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Review Dual targeting of CCR2 and CCR5: therapeutic potential for immunologic and cardiovascular diseases Qihong
Review Dual targeting of CCR2 and CCR5: therapeutic potential for immunologic and cardiovascular diseases Qihong

Review

Dual targeting of CCR2 and CCR5:

therapeutic potential for immunologic and cardiovascular diseases

Qihong Zhao 1

Research and Development, Bristol-Myers Squibb, Princeton, New Jersey, USA

RECEIVED OCTOBER 9, 2009; REVISED FEBRUARY 19, 2010; ACCEPTED FEBRUARY 22, 2010. DOI: 10.1189/jlb.1009671

ABSTRACT

A cardinal feature of inflammation is the tissue recruit- ment of leukocytes, a process that is mediated pre- dominantly by chemokines via their receptors on mi- grating cells. CCR2 and CCR5, two CC chemokine re- ceptors, are important players in the trafficking of monocytes/macrophages and in the functions of other cell types relevant to disease pathogenesis. This re- view provides a brief overview of the biological actions of CCR2 and CCR5 and a comprehensive summary of published data that demonstrate the involvement of both receptors in the pathogenesis of immunologic dis- eases (RA, CD, and transplant rejection) and cardiovas- cular diseases (atherosclerosis and AIH). In light of the potential for functional redundancy of chemokine re- ceptors in mediating leukocyte trafficking and the con- sequent concern over insufficient efficacy offered by pharmacologically inhibiting one receptor, this review presents evidence supporting dual targeting of CCR2 and CCR5 as a more efficacious strategy than target- ing either receptor alone. It also examines potential safety issues associated with such dual targeting. J. Leukoc. Biol. 88: 41–55; 2010.

Introduction

Tissue recruitment of leukocytes, a cardinal feature of inflam- mation, is mediated mainly by chemokines (chemotactic cyto- kines) via their receptors. The chemokine superfamily can be categorized into four groups (CC, CXC, CX3C, and C), ac- cording to the number and spacing of conserved cysteines in

Abbreviations: AIA adjuvant-induced arthritis, AIH accelerated intimal hy- perplasia, ApoB tg apolipoprotein B transgenic, ApoE apolipoprotein E, BOS bronchiolitis obliterans syndrome, CAV cardiac allograft vasculopa- thy, CD Crohn’s disease, CIA collagen-induced arthritis, CsA cyclosporine A, DNBS dinitrobenzenesulfonic acid, DSS dextran sodium sulfate, GMME1 GM-CSF fused to an N-terminal truncated form of MCP-1, HFD high fat diet, KO knockout, LDLR low-density lipoprotein receptor, LIA LPS-induced arthritis, MMP matrix metalloprotease, PNA CCR5 antisense peptide nucleic acid, RA rheumatoid arthritis, SCWA streptococcal cell wall-induced arthritis, shRNA short hairpin RNA, TBEV tick-borne encephalitis virus, VSMC vascular smooth muscle cell, WNV West Nile virus

0741-5400/10/0088-0041 © Society for Leukocyte Biology

the amino acid sequence [1– 4]. The effects of chemokines on their target cells are mediated by receptors that belong to the seven-transmembrane-spanning G protein-coupled receptor family [1– 4]. Besides their well-recognized role in leukocyte recruitment, some chemokines and chemokine receptors im- pact other cellular functions such as activation, proliferation, and differentiation [1– 4]. In addition, a subset of chemokine receptors plays a nonredundant role in infectious diseases, as demonstrated by resistance to HIV/AIDS in people homozy- gous for CCR5 32 (a loss of function mutation) [5–9] and resistance to Plasmodium vivax-induced malaria in people carry- ing a loss-of-expression mutation (T46C) in the gene promoter of the Duffy antigen receptor for chemokines [10, 11]. As che- mokine receptors are G protein-coupled receptors, many phar- maceutical/biotech companies have invested appreciable time and efforts in developing small-molecule antagonists [12, 13]. As a result, two such antagonists have been approved by the U.S. Food and Drug Administration: Maraviroc (a CCR5 antag- onist) for the treatment of HIV/AIDS [14] and Plerixafor (a CXCR4 antagonist) for use in combination with G-CSF to mo- bilize hematopoietic stem cells to the peripheral blood for col- lection and subsequent autologous transplantation in patients with non-Hodgkin’s lymphoma and multiple myeloma [15]. However, for chronic inflammatory diseases, clinical trials with antagonists of a single chemokine receptor (e.g., CCR1, CCR2, or CCR5) have not proved successful [12, 13]. Considering the complexity in the pathogenesis of these diseases and the po- tential for functional redundancy of chemokine receptors, these failed clinical studies suggest that targeting a single re- ceptor may not be adequate for efficacy in these chronic con- ditions. CCR2 and CCR5 are two CC chemokine receptors that are important players in the trafficking of monocytes/macro- phages and in the functions of other cell types relevant to dis- ease pathogenesis [16, 17]. Considering that several reviews about targeting CCR2 or CCR5 alone have been published previously, and clinical trials with agents against either recep- tor have, so far, afforded little efficacy in patients with chronic

1. Correspondence: Bristol-Myers Squibb, Rt. 206 and Province Line Rd., Princeton, NJ 08543, USA. E-mail: qihong.zhao@bms.com

Volume 88, July 2010 Journal of Leukocyte Biology 41

inflammatory diseases, this review focuses on dual targeting of CCR2 and CCR5. It provides a

inflammatory diseases, this review focuses on dual targeting of CCR2 and CCR5. It provides a brief overview of the biological functions of CCR2 and CCR5 and a comprehensive summary of published data demonstrating the involvement of both re- ceptors in five immunologic and cardiovascular diseases. In light of the potential for functional redundancy of chemokine receptors in mediating leukocyte trafficking and the conse- quent concern over insufficient efficacy offered by pharmaco- logically inhibiting one receptor, the review presents evidence supporting dual targeting of CCR2 and CCR5 as a more effica- cious strategy than targeting either receptor alone. In addi- tion, it examines potential safety issues associated with dual targeting of CCR2 and CCR5.

BIOLOGICAL ACTIONS OF CCR2 AND

CCR5

The chemokine receptor CCR2 is expressed abundantly on the so-called “inflammatory” subset of blood monocytes [18]. It is also expressed on, and functions in, other immune/inflamma- tory cell types such as dendritic cells and memory Th1 cells [19, 20]. CCR2 is a G protein-coupled receptor that binds multiple ligands, known as macrophage chemoattractant pro- teins, including CCL2 (MCP-1), CCL8 (MCP-2), CCL7 (MCP- 3), and CCL13 (MCP-4). The relative contribution of each of these ligands to CCR2-mediated in vivo function remains to be elucidated [2, 3]. Of these ligands, MCP-1 is studied most ex- tensively, and CCR2 is considered to be the exclusive receptor for MCP-1 [2, 3]. The essential role of the CCR2/MCP-1 axis in the tissue recruitment of monocytes/macrophages has been demonstrated by studies in which CCR2 or MCP-1 has been genetically ablated [21–24], as well as studies in which CCR2 or MCP-1 is pharmacologically inhibited [25, 26]. KO mice of CCR2 or MCP-1 are apparently healthy but exhibit an im- paired ability to recruit monocytes/macrophages to sites of inflammation [27–30] and to produce cytokines such as TNF- [31, 32]. The impaired recruitment of monocytes in CCR2 KO mice or mice treated with a CCR2 antagonist can be explained by reduced emigration of inflammatory monocytes from bone marrow to blood [32] and reduced immigration of blood monocytes from blood to inflamed tissues [33–36]. Notably, the CCR2/MCP-1 axis is not important for monocyte adhesion but for migration into the inflamed tissue, such as atheroscle- rotic arteries [37]. Consistent with its key role in monocyte trafficking, CCR2 has been shown to drive inflammation in a number of animal models of diseases (see below). These in- clude, but are not limited to, immunologic disorders such as RA, CD, and transplant rejection, as well as cardiovascular dis- eases, including atherosclerosis and AIH. Compared with CCR2, CCR5 is expressed predominantly on macrophages differentiated from blood monocytes and Th1 cells activated in response to inflammatory stimuli [38]. It is also expressed on nonimmune cells such as osteoclasts [39] and VSMCs [40]. CCR5 is a G protein-coupled receptor that binds multiple ligands, including CCL4 (MIP-1 ), CCL5 (RANTES), CCL3 (MIP-1 ), CCL8 (MCP-2), and CCL3L1 (MIP-1 /LD78 ). Of these ligands, MIP-1 and MIP-1 / LD78 are considered to be selective to CCR5, yet RANTES is

42 Journal of Leukocyte Biology Volume 88, July 2010

the most extensively studied [4]. The relative contribution of each of these ligands to CCR5-mediated in vivo function re- mains to be elucidated. Relative to CCR2, the in vivo function of CCR5 is less well defined. CCR5 has been shown to contrib- ute to the survival of macrophages during inflammation and infection [41]. It may function to retain tissue macrophages in inflamed tissue [42]. Also, CCR5 is important in Th1 cell re- cruitment and activation in inflammation [43]. For nonleuko- cyte cell types, CCR5 is important for the formation of oste- oclasts [39], suggesting its contribution to RA pathogenesis by affecting osteoclast function. In addition, engagement of CCR5 on VSMCs by MIP-1 leads to their activation as mea- sured by increased calcium flux and secretion of tissue factor [40], suggesting its role in atherosclerosis and AIH. Consistent with its role in these cell types, CCR5 has been shown to be an important player in animal models of RA, CD, transplant rejec- tion, atherosclerosis, and AIH.

PATHOGENIC ROLES OF CCR2 AND CCR5 IN DISEASES

CCR2 and CCR5 modulate functions of monocytes/macro- phages and Th1 cells as well as nonimmune cells and are both involved in the pathogenesis of animal models of RA, CD, transplant rejection, atherosclerosis, and AIH. For each of these diseases, a brief overview of its pathogenesis and a sum- mary of preclinical evidence, genetic and pharmacologic, for CCR2 and CCR5 are provided in the following section. Where available, clinical data related to intervention of CCR2 or CCR5 are also discussed.

RA

RA is characterized clinically by joint pain, stiffness, and swell- ing as a result of synovial inflammation. Despite advances in biologic therapies, such as TNF- blockers, significant unmet medical need remains, including a significant percentage of nonresponders and inadequate responders to each therapy and the development of secondary and even tertiary resistance to these therapies. Normal synovium contains a few cells and is composed mostly of fibroblast-like cells, plus a smaller portion of macrophage-like cells and other bone marrow-derived cells. In contrast, the synovium in RA patients expands into a vascu- larized, invasive inflammatory mass (the pannus) that destroys cartilage. Immune cell infiltrates in the pannus are predomi- nantly CD4 T cells and macrophages, both of which exhibit an activated phenotype. CD4 T cells are thought to contrib- ute to the initiation of the disease, and macrophages contrib- ute to the perpetuation of the disease. These cells produce cytokines and MMPs, contributing to pain and inflammation in the affected joints, as well as to the destruction of cartilage matrix and bone. Consistent with the role of CCR2 and CCR5 in the tissue recruitment of monocyte/macrophages and Th1 cells and that of CCR5 in osteoclast formation, genetic and pharmacological intervention of either receptor has been shown to reduce disease in multiple preclinical models of RA.

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CCR2 inhibition in RA

Interventions, pharmacological and genetic, have been used to validate the CCR2/MCP-1 axis in preclinical models of RA

( Table 1). Pharmacological inhibitors included antagonists of

CCR2 (peptidic and small-molecule), antibodies against CCR2 and MCP-1, and a mutant MCP-1, which inhibits binding of endogenous MCP-1 to the endothelial surface. CCR2 KO mice have also been used as a genetic approach. Independent studies using three different antagonists of CCR2 demonstrated the importance of the CCR2-MCP-1 axis in mouse models of RA [26, 44, 45]. The first study used a peptide antagonist of CCR2, 7-ND, a mutant MCP-1, in which the N-terminal amino acids 2– 8 have been deleted [44]. Ad- ministration of 7-ND substantially reduced the incidence and progression of spontaneous arthritis enhanced by CFA in MRL/lpr mice. The reduction in disease severity was seen in preventative and therapeutic dosing of this antagonist, al- though less with therapeutic dosing. Disease reduction corre- lated with reduction in histopathological parameters: synovial inflammation, synovial hyperplasia, pannus formation, and bone destruction. The second study used an engineered GM- CSF-MCP-1 fusokine GMME1, which potently inhibits CCR2- mediated function [45]. GMME1 robustly reduced the inci- dence and severity of mouse CIA as well as synovial inflamma- tion and cartilage erosion when administered therapeutically. The third study used a small-molecule CCR2 antagonist INCB3344 [26]. Therapeutic dosing of INCB3344 reduced dis- ease severity of AIA in the rat by 75%, as assessed by the clini- cal score of swollen joints. This dosing scheme provided plasma drug levels at trough that covered the chemotaxis IC 70 . Histological evaluation showed significant inhibition of joint inflammation (82%) and bone erosion (64%) in these ani- mals. Consistent with the results from studies with antagonists of CCR2, blockade of MCP-1 following MCP-1 DNA vaccination [46] or direct administration of anti-MCP-1 antibody [47–50]

Zhao Therapeutic potential of CCR2/CCR5 dual targeting

further demonstrated the importance of the MCP-1/CCR2 axis in the pathology of experimental arthritis (Table 1). MCP-1 DNA vaccination robustly inhibited AIA in rat [46]. Anti- MCP-1 antibodies were efficacious in multiple models: CIA in rat, SCWA in rat, and LIA in rabbit [47–50]. These anti-MCP-1 antibodies inhibited arthritis when dosed preventatively or therapeutically. Reduction in clinical score correlated with that in histopathological parameters such as synovial inflammation. Furthermore, the importance of MCP-1 in the rat AIA model was demonstrated with P8A-MCP-1, a mutated MCP-1, in which proline at position 8 was changed to alanine, which displaces endogenous MCP-1 from the endothelial surface and inhibits monocyte infiltration into inflamed tissues [51]. Therapeutic dosing of P8A-MCP-1 reduced clinical signs and histopathology as measured by joint destruction, synovial lining, macrophage infiltration, and bone erosion. Although there is substantial pharmacological data support- ing CCR2 in the pathogenesis of experimental arthritis, re- ports with CCR2 KO mice and anti-mouse CCR2 antibody MC-21 provided contrasting results. Reports from the same laboratory have described worsening of arthritis in three differ- ent models in CCR2-deficient mice relative to wild-type con- trols [86, 87]. The three models are CIA, collagen antibody- induced arthritis, and Mycobacterium avium-induced arthritis. The mechanism underlying the increased disease in CCR2- deficient mice is not clear but was attributed by the authors to the reduction of activation-induced cell death as a result of CCR2 deficiency [86]. A separate study with the anti-mouse CCR2 antibody MC-21 showed that i.p. injection of MC-21 at 500 g/mouse during the disease-initiation phase (Days 0 –15) improved clinical signs and histological scores of mouse CIA, but the same treatment during the progression phase (Days 21–36) exacerbated the disease [88]. The mechanism underly- ing the disease worsening by therapeutic dosing of MC-21 at this dose is not clear and was attributed initially by the authors to the inhibition of the so-called CCR2 regulatory T cell

Table 1. A Summary of Preclinical Studies Supporting a Role of CCR2/MCP-1 Axis in Diseases

Disease

Species

Model

Intervention

Reference

RA

mouse, rat, rabbit

MRL/lpr arthritis, CIA, AIA, SCWA, LIA

7-ND MCP-1, GM-CSF-CCL2, anti-CCR2 Ab, anti-MCP-1 Ab,

[26, 44–52]

 

P8A-MCP1

CD

mouse

DSS colitis, DNBS colitis Heart allograft, islet allograft

CCR2 KO, MCP-1 KO CCR2 KO, anti-MCP-1 Ab plus rapamycin, CCR2 KO plus rapamycin 7-ND MCP-1, CCR2 KO, MCP-1 KO, anti-MCP-1 Ab CCR2 KO, MCP-1 KO, 7-ND MCP-1, propagermium

[53, 54]

Transplant rejection

mouse

[55, 56]

(acute)

Transplant rejection (chronic)

mouse, rat

CAV, BOS

[57–60]

Atherosclerosis

mouse, rabbit, pig

ApoE / mouse (HFD or normal chow), LDLR / mouse (HFD), ApoB tg (HFD), LDLR / rabbit (normal chow), pig (HFD) Wire injury, vein grafting, cuff placement balloon injury, stenting

[61–71]

AIH

mouse, rat, rabbit, dog, monkey

CCR2 KO, MCP-1 KO, shRNA CCR2, 7-ND MCP-1, anti-MCP-1 Ab, anti-CCR2 Ab

[72–85]

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Volume 88, July 2010 Journal of Leukocyte Biology 43

function [88]. However, 3 years later, the same authors re- ported that the worsening of

function [88]. However, 3 years later, the same authors re- ported that the worsening of mouse CIA was a result of activa- tion of CCR2-expressing basophils via CCR2 cross-linking by MC-21, administered at high dose but not low dose [52]. In contrast to high-dose MC-21, low-dose MC-21 reduced the dis- ease when dosed therapeutically [52]. This is consistent with the effect of other inhibitors of the CCR2/MCP-1 axis on ex- perimental arthritis, when they are dosed therapeutically. In summary, the majority of preclinical data from multiple stud- ies supports the notion that pharmacological inhibition of CCR2 reduces arthritis. Clinically, three separate clinical studies in RA patients using different interventions of CCR2-MCP-1 axis have been re- ported. The agents include Novartis’s anti-MCP-1 antibody (ABN912) [89], Millennium’s humanized anti-CCR2 antibody (MLN1202) [90], and Merck’s small-molecule antagonist (MK812). All three studies failed to demonstrate a clinical benefit in RA patients. The basis for these failures is not com- pletely clear. However, in the study with ABN912, a dose-de- pendent increase of plasma MCP-1 was observed [89]. This increase was thought to be a result of the reduced clearance of MCP-1 bound to ABN912, arguing that the agent was not appropriate for testing the hypothesis. In the study with MLN1202, the high dose (4 mg/kg) reached a trough plasma concentration that provided 94% of CCR2 occupancy over the treatment period yet offered no efficacy [90]. This suggests that CCR2 is an irrelevant mechanism for RA, or even greater receptor occupancy is needed. The details of the MK812 clini- cal study have not been published.

CCR5 inhibition in RA

Genetic (CCR5 KO) and pharmacological (CCR5 antagonist) approaches were used to validate CCR5 in models of RA (Ta- ble 1). Two studies evaluated the effect of CCR5 deficiency on mouse CIA. One study showed that loss of CCR5 did not affect the incidence or severity of the disease [86], and the other showed that loss of CCR5 reduced the incidence significantly but not the severity of the disease [91]. The incidence reduc- tion correlated with the lower plasma levels of IgG, especially IgG2a and IgG2b, against collagen type II [91]. Notably, treat- ment with the CCR5 antagonist SCH-X immediately following CIA induction reduced the clinical score, joint inflammation, and bone/cartilage erosion significantly in a nonhuman pri- mate (rhesus monkey) CIA model [92]. The treatment also reduced systemic inflammation, as measured by C-reactive pro- tein, and altered antibody response to type II collagen. Clinically, the effect of CCR5 intervention has been studied using gene polymorphism (CCR5 32) and CCR5 antagonists. CCR5 32 is a 32-bp deletion within the coding sequence of CCR5 that results in a complete loss of function of the recep- tor in homozygous individuals [93, 94]. Several studies have shown that loss of function of CCR5 reduces incidence and/or severity of human RA [95–99]. Recent meta-analysis has con- firmed the negative association between CCR5 32 and human RA (odds ratio 0.65; P 0.0001) [100]. Also, a recent report found a negative association between two functional polymor- phisms in the CCR5 gene ( 32 and C-1835T) and juvenile RA [101]. Importantly, although the frequency of homologous

44 Journal of Leukocyte Biology Volume 88, July 2010

32 deletion is low in RA patients versus normal controls, two typical RA patients with homologous 32 deletion were identi- fied in one study [99], raising a question of whether pharma- cologic targeting of CCR5 will be effective in RA. Consistent with this concern, two trials evaluating CCR5 antagonists in RA have been reported to be ineffective in patients with RA. De- tails about the study design of these trials have not been pub- lished.

CCR2 and CCR5 dual inhibition in RA

Consistent with the notion that CCR2 and CCR5 are important players in preclinical models of RA, mice treated with a small-

molecule antagonist of mouse CCR2, CCR5, and CXCR3 (TAK-779) were protected from CIA [102]. In addition, infil- tration of leukocytes (including T cells) into the joints was ro- bustly inhibited in TAK-779-treated mice. IC 50 (nM) of TAK- 779 in inhibiting functions mediated by CCR2, CCR5, and CXCR3 in mouse was reported to be 24, 369, and 236, respec- tively [103, 104]. One caveat with this study is that no pharma- cokinetic/exposure data were provided, and therefore, the relative inhibition of each of the three receptors by TAK-779

was unknown. This caveat applies to all other studies using TAK-779 discussed in this review.

CD

CD is an uncontrolled, chronic mucosal inflammation in the

gastrointestinal tract. Despite advances in biologic therapies, such as TNF- blockers, a significant, unmet medical need re- mains, as these therapies are suboptimal, not only for induc- tion but also for maintenance and remission. In addition to concerns over malignancy and serious infection associated with administration of TNF- blockers, concomitant use of potent immunosuppressants for helping maintain efficacy adds to the safety burden. CD is a prototypic Th1-type granulomatous dis- ease characterized by accumulation and activation of memory- effector T cells and macrophages in the gut epithelium. Con- sistent with the role of CCR2 and CCR5 in the tissue recruit- ment of monocytes/macrophages and Th1 cells, genetic and pharmacological interventions have demonstrated the impor- tance of CCR2 and CCR5 in preclinical models of CD.

CCR2 inhibition in CD

CCR2 KO and MCP-1 KO mice have been used to demon-

strate the importance of the CCR2/MCP-1 axis in models of

CD (Table 1). The models included DSS-induced acute colitis,

in which the immune cell infiltrates are predominantly neutro- phils and monocyte/macrophages, as well as DNBS-induced subacute colitis, where monocyte/macrophages and T cells predominate. In one study [53], CCR2 KO mice were pro- tected from DSS-induced intestinal adhesions, mucosal ulcer- ations, and colonic inflammation by Day 3, but the effect dis- appeared by Day 7. In these mice, increased levels of IL-10 and decreased levels of IFN- may have contributed to re- duced inflammation. In a separate study about DNBS-induced colitis [54], MCP-1 KO mice showed markedly reduced severity of colitis, macroscopically and histologically, and decreased

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mortality. This disease reduction was correlated with a down- regulation of myeloperoxidase activity, decreased levels of IL- 1 , IL-12p40, and IFN- , and reduced infiltration of CD3 T cells, F4/80 macrophages, and 5-hydroxyltryptamine-express- ing enterochromaffin cells in the colonic mucosa.

CCR5 inhibition in CD

Compared with wild-type mice, CCR5 KO mice are protected from DSS-induced colitis macroscopically and histologically [53] (Table 2). In these mice, mucosal mRNA expression of IL-4, IL-5, and IL-10 was increased, whereas that of IFN- was decreased, corresponding to a shift to a Th2 pattern of T cell activation.

CCR2 and CCR5 dual inhibition in CD

Consistent with the notion that CCR2 and CCR5 are important players in preclinical models of CD, mice treated with a small- molecule antagonist of mouse CCR2, CCR5, and CXCR3 (TAK-779) were protected from DSS-induced colitis [104]. Fur- thermore, infiltration of monocyte/macrophages into the lam- ina propria was inhibited almost completely, and expression of colonic IL-1 and IL-6 was decreased significantly in TAK-779- treated mice.

TRANSPLANT REJECTION

Immunosuppressive drugs and ancillary care have led to out- standing short-term (1- to 3-year) patient and graft survival rates. However, a significant, unmet medical need remains as a result of such problems as poor, long-term ( 5 year) graft sur- vival rates (e.g., 50% in the case of heart transplant) and significant toxicities of the currently available immunosuppres- sive drugs. The mechanism for acute rejection is the ability of T cells to recognize, via their antigen receptors, polymorphic versions of a variety of proteins, such as the MHC. Chronic allograft rejection is mediated by antigen-specific cellular and humoral immunity and importantly, is often associated with considerable transplant vasculopathy, one form of AIH that limits the long-term survival of allografts. Consistent with the

Zhao Therapeutic potential of CCR2/CCR5 dual targeting

role of CCR2 and CCR5 in the tissue recruitment of mono- cyte/macrophages and Th1 cells and that of CCR5 in activat- ing VSMCs, genetic and pharmacological interventions have demonstrated the importance of the two receptors in models of allograft rejection, acute and chronic.

CCR2 inhibition in transplant rejection

Acute rejection. Genetic (CCR2 KO) and pharmacologi- cal (anti-MCP-1 antibody) approaches were used to support the importance of the CCR2/MCP-1 axis in acute rejection of allograft transplants (Table 1). In one study with the car- diac allograft model [55], CCR2-deficient mice had a mod- est prolongation of graft survival (13 days) relative to wild- type control (8 days), with an associated reduction of IFN- and increase of IL-4, suggesting that allograft survival is re- lated to the shift of Th1 to Th2 function. This study also evaluated the effect of CCR2 deficiency on islet allograft survival in a heterotopic islet cell allograft rejection model, in which pancreatic islet allografts are placed under the kid- ney capsule of recipient mice rendered diabetic by strepto- zotocin [55]. When islet cells were transplanted into CCR2- deficient recipients, a prolonged allograft survival (24 days) was observed relative to that of wild-type mice (12 days). Of note, this study did not examine the added effect of an im- munosuppressant (e.g., CsA or rapamycin) on the survival of heart or islet allograft. In a separate study using a similar heterotopic islet cell allograft rejection model [56], islet allograft survival in CCR2-deficient mice was not signifi- cantly different from that in wild-type littermates. However, when CCR2-deficient recipients were treated with a sub- therapeutic dose of rapamycin, the islet allograft was en- grafted permanently ( 120 days). Wild-type littermates treated with the same protocol did not exhibit any long- term graft survival; rejection occurred within 20 days [56]. Permanent engraftment ( 120 days) was also observed when wild-type mice were treated with anti-MCP-1 antibody plus rapamycin [56]. These data suggest a potential additive benefit for intervention of the CCR2/MCP-1 axis with stan- dard immunosuppressant treatment.

Table 2. A Summary of Preclinical Studies Supporting a Role of CCR5/RANTES Axis in Diseases

Disease

Species

Model

Intervention

Reference

RA

mouse, monkey

CIA

CCR5 KO, CCR5 antagonist CCR5 KO

[91, 92]

CD

mouse

DSS colitis

[53]

Transplant rejection (acute)

mouse, monkey

Heart allograft, islet allograft CCR5 KO, CCR5 KO plus CsA, CCR5 KO plus rapamycin, CCR5 antagonist (small-molecule)

[105–111]

Transplant rejection (chronic)

mouse, monkey

CAV

CCR5 KO plus CsA, CCR5 KO plus rapamycin, CCR5 KO plus anti-CXCR3 Ab, Met-RANTES, CCR5 antagonist CCR5 KO, Met-RANTES,

[105, 106, 112–114]

Atherosclerosis

mouse

ApoE / mouse (HFD or normal chow), LDLR / mouse (HFD) Wire injury

[115–118]

 

[

44 AANA 47 ]-RANTES

AIH

mouse

CCR5 KO, Met-RANTES

[119–121]

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Volume 88, July 2010 Journal of Leukocyte Biology 45

Chronic rejection. The importance of the CCR2-MCP-1 axis in models of chronic rejection of transplant

Chronic rejection. The importance of the CCR2-MCP-1 axis in models of chronic rejection of transplant has been studied using genetic deletions of CCR2 and MCP-1, as well as pharmacological interventions (CCR2 antagonism with 7-ND- and anti-MCP-1-neutralizing antibody; Table 1). Two separate studies evaluated the effect of intervention against the CCR2/ MCP-1 axis in two models of chronic rejection: cardiac allo- grafts and aortic allografts. In the study with cardiac allografts [57], intramuscular gene transfer of 7-ND MCP-1 reduced inti- mal hyperplasia by 39% and decreased infiltrates of F4/80 and CCR2 mononuclear cells. In contrast, in the study with aortic allografts, CCR2 deficiency or MCP-1 deficiency did not reduce intimal hyperplasia [58]. The basis for the difference in the effects on neointima formation between the two studies is not clear and is likely related to the difference in arterial sites between the two models. This supposition is consistent with the finding that CCR2 deletion reduced atherosclerotic lesions in the aortic root but not in the thoracoabdominal aorta (see Atherosclerosis below). Alternatively, potential Th2 switch, resulting from genetic deficiency of MCP-1 or CCR2 but not from pharmacological inhibition of the CCR2/MCP-1 axis, may account for the difference; a Th2-cytokine milieu is considered to be detrimental to aortic allograft survival [59]. Two separate studies about chronic rejection of lung allo- graft evaluated the effects of intervention against the CCR2/ MCP-1 axis on BOS, the leading cause of mortality following lung transplantation and contributing to the low allograft sur- vival rate. The syndrome is characterized by a perivascular/ bronchiolar recruitment of mononuclear cells that ultimately destroys the airway. In one study [60], following tracheal trans- plantation, recruitment of mononuclear cells was reduced sig- nificantly, and BOS was attenuated in CCR2-deficient mice and in wild-type mice treated with a neutralizing anti-MCP-1 antibody. This finding was corroborated by another study us- ing a rat heterologous s.c. model of BOS [122], in which neu- tralizing anti-MCP-1 antibody treatment reduced airway ob- struction and airway epithelium loss by 80% and 50%, re- spectively.

CCR5 inhibition in transplant rejection

Acute rejection. Genetic and pharmacological interven- tions demonstrated the importance of CCR5 in acute rejection of allografts (Table 2). With the cardiac allograft model, four studies were reported. The first study showed that CCR5 defi- ciency or treatment with a neutralizing antibody against CCR5 prolonged allograft survival from 7 to 20 days and markedly reduced infiltrating, activated mononuclear cells [105]. When CsA was given to CCR5-deficient mice, permanent engraftment ( 100 days) was observed. This is in contrast to the survival of only 2–3 days observed when wild-type mice were given CsA alone [105]. In the second study using a similar cardiac allo- graft model [106], mice treated with an antibody against CCR5 combined with CsA had prolonged allograft survival (83 days) relative to CsA treatment alone (6 days). This effect was associated with significant reduction of mononuclear cells in the graft. In the other two studies from the same laboratory using a model of heterotopic heart allograft transplantation [107, 108], CCR5 deficiency only modestly prolonged the sur-

46 Journal of Leukocyte Biology Volume 88, July 2010

vival of cardiac allograft (by 2–3 days) but substantially de- creased T cell and macrophage infiltrates at the time of rejec- tion. The authors attributed this modest effect to the in- creased production of alloreactive antibodies in CCR5- deficient mice [107, 108]. It is important to note that treatment with CsA was not conducted in these two studies. With the allograft pancreatic islet cell heterotopic model, two studies were reported. In the first study [109], islet allo- grafts, transplanted into CCR5-deficient mice, survived longer (35 days) than those into wild-type littermates (10 days). The prolonged allograft survival in CCR5-deficient mice was associ- ated with a shift from a Th1 to a Th2 phenotype. In the sec- ond study [110], CCR5 function was blocked with PNA CCR5, an PNA CCR5, in which the sugar phosphate backbone of the natural nucleic acid has been replaced with a synthetic peptide backbone. Islet allografts in mice treated with PNA CCR5 sur- vived significantly longer (12 days) than those treated with control (6.5 days). This prolongation was associated with de- creased CCR5 expression and reduced T cell proliferation. Notably, in a primate model of heterotopic allo-heart trans- plantation, monotherapy with a CCR5 antagonist from Merck (Whitehouse Station, NJ, USA; CMPD 167) markedly attenu- ated recruitment of CCR5-positive leukocytes into the graft, decreased peri-operative stress responses (e.g., fever and di- minished activity), yet only marginally prolonged allograft sur- vival [111]. Chronic rejection. The importance of CCR5 in chronic rejection, especially CAV, is highlighted in four studies in mouse and one study in nonhuman primate (Table 2). In the first mouse study using a cardiac allograft transplant model [105], treatment of CCR5-deficient mice with CsA at the clinical dose caused permanent allograft survival ( 100 days), compared with only 2- to 3-day survival in the wild- type mice. Histological examination at Day 100 post-trans- plantation revealed no sign of vasculopathy in the CCR5- deficient recipients, as measured by leukocyte infiltration, interstitial fibrosis, and intimal hyperplasia [105]. In the second mouse study using a similar model [106], allografts treated with anti-CCR5-neutralizing antibody plus rapamycin exhibited significantly prolonged survival (83 days), as com- pared with allografts treated with a control antibody plus rapamycin (6 days). Treatment with an anti-CCR5 antibody plus rapamycin also reduced the progression of chronic re- jection significantly, as measured by lack of intimal lesions at Day 90. In the other mouse studies, the effect of dual in- tervention of CCR5 and CXCR3 or of CCR5 and CCR1 on CAV was evaluated. In a fully MHC-mismatched murine car- diac allograft model [112], treatment of CCR5 / mice with anti-CXCR3 antibody led to permanent graft survival ( 100 days) of the donor hearts with complete lack of inti- mal lesions. In a model in which B6.CH-2 bm12 strain donor hearts were transplanted heterotopically into C57BL/6 mice (myosin heavy chain II mismatch) [113], blockade of CCR5 and CCR1 with Met-RANTES reduced intimal thickening significantly with markedly decreased infiltration of CD4 and CD8 T cells and MOMA-2 monocytes/macrophages in the allografts. In the nonhuman primate (cynomolgus monkey) study using heterotopic allo-heart transplantation

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described above [111], treatment with the CCR5 antagonist CMPD 167 plus CsA delayed alloantibody production and suppressed vasculopathy up to Day 54, as measured by a CAV scoring system for intimal lesions. In humans, graft survival was significantly longer in patients homozygous for CCR5 32 than those homozygous CCR5 wild-type and het- erozygous CCR5 32 (hazard ratio 0.367; P 0.033) [114].

CCR2 and CCR5 dual inhibition in acute and chronic rejection

Consistent with the role of CCR2 and CCR5 in models of acute and chronic rejection, wild-type mice treated with the small-molecule antagonist TAK-779 or CCR2-deficient mice treated with TAK-779 exhibited prolonged survival of heart and islet allografts. In the first study, TAK-779 treatment pro- longed survival of both types of allografts and robustly inhib- ited CD4 , CD8 , and CD11c cellular infiltration [123]. Fur- thermore, TAK-779 treatment substantially attenuated the de- velopment of CAV, fibrosis, and cellular infiltration. The second study evaluated the effects of CCR2 deficiency, CCR5 deficiency, and a combination of CCR2 deficiency with rapa- mycin or TAK-779 in a model of islet allografts [124]. CCR2 or CCR5 deficiency modestly prolonged the survival from 11 to 16 days. The combination of CCR2 deficiency with rapamycin further prolonged the survival (38 days) relative to either treatment alone (16 days for CCR2 deficiency and 17 days for rapamycin) [124]. However, the combination of CCR2 defi- ciency with TAK-779 did not prolong allograft survival, and the authors concluded that dual inhibition of CCR2 and CCR5 had no synergistic effect [124]. Adding CsA or rapamycin to a combination of CCR2 deficiency and TAK-779 was not con- ducted but would provide further proof of concept.

ATHEROSCLEROSIS

Cardiovascular disease continues to be the principal cause of death in the Western world despite changes in lifestyle and the increasing use of therapies to lower plasma lipids. Compel-

ling evidence exists that vascular inflammation plays a key role

in

atherosclerosis. Immune cells, particularly macrophages and

T

cells, dominate in atherosclerotic lesions. In experimental

models of atherosclerosis, recruitment of macrophages and Th1 cells plays a crucial role in the progression of atheroscle- rosis and destabilization of the vulnerable plaque. Consistent with the role of CCR2 and CCR5 in the tissue recruitment of monocyte/macrophages and Th1 cells and that of CCR5 in the activation of VSMCs, genetic and pharmacological evi- dence exists, demonstrating the importance of the two recep- tors in multiple models of atherosclerosis.

CCR2 inhibition in atherosclerosis

With genetic and pharmacologic intervention, numerous stud- ies in preclinical models of atherosclerosis have shown that targeting the CCR2-MCP-1 axis reduces aortic root lesion size, decreases macrophage infiltration, and also increases plaque stability (Table 1). Notably, substantial reduction of lesional macrophage content but not of plasma levels of lipids and li-

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Zhao Therapeutic potential of CCR2/CCR5 dual targeting

poproteins was observed consistently when the CCR2-MCP-1 axis was inhibited. Genetically, CCR2 / and MCP-1 / mice were used to validate the CCR2-MCP-1 axis in mouse models of atheroscle- rosis: ApoE / mice, LDLR / mice, and ApoB tg mice (Ta- ble 1). Two independent studies have shown that relative to ApoE / controls, CCR2 / /ApoE / mice fed with a HFD or normal chow have reduced aortic root lesions with de- creased cellular infiltration [61, 62]. Smaller yet statistically significant decreases in the size of early lesions were also ob- served in CCR2 / /ApoE / mice, indicating a gene-dosage effect [61, 62]. Interestingly, relative to CCR2 / /ApoE / mice, CCR2 / /ApoE / mice exhibited significantly re- duced lesions at the aortic root but not at the thoracoabdomi- nal aorta [63]. Deficiency of MCP-1 was shown to markedly reduce lesion size and macrophage infiltrates in two models of atherosclerosis: the LDLR / and ApoB tg mice fed with a HFD [64, 65]. Conversely, MCP-1 overexpression in ApoE / mice increased the macrophage infiltrate and size of lesion

[125].

Pharmacologically, the importance of the CCR2-MCP-1 axis in experimental atherosclerosis was demonstrated following gene transfer of 7-ND in ApoE / mice in three separate studies [66 – 68]. HFD, normal chow, and normal chow plus treatment with angiotensin II were used to modulate the for- mation of the atherosclerotic lesion, respectively. These studies demonstrated consistently that 7-ND gene transfer reduced lesions with decreased macrophage infiltration. Importantly, in the 20- to 30-week-old ApoE / mice with more advanced le- sions, 7-ND gene transfer did not reverse the lesion, but it al- most blocked its progression completely. Furthermore, it also increased lesional collagen and elastin content, suggesting that inhibition of the CCR2-MCP-1 axis by 7-ND stabilizes the plaque. The importance of the CCR2-MCP-1 axis in models of ath- erosclerosis was also demonstrated following treatment with propagermanium, a polymer that selectively inhibits in vitro chemotaxis in response to MCP-1 [126]. Although its exact mechanism of action is not clear, it may target GPI-anchored proteins that are functionally important for CCR2 [126]. This compound reduced monocyte/macrophage infiltrates in thio- glycollate-induced peritonitis in mice, a model for measuring CCR2-dependent monocyte/macrophage recruitment [69]. Three studies evaluated the effect of propagermanium in three different models of atherosclerosis: the ApoE / mouse, the LDLR / rabbit, and the HFD-fed pig, and provided con- sistent evidence that propagermanium treatment reduces le- sions with decreased macrophage infiltration [69 –71]. Although there are robust genetic and pharmacological data supporting the importance of CCR2 in experimental athero- sclerosis, a study with CCR2 / /ApoE / mouse bone mar- row transplantation reached a different conclusion. The study was designed to test the ability of CCR2 / leukocytes to re- verse established atherosclerotic lesions in the ApoE / back- ground [127]. Bone marrow from CCR2 / /ApoE / mice was transferred into the ApoE / mice (16 weeks old) that had preformed lesions. Assessment of lesion size 9 weeks post- transplantation showed no effect of CCR2 deletion on the size

Volume 88, July 2010 Journal of Leukocyte Biology 47

of advanced lesion, indicating that bone marrow progenitor cell-derived CCR2 did not influence the progression

of advanced lesion, indicating that bone marrow progenitor cell-derived CCR2 did not influence the progression of estab- lished lesions. A caveat with this study is that only cells newly derived from bone marrow were examined. However, the re- sults suggest that the CCR2-MCP-1 axis may have a diminished role in advanced lesions in which the inflammatory process may involve other pathways (e.g., CCR5-RANTES axis). Clinical evidence has yet to be generated that inhibiting the CCR2-MCP-1 axis provides a benefit, i.e., reduced morbidity and mortality in patients with atherosclerosis. Encouragingly, an initial analysis showed that MLN1202, a humanized anti- CCR2 antibody, was well tolerated and met its primary end- point of reducing C-reactive protein levels for months after a single dose in 108 patients who were at high risk for athero- sclerosis [128]. It will be interesting to see if continued treat- ment with MLN1202 provides clinical benefit for patients with atherosclerosis.

CCR5 inhibition in atherosclerosis

Multiple lines of evidence have demonstrated that inhibition of the CCR5-RANTES axis reduces lesion size, decreases mac- rophage infiltration, and increases plaque stability in animal models of atherosclerosis (Table 2). Several studies have shown that CCR5 deficiency reduced atherogenesis in ApoE / mice fed HFD or normal chow. One study evaluated two separate lines of CCR5 / /ApoE / mice [115]. Relative to ApoE / control mice, CCR5 / /ApoE / mice exhibited a reduction of lesion size at the aortic root by 50% and in the thoracoabdominal aorta by 60% after 10 –12 weeks of a high- cholesterol diet, and the reductions were 50% and 80%, re- spectively, after 22 weeks of a high-cholesterol diet. The CCR5 / /ApoE / mice also exhibited reduced lesional mac- rophage content, decreased lesional expression of CD4, de- creased Th1 T cell Ig and mucin domain 3 expression, in- creased expression of the anti-inflammatory cytokine IL-10, and increased smooth muscle cell content. Consistent with this, another study showed that relative to ApoE / control mice, CCR5 / /ApoE / mice fed with normal chow for 26 weeks, 36 – 45 weeks, and 65– 80 weeks, CCR5 deletion reduced the aortic lesion by 13%, 46%, and 58%, respectively [116]. Yet, in a separate study, relative to ApoE / control mice, CCR5 / /ApoE / mice fed with normal chow for 16 weeks did not exhibit significant reduction in the size of the aortic root lesions [129]. One possible explanation for this discrep- ancy is the difference in the length of the feeding with normal chow and/or the stage of the lesions, especially considering that the inhibitory effect of CCR5 deletion on atherogenesis appears to be more pronounced on late-stage/advanced le- sions. In summary, following HFD and normal chow, CCR5 deletion inhibits atherogenesis, especially at a late stage, and this inhibition applies to lesion formation at aortic root and thoracoabdominal aorta; in contrast, CCR2 deletion appears to impact atherogenesis at the aortic root but not the thoracoab- dominal aorta. Despite these preclinical data, the Offspring Cohort of the Framingham Heart Study found no statistically significant association between CCR5 32 homozygosity or het- erozygosity and a lower risk of cardiovascular disease [130].

48 Journal of Leukocyte Biology Volume 88, July 2010

Pharmacologically, Met-RANTES, an antagonist of mouse CCR5 and mouse CCR1 [131], reduced atherosclerosis in LDLR / mice fed with HFD. Given that CCR1-deficient mice showed increased lesions in ApoE / or in LDLR / back- ground [132, 133], the inhibitory effect of Met-RANTES on atherosclerosis was most likely mediated by CCR5 antagonism. Consistent with the results obtained with CCR5 KO studies, Met-RANTES reduced lesions in the aortic root by 40% and

those in the thoracoabdominal aorta by 60% in LDLR / mice fed a high-cholesterol diet for over 14 weeks [117]. Met- RANTES also reduced lesional Mac-1 macrophage and CD4

T cells and increased smooth muscle cells, again in agreement

with the results of studies with CCR5 KO mice. In addition,

increased interstitial collagen content and reduced expression

of

MMP-9 within the lesion suggest an added beneficial effect

of

Met-RANTES (presumably via the CCR5 antagonism) on

the stability of atherosclerotic plaques. The effect of Met- RANTES on the lesion in LDLR / mice was corroborated further by a more recent study using [ 44 AANA 47 ]-RANTES, an inhibitor of RANTES oligomerization [118].

One study transplanted CCR5 / and CCR5 / bone mar- row into lethally irradiated LDLR / mice to assess the effect of CCR5 deficiency on atherosclerosis [134]. CCR5 deficiency

in bone marrow-derived cells reduced macrophage content

and MMP-9 and increased IL-10 and collagen content in early atherosclerosis. However, these effects were not sustained in advanced atherosclerosis. Although this study questions the importance of CCR5 in a late stage of atherosclerosis, two ca- veats should be considered. First, lethal irradiation kills only high-proliferating cells, such as bone marrow-derived cells, but not resident lesional macrophages, which are thought to ex- press high levels of CCR5. Second, this procedure did not af- fect the function of CCR5-expressing cells that are not bone marrow-derived, including CCR5-expressing VSMCs, which may contribute to atherosclerosis.

CCR2 and CCR5 dual inhibition in atherosclerosis

No study directly evaluating the effect of dual inhibition of CCR2 and CCR5 on atherosclerosis has been reported pre- clinically or clinically. Two related studies have been reported:

one with TAK-779, a small-molecule antagonist for mouse CCR2, CCR5, and CXCR3 (a CXC chemokine receptor), and a study with combined intervention of MCP-1, CX3CR1 (a recep- tor for fractalkine CX3CL1), and CCR5. Treatment of LDLR / mice on HFD with TAK-779 reduced the lesion in the aortic root by 43% and in the carotid arteries by 68% [135]. The number of T cells in the lesions was reduced by 95%, concurrent with 98% reduction in IFN- . In another study with ApoE / mice [136], a combined deficiency of MCP-1 and CX3CR-1, together with Met-RANTES treatment, led to abrogation of bone marrow monocytosis and to a marked and additive 90% inhibition of atherogenesis at the aortic sinus. Additionally, comparing the effect of dual defi- ciency of MCP-1 and CX3CR1 with either single deficiency, the study showed an added effect with dual intervention [136]. The results from this study agree with those from an earlier report showing that monocyte subsets differentially use CCR2, CCR5, and CX3CR1 to accumulate with atherosclerotic

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plaques in ApoE / mice [137]. Overall, these results support the notion that each the three axes (MCP-1/CCR2, RANTES/ CCR5, and CX3CL1/CX3CR1) impacts the pathogenesis of atherosclerosis independently. Notably, the importance of CX3CR1 in atherosclerosis has been demonstrated in mouse [138, 139] and in human [130]. Mechanistically, unlike CCR2 and CCR5, CX3CR1 functions as an adhesion molecule to tether leukocytes to the inflamed vessels, and intervening CX3CR1 reduces this tethering function, thus providing the protective effect against atherosclerosis [130].

AIH

Intimal hyperplasia is the thickening of the intima of a blood vessel as a result of smooth muscle cell migration from the media and their subsequent proliferation in re- sponse to mechanical injury and altered hemodynamics. It occurs pathologically in a variety of disease settings (e.g., atherosclerosis). Intimal hyperplasia also occurs after arte- riovenous fistula for dialysis, coronary artery bypass grafting, and allograft transplantation. Under these conditions, it is now referred to as AIH, which is the major cause not only for long-term vascular graft failure associated with these sur- gical procedures but also for chronic rejection of allografts. The unmet medical need for AIH remains high, as no via- ble therapies are currently available. It has become increas- ingly evident that subendothelial inflammation, including infiltration of monocytes/macrophages, is an integral part of the pathogenesis of AIH. Consistent with the function of CCR2 and CCR5 in the tissue recruitment of monocyte/ macrophages and that of CCR5 in the activation of VSMCs, genetic and pharmacological evidence exists, demonstrating the importance of CCR2 and CCR5 in multiple models of AIH.

CCR2 inhibition in AIH

The role of CCR2 and MCP-1 has been studied extensively us- ing genetic and pharmacological approaches in animal models of AIH. These studies have provided consistent evidence that inhibition of CCR2 reduces intimal hyperplasia (Table 1). Ge- netically, CCR2 / and MCP-1 / mice have been used to demonstrate the importance of the CCR2/MCP-1 axis in mouse models of AIH. Two studies using wire injury of the femoral artery— one with normolipidemic CCR2 / mice and the other, hyperlipidemic CCR2 / /ApoE / mice—have demonstrated that deletion of CCR2 reduces intimal hyperpla- sia (neointima) by 61% and 47%, respectively [72, 73]. Mecha- nistically, significant reduction of monocyte/macrophage con- tent in the neointima was observed in one study. However, in the other study, robust reduction of VSMC proliferation, but not the monocyte/macrophage content, was detected. The basis for this lack of reduction in the lesional content of monocytes/macrophages in this study is not clear but is likely related to the normolipidemic nature of the model, in which there are too few macrophages to measure accurately. In the same model of wire injury, MCP-1 deficiency reduced the neo- intima formation by 30% and appeared to reduce VSMC

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Zhao Therapeutic potential of CCR2/CCR5 dual targeting

proliferation [74]. Most recently, in a vein-grafting model (ve- nous interposition in the carotid artery) in ApoE*3-Leiden mice fed with a HFD, local infection of the vein graft with len- tivirus expressing shRNA-silencing CCR2 reduced vein-graft thickening by 38% and was associated with reduced smooth muscle cell migration and proliferation [75]. Although this study provides interesting evidence that local inhibition of CCR2 function can reduce AIH, it does not address the role of CCR2 in the emigration of inflammatory monocytes from bone marrow to blood. Pharmacologically, a number of studies in multiple species have evaluated the effect of intervening in the CCR2/MCP-1 axis in different models of AIH. In mice, two independent studies have evaluated the effect of intervening in the CCR2/ MCP-1 axis on two different models of AIH: cuff placement on femoral artery and vein graft [76, 77]. In both studies, gene transfer of 7-ND MCP-1 (a peptide antagonist for CCR2) into skeletal muscles reduced the neointima by 60% and 51%, re- spectively, and reduced neointimal monocyte/macrophage content, cytokine production, and VSMC proliferation signifi- cantly. In rat normolipidemic models of balloon injury of the carotid artery, two studies using gene transfer of 7-ND MCP-1 or anti-MCP-1 antibodies demonstrated reduction of neointima of 60% and 56%, respectively, with significantly reduced neo- intimal monocyte/macrophage content, local cytokine produc- tion, and VSMC proliferation [78, 79]. In the rabbit, three studies evaluated the effect of intervening in the CCR2/MCP-1 axis on AIH in two different hyperlipidemic models: balloon injury of the carotid artery and stenting of the iliac artery. In the balloon injury model [80], gene transfer of 7-ND MCP-1 into skeletal muscles reduced neointima by 40%. In the stent- ing model, coating stents with a biocompatible polymer con- taining a plasmid expressing the 7-ND MCP-1 gene reduced the neointima by 23% and 44% in the two separate studies [81, 82]. In all of these studies, significant reduction of neoin- timal monocyte/macrophage content was observed [80 – 82]. In the dog normolipidemic model of autologous vein graft, jugular vein grafts were first transfected with adenovirus con- taining the 7-ND gene or a control vector and then interposed into the carotid arteries. Gene transfer with 7-ND reduced the neointima by 65% relative to vector controls and reduced monocyte/macrophage content and VSMC proliferation signif- icantly [83]. In the monkey, five studies evaluated the effect of intervening in the CCR2/MCP-1 axis using an anti-CCR2 anti- body or with 7-ND gene transfer on AIH. In the balloon injury model, the anti-CCR2 antibody did not inhibit neointima for- mation, and in the model of stenting of the iliac artery, it re- duced neointima by 46% and reduced macrophage content significantly in the neointima [84]. Four additional monkey studies evaluated the effect of 7-ND gene transfer on different models of AIH, including arterial injury-induced cuff [76], bal- loon [79], and stenting of the iliac artery [82, 85]. 7-ND gene transfer reduced neointima formation by 75%, 85%, 25–30%, and 50%, respectively, and reduced local inflammatory param- eters significantly. The different magnitude of reduction in neointima is likely related to the differences in how the arte- rial injury was induced, how long each study was carried out, and how the 7-ND gene was delivered (Table 1). In summary,

Volume 88, July 2010 Journal of Leukocyte Biology 49

genetic deletion and pharmacological inhibition of the CCR2/ MCP-1 axis reduced neointima in normolipidemic and

genetic deletion and pharmacological inhibition of the CCR2/ MCP-1 axis reduced neointima in normolipidemic and hyper- lipidemic models of AIH, induced by such insults as mechani- cal injuries, vein graft, and allograft vasculopathy in mouse, rat, rabbit, dog, and monkey. Furthermore, the inhibitory ef- fect of intervening on this axis on AIH (vasculopathy) was also demonstrated in the context of chronic rejection (see Trans- plant Rejection above).

CCR5 inhibition in AIH

The importance of CCR5 in AIH pathogenesis was demon- strated with genetic (CCR5 KO) and pharmacological (Met- RANTES) approaches (Table 2). In two studies using a hyper- lipidemic (ApoE / ) mouse model of wire-induced injury of left carotid arteries, CCR5-deficient mice showed an 60% reduction in neointima formation relative to the wild-type lit- termates [119, 120]. Also, CCR5 deficiency decreased neointi- mal macrophages and T cells substantially and led to a greater than twofold increase in levels of the anti-inflammatory cyto- kine IL-10 in the neointima [119]. Pharmacologically, systemic administration of Met-RANTES, a peptide antagonist for mouse CCR5 and mouse CCR1 [131], reduced neointima for- mation by 35% and macrophage infiltrates by 50% in a similar model of wire-injured carotid arteries [121]. Given that CCR5 deficiency, but not CCR1 deficiency, reduced neointima formation in the same study [119], the inhibitory effect of Met-RANTES on neointima formation was most likely medi- ated by a CCR5 antagonism and not a CCR1 antagonism. Taken together, genetic and pharmacologic evidence exists demonstrating the importance of the CCR5/RANTES axis in AIH. Furthermore, compelling evidence also exists in support of a key role of this axis in AIH in the context of chronic re- jection (see Transplant Rejection above).

RATIONALE FOR DUAL TARGETING OF CCR2 AND CCR5

Complementary cellular distribution and differential cellular functions of CCR2 and CCR5 provide a rationale that dual tar- geting of the two receptors will potentially provide greater effi- cacy than targeting either receptor alone. CCR2 and CCR5 are expressed on different cell types in a complementary manner. For monocyte/macrophages and T cells, the expression pat- tern for the two receptors appears to be complementary. CCR2 is expressed predominantly on blood monocytes [18] and memory T cells [20], whereas CCR5 is expressed on tissue macrophages and activated T effector cells [38]. Recent stud- ies have also shown that CCR2 and CCR5 are expressed on separate subsets of blood monocytes [140]. In the case of non- leukocyte cell types, the expression pattern of the two recep- tors is distinct in that CCR5, but not CCR2, is expressed on and functionally important for osteoclasts [39] and VSMCs [40], which are considered to contribute to RA and atheroscle- rosis/AIH, respectively. The complementary expression of the two receptors is sup- ported further by recent in vitro and ex vivo evidence demon- strating a reciprocal pattern of expression and function for

50 Journal of Leukocyte Biology Volume 88, July 2010

CCR2 versus CCR5 during the differentiation of monocytes into macrophages. In vitro studies have shown a down-regula- tion of CCR2 expression and an up-regulation of CCR5 ex- pression during monocyte differentiation into macrophages [141, 142]. This reciprocal pattern of expression of the two receptors is paralleled by the reciprocity in their function; i.e., calcium flux and chemotaxis in response to MCP-1 are de- creased, but that in response to RANTES or MIP-1 is in- creased [141, 142]. Furthermore, ex vivo studies with samples from RA patients have shown substantially higher expression of CCR2 than CCR5 on monocytes in peripheral blood [143]. In contrast, substantially higher expression of CCR5 relative to CCR2 is observed on macrophages in synovial fluid [144]. Thus, this suggests down-regulation of CCR2 and up-regula- tion of CCR5, as monocytes migrate from blood into synovial fluid and differentiate into synovial-fluid macrophages. The reciprocal pattern of expression and function for CCR2 versus CCR5 during monocyte differentiation implies that the two receptors exert different effects on monocytes/macrophages, and dual targeting may offer greater inhibition of their func- tion than targeting either receptor alone. As discussed previ- ously, CCR2 plays an essential role in mediating the migration of monocytes from bone marrow to blood and from blood to tissues. CCR5 functions to modulate the activation, survival, and possible retention of macrophages in the tissue. As discussed in Atherosclerosis above, a compelling example of spatially dependent differences in the functions of the two receptors has been provided by examining the effect of CCR2 deficiency and CCR5 deficiency on the atherosclerotic lesions in aortic root versus the thoracoabdominal aorta in ApoE / mice fed with HFD, one commonly used model of atheroscle- rosis. CCR2 deficiency reduced lesions in the aortic root by 50 –75% by Weeks 5–13 of HFD [61, 62] but did not reduce lesions in the thoracoabdominal aorta at all after 10 weeks of HFD [63]. In contrast, following 10 or 12 weeks of HFD, CCR5 deficiency reduced thoracoabdominal aortic lesions by 60% and aortic root lesions by 50%, respectively [115]. Practically, this rationale is supported further by the reports of successful identification of potent dual antagonists against CCR2 and CCR5 [13]. Fortuitously, the process of discovering such dual inhibitors has been helped by the high molecular homology (71% identical residues) shared between the two receptors [144].

SAFETY CONCERNS OVER DUAL TARGETING OF CCR2 AND CCR5

From the perspective of drug safety, data gleaned thus far from the literature have suggested that a dual antagonist of CCR2 and CCR5 may have several potential issues. First, CCR5 acts as one of the coreceptors for HIV entry into T cells and macrophages [145], and CCR5 antagonists (e.g., Maraviroc) have proven to be effective for the treatment of HIV [14]. This class of agents works specifically against CCR5-tropic vi- ruses and is inactive against viruses that use CXCR4 as the co- receptor [14]. During infection, HIV starts out as CCR5-tropic and only later does it switch to use CXCR4. Thus, concern with this class of agents, albeit not yet realized, is that treat-

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ment with an antagonist against CCR5 or CCR2/5 can result in coreceptor switching to CXCR4 [14]. To mitigate this po- tential risk, appropriate screening for HIV positivity and exclu- sion criteria should be implemented in the clinical studies with a CCR2/5 dual antagonist. Second, an association be- tween CCR5 deficiency and increased neuro-invasive diseases induced by flaviviruses, WNV and TBEV, has been reported from studies about CCR5-deficient mice (WNV) [146] and hu- mans carrying a CCR5 32 homozygous allele (WNV and TBEV) [147–149]. This raises concern over the increased sus- ceptibility to flaviviral infection following long-term use of an antagonist of CCR5 or of CCR2/5. Although this increased susceptibility has not yet been reported in HIV patients taking a CCR5 antagonist as part of an anti-HIV regime, the conse- quences of long-term pharmaceutical intervention on CCR5 or CCR2/5 should be assessed carefully through rigorous post- marketing surveillance. Third, it has been reported that defi- ciency of CCR2 and/or CCR5 can increase susceptibility to several other microbial infections in mice summarized in two recent reviews [150, 151]. It is important to note, however, that most of these studies were conducted with microbial inoc- ula, which are very high relative to naturally occurring infec- tions. Clinical translation from these preclinical findings will most likely be obtained empirically. Finally, a concern has also been raised over a potential linkage between CCR2 and/or CCR5 and hepatotoxicity as a result of reports of reduced liver regeneration in mice deficient in CCR2 [152, 153] and CCR5 [154, 155], as well as increased serum liver enzymes in humans treated with some CCR5 antagonists [156]. However, more extensive clinical experience with CCR2 inhibitors (Phase II) [90] and with a marketed CCR5 antagonist (Maraviroc) and other late-stage CCR5 inhibitors has called into question any direct linkage between CCR2/5 antagonism and hepatotoxic- ity.

CONCLUSION AND FUTURE PROSPECTS

Summarized in this review is a large body of preclinical litera- ture/data providing genetic and pharmacologic evidence that CCR2 and CCR5 are important players in the pathogenesis of immunologic and cardiovascular diseases. Although clinical proof-of-concept for a dual antagonist of the two receptors is yet to be obtained, developing such a dual antagonist for its broad clinical use holds great promise. One intriguing question in studying chemokine receptor- mediated recruitment of leukocytes is why so many chemo- kines and chemokine receptors are needed for the recruit- ment of one leukocyte type. A simple answer to this “apparent redundancy” question is that these receptors are required in a spatially and temporally dependent manner to fine-tune the recruitment of a leukocyte type into inflamed tissue. From the perspective of drug discovery, targeting one chemokine recep- tor may not be sufficient to provide clinical benefit for pa- tients with chronic inflammatory diseases, as has been sug- gested by failed clinical studies with selective inhibitors of the CCR2-MCP-1 axis in RA. As presented in this review, strong preclinical evidence exists for the spatially and temporally de- pendent modulation of expression and function of CCR2 and

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Zhao Therapeutic potential of CCR2/CCR5 dual targeting

CCR5 and for the importance of both receptors in the patho- genesis of multiple diseases. Therefore, dual targeting of CCR2 and CCR5 should provide greater efficacy than targeting CCR2 or CCR5 alone, and a CCR2/CCR5 dual inhibitor has the po- tential for broad, clinical use. Not surprisingly, this opportu- nity is being seized by the pharmaceutical industry [13], so it is reasonable to expect that such dual antagonists will soon be evaluated in patients for efficacy and safety.

ACKNOWLEDGMENTS

The author thanks Drs. Phil Murphy, Paul Davies, Gary Schieven, Luisa Salter-Cid, Timothy Reilly, Megan Wind-Ro- tolo, and Julie Carman for their comments about the manu- script.

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date on chemokine receptor nomenclature. Pharmacol. Rev. 54, 227–229.

2. Gerard, C., Rollins, B. J. (2001) Chemokines and disease. Nat. Immunol. 2, 108 –115.

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KEY WORDS:

chemokines chemokine receptors disease pathogenesis mono- cyte/macrophage recruitment

Volume 88, July 2010 Journal of Leukocyte Biology 55