Targeting chemokine receptors in allergic disease

Biochem. J. (2011) 434, 11–24 (Printed in Great Britain) doi:10.1042/BJ20101132 11

REVIEW ARTICLETargeting chemokine receptors in allergic diseaseJames E. PEASE1

Leukocyte Biology, and MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, NHLI Division, Faculty of Medicine, Imperial College London, London SW7 2AZ, U.K.

The directed migration of cells in response to chemical cues isknown as chemoattraction, and plays a key role in the temporal andspatial positioning of cells in lower- and higher-order life forms.Key molecules in this process are the chemotactic cytokines, orchemokines, which, in humans, constitute a family of approx. 40molecules. Chemokines exert their effects by binding to specificGPCRs (G-protein-coupled receptors) which are present on a widevariety of mature cells and their progenitors, notably leucocytes.The inappropriate or excessive generation of chemokines is akey component of the inflammatory response observed in severalclinically important diseases, notably allergic diseases such asasthma. Consequently, much time and effort has been directedtowards understanding which chemokine receptors and ligands

are important in the allergic response with a view to therapeuticintervention. Such strategies can take several forms, although,as the superfamily of GPCRs has historically proved amenableto blockade by small molecules, the development of specificantagonists has been has been a major focus of several groups.In the present review, I detail the roles of chemokines and theirreceptors in allergic disease and also highlight current progress inthe development of relevant chemokine receptor antagonists.

Key words: allergy, asthma, chemokine, G-protein-coupledreceptor (GPCR), inflammation, leucocyte.

INTRODUCTION

Allergic diseases such as asthma, allergic rhinitis and atopicdermatitis are typified by an undesirable reaction to antigens(allergens) and are characterized by an influx of eosinophils,lymphocytes, basophils and mast cells to the inflamed tissue. In thelast two decades, the incidence of allergic diseases such as asthmahas reached epidemic proportions within Western industrializedcountries [1]. In the U.K. alone, over 5 million people currentlyreceive treatment for asthma, resulting in increased morbidity andpoorer quality of life for the afflicted and a significant financialburden upon healthcare structures [2]. Consequently, much timeand effort has been put into dissecting the allergic response in bothhuman and rodent models of disease, with a view to discoveringnovel therapeutic targets. In the present review, I discuss the rolesof chemokines and their receptors in the allergic response anddiscuss efforts in targeting chemokine receptors with chemokinereceptor antagonists.

CHEMOKINES AND THEIR RECEPTORS

Pivotal to leucocyte trafficking within allergic tissues are thechemotactic cytokines, or chemokines, and their receptorswhich finely tune leucocyte recruitment to both inflammatorysites and secondary lymphoid organs. Chemokines are smallproteins, typically of approx. 8−10 kDa, and induce a varietyof intracellular signals following binding to their cell-surfacereceptors. These signals serve to direct cell migration towardsthe source of chemokine in a process known as chemotaxis [3].The chemotactic property of chemokines is not specific to immunesystem cells. For example, in embryogenesis, the temporal and

spatial positioning of progenitors is finely tuned by the actionof the chemokine stromal-cell-derived factor 1 (CXCL12) andits specific receptor CXCR4 [4]. In addition to cell recruitment,chemokines also play important roles in angiogenesis, wherethe activation of chemokine receptors on endothelial cells canpromote both angiogenic and angiostatic responses [5].

In humans, the chemokine family consists of over 40 membersdivided into two major and two minor families on the basis of thelocation of two N-terminal cysteine residues. In the CC family,these cysteine residues are adjacent, whereas, in the CXC family,they are separated by a single amino acid. Two other minorclasses, the CX3C and C chemokine families exist which havethree members between them. A systemic nomenclature is nowin operation in which chemokines are given the prefix CCL (CCligand), CXCL (CXC ligand), CX3CL (CX3C ligand) and XCL(C ligand), together with an identifying number, updating theanecdotal method of defining a chemokine upon its function [6].Chemokines exert their biological effects by binding to specificGPCRs (G-protein-coupled receptors), which, in humans, number19. The prefixes CCR and CXCR are used to define receptors forCC and CXC chemokines respectively (Table 1). Promiscuityis rife among the receptors with each receptor typically havingseveral chemokine ligands, although the ligand repertoire ofreceptors is class-restricted, i.e. CC chemokines have agonistactivity only at CC chemokine receptors and, likewise, CXCchemokines only activate CXC chemokine receptors.

LEUCOCYTE TRAFFICKING DURING THE ALLERGIC RESPONSE

The allergic response can be broadly broken down into two com-ponents, the first or early-phase response which peaks at approx.

Abbreviations used: AHR, airways hyperresponsiveness; ASM, airway smooth muscle; BALF, bronchioalveolar lavage fluid; BALT, bronchus-associatedlymphoid tissue; CNV, choroidal neovascularization; DC, dendritic cell; FcεRI, high-affinity IgE receptor; HPSC, haemopoietic pluripotent stem cell; IFNγ,interferon γ; IL, interleukin; PBMC, peripheral blood mononuclear cell; plt , paucity of lymph node T-cells; SCID, severe combined immunodeficiency; Th,T-helper; Treg, regulatory T-cell.

1 email [email protected]

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Figure 1 Cellular and molecular events in the allergic immune response

(A) Events of the early phase of the allergic response in which IgE bound by FcεRI on mast cells is cross-linked by allergen (red). The ensuing mast cell activation and degranulation releases avariety of cytokines and mediators with paracrine effects on other cells, such as the stimulation of IgE production by B-cells, the enhanced survival of eosinophils (Eo) and the contraction of smoothmuscle. PAF, platelet-activating factor. (B) Events of the late-phase reaction in which antigen uptake by DCs and subsequent presentation to Th2 cells induces the expression of more cytokines andmediators with activities on additional leucocytes [e.g. basophils (Bo) and non-leucocytes (e.g. epithelial cells)]. TGF-β1, transforming growth factor β1.

Table 1 Human chemokine receptors and their expression by leucocytes

Abbreviations: B, B-lymphocyte; Bo, basophil; Eo, eosinophil; Mc, mast cell; Mo, monocyte; NK,natural killer cell; No, neutrophil; PMN, polymorphonuclear cell; T, T-lymphocyte. For simplicity,only receptor/ligand pairs reported to induce intracellular signalling are shown.

Chemokine receptor Chemokine ligands Leucocyte expression

CCR1 CCL3, CCL3L1, CCL5, CCL7 CCL14,CCL16, CCL23

Mo, DC, Eo, Bo, T, PMN, NK

CCR2 CCL2, CCL5, CCL7, CCL13 Mo, DC, T, BoCCR3 CCL5, CCL7, CCL8, CCL11, CCL13,

CCL15, CCL24 CCL26 CCL28Eo, T, Bo, Mc

CCR4 CCL17, CCL22 DC, T, Bo, NKCCR5 CCL3, CCL3L1, CCL4, CCL5, Mo, DC, TCCR6 CCL20 DC, TCCR7 CCL19, CCL21 DC, T, B, NKCCR8 CCL1 Mo, T, NKCCR9 CCL25 TCCR10 CCL27, CCL28 TCXCR1 CXCL6, CXCL8 No, MoCXCR2 CXCL1, CXCL2, CXCL3 CXCL5,

CXCL6,CXCL7, CXCL8No, Mo

CXCR3 CXCL9, CXCL10, CXCL11 T, BCXCR4 CXCL12 T, B, DC, MoCXCR5 CXCL13 T, BCXCR6 CXCL16 TCXCR7 CXCL11, CXCL12 NoneXCR1 XCL1 T, NKCX3CR1 CX3CL1 T, NK, DC, Mo

15 min after allergen challenge and is initiated by leucocytesknown as mast cells. Mast cells are derived from pluripotentstem cells which reside in the bone marrow. They are releasedinto the blood as progenitors which are recruited into tissues,where they mature into long-lived resident mast cells under theinfluence of specific growth factors and cytokines. Patients with

allergic rhinitis can have 50-fold more mast cells in the nasalmucosa in the pollen season when compared with the winterseason [7,8], whereas mast cell numbers can be 10-fold higherin the lungs of asthmatic patients when compared with controls[9]. An increase in circulating mast cell progenitors in asthmaticpatients has also been reported [10], suggesting the existenceof mechanisms responsible for the both recruitment and localmaturation of mast cell progenitors to maintain basal populationsof mast cells in tissues, and also additional mechanisms to increaselocal mast cell numbers in the allergic setting.

The mast cell is capable of mounting allergen-specificresponses conferred by an IgE antibody bound to high-affinityIgE receptors (FcεRI) which is achieved following allergen cross-linking of IgE (Figure 1A). This triggers the degranulation of themast cells and results in a release of pre-formed mediators, suchas histamine, and rapidly synthesized mediators such as LTC4

(leukotriene C4) and PGD2 (prostaglandin D2). These mediatorsact on structural cells to induce a variety of effects, includingsmooth muscle contraction, increased microvascular permeabilityand, in the case of allergic airways, increased mucus production(Figure 1). IL (interleukin)-5 is an important survival factorfor eosinophils [11,12] and induces the release of eosinophilprogenitors from the bone marrow [13], whereas IL-4 and IL-13 induce naive B-cells to switch to IgE production, driving theallergic response forwards [14,15].

The early phase of the allergic reaction is followed severalhours later by a late-phase reaction which is remarkable foran influx of leucocytes to the effected tissue, including T-cells,eosinophils and basophils. Key to the late-phase reaction andsubsequent leucocyte recruitment is the interplay between avariety of structural and immune cells, notably that betweenDCs (dendritic cells) and effector CD4+ Th (T-helper) cells(Figure 1B). DCs act as immune system sentinels with a voraciousappetite for antigen. On encountering and taking up antigen, DCsundergo a process of maturation in which they relocate to local


Chemokine receptors in allergic disease 13

Figure 2 Chemoattractants and their receptors orchestrate leucocyte trafficking in the allergic immune response

Gradients of chemokine are generated in the tissues following exposure to allergen and subsequent interplay between leucocytes and structural cells. Chemokines induce the release of leucocyteprogenitors from the bone marrow to the peripheral blood and guide their recruitment along a concentration gradient to the site of allergic inflammation and chemokine generation. Key to thisrecruitment is the dynamic cell-surface expression of a wide repertoire of chemokine receptors (depicted as serpentine structures) which also mediate DC and T-cell trafficking to secondary lymphoidorgans in the adaptive immune response. Some chemokines such as the Th1-associated CXCR3 ligands can inhibit Th2 cell recruitment, thereby fine-tuning the immune response. Eo, eosinophil;MC, mast cell.

lymph nodes via the lymphatic system and present processedantigen to resident T-cells, resulting in T-cell activation anddifferentiation. In both humans and rodents, distinct subsets of Thcells mediate specific immune responses tailored to the microbeencountered. The presence of specific cytokines dictates whichlineage of Th cell will be produced. IFNγ (interferon γ ) and IL-12drive naı̈ve T-cell differentiation towards cells of the Th1 lineagewhich themselves produce IFNγ and are critical for responsesagainst intracellular pathogens and viruses. IL-4 is a key cytokinefor the production of Th2 cells which themselves produce IL-4 and are key for responses to intracellular pathogens such ashelminths and the promotion of Ig production by B-cells. BothIFNγ and IL-4 form autocrine positive-feedback loops to amplifyTh1 and Th2 differentiation respectively and antagonize eachother’s differentiation by a number of mechanisms [16]. Arecently discovered Th17 subset of cells (so-called because theyproduce IL-17) is thought to govern responses to fungi andintracellular bacteria [17]. Th responses are kept in check by theactivities of another subset of T-cells known as Tregs (regulatoryT-cells) which secrete IL-10 with potent immunosuppressiveproperties.

Allergic reactions are thought to be an aberrant anti-helminthresponse, and Th2 cells dominate at the site of allergensensitization. The subsequent secretion of Th2 cytokines such asIL-4 and IL-13 induces the generation of chemokines by epithelialcells which recruit effector cells such as eosinophils and basophils(Figure 2). Th2-produced IL-5 also promotes eosinophil survival.In the case of an airway from an asthmatic patient, the leukotrienesproduced by the incoming eosinophils induce bronchoconstrictionand mucus hypersecretion, whereas eosinophil-derived TGF-β1(transforming growth factor β1) drives a pro-fibrotic response. Inthe case of repeated provocation with allergen, the excessive repairprocesses result in increased ASM (airway smooth muscle) mass,

collagen deposition, angiogenesis and subsequent thickening ofthe airway wall in a process known as remodelling [18−20].

Although they account for less than 1% of circulatinggranulocytes in the peripheral blood, basophils are importanteffector cells in IgE-mediated allergic reactions, producinghistamine, cytokines and lipid mediators following FcεR1 cross-linkage by antigen [21]. They arise from the same progenitorcells as mast cells [22] and are thought to be part of a positive-feedback loop for IgE-mediated immediate-type hypersensitivityreactions [23]. Basophils have been shown to be a predominatingantigen-presenting cell in Th2 reactions against both helminthsand allergens, presenting peptide in conjunction with MHC classII molecules to naive CD4+ T-cells and producing IL-4 in theprocess [24−26].

CC CHEMOKINE RECEPTORS IMPLICATED IN THE ALLERGICRESPONSE

CCR1

As its name suggests, CCR1 was the first of the CC chemokinereceptors to be identified and is expressed by mast cells [27],eosinophils [28,29] basophils [30,31] and NKT (natural killerT-) cells [32]. CCR1 binds a variety of chemokine ligands,notably CCL3 and CCL5, both of which are found in the BALF(bronchioalveolar lavage fluid) from allergen-challenged lungsfrom an asthmatic patient [33,34] and in nasal secretions fromallergen-challenged allergic rhinitic patients [35,36]. Likewise,in allergen-induced late-phase reactions in the skin, CCL3 isproduced by influxing neutrophils and basophils [37]. CCR1 andFcεRI are co-localized within the plasma membrane of mast cells[38], and co-stimulation of both receptors results in an enhanceddegranulation response and decreased chemotaxis to CCL2. This


14 J. E. Pease

is postulated to maintain mast cell numbers at sites of allergicinflammation, thereby focusing the immune response [39]. Ina murine model of allergic conjunctivitis, CCR1 deficiency orCCL3 blockade results in a reduction in disease score followingco-stimulation of CCR1 and FcεRI, suggesting a potential use foranti-CCR1 therapeutics in allergy [40].

CCR2

The chemokine receptor CCR2 binds all four members ofthe MCP (monocyte chemoattractant protein) family (CCL2,CCL7, CCL8 and CCL13) and is expressed by monocytes[41−45], Th1 cells [46,47], basophils [31] and mast cells[48]. Three of the four CCR2 ligands have been reported toinduce basophil degranulation directly, namely CCL2, CCL7and CCL13 [49−51]. CCR2 appears to be vital for an effectiveTh1 response, with deletion of CCR2 resulting in impairedresponses to intracellular pathogens [52−54], thought to be due todefective trafficking of monocytes with Th1-polarizing potential.As might be anticipated with the reciprocal nature of Th1/Th2responses, CCR2-deficient mice show enhanced Th2 responsesfollowing challenge with ovalbumin [55] and Aspergillus [56].Conversely, deletion of CCL2, the principal CCR2 ligand, resultsin reduced IL-4 and IL-5 production and an inability to undergoIg class switching following ovalbumin challenge of the airways[57,58]. This suggests a role for CCL2 in controlling Th2polarization, supported by the finding that depletion of murineCCL2 reduced AHR (airways hyperresponsiveness) in ovalbuminand cockroach allergen challenge models [59,60]. The preciserole for receptor and ligand in inflammation, however, is notstraightforward. Similar studies in CCR2-deficient mice haveshown enhanced Th2 responses to ovalbumin [55] and Aspergillus[56], whereas a direct comparison of CCR2- and CCL2-deficientmice reported intact Th2-mediated responses and lung fibrosisin both animals following challenge with Aspergillus [61]. In anon-human primate model of allergic airways disease followingchallenge with Ascaris suum antigen, CCR2 blockade by a specificmonoclonal antibody resulted in reduced AHR and macrophageand eosinophil recruitment to the lung, although it is noteworthythan no significant T-cell recruitment was detectable in this model[62].

CCL2 has been shown to stimulate degranulation of mastcells in a murine model of allergic conjunctivitis with CCR2blockade significantly reducing the disease score [63]. A recentreport also highlighted a complex role for the CCL2/CCR2 axisin the recruitment of mast cell progenitors to the allergic lung[64]. In this study, sensitization followed by allergen challengeresulted in mast cell progenitor recruitment to the lung whichcorrelated with increased CCL2 levels in the BALF and whichwas significantly reduced in CCR2- and CCL2-deficient mice.In vitro culture of mast cell progenitors revealed that CCR2 wasa functional receptor, but became uncoupled from chemotaxison maturing cells, despite maintaining surface expression levels.Reconstitution of CCL2-deficient mice with wild-type or CCL2-deficient bone marrow suggests that CCL2 produced by both bonemarrow-derived and lung stromal cells is required for mast cellprogenitor recruitment to the allergic lung. It remains to be seenwhether the corresponding chemokine/receptor axes direct mastcell recruitment in humans.

CCR3

CCR3 is the principal chemokine receptor expressed byeosinophils [65], and is also expressed by Th2 cells [66], basophils[67] and mast cells [68,69]. CCR3 binds the eotaxin family of

chemokines comprising CCL11 [70], CCL24 [71,72] and CCL26[73,74]. The accumulation of eosinophils within the bronchialwall is a characteristic feature of allergic airways inflammation,although the precise role of the eosinophil in asthmatic diseaseremains complex and controversial (see below). A critical rolefor CCR3 in orchestrating eosinophil migration is supported bythe use of CCR3-deficent mice which have reduced numbersin the gut and traffic in reduced numbers to the lungs followingallergen challenge [75]. Interestingly, in the airways, the trackingdefect lies predominantly at the point of migration from thesubendothelial space into the lung parenchyma, suggesting thatother chemokines may co-operate in general recruitment from thecirculation [75].

The route of sensitization is an important consideration inthis model as intradermal sensitization favours the developmentof increased AHR in response to the bronchoconstrictormethacholine which is absent from CCR3-deficient mice [76].In contrast, in CCR3-deficient mice, intraperitoneal sensitizationresults in increased numbers of intraepithelial mast cells in thetrachea compared with wild-type mice which was accompaniedby increased AHR [75]. This suggests that the CCR3/eotaxinaxis does not play a major role in mast cell homing to the lungand subsequent differentiation and that, in the absence of CCR3,mast cell progenitors are recruited and retained in the airwayepithelium. This is in keeping with a recent report suggesting thatimmature and mature bone marrow-derived mast cells are unableto migrate in vitro to the CCR3 ligands CCL11 and CCL24 [77].Similarly, skin mast cell numbers were normal in CCR3-deficientmice following epicutaneous ovalbumin sensitization, suggestingthat CCR3 does not play a role in mast cell homing to the skin [76].However, in a murine model of allergic conjunctivitis, antagonismof CCR3 was reported to impair the early-phase reaction thoughtto be due to the stabilization of mast cells, perhaps suggesting arole for the receptor in mast cell degranulation [78].

In humans, CCL11 plasma levels have been reported to beelevated in acute compared with stable asthmatic patients [79],and increased expression of CCL11 and CCL24 is observed in theallergic lungs of asthmatic patients [80,81] and in their sputum[82,83]. In mice, although functional orthologues of CCL11and CCL24 are found, CCL26 is apparently a pseudogene [84].Studies of an ovalbumin-sensitization model of airway diseaseemploying mice either singly or doubly deficient in CCL11 andCCL24 suggest that both ligands need to be depleted for ablationof pulmonary tissue eosinophilia to levels observed in CCR3-deficient mice [85].

CCR4

CCR4 specifically binds the chemokines CCL22 and CCL17[86]. DC maturation is accompanied by the production of CCL17and CCL22, and elevated levels of both chemokines have beendetected in the skin lesions of atopic dermatitis patients [87−89],and in the lungs of both healthy volunteers and asthmatic patientsfollowing allergen challenge [90,91]. CCR4 itself is expressedby mast cells [27], monocytes, DCs and natural killer cells [92],and is notably induced on Th2 cells following their polarizationin vitro [46,47]. Such polarization is also observed in vivo withIL-4-producing cells recovered from the BALF of asthmatic andhealthy subjects shown to be CCR4+ [93].

Several studies support a role for CCR4 and its ligands CCL17and CCL22 in T-cell trafficking to the allergic murine lung.Antigen-specific Th2 cells from CCR4-deficient mice fail to trafficin significant numbers to the allergic lung of wild-type micefollowing adoptive transfer [94], whereas antibody neutralizationof CCL22 proved effective in the long-term blockade of Th2



cell recruitment following repeated stimulation with allergen[95]. Similarly, neutralization of CCL17 decreased CD4+ andeosinophil recruitment to the BALF, coupled with reductionsin AHR and Th2 cytokine production [96]. In contrast withthese studies, CCR4-deficient mice showed no protection againstairway inflammation [97]. Likewise, neutralization of CCR4 inguinea pigs with a monoclonal antibody was ineffective in termsof modulation of the allergic response [98]. More recently, CCR4blockade in a human PBMC (peripheral blood mononuclearcell)-reconstituted SCID (severe combined immunodeficiency)mouse model via a small-molecule antagonist was reported toabolish many of the features of inflammation, including airwayeosinophilia, goblet cell hyperplasia, IgE synthesis and bronchialhyperreactivity [99]. The same small-molecule antagonist has alsoproved effective in the in vitro blockade of responses from CCR4+

T-cells, a population increased in numbers in nasal biopsies ofrhinitic patients following allergen challenge, suggesting anotherpotential therapeutic avenue for CCR4 blockade [100].

CCR7

CCR7 binds the chemokines CCL19 and CCL21 [101−103] andplays a key role in the homoeostatic trafficking of B-cells, T-cellsand DCs to secondary lymphoid organs [104,105]. The DDD/1mouse carries an autosomal recessive mutation known as paucityof lymph node T-cells (plt) [106] in which the gene encodingCCL19 and one of the two genes encoding CCL21 has beendeleted, resulting in a lack of CCL19 and CCL21 expressionin lymphoid organs, but expression of CCL21 in non-lymphoidorgans [107−109]. Together with CCR7-deficient mice, they haveproved valuable tools to dissect the role of CCR7 ligands inhomoeostasis and inflammation, revealing many roles for ligandand receptor in leucocyte function.

CCR7-deficient mice exhibit a host of defects, includingdefective migration of positively selected thymocytes fromthe cortex to the medulla [105], which is essential for theestablishment of central tolerance in the thymus [110]. Theyalso have impaired leucocyte migration and poorly formedsecondary lymphoid organs [111], although they do develophighly organized BALT (bronchus-associated lymphoid tissue)[112]. Notably, they possess dramatically reduced numbers ofTregs in the lung-draining bronchial lymph nodes, suggesting thatTregs control BALT formation and rely upon CCR7 for homing tothe peripheral lymph nodes [112]. CCR7 signalling also appearsto have a positive influence on the intranodal motility of T-cells,as CCR7 or plt mice exhibit impaired motility as assessed byintravital two-photon microscopy [113]. Similarly, there is a rolefor CCR7 in egress from tissues as, although CCR7+ and CCR7−

effector T-cells can be recruited to the allergic murine lung, onlyCCR7+ T-cells are able to exit the lung and enter the afferentlymphatics [114].

DCs play an key role in sensitization to inhaled allergens astheir depletion results in a loss of leucocyte recruitment and Th2cytokine secretion which can be restored by adoptive transfer ofDCs [115]. Migration of mature DCs to the draining lymph nodesis heavily dependent upon CCR7 as plt mice and CCR7− /− strainshave severely impaired DC trafficking from the skin to the lymphnodes following allergen challenge [107,111,116]. Likewise, in ahuman PBMC-reconstituted SCID mouse model, neutralizationof the CCR7 ligand CCL21 reduced the pulmonary inflammatoryresponse following allergen challenge of mice repopulated withT-cells from allergic donors [117]. DC migration from the lung tothe draining bronchial lymph nodes under steady-state conditionsis also impaired by CCR7 deletion, explaining the inability of

CCR7-deficient mice to induce tolerance to intratracheal instilledovalbumin [118].

Curiously, in models of airways inflammation in responseto inhaled ovalbumin, plt mice exhibit enhanced airwayinflammation and AHR compared with controls, despite an initialdelayed IgE-specific response [119,120]. This may suggest anadditional role for CCR7 in the resolution of inflammation andtogether with the impaired tolerance and Treg trafficking observedin CCR7-deficient mice might argue against antagonism ofCCR7 in allergic disease treatment. ASM has also been reportedto express CCR7, and the increased CCL19 found in bronchialbiopsies of asthmatic patients may suggest a role for theCCR7−CCL19 axis in promoting the ASM hyperplasia observedin asthma [121].

CCR8

CCR8 is one of the few monogamous receptors in the chemokinesystem, with only CCL1 able to induce its activation. It does haveadditional suitors among the viral chemokines, however, with theHHV-8 (human herpesvirus 8)-encoded chemokine vMIP-I (viralmacrophage-inflammatory protein I) acting as a CCR8 agonist[122,123]. Polarization of T-cells in vitro to Th2 subsets results inexpression of CCR8 mRNA [46,47]. Conflicting reports exist inthe literature as to whether or not increased expression of CCL1 isobserved in the lungs of asthmatic patients compared with healthycontrols [124−128] and also whether or not increased numbersof CCR8+ T-cells are recruited to the allergic lung. The latter hasbeen problematic due to the lack of a seemingly reliable antibodyagainst human CCR8. For example, in one study examiningCCR8 expression on a population of CD4+ CD25+ Tregs, CCR8surface staining was undetectable using a commercially availableantibody, although the cells evidently expressed functionalCCR8, as assessed by CCL1-driven actin polymerization[129].

Studies in mice aimed at validating CCR8 as a target forallergic airways disease suggest that, as is the case with otherligand/receptor axes in the chemokine system, the precise roleof CCR8 and CCL1 in disease is complex. Initial studies usingknockout mice suggested that the role of the receptor was minimal,as CCR8-deficient mice did not exhibit impaired inflammation ofthe airways following allergen challenge [130,131]. The findingthat CCR8 is also expressed by CD4+ CD25+ Tregs may helpto interpret these data, as the inability of Tregs to traffic to sitesof inflammation is likely to be disadvantageous [132,133]. Incontrast, the CCR8/CCL1 axis appears to be important for therecruitment of eosinophils to the murine lung [134]. Notably, invitro studies of mast cells report that CCL1 is the predominantchemokine secreted following their activation, and subsequentstudies with CCL1 blockade or CCR8-deficient mice have showna reduction in lung inflammation similar to that detected inmast cell-deficient mice, leading to the hypothesis that that mastcell-derived CCL1 serves to recruit CCR8-expressing CD4+ T-cells to the inflamed lung [127]. CCR8-deficent mice sensitizedwith Aspergillus fumigatus antigens were shown to clear fungalmaterial more rapidly from the lung than littermates, suggestingthat CCR8 may be a useful target in fungal-associated pulmonarydiseases [135].

CXC CHEMOKINE RECEPTORS IN ALLERGIC DISEASE

CXCR1 and CXCR2

CXCR1 and CXCR2 were the first chemokine receptors tobe identified at the molecular level and bind a number of


16 J. E. Pease

CXC chemokines, notably CXCL8 [136,137]. Both receptorsare principally expressed by neutrophils and, in patients withsevere asthma exacerbations, increased neutrophil recruitment tothe bronchial mucosa is accompanied by increased expressionof CXCR1 and CXCR2, together with the ligands CXCL5 andCXCL8 [138].

CXCR2 ligands form a subset of CXC chemokines containingan ELR (Glu-Leu-Arg) motif at their N-terminus and have knownangiogenic properties. Ex vivo culture of human microvascularendothelial cells from healthy lung in a cocktail containing IL-4and TNFα (tumour necrosis factor α) has been reported to promotetube formation in a CXCR2-dependent manner [139], whereasa recent study reported that the recruitment of bone marrow-derived endothelial progenitor cells to the lungs following allergenchallenge could be significantly impaired by CXCR2 blockade[140]. Both studies suggest that CXCR2 may be an attractivetarget for manipulating the airway remodelling associated withasthma.

Similarly, in a murine model of A. fumigatus-induced asthma,CXCR2 deficiency results in significantly reduced production ofTh2 cytokines in the allergic lung, together with reduced numbersof eosinophils and T-cells [141]. Notably, the recruitment ofneutrophils was unimpaired in these mice and was dependenton the production of two other CXC chemokines, CXCL9 andCXCL10. Neither of these chemokines are known neutrophilattractants, suggesting that they act by inducing the expressionof other neutrophil chemoattractants [141]. CXCR2 also appearsto play a role in the homing of murine mast cell progenitors to thesmall intestine, as CXCR2-deficient mice have reduced numbersof intestinal mast cell progenitors [142].

CXCR3

CXCR3 is expressed by approx. 40% of freshly isolated T-cells from peripheral blood and is rapidly up-regulated uponpolarization to the Th1 phenotype [143,144]. CXCR3 mediateschemotaxis in response to the chemokines CXCL9, CXCL10and CXCL11 [145,146], and all three chemokines are inducedby the archetypal Th1 cytokine IFNγ and their production istypically associated with responses to intracellular pathogenssuch as viruses. Although atopic dermatitis is thought to be aTh2-type disease, IFNγ is produced in chronic disease and allthree CXCR3 ligands have been detected in the lesional skinfrom atopic dermatitis patients and also skin biopsies exhibitingallergic contact dermatitis reactions [147−149].

CXCR3 ligands are also up-regulated in both human [91] andmurine [150] lungs following allergen challenge, but their preciserole in disease is unclear. In a study by Thomas et al. [151]of asthmatic patients undergoing segmental allergen challenge,virtually all CD4+ cells recovered from the BALF pre-challengewere CXCR3+, which was rapidly down-regulated followingallergen challenge, suggesting that the receptor underwentendocytosis following engagement with ligand [151]. We andothers have shown in vitro that CXCR3 ligands are naturalantagonists of CCR3-mediated responses such as Th2 cellrecruitment [152,153], which, bearing in mind the expression ofCCR3 on Th2 cells, may serve to finely tune T-cell polarizationand the subsequent immune response in vivo.

CXCR4

CXCR4 is another monogamous chemokine receptor, binding thechemokine CXCL12 [154,155] and, unlike most other chemokinereceptors, its expression is almost ubiquitous, particularly during

embryogenesis where it plays a critical role in haemopoiesis,angiogenesis, neurogenesis and cardiogenesis, as is evident fromthe lethality of CXCR4 deletion in the mouse [156−158]. Bothligand and receptor are also vital for the homing and retentionof HPSCs (haemopoietic pluripotent stem cells) within the bonemarrow [159,160]. In the context of allergic disease, allergenchallenge of asthmatic patients has been reported to induce adown-regulation in CXCR4 expression on HPSCs and a reductionin levels of bone marrow CXCL12 levels which may promoteegress of cells to the periphery perhaps mediated via increasedlevels of the CCR3 ligand CCL11 present in the circulation[161]. Cell-surface levels of CXCR4 on Th2 cells have beenreported to be up-regulated by the Th2 cytokine IL-4 [162], andeosinophils recovered from the BALF of asthmatic patients haveincreased levels of cell-surface CXCR4 [163], suggesting thatthe receptor plays a role in leucocyte homing to the inflamedlung. Neutralization of either CXCR4 or CXCL12 by blockingmonoclonal antibodies was observed to reduce lung eosinophiliaand AHR in murine models of allergic airway inflammation[164].

Since CXCR4 is highly conserved across species, antagonistsdeveloped against the human receptor have excellent potency atmurine CXCR4, allowing them to be used in disease models.One such compound, named AMD3100, has been reportedto show efficacy in a murine model of cockroach allergen-induced inflammation, significantly reducing AHR and associatedinflammation [165]. A second-generation derivative, AMD3465,was also reported by the same group to have efficacy in a murinemodel of Th2-cell-mediated hypersensitivity to Schistosomamansoni egg antigen-coated beads [166]. AMD3100 (also knownas Plerixafor or Mozobil®) recently obtained FDA (U.S. Foodand Drug Administration) approval for use as an HPSC mobilizer,used in combination with G-CSF (granulocyte colony-stimulatingfactor) for the treatment of multiple myeloma, and it remainsto be seen whether it will have any clinical use in the allergicsetting.

ANTAGONIZING CHEMOKINE RECEPTORS IN ALLERGIC DISEASE

Chemokine receptors are members of the GPCR superfamily,and have historically proved amenable to antagonism by smallmolecules, with approx. 50% of currently prescribed drugs actingat GPCRs [167]. This has led to a hive of activity by thepharmaceutical industry to identify potent chemokine receptorantagonists. Chemokine binding to receptors appears to be acomplex process involving interactions of the chemokine with ahighly negatively charged receptor N-terminus, often rich in acidicresidues, sulfated tyrosine resides and O-linked glycosylation[168−170]. This interaction serves to tether the chemokineligand to the receptor and promotes secondary interactionswith other receptor regions, ultimately stabilizing a particularreceptor conformation, leading to the activation of G-proteins andintracellular signalling (Figure 3). Studies of several receptorshave implicated interactions involving the N-terminus of thechemokine with a hydrophobic binding pocket site composed ofthe receptor transmembrane helices [171−175]. Such pockets are‘highly druggable’ by small-molecule antagonists, and severalchemokine receptor antagonists have been shown to act asantagonists by binding within these regions [176−180]. Morerecently, a second class of antagonist-binding site has beendescribed, involving intracellular access of the antagonist to thereceptor C-terminus [181]. Although the precise residues makingup the binding site remain undetermined, this region contains ahighly conserved eight-helix which has been shown to influence



Figure 3 Molecular mechanisms underlying chemokine receptor activation and antagonism by small molecules

(A) The two-step model of chemokine receptor activation in which a chemokine (red) is tethered by the N-terminus of a receptor which it subsequently activates by insertion of the chemokineN-terminus into an intrahelical pocket. (B) A classical intrahelical binding site for the majority of small-molecule antagonists (green) including CCR3 antagonists [180]. These act as allostericmodulators allowing chemokine binding, but blocking activation. (C) A recently described novel intracellular binding site to which a subset of CCR4 antagonists have been postulated to bind [181].Access to this intracellular site is thought to rely upon the lipophilicity of the antagonist.

chemokine binding to the viral chemokine receptor ORF74 [182]and also β-arrestin translocation following activation of therelated formyl peptide receptor [183].

One initial hurdle in drug development has been the inabilityof human antagonists to hit the rodent orthologue withreasonable potency, making target validation in disease modelsrelatively difficult. As a consequence, despite several publicationsdescribing in vitro efficacy, descriptions of antagonist efficacy invivo lag well behind. Table 2 lists small-molecule antagonistswhich have been reported in the literature to possess efficacy inboth in vitro and in vivo models of allergic disease [165,184−196].Although several compounds have been described with efficacyin vivo, not all have gone on to feature in clinical trials.Reasons for this may include poor pharmacokinetic profiles andsafety issues. A notable problem with a variety of prototypicchemokine receptor antagonists has been that they often containa key quaternary nitrogen residue, postulated to interact with aconserved glutamate residue in the seventh transmembrane helixof the receptor [176,177,197]. Unfortunately, this also often givesthem a pharmacophore similar to that observed in blockers ofthe human ether-a-go-go-related gene, which inhibit a cardiacpotassium channel and may induce a fatal sudden cardiacarrhythmia [197,198]. Current antagonist screens utilize a varietyof techniques to try to weed out such cross-reactivity at an earlystage of drug development [200].

It can be argued that, in many instances, antagonist discoveryand progression to clinical trial has steamrollered aheadin the absence of a detailed understanding of the role ofchemokines and their receptors in disease. As our knowledgeof the chemokine system has increased, solutions to seeminglyintractable paradoxes have been forthcoming. For example, asmentioned above, CCR2-deficient mice show an impaired Th1response, yet mice deficient in the principal CCR2 ligand CCL2can mount perfectly robust Th1 responses. Similarly, in plt mice,although migration of DCs to the draining lymph nodes is ablated,Th1 and Th2 responses are apparently enhanced [201]. Bothparadoxes have recently been shown to unite around a subsetof CD11c+CD11bhiGr-1+ inflammatory DCs which are ableto capture large amounts of antigen and whose numbers are

substantially increased in plt mice compared with controls. Theseinflammatory DCs enter the lymph nodes directly from the bloodin a CCR2-dependent fashion, therefore bypassing CCR7 homingvia the lymphatics [202]. In CFA (complete Freund’s adjuvant)-immunized ccr2− /− mice, CD11c+CD11bhiGr-1+ DCs are ableto migrate to the draining popliteal lymph nodes (where CCL8 ishighly expressed), but are unable to traffic to inflamed footpads(where CCL2 is the most abundant chemokine). Likewise, sincewe know now that a substantial number of Tregs express CCR4and CCR8, targeting either receptor may have unwanted sideeffects [133,203,204]. A greater understanding of such intricaciesin the chemokine system might present clues as to why, to date,blockade of seemingly logical targets has been unsuccessful inthe clinical treatment of inflammatory disease [205,206].

To date, the only small-molecule chemokine receptor antagonistreported to have featured in any clinical trial of allergic diseaseis the CCR3 antagonist GSK766994 from GlaxoSmithKline.Despite being orally active in a brown rat model of asthma andpossessing a reasonable half-life, this compound sadly did notshow efficacy in a Phase III clinical trial for the treatment ofallergic rhinitis [192]. One reason put forward for the failureof this and other chemokine antagonists in the clinic is theapparent redundancy in the chemokine system, meaning thatblocking a single chemokine receptor in heterogeneous diseasessuch as asthma may prove unfruitful. This is particularly true inhumans where immune responses are likely to be much moreheterogeneous that in those observed using inbred rodent strains.In such a scenario, the use of a broad-spectrum antagonist hittingseveral receptors may be appealing. For example, the moleculeUCB35625 antagonizes both CCR1 and CCR3 [207], withboth receptors implicated in mast cell and eosinophil activation[31,41,42,78]. The activity of broad-specificity antagonists neednot be limited to chemokine receptors. For example, a dualantagonist of CCR3 and the H1 histamine receptor has beendescribed which may impede both leucocyte recruitment andthe vasodilatory and bronchoconstrictory properties of histamine[208]. Such compounds may provide the basis of novel therapeuticopportunities for the treatment of allergy, allowing the sparing ofcorticosteroids with obvious benefits for patients.


18 J. E. Pease

Table 2 Chemokine receptor antagonists with both in vitro efficacy and in vivo efficacy in models of allergic disease

Receptor Compound (company) In vitro potency In vivo efficacy Trial status Reference

CCR2 7a (Johnson & Johnson) Inhibition of CCL2 binding(IC50 = 4 nM)

A 100 mg/kg dose resulted in 36 % reduction in AHRand 83 % reduction in the total cellular influx inBALF of mice

[184]

CCR4 22 (Bristol-Myers Squibb) Inhibition of CCL22-inducedchemotaxis (IC50 = 3 nM)

A 30 mg/kg dose effective in reducing eosinophilnumbers into murine BALF

[185]

8c (Astellas Pharma Inc) Inhibition of CCL22-inducedchemotaxis (IC50 = 23 nM)

A 30 mg/kg dose resulted in inhibition of ear swellingin a murine contact hypersensitivity model

[186]

RS-1154 (Daiichi SankyoCo.)

Inhibition of CCL17-inducedchemotaxis (IC50 = 5.5 nM)

Effective at reducing ovalbumin-induced ear swellingat 30 mg/kg in a murine model.

[187]

K327 (Kyowa Hakko KirinCo.)

Inhibition of CCL17 binding(IC50 = 72 nM)

Inhibited the recruitment of CCR4+ CD4+ T-cells tothe murine lung in an ovalbumin-challenge modelat a dose of 44 mg/kg, twice daily.

[188]

CCR3 DPC 186 (Bristol-MyersSquibb)

Inhibition of CCL11-inducedchemotaxis at hCCR3(IC50 = 10−60 pM) andmCCR3 (IC50 = 41 nM)

Dose-dependent reduction in eosinophil recruitmentin a murine model of allergic airway inflammation

Entered into Phase I clinicaltrials

[189]

A-122057 A-122058 (AbbottLaboratories)

Inhibition of CCL11 binding (IC50

values of 600 and 975 nM)Oral dosage of 10 mg/kg caused a reduction in

CCL11-induced peritoneal eosinophilia in mice[190]

GW-766904(GlaxoSmithKline)

None published Described as having good selectivity,pharmacokinetic properties in the rat and dog andno significant cytochrome P450 inhibition

Subsequently entered clinicaldevelopment

[191]

GW701897B(GlaxoSmithKline)

None published Prevention of antigen-induced clustering ofeosinophils along the vagus nerves andhyperresponsive to vagal stimulation followingantigen inhalation in a guinea-pig model of airwayinflammation

[192]

GSK766994(GlaxoSmithKline)

None published Good pharmacokinetics in dogs with a reported 89 %bioavailability, a half-life of 2.8 h and reducedclearance. Orally active in a brown rat model ofasthma

No efficacy demonstrated in aPhase III allergic rhinitis trial

[193]

14n (Schering Plough) Inhibitor of CCL11 binding(K i = 3.5 nM) and humaneosinophil chemotaxis(IC50 = 160 nM)

Reasonable AUC (area under the curve) of 1341 ng/hper ml at 10 mg/kg by mouth in a ratpharmacokinetic study, although lower affinity forthe rat receptor resulted in significant challengeswith in vivo profiling

Produced an undesired 85 %inhibition at 1 μM in thehERG (humanether-a-go-go-related gene)voltage clamp assayprecluding furtheradvancement

[194]

YM-344031 (YamanouchiPharmaceutical Co.)

Inhibition of chemotaxis of humanCCR3-expressing cells(IC50 = 19.9 nM)

Oral administration to macaques (1−10 mg/kg)significantly inhibited CCL11-induced eosinophilshape change in whole blood. Oral administrationto mice (100 mg/kg) prevented both immediate-and late-phase allergic skin reactions

[195]

YM-355179 (YamanouchiPharmaceutical Co.)

Inhibtion of intracellular Ca2+

influx, chemotaxis, andeosinophil degranulation(respective IC50 values of 8.0,24 and 29 nM)

Oral administration (1 mg/kg) inhibitedCCL11-induced shape change of eosinophils inmacaques. Intravenous injection (1 mg/kg) alsoinhibited eosinophil infiltration into macaqueairways following segmental bronchoprovocationwith CCL11

[196]

CXCR4 AMD3100 (Genzyme) Inhibtion of intracellular Ca2+

(IC50 = 1−10 ng/ml)Significant reduction in murine AHR following

cockroach antigen challenge[165]

In asthma, efforts have centred upon CCR3 in the belief thattargeting eosinophil recruitment is likely to be beneficial in diseasetreatment. However, the precise role of the eosinophil in asthmaticdisease remains complex and controversial. Although deletion orneutralization of the eosinophil survival factor IL-5 suggested alink between eosinophil activation and AHR in murine studies[209,210], subsequent neutralization of IL-5 in mild asthmaticpatients was without effect on airways function [211], pouringcold water on the notion that targeting eosinophil recruitmentwould be beneficial as an asthma treatment and leading to a lossof interest in targeting CCR3 by the pharmaceutical industry.A role for eosinophils in the airways-remodelling process wassubsequently established by the use of eosinophil-deficient mice[212,213], which has since been shown to translate to humans[214]. Since a previous trial of corticosteroid treatment suggested

that reducing the number of sputum eosinophils was associatedwith a reduction in the frequency of asthma exacerbations, itwas a logical step to reassess the efficacy of IL-5 neutralizationin patients with defined eosinophilic asthma [215]. The resultsof two such trials found IL-5 treatment effective in reducing boththe levels of circulating and sputum eosinophils and the frequencyof asthma exacerbations [216,217]. As exacerbations within thispopulation of asthmatic patients are associated with significantmorbidity, mortality and health care costs, this has led to a renewedinterest in targeting the eosinophil in disease, provided that anappropriate cohort of patients is chosen for treatment. CCR3stands out as an obvious candidate molecule and, since it is alsoexpressed on other cell types involved in the allergic response,such as basophils [67], mast cells [68–69] and Th2 lymphocytes[66], targeting CCR3 may provide ‘added value’.



Of note is the fact that CCR3 activation has beenrecently implicated in AMD (age-related macular degeneration),with a recent high-profile study suggesting that CCR3signalling in retinal endothelial cells drives CNV (choroidalneovascularization) in both humans and mice in the distinctabsence of inflammation and leucocyte recruitment [218]. Useof a prototypic CCR3 antagonist in a murine model of CNV was anotably more effective inhibitor of CNV than the current clinicallyapproved anti-VEGF (vascular endothelial growth factor)-Atreatment, highlighting the point that antagonist development inone field of research may have promising applications in another[218]. The fact that two chemokine receptor antagonists have todate received regulatory approval (Miraviroc has been licensed forthe treatment of HIV-1 infection [219] and Plerixafor/Mozobil®

has been approved for haemopoietic stem cells mobilization[220]) has no doubt given ‘a shot in the arm’ to the field and revivedoptimism that antagonists showing efficacy in the inflammatorysetting will soon be forthcoming.

FUNDING

I am grateful to the Medical Research Council and Asthma UK for funding our research inthis area.

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Received 26 July 2010/20 October 2010; accepted 27 October 2010Published on the Internet 27 January 2011, doi:10.1042/BJ20101132


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Targeting chemokine receptors in allergic disease

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