-shaped concentration responsecurve. Both neutrophils and monocytes predominantlyexhibit bell-shaped curves when migrating towards achemotactic stimulus. Studies have revealed that monocytespolarize and move within the first few minutes followingstimulation with chemotactic factors [32]. Cells in optimaland uniform concentrations of a chemokine then moverapidly and show persistent paths, because the ligand-activated signalling is slow enough to allow the cell todistinguish a point of first stimulation on its surface [32].The cell then redistributes receptors to that point, whichbecomes the head of the cell, as the cell polarizes and moves[32]. Anterior distribution of functional receptors thenfavours persistence of movement in that direction. If theligand (chemokine) is presented in a concentration gradientthen most cells will polarize with their heads facing thegradient source, and will thus move towards that source[32]. However, at high concentrations of chemokine, it bindsrapidly at many points on the cell surface and the cell failsto establish a polarity and thus cannot move efficiently [32].This model also applies to neutrophil chemotaxis and thistheory of migration in response to chemotactic stimulisupports the bell-shaped curve response. At low

concentrations of chemokine, the chemokine does not bindto the cells quickly enough for the cell to establish polarityand move effectively, resulting in the first part of the curve(A) (Fig. 4). Then, as the concentration of chemokinebecomes optimal, migration reaches a peak (B) (Fig. 4)before slowing down when the concentration of chemokinesaturates the receptors (C) (Fig. 4).

CHEMOKINES IN COPD

Chemokines have been implicated in the pathogenesis ofCOPD due to their ability to recruit inflammatory cells tosites of inflammation. COPD is associated with an increasedinflux of inflammatory cells, which are responsible for thetissue destruction associated with this disease. There are alsostudies which have indicated that specific chemokines areelevated in COPD. The lack of successful, traditional,therapeutics for this disease has resulted in chemokines andtheir receptors being a current focus of attention for potentialtreatments for COPD. This coupled with a gradual increasein our understanding of the mechanisms by whichchemokines operate make them an extremely interestingtarget of investigation in COPD.

Fig. (4). Typical bell shaped curve for monocyte migration towards a chemoattractant [32].

At low concentrations of chemoattractant the concentration of chemoattractant is too low for the cell to move effectively (A). Atoptimal concentrations binding of the chemoattractant is slow enough to allow the cell to establish polarity and move towards thechemoattractant (B). At high concentrations of chemoattractant binds rapidly at many points on the cell surface and the cell fails toestablish polarity and movement is not efficient (C) [32].

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CXC CHEMOKINES proposal that the difference between patients with COPD and‘healthy smokers’ could be related to the amount of IL-8produced and released by airway epithelial cells [41], thusmaking it a good target for therapeutics in COPD.

The first CXC chemokine to be characterized was plateletfactor 4 (PF4) in 1977, 10 years before the discovery of IL-8[33]. CXC chemokines are divided into ELR+ and ELR-chemokines. The ELR+ chemokines have an uniformity offunction which allows their classification as a family ofneutrophil chemoattractants and activators, whereas the ELR-CXC chemokines display a mixture of disparate activities,with, as yet, no clear underlying theme [23].

GRO

GROα (CXCL1) was at first reported as an endogenousgrowth factor for human melanoma cells and namedmelanoma growth stimulatory activity (MGSA) [42]. TheGROα gene was identified in transformed Chinese hamsterand human fibroblasts, where its expression was found to begrowth-regulated and subsequently shown to be expressed bymelanoma and glioblastoma cells as well as renal, prostaticand bladder carcinoma cells [42]. Monocytes, endothelialcells, fibroblasts and synovial cells also produce GROα afterstimulation with lipopolysaccharide (LPS), IL-1β or tumournecrosis factor (TNF)α [42]. GROα is structurally related toIL-8 and is a powerful activator of neutrophils withchemotactic ability for neutrophils, basophils, freshlyisolated T-lymphocytes [43, 44] and monocytes [36]. GROβand GROγ were identified subsequently using cDNAlibraries from activated monocytes and neutrophils. Theyshare 90 and 86% amino acid similarity with GROαrespectively, and all three GRO proteins share 33-40%identity with IL-8. GROβ and GROγ can also induceleukocyte chemotaxis, shape change and a transient increasein cytosolic intracellular calcium, granule exocytosis and therespiratory burst [42]. There are elevated levels of GROαpresent in the induced sputum from patients with COPDwhen compared to smokers and non-smokers [26]. Theimportance of elevated GROα levels in COPD ishighlighted by the observation that peripheral bloodmononuclear cells (PBMC) and monocytes from patientswith COPD migrate in higher numbers towards GROα [36].This appears to be due to an increase in the concentration ofGROα required for a maximal response. Thus, not only isthere an increase in the numbers of monocytes migratingtowards GROα at low concentrations, at high concentrationswhen the cells would normally arrest (at the top of the bell-shaped curve), monocytes continue to migrate. The net resultis an increased monocyte/macrophage load, which couldaccount for the increase in macrophage numbers in COPD.Consequently, not only can GROα act as a neutrophilchemoattractant [43] it is also important in the developmentof the increased macrophage burden in COPD. Thesefindings signify an important role for this chemokine in therecruitment of inflammatory cells in COPD, and a candidatetarget for a potential treatment for this disease.

ELR+ CHEMOKINES

IL-8

IL-8 (CXCL8) was identified as a protein of 72 aminoacids with a molecular weight of 8.3 kDa and was initiallyisolated from the culture supernatants of stimulated humanblood monocytes. It exists in two forms, a 72-amino acidprotein which predominates in cultures of monocytes andmacrophages, and a 77-amino acid protein whichpredominates in cultures of tissue cells such as endothelialcells and fibroblasts [33]. Lymphocytes, granulocytes,bronchial epithelial cells, smooth muscle cells,keratinocytes, hepatocytes, mesangial cells and chondrocytesalso produce IL-8. It is an inflammatory cytokine, whichfunctions mainly as a neutrophil chemoattractant andactivating factor. IL-8 is a potent neutrophil activator, withthe capacity to upregulate adhesion molecule expression onthe surface of neutrophils, enhancing LTB4 production,inducing neutrophil chemotaxis and increasing neutrophiladherence to endothelial and epithelial cells [34]. IL-8accounts for a considerable amount of neutrophilsequestration in a variety of diseases, one of which mayinclude COPD [34]. Cigarette smoke, one of the causalfactors of COPD, stimulates IL-8 release from bronchialepithelial cells [34] and alveolar macrophages [35] in vitro.Elevated levels of mRNA have been measured in bronchiolarwalls, alveolar epithelium and airway secretions of patientswith COPD [34]. High levels of IL-8 are found in acuteinflammatory conditions of the lung, including emphysemaand adult respiratory distress syndrome (ARDS), in whichIL-8 and neutrophil numbers are reported to correlate withmortality [33]. IL-8 expression is increased in the inducedsputum from patients with COPD [10, 25] and it has beenshown that neutrophils from patients with COPD migrate inhigher numbers towards IL-8 than neutrophils from non-smokers [27], emphasising the importance of this chemokinein COPD. Monocytes can also migrate towards IL-8 [36,37]. The observation that IL-8 enhances the production ofLTB4 [34], which is also a neutrophil chemoattractantpresent in induced sputum [38], highlights an indirectmanner in which IL-8 can recruit neutrophils to the airwaysin COPD. IL-8 stimulation of neutrophils via CXCR1 andCXCR2 receptors causes an immediate increase inintracellular calcium [39]. Studies by Goodman et al. 1998[40] have shown that IL-8 is produced in higher quantitiesthan other CXC chemokines by human alveolar macrophagesupon stimulation with LPS, suggesting that this may be onesource of enhanced levels of IL-8. Basal levels of IL-8 arealso produced in increased amounts by alveolar macrophagesfrom patients with COPD when compared to smokers [35].The numerous functions of IL-8 in COPD have led to the

NAP-2

Neutrophil activating peptide (NAP)-2 (CXCL7) wasoriginally isolated from stimulated mononuclear cells [45].NAP-2 is the only neutrophil activating chemokine that isnot induced by gene activation as it is generated byproteolytic processing from inactive precursors released fromplatelet α-granules and occurs in the presence of monocytes,and purified proteases, such as cathepsin G [45]. Theformation of NAP-2 therefore depends on the presence ofplatelets in monocyte cultures [45]. NAP-2 is chemotacticfor neutrophils in vitro and in vivo and can induce cytosolic

Chemokines and their Receptors as Targets for the Treatment of COPD Current Respiratory Medicine Reviews, 2005, Vol. 1, No. 1 21

calcium increases, exocytosis and respiratory burst [45].NAP-2 stimulates neutrophil chemotaxis with a similarpotency to IL-8, however, whereas IL-8 induced migration ischaracterised by a bell-shaped curve, NAP-2 retainschemotactic activity at high concentrations [46]. Thesefindings are enhanced by the observation that NAP-2 inducestwo optima of neutrophil chemotaxis. The first optimumwas elicited within a low concentration range (7.6-100ng/ml), whereas, the second optimum occurred atconcentrations more than 200-fold higher (760-7600ng/ml)[47]. These results provide evidence that both CXCR1 andCXCR2 are involved in NAP-2 induced neutrophilchemotaxis, with CXCR2 rendering the cells responsive tolow concentrations of NAP-2, while CXCR1 extends theirresponsiveness to much higher concentrations of NAP-2[47]. NAP-2 is also chemotactic for PBMC and monocytes,with elevated migration observed with cells from patientswith COPD [36]. The observation that NAP-2 ischemotactic for monocytes conflicts with other studies thatsuggested that CXC chemokines are not chemotactic formonocytes [42, 45]. As these chemokines are thought toexert their effects via the same receptors [18, 48] this conflictwithin the literature is surprising. These findings indicatethat NAP-2 may be an important chemokine in COPD as ithas the potential to recruit both neutrophils and monocytesto the sites of inflammation. However, in order to enhancethese observations the levels of NAP-2 in COPD need to beelucidated.

cells [52]. A number of cell types including mononuclearcells, keratinocytes, fibroblasts, endothelial cells, bronchialepithelial cells, smooth muscle cells [53] and T-lymphocytescan express IP-10 following stimulation with IFN-γ [52,54]. As with other ELR-negative chemokines IP-10 is a poorneutrophil activator and chemoattractant [55]. IP-10 exertsits effects via CXCR3, which is present on T-lymphocytes,natural killer cells [28, 56, 57], and B-lymphocytes [57]. IP-10 stimulation of this receptor results in the recruitment andactivation of these cells [28, 56]. IP-10 is present in bothBAL fluid and induced sputum from patients with COPDbut not at elevated levels when compared with samples fromnon-smokers or smokers [58]. IP-10 is also a potent naturalantagonist for CCR3, a receptor that promotes Th2 cellmigration [59]. Hence, expression of these chemokineswould favour a more polarised Th1 response that has beendemonstrated in COPD [60, 61]. Enhanced levels of CXCR3expression associated with increased numbers of CD8+ cellshas been reported in the lungs of patients with COPD [62]indicating that this may be one mechanism of CD8+ cellrecruitment into the lungs of these patients. This evidencesupports a mechanism by which the airway inflammation inthe lungs of patients with COPD is maintained andamplified [14].

MIG

Monokine induced by IFN-γ (MIG) (CXCL9), is also anIFN-γ inducible protein isolated from macrophages [63].MIG is present in both BAL fluid and induced sputum frompatients with COPD, with elevated levels in the sputumsamples from patients with COPD when compared withsamples from non-smokers (Hall, Z, unpublished data). MIGalso exerts its effects via CXCR3, which is present on T-lymphocytes, natural killer cells [28, 56, 57], and B-lymphocytes [57]. Stimulation of this receptor results in therecruitment and activation of these cells [28, 56],highlighting a role for this chemokine in recruiting T-lymphocytes to sites of inflammation.

ENA-78

Epithelial-derived neutrophil activating peptide (ENA)-78(CXCL5) was first isolated from the human type II-likeepithelial cell line, A549 [49]. It is a 78-amino acid protein[50], with a molecular weight of 8.4 kDa [49]. Unstimulatedepithelial cells produce low levels of ENA-78, although inthe presence of TNFα or IL-1β, a rapid induction of ENA-78is observed [49]. ENA-78 mRNA is produced by lungfibroblasts, monocytes, endothelial cells and mesothelialcells following stimulation with LPS or IL-1β [49].Furthermore, LPS can also induce ENA-78 peptideproduction from monocytes [49]. ENA-78 has only 22%sequence similarity to IL-8, but shares 53% and 52%sequence identity with NAP-2 and GROα, respectively [51].ENA-78 is a potent activator of neutrophil function [50],inducing concentration-dependent chemotaxis between 0.1and 100nM in vitro [49]. This response is identical to thatof NAP-2, but somewhat reduced to that of IL-8 [49].However, when ENA-78 is released from type II-likeepithelial cells it can be processed by alveolar macrophagesthrough the release of cathepsin G, to molecules that areequipotent with IL-8. ENA-78 may contribute significantlyto the recruitment of neutrophils into the lung [49],however, ENA-78 is also chemotactic for monocytes [36,37], therefore this chemokine is important in sustainingairway inflammation.

I-TAC

IFN-inducible T cell α chemoattractant (I-TAC)(CXCL11) is the most potent and efficacious of all theCXCR3 chemokines [57, 64]. I-TAC mRNA is up-regulatedin monocytes, bronchial epithelial cells, neutrophils,keratinocytes and epithelial cells treated with IFN-γ , thusimplying a role in T-lymphocyte recruitment to areas ofinflammation [57]. I-TAC has greatest sequence homologywith IP-10 and MIG and binds to CXCR3 with higheraffinity than IP-10 and MIG [64]. It appears that there areboth a high affinity and low affinity binding site for I-TACon CXCR3 [64]. I-TAC is present at elevated levels in BALfluid and induced sputum from patients with COPD whencompared with samples from non-smokers [58]. I-TAC maytherefore be an important chemokine in the recruitment of T-lymphocytes as it has potent chemotactic activity for IL-2simulated T-lymphocytes [28, 64]. Its unique selectivity foreffector T-lymphocytes highlights the importance of I-TACin COPD as T-lymphocytes have been implicated in thepathogenesis of this disease.

ELR- CHEMOKINES

IP-10

Interferon (IFN)-γ -inducible protein (IP)-10 (CXCL10),is a product of an IFN-γ inducible gene cloned from U937

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All three CXCR3 chemokines can act as naturalantagonists at CCR3 [57, 65]. They inhibit CCR3 responsesto eotaxin, eotaxin-2 and eotaxin-3 [65], with I-TAC beingthe most efficacious inhibitor in chemotaxis assays [65]. I-TAC also has an antagonistic effect on CCR5, reducing itsactivities such as the release of free intracellular calcium andactin polymerisation to minimal levels [57]. These factorsmake the CXCR3 chemokines important therapeutic targetsin COPD.

thought of as a ‘housekeeping’ chemokine rather than aninflammatory chemokine [71].

CXCL16

CXCL16 and the scavenger receptor forphosphatidylserine and oxidised low-density lipoprotein(SR-PSOX) have recently been found to be one and the same[73, 74]. CXCL16 was identified by two groups as theligand for the orphan receptor CXCR6 [73, 74]. It is atransmembrane chemokine, the second discovered, which hassignificant structural homology with fractalkine [73, 74].SR-PSOX was independently identified through expressioncloning designed to identify receptors that could mediate celladhesion to phosphatidylserine-coated surfaces [73, 74]. SR-PSOX is expressed by macrophages in vitro, while CXCL16is expressed by dendritic cells, macrophages [73, 74] and B-lymphocytes [73]. In its soluble form, CXCL16 causes themigration of activated CD4+ and CD8+ T-lymphocytes viaCXCR6 [73]. It appears that CXCL16 is synthesized as anintracellular precursor that is transported to the cell surfacewhere it is cleaved by the metalloproteinase, ADAM10 [73].This conversion from a cell surface protein into a solubleform may be relevant in COPD as CXCL16 is chemotacticfor T-lymphocytes. If CXCL16 were present in COPD thenblocking its conversion to a soluble, and thuschemoattractant form, may reduce T-lymphocyteaccumulation in the airways of COPD patients. Thepotential role of CXCL16 in COPD is emphasised by theobservation that CXCL16 is produced by macrophages [73].Macrophage numbers are increased in the airways of patientswith COPD [9, 10], which could lead to an increasedCXCL16 presence in the airways resulting in increasedrecruitment of T-lymphocytes in COPD. CXCL16 alsoactivates NF-κB and induces κB-dependent proinflammatorygene transcription [75] which is an important signaltransduction pathway crucial to the regulation of manyinflammatory genes including chemokines.

CXC CHEMOKINES WITH POTENTIAL ROLES INCOPD

GCP-2

Granulocyte chemotactic protein (GCP)-2 was initiallyisolated from cytokine-stimulated cell culture supernatants[66, 67]. GCP-2 shares 77% amino acid sequence similaritywith another CXC chemokine ENA-78, yet only 31, 44 and46% with IL-8, GROα and NAP-2 respectively [66].However, in spite of its low sequence similarity with IL-8,it is the only other CXC chemokine which binds to bothCXCR1 and CXCR2 at nanomolar concentrations [66].Stimulation of neutrophils with GCP-2 results inchemotaxis, enzyme release and calcium mobilisation [66].Although CXCR1 and CXCR2 are present on mononuclearcells, no chemotactic activity has been reported formonocytes or lymphocytes indicating that GCP-2 is aneutrophil-specific chemokine [67]. It is possible that GCP-2 has a role as a neutrophil chemoattractant in COPD andthat it may be responsible in part for the increased influx ofneutrophils into COPD airways. However there is noinformation regarding the levels of this chemokine and itsbiological role in COPD.

SDF-1

Stromal-cell-derived factor (SDF)-1 (CXCL12) isubiquitously expressed [14, 68] and binds to CXCR4 [69].SDF-1 is 10-fold more potent than the other T-lymphocytechemoattractants [70]. It can also stimulate actinpolymerisation [70] and is active on both naïve and activatedT-lymphocytes, [64], monocytes [64, 70], B-lymphocytes[71, 72], but not neutrophils [70] or mature B-lymphocytes[72] in vitro. In vivo, SDF-1 is a highly efficacious andpotent mononuclear cell chemoattractant [70], indicating arole for this chemokine in the recruitment of inflammatorycells. SDF-1 is classed as a ‘housekeeping chemokine [71],therefore, the levels of SDF-1 need to be determined inCOPD so that the full role of this chemokine can beelucidated in this disease.

CC CHEMOKINES

The first CC chemokine was identified after cloning bydifferential hybridisation from human tonsillar lymphocytesand was termed LD78 [33]. Several cDNA isoforms of aclosely related chemokine, Act-2 were later identified. Twosimilar proteins, MIP-1α and MIP-1β, were later purifiedfrom the culture medium of murine macrophages stimulatedwith LPS and subsequently cloned [33]. The murineproteins are considered as the homologues of LD28 and Act-2 because of their amino acid identity of more than 70% andthe terms human MIP-1α and MIP-1β are now commonlyused instead of LD28 and Act-2 [33]. The best-characterisedCC chemokine is MCP-1, which was purified from culturesupernatants of blood mononuclear cells, as well as gliomaand myelomonocytic cells lines and was cloned fromdifferent sources [33]. Other CC chemokines such as I-309,RANTES and MCP-2 were purified or cloned as products ofactivated T-lymphocytes. MCP-2 was purified along withMCP-3 from cultures of osteosarcoma cells [33]. Eotaxin isa potent stimulus for eosinophils, inducing eosinophilmigration in vitro and accumulation in vivo [76].

BCA-1

B-Cell-attracting Chemokine (BCA)-1 shares 24-34%sequence homology with the CXC chemokines, in spite ofthis, it is not closely related to any other specific group ofchemokines [72]. It is the most efficacious B-cell attractantwhich selectively binds to CXCR5 [71, 72]. BCA-1 isinactive on freshly isolated and IL-2 stimulated T-lymphocytes, monocytes and neutrophils [72]. BCA-1 is

Chemokines and their Receptors as Targets for the Treatment of COPD Current Respiratory Medicine Reviews, 2005, Vol. 1, No. 1 23

MCP mechanism whereby two opposing chemokine gradients cancooperate in driving out monocytes from blood vessels intothe tissues [86]. Eotaxin is present in the serum of patientswith COPD and the percentage fall in forced expiratoryvolume in one second (FEV1) correlated with eotaxin levels[87]. The levels of eotaxin are not elevated in bronchialbiopsies from patients with COPD indicating that it is amore important chemokine in Th-2 driven diseases (e.g.asthma) rather than Th1 driven diseases (e.g. COPD) [60]. Ifeotaxin is present in the airways of COPD patients atfunctional levels, it is possible that this chemokinecombined with the elevated levels of MCP-1 found inCOPD could combine to cause increased monocytemigration into the airways thus resulting in the increasedmacrophages in the tissues associated with COPD. Eotaxin-3 has also been found to be antagonistic for CCR1 andCCR5, indicating that it has a modulatory rather thaninflammatory function [85]. However further investigationsare needed to confirm a role for the eotaxins in COPD.

MCP-1 (CCL2) is an 8.7 kDa chemokine produced bymonocytes, T-lymphocytes, fibroblasts, endothelial cells,epithelial cells, smooth muscle cells and keratinocytes [39].It was first purified from conditioned medium of baboonaortic smooth muscle cells in culture on the basis of itsability to attract monocytes but not neutrophils in vitro [23].MCP-1 is a potent chemoattractant for monocytes in vitroand can induce the expression of integrins required forchemotaxis [23]. In transendothelial migration assays, MCP-1 is a equipotent for activated both CD4+ and CD8+ T-lymphocytes [23], but not B-lymphocytes or natural killercells [23]. MCP-1 has been generally observed to be a morepotent and efficacious monocyte chemoattractant than MCP-2, MCP-3 [16] or MCP-4 [23]. MCP-5 has so far only beenidentified in mice [23]. MCP-1 is expressed in varioustissues including human lungs, where it is expressed bymacrophages, endothelial, bronchial epithelial, and smoothmuscle cells [7]. MCP-1 mediates its cellular effects bybinding to CCR2 [77] which is mainly expressed bymonocytes, macrophages and T-lymphocytes [7]. MCP-1activation of monocytes results in a calcium flux [39], andhas been implicated in diseases that have a mononuclear cellinflammatory component [78]. COPD is associated with anincrease in inflammatory cells, in particular, macrophages[9]. Monocytes are the precursor cells for macrophages [79]and MCP-1 levels are increased in the induced sputum frompatients with COPD [26]. This coupled with the observationthat MCP-1 is chemotactic for monocytes [36, 37]highlights the importance of this chemokine in COPD.MCP-2, MCP-3 and MCP-4 are potent chemoattractants foreosinophils [80], which have been shown to be central to theairway inflammation associated with asthma [81] as opposedto COPD. However, MCP-3 has been identified as a potentantagonist at CCR5 [57], but an agonist at CCR1, CCR2and CCR3.

SLC

Secondary lymphoid-tissue chemokine (SLC) (CCL21)is constitutively expressed by cells throughout the T-cellzones of the spleen, lymph nodes [88, 89] and Peyer’spatches [88]. SLC exerts its effects via CCR7 [69, 90], andhas chemotactic activity for Th1 lymphocytes and maturedendritic cells [89]. SLC appears to play a crucial role indirecting T-lymphocyte and dendritic cell movements [88]and its effect on Th1 cells implicates a role for thischemokine in COPD.

OTHER CC CHEMOKINES WITH A POTENTIALROLE IN COPD

There are a great number of CC chemokines which as yethave no defined role in COPD. Some however have anumber of qualities which if they were discovered to bepresent in the airways of COPD patients would make themimportant considerations for therapeutic targets in COPD.

Eotaxin

Eotaxin (CCL11) was first described as an innovativeeosinophil chemoattractant, and initially found in the BALfluid of allergic guinea-pigs [82]. Subsequently it was alsofound in the BAL fluid of asthmatic subjects [83]. Theeotaxins are a group of three chemokines that mediate therecruitment of eosinophils via interaction with CCR3 [84,85]. Increased expression of mRNA and protein for eotaxinand eotaxin-2 (CCL24) can be demonstrated in bronchialbiopsies from asthmatic subjects when compared withcontrols, indicating that it is indicative of an ongoing airwayinflammatory response [24]. Eotaxin-3 (CCL26) ischemotactic for eosinophils, basophils [84, 85] and Th2 T-lymphocytes [85] but is 10-fold less potent than the othertwo eotaxins [84]. The role of the eotaxins in COPD isunclear but these compounds can act as natural antagonists.Eotaxin and eotaxin-3 are antagonistic for CCR2 [76, 85].This observation itself has important implications in COPD,as monocytes are recruited to the airways by MCP-1 whichmediates its effects via CCR2. While eotaxin-3 can inhibitMCP-1 mediated responses in monocytes, it can alsopromote the movement of monocytes away from a gradientof eotaxin-3 in vitro [86]. This effect is further increased bythe addition of MCP-1 [86]. These findings give rise to a

MIP

MIP-3α (CCL20), which binds CCR6, is increased afterallergen challenge in sensitised mice. However, CCR6deficient mice have an attenuated airway inflammatoryresponse with reduced eosinophil infiltration, decreased IL-5in the lung and serum levels of IgE [14], suggesting thatCCR6 and MIP-3α are important in airway inflammationthat is more reflective of the asthmatic response rather thanthat observed in COPD. MIP-1α (CCL3) and MIP-1β(CCL4) were purified from LPS treated monocytic cell lines.MIP-1α attracts and activates monocytes more efficientlythan MIP-1β but less so than MCP-1 [23]. However, theMIP proteins may still be important in airway inflammationas they can recruit inflammatory cells including dendriticcells, NK cells, basophils and eosinophils [23].

RANTES

RANTES (CCL5) is elevated in the bronchial biopsiesfrom mild asymptomatic asthmatic subjects compared with

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controls [24]. The number of cells which express mRNA forRANTES is significantly increased in mild to moderatesymptomatic asthmatic subjects compared with controls[24]. RANTES is also chemoattractant for memory T-lymphocytes in vitro , however it is most the most effectivebasophil chemoattractant [80]. At present, RANTES doesnot appear to play a role in COPD.

I-309

I-309 (CCL1) is produced by T-lymphocytes, stimulatedmonocytes and epithelial cells [99]. It binds to CCR8 [100]and can cause the migration of monocytes in umbilical cordvein endothelial cells by lipoprotein A [99] as well as bloodCD4+ and CD8+ cutaneous T-lymphocytes [100]. This dataimplicates a role of I-309 in vessel wall biology [99] inaddition to the trafficking of cutaneous T-lymphocytes to theskin [100].

DC-CK1

Dendritic cell-derived CC chemokine (DC-CK1)(CCL18) is, as its name suggests, specifically expressed byhuman dendritic cells at high levels [91]. It is expressed in Tand B-lymphocyte areas of secondary lymphoid organs [92]and attracts naïve T-lymphocytes [91, 92], suggesting thatDC-CK1 has a role in the induction of immune responses[91]. There is no evidence that this chemokine has a role inCOPD.

MEC

Mucosae-Associated Epithelial Chemokine (MEC)(CCL28) is expressed by epithelia in mucosal tissues [101].Its mRNA is most abundant in salivary glands, with strongexpression in the colon, trachea and mammary gland [102].It binds to CCR10 [101, 102], and attracts subsets ofmemory T-lymphocytes as well as eosinophils [101].

TARCCTACK

The expression of thymus- and activation-regulatedchemokine (TARC) (CCL17) is the ligand for CCR4 whichis expressed on T-helper/Th2 CD4+ cells [93, 94]. TARC isproduced by activated PBMC, bronchial epithelial cells andkeratinocytes [94]. The expression of TARC is increased inbronchial epithelial cells in response to TNFα and thenfurther increased with IFN-γ [93]. IL-13 was found to be themost potent inducer of TARC expression and proteinproduction in PBMC, though the production of TARC canalso be induced by GM-CSF, PHA, IL-3 and IL-4 [94].TARC has been implicated in the pathogenesis of allergicasthma [93], but as yet has no defined role in COPD.

Cutaneous T-cell-attracting chemokine (CTACK)(CCL17) is expressed by skin keratinocytes [103, 104]. Thepresence of this chemokine results in homing of T-lymphocytes to the skin [103, 104] via CCR10 [103],therefore this chemokine is unlikely to be involved in thepathogenesis of COPD.

TECK

Thymus-expressed chemokine (TECK) (CCL25) is foundin the small intestine and thymus [105, 106]. It exerts itseffects via CCR9 [105-107], and has chemotactic activity fordendritic cells, thymocytes [69, 107] and activatedmacrophages [107]. Although this chemokine causes themigration of macrophages there is no defined role for TECKin COPD.

MDC

Macrophage derived chemokine (MDC) (CCL22), wasdiscovered by two separate groups [95]. It is located onchromosome 13, and is produced by macrophages,monocytes, dendritic cells, activated T-lymphocytes andepithelial cells [95]. MDC is chemotactic for macrophages,monocytes, natural killer cells, and activated Th2lymphocytes [95, 96]. Like TARC, MDC exerts its effectsvia CCR4, and appears to have a role in asthma [95, 96], butnot COPD.

CHEMOKINE RECEPTORS

Chemokines exert their effects by binding to specific cellsurface receptors [48]. Chemokine receptors comprise a largefamily of seven transmembrane domain GPCR differentiallyexpressed in diverse cell types [18]. To date, there are over600 members of the GPCR family which have beenidentified and classified into sub-families [108]. Chemokinebinding initiates a cascade of intracellular events that resultsin biological effects [109]. When a chemokine binds to areceptor, a conformational change occurs which leads to thedissociation of the receptor associated heterotrimeric Gproteins into α and βγ subunits [48]. In generally, these G-protein subunits activate various effector enzymes [48].Inositol triphosphate (IP3) is generated, intracellular calciumis released and protein kinase C (PKC) is activated [31].Chemokine receptor signalling also activates smallguanosine triphosphate-binding proteins of the Ras and Rhofamilies [110]. Rho proteins are involved in cell motility viaregulation of actin-dependent processes such as membraneruffling, pseudopod formation, and assembly of focaladhesion complexes. This leads to the activation of

ELC

Epstein-Barr-Virus-induced Molecule 1 (EBI-1) ligandchemokine (ELC) is also known as MIP3β (CCL19) [97,98]. It is highly expressed in lymphoid tissues, with theappendix and thymus containing the highest levels [88, 97,98]. Monocytes that have been activated by LPS or IFNγ inthe presence of IL-10 antibodies are the only cell type toexpress significant amounts of ELC [97, 98]. ELC exerts itseffect via CCR7 and has chemoattractant activity for the T-lymphocyte lymphomas line HUT78, resting and activatedmouse T-lymphocytes [97], as well as resting CD4+ andCD8+ T-lymphocytes and B-lymphocytes [98]. ELC appearsto have a homeostatic role, as well as a role in the migrationof a broad range of lymphocytes into lymphoid tissues [98].At present there is no documented role for ELC in COPD.

Chemokines and their Receptors as Targets for the Treatment of COPD Current Respiratory Medicine Reviews, 2005, Vol. 1, No. 1 25

Fig. (5). An overview of intracellular signalling when an agonist binds to its receptor.

Agonist binding to the receptor, results in dissociation of G-protein α and βγ subunits. These subunits activate various effectorenzymes including phospholipases, which leads to inositol triphosphate (IP3) production. Intracellular calcium is increased andprotein kinases are activated. This leads to chemotaxis, an increase in respiratory burst, phagocytosis, degranulation and lipidmediated synthesis. The abbreviations are as follows: Ca, calcium; DAG, diacylglycerol; IP3, inositol-1, 4, 5-triphosphate; PIP2,phosphatidylinositol-4, 5-biphosphate; PLC, phospholipase C; R, receptor; TK, tyrosine kinase.

chemotaxis as well as a wide range of functions in differentleukocytes such as an increase in respiratory burst,degranulation, phagocytosis and lipid mediated synthesis[48] (Fig. 5).

aimed at characterising the ligand binding profiles of thesereceptors have shown that both CXCR1 and CXCR2 bindIL-8 with high affinity, whereas CXCR2 binds GROα,NAP-2 and ENA-78 with high affinity [47, 113]. Damaj etal. 1996 [114] demonstrated that neutrophils have the abilityto discriminate between IL-8 and GROα interacting withCXCR2. They concluded that the functional consequences ofIL-8 and GROα occupying CXCR2 are not equivalent butare agonist dependent [113, 114]. CXCR1 and CXCR2 mayhave differing roles in neutrophils in vitro. CXCR1 appearsto be dominant for chemotaxis in response to IL-8, whereasCXCR2 appears to mediate neutrophil chemotaxis to GROαat low concentrations [115]. However, the CXC chemokines

CXC CHEMOKINE RECEPTORS

CXCR1 and CXCR2

CXCR1 and CXCR2 were the first chemokine receptorsubtypes to be defined [111]. They share 77% identity [112],and are expressed on a wide range of cell types including T-lymphocytes, monocytes and neutrophils [48]. Studies

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are also chemotactic for monocytes and these effects are alsomediated via CXCR1 and CXCR2 [36, 37].

subacute inflammation where CCR5 and CXCR3 ligandsplay a prominent role [122]. This may be important inCOPD where CXCR3 ligands are present in BAL andsputum, indicating CXCR3 as a potential target fortherapeutics in COPD. A non-peptide chemokine receptorantagonist for CXCR3 and CCR5 effectively blocks themigration of T-lymphocytes expressing these receptors tosites of inflammation and therefore may have therapeuticbenefit in COPD [123].

Neutrophils and monocytes from patients with COPDshowed elevated levels of migration towards IL-8 [27],GROα and NAP-2 respectively [36], however this increasedmigration is not due to an increase in receptor expression onthese cells [36]. It is possible that differences in CXCR2recycling when they are stimulated by different CXCchemokines may account for the increased migration seenwith monocytes from patients with COPD [36]. Studies byPetersen et al. 1994 [116], have indicated that althoughneutrophils express a limited number of common receptorsfor IL-8 and NAP-2 these cytokines can induce differentbiological responses by discrete mechanisms of action. Highaffinity binding of IL-8, GROα and NAP-2 to CXCR2involves interaction with specific and different amino acidresidues of CXCR2 [113]. This suggests that CXCR2 aminoacid residues required for cell activation are not necessarilythose required for ligand binding [113]. Therefore, theapparent redundancy of chemokines and chemokine receptorsmay be explained by this observation since differentchemokines may bind to a single receptor, but exert differentfunctions. Furthermore, coupling of CXCR1 and CXCR2 toidentical G-proteins triggers differing cellular responsesdependent on receptor phosphorylation [117]. Thesereceptors would make good therapeutic targets as inhibitionof these receptors could reduce the numbers of monocytesand neutrophils entering the airways thus reducing theinflammatory load and consequently the tissue damageassociated with COPD. At present there are a number ofsmall molecule antagonists which can reduce the migrationof inflammatory cells via these receptors [36], one of themost promising is the CXCR2 antagonist SB 332235 thathas proved to be very efficacious in both in vitro and in vivostudies and is currently undergoing clinical trial analysis.

CXCR3 also binds eotaxin highlighting the possibilitythat it may also act as a decoy receptor which sequesterseotaxin resulting in the down-regulation of eotaxin responseson CCR3 expressing cells [65]. This may help explain theTh1 mediated response in COPD as opposed to the Th2 typeresponses observed in asthma where eotaxin and eosinophilrecruitment are characteristic features. CXCR3 is up-regulated on endothelial cells and CD4+ (Th1) infiltratinglymphocytes indicating an immunoregulatory role for thisreceptor. By causing the sequestration of eotaxin, CXCR3may reduce the development of a Th2 response resulting indominance of a Th1 response [65]. It is thought that Th1 T-lymphocytes are the predominant CD4+ T-lymphocytes inCOPD [61], thus highlighting the importance of thisreceptor and thus its potential as a target for the treatment ofCOPD.

OTHER CXC CHEMOKINE RECEPTORS

CXCR4

CXCR4 is expressed constitutively in a wide range oftissues and is essential for normal development [68].CXCR4 is a critical component of the inflammatory processinvolved in a mouse model of allergic airway inflammation[68]. CXCR4 expression on T-lymphocytes is upregulatedby IL-4, suggesting a predominant expression on Th2 cells[14]. CXCR4 is also expressed on freshly isolated B-lymphocytes [71]. The role of this receptor has not yet beenelucidated in COPD, but as it is predominantly expressed onTh2 cells this receptor may have more relevance in allergicdiseases such as asthma.

CXCR3

CXCR3 has been shown to play a key role in themigration of T-lymphocytes to sites of inflammation [118].CXCR3 exists as two separate isoforms namely the ‘classic’form CXCR3-A and the splice variant CXCR3-B [119].CXCR3 is highly expressed in IL-2-activated T-lymphocytes[64], natural killer cells [28, 65], but it is not detectable inresting T-lymphocytes, B-lymphocytes, monocytes orgranulocytes. However, others have found CXCR3 to bepresent on blood T-lymphocytes, B-lymphocytes, naturalkiller cells [54, 120] and epithelial cells [119]. CXCR3expressing T-lymphocytes from the blood resemble T-cellsthat infiltrate inflammatory lesions [120] indicating a rolefor this receptor in COPD. CXCR3 mediates IP-10, MIGand I-TAC stimulated calcium mobilisation and chemotaxisyet it is completely unresponsive to stimulation by ELR+CXC chemokines and CC chemokines, suggesting that thisreceptor is involved in the selective recruitment of effector T-cells [121]. Approximately 25% of circulating memoryCD4+ (Th1) T-lymphocytes co-express CXCR3 and CCR5[118, 122], though it is unclear whether these receptors areinduced after tissue entry [122]. It is possible that theupregulation of these receptors on tissue lymphocytes mayenhance the ability of these cells once they have returned tothe circulation to be subsequently recruited to active sites of

CXCR5

CXCR5 is present on blood B-lymphocytes and isresponsible for their migration towards BCA-1 [71].However as BCA-1 is a ‘housekeeping’ rather than aninflammatory chemokine, this receptor is unlikely to be apotential target for the treatment of COPD.

CXCR6

The CXCR6 receptor, also known as Bonzo, is thereceptor for CXCL16 [124]. It was previously described as afusion cofactor for HIV-1 and SIV [124]. CXCR6 isexpressed by subsets of T-lymphocytes [73, 74, 124, 125],human airway smooth muscle cells [75] and natural killercells [73, 74, 125] but not by B-lymphocytes, monocytes ordendritic cells [124]. CXCR6 is expressed by 2-6% of CD4+cells and 4-12% of CD8+ cells suggesting its presence onfunctionally specialised T-lymphocytes within the total

Chemokines and their Receptors as Targets for the Treatment of COPD Current Respiratory Medicine Reviews, 2005, Vol. 1, No. 1 27

lymphocyte fraction [124]. These ‘Bonzo’ T-lymphocytes areassociated with inflamed tissues and may have a possiblerole in T-lymphocyte homing to chronically inflamed tissues[124]. CXCR3 and CCR5 are also present on ‘Bonzo’ T-lymphocytes thus enhancing the homing of these cells totissue-effector sites [124]. This effect may be furtheramplified by the notion that ‘Bonzo’ T-lymphocytes mayself-generate once they have established themselves withinthe tissues [124]. Half of the CD4+ ‘Bonzo’ T-lymphocytescan produce IFN-γ [124, 126], which causes the induction ofthe CXCR3 chemokines thus potentiating the recruitment ofT-lymphocytes to sites of inflammation. These findingscoupled with the observation that the majority of CD8+‘Bonzo’ T-lymphocytes contain granzyme A [124] and alsoproduce IFN-γ [124, 126], has implications in COPD.CD8+ T-lymphocytes are up-regulated in COPD [11, 127];if these CD8+ T-lymphocytes are also ‘Bonzo’ T-lymphocytes, then they may up-regulate IFN-γ , leading toan increase in CXCR3 chemokines which in turn would leadto increased recruitment of T-lymphocytes thus furthering aninflammatory cycle in COPD. These findings substantiateCXCR3 as a potential target for the treatment of COPD butgive rise to the possibility of perhaps a combined CXCR3,CXCR6 antagonist. It has been noted that the down-regulation on CXCR6 by T-lymphocyte activation signalsthat involve the Ca2+-dependent calcineurin pathway [125],which has the potential to be exploited in the search forsuitable antagonists for the treatment of COPD.

MCP-1 responsiveness is accompanied by an increase inCCR1 and CCR5 expression [132]. This observation mayhave some relevance in COPD. There are increased levels ofMCP-1 in the airways of patients with COPD [26]. MCP-1is responsible in part for monocyte migration into theairways [36] which results in an increase in macrophageburden in COPD. If receptor expression changes uponmonocyte to macrophage differentiation [132] themacrophages may be retained in the tissues as they cannot berecruited back out of the tissues via CCR2 and contribute tothe airway destruction associated with COPD. The increasein CCR1 and CCR5 expression results in macrophagesresponding to both MIP-1α and eotaxin predominantly. I-TAC is antagonistic for CCR5, therefore the actions of MIP-1α would be inhibited by this chemokine [57]. At present itis unclear how these chemokines interact and the effects theyhave on macrophages in COPD, further research into thesereceptors is needed to clarify their role in COPD.

CCR3

CCR3 is present on eosinophils, basophils, mast cellsand Th2 T-lymphocytes [65]. The eotaxin family areagonists for CCR3 but the CXCR3 ligands are antagonistic[65]. It is thought that the actions of the CXCR3 ligands onCCR3 might enhance Th1 mediated responses and thus Th1mediated diseases [65], such as COPD [60].

OTHER CC CHEMOKINE RECEPTORSCC CHEMOKINE RECEPTORS

The remaining CC chemokine receptors, CCR4, CCR6,CCR7, CCR8, CCR9, CCR10 and CCR11 do not bindchemokines that have a defined role in COPD. A greaterunderstanding of the pathogenesis of this disease mayhighlight the importance of these receptors but at presentthey are not considered potential targets for therapeutics inCOPD.

CCR2

CCR2 is the only leukocyte MCP-1 receptor so faridentified. It is important in inflammation as it has thecapacity to recruit monocytes to sites of inflammation [18].CCR2 cDNA encodes two MCP-1 specific receptors,CCR2(a) and CCR2(b) [128]. CCR2(b) appears to beexpressed predominantly and plays a role in chronicinflammation including atherosclerosis and multiplesclerosis. The mRNA for CCR2 is detectable in monocytes,blood-derived dendritic cells, NK cells and T-lymphocytesbut not in neutrophils [129, 130]. Antibody studies haveshown that CCR2(b) is expressed in monocytes, activatedmemory T-lymphocytes, and B-lymphocytes [129, 130].CCR2 makes a good therapeutic target for COPD asinhibition of this receptor would reduce the number ofmonocytes and thus macrophages in the airways of patientswith COPD. Monocyte migration towards MCP-1 isinhibited by the presence of an anti-CCR2 antibody (Traves,S.L unpublished data), and treatment with MCP-1neutralising antibodies and antagonists reducedinflammation indicating that modulation of MCP-1expression or activity may be beneficial in treatinginflammatory diseases [131]. There are a number of smallmolecule antagonists currently under examination.

THERAPEUTIC TARGETS IN COPD

The observation that there are twice as many chemokinesas receptors led to early speculation that a single receptorwould bind a number of chemokines indicating a level ofredundancy. This resulted in the question as to how thechemokines co-operate to bring about an inflammatoryresponse. Initially it was suggested that a single chemokinecould attract a specific cell type [133], however this isunlikely [18, 48] as it is known for example, that CXCR1and CXCR2, which bind ELR+ CXC chemokines, arepresent on both neutrophils and monocytes [36, 37]. Thesereceptors are functional on both cell types and areresponsible for the observed migration of neutrophils [47]and monocytes [36, 37]. This is important when identifyingtherapeutic targets for COPD, as the inhibition of a singlereceptor or chemokine may reduce the influx of a number ofinflammatory cell types. Deciding on a single receptor orchemokine as a therapeutic target for reducing theinflammatory load in the lung is difficult. The matter isfurther complicated by the observation that chemokines mayalso act as functional antagonists at other, distinct receptors,thus may play a role in reducing airway inflammation ororchestrating a particular type of inflammatory response, for

CCR1 and CCR5

Monocytes predominantly express CCR2 but during theirdifferentiation into macrophages CCR2 expression is down-regulated [132]. This down-regulation in CCR2 and thus

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example Th1 vs Th2. This review however has highlighted anumber of potential therapeutic targets for the treatment ofCOPD.

INHIBITION OF CHEMOKINES

Another possible route of reducing the inflammatoryinflux into the airways is to inhibit chemokine production,or to inhibit the signalling pathways stimulated uponchemokine-receptor binding. Inhibition of these pathwayswould lead to lower chemokine levels which, in turn, willreduce the number of cells migrating into the airways.Blocking antibodies are one way of achieving this effect.Currently, there are blocking antibodies available for IL-8and related chemokines which reduce neutrophilicinflammation in animal models and the chemotactic activityof induced sputum from patients with COPD for neutrophils[139]. There is currently a human monoclonal antibody forIL-8 in COPD clinical trials [139], however there are otherCXC chemokines involved in the pathogenesis of COPD,therefore these studies will be of benefit in determining thelevel of redundancy in this system.

RECEPTOR ANTAGONISM

The most relevant therapeutics for the treatment ofCOPD would be compounds that inhibit key receptors, inorder to decrease the inflammatory cell influx into theairways and thus reduce the amount of damage to the lungs.The ideal receptors to target would appear to be CXCR2 andCXCR3. Inhibition of these receptors would reduce thenumber of neutrophils, T-lymphocytes and monocytes, andtherefore macrophages entering the airways. Completeinhibition of these receptors may result in such a reducedlevel of inflammatory cells reaching the airways thathomeostasis is not maintained, leaving the patientimmunosuppressed. Partial inhibition of these receptorscould reduce the inflammatory load in the lung to thatobserved in healthy subjects, leading to a reduced level oftissue destruction associated with COPD, but maintaininghomeostasis.

Nuclear factor (NF)κB is a transcription factor which isactivated in macrophages and epithelial cells in COPD andregulates the expression of inflammatory chemokines [139].Small molecule inhibitors of IKK2 upstream of NFκB, cansubdue the release of chemokines from alveolar macrophages[139]; these inhibitors could be potential therapeutics forCOPD. One disadvantage of using these as long-termtherapeutics again is the possibility of immunosuppression[139]. Another potential signal transduction pathway that isof interest is the p38 MAPK pathway. Small moleculeinhibitors of this pathway have a wide spectrum of anti-inflammatory effects [139]. SB239063 has been successful atreducing neutrophilic infiltration and as well as decreasingthe concentrations of IL-6 and MMP-9 in BAL fluid in ratmodels, highlighting it as a potential treatment for COPD[139].

A range of selective small-molecule antagonists forCXCR2 have been tested using a rabbit model as theirperipheral blood neutrophils express both CXCR1 andCXCR2 [134]. These experiments have led to the discoveryof a potent CXCR2 antagonist which is effective in both thissystem and human neutrophils in vitro [134]. This indicatesa role for this compound in the treatment of COPD. Thereare however a number of factors that should be taken intoconsideration. CXCR2 receptor knockout mice have beenshown to be more susceptible to infections [135]. The samemay apply to humans, where by receptor blockade may leadto an imbalance in the normal homeostatic functions ofinflammatory cells. A combination of receptor antagonists,for example a CXCR2 and CXCR3 antagonist may be ofadditional benefit as multiple cell types could be targeted.

The main obstacle for the discovery of successfultherapeutics for the treatment of COPD is that the role ofchemokines in airway inflammation is extremely complexand requires further investigation. The elucidation of thepathways controlling the expression of cytokines andchemokines will not only improve our understanding of themechanisms underlying inflammatory lung diseases but willalso aid in the design of novel therapies for the treatment ofthese diseases. There are a number of candidate compoundsundergoing clinical trials at the present time though whetherone compound alone is enough to halt the progression ofthis fatal disease remains to be elucidated.

Another potential therapeutic target is CCR2, asinhibition of this receptor would reduce the number ofmonocytes entering the airways. This is turn would reducethe number of macrophages which can cause all thepathological effects associated with COPD. Again CCR2knockout mice are more susceptible to infection, possiblydue to a defect in effective antigen presentation to T-lymphocytes [136]. CCR2 knockout mice also show signsof exacerbated disease in some models [137]. The reasons forthis is unknown but indicates that chemokines and theirreceptors have a role in down-regulating inappropriateimmune responses under normal conditions [138].

REFERENCES

[1] Pauwels RA, Rabe KF. Burden and clinical features of chronicobstructive pulmonary disease (COPD). Lancet 2004; 364: 613-620.

Another way of reducing the inflammatory influx intothe airways could be to block adhesion molecules. E-selectinpresent on endothelial cells interacts with sialyl-LewisX onneutrophils, a compound, TBC1269 has been developedwhich imitates sialyl-LewisX [139]. This compound canblock selectins and inhibit granulocyte adhesion onneutrophils; however, again there are concerns with reducingneutrophil numbers to such an extent that the neutrophilicresponse is compromised [139]. A more appropriate targetmay be MAC1 (CD11b/CD18) which is expressed onneutrophils, monocytes and macrophages [139], althoughlittle is known about the effect of MAC1 in COPD.

[2] Lopez AD, Murray CC. The global burden of disease, 1990-2020.Nat Med 1998; 4: 1241-1243.

[3] Barnes PJ, Kleinert S. COPD--a neglected disease. Lancet 2004;364: 564-565.

[4] GOLD. Global Initiative for Chronic Obstructive PulmonaryDisease. Global Strategy for the Diagnosis, Management, andPrevention of Chronic Obstructive Pulmonary DiseaseNHLB/WHO Workshop Report. 2001; NIH Publication No 2701:1-100.

[5] Barnes PJ. Chronic obstructive pulmonary disease. N Engl J Med2000; 343(4): 269-280.

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Received: October 23, 2004 Accepted: November 1, 2004