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The Journal of Immunology, 2007, 179, 2289 -2299
Copyright © 2007 by The American Association of Immunologists, Inc.
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CCR5 Blockade Modulates Inflammation and Alloimmunity in Primates1

Carsten Schröder2,*,{dagger}, Richard N. Pierson, III2,3,*,{dagger}, Bao-Ngoc H. Nguyen2,*,{dagger}, Douglas W. Kawka{ddagger}, Laurence B. Peterson{ddagger}, Guosheng Wu*,{dagger}, Tianshu Zhang*,{dagger}, Martin S. Springer{ddagger}, Sal J. Siciliano{ddagger}, Susan Iliff{ddagger}, Julia M. Ayala{ddagger}, Min Lu{ddagger}, John S. Mudgett{ddagger}, Kathy Lyons{ddagger}, Sander G. Mills{ddagger}, Geraldine G. Miller§, Irwin I. Singer2,{ddagger}, Agnes M. Azimzadeh*,{dagger} and Julie A. DeMartino2,{ddagger}

* Division of Cardiac Surgery, Department of Surgery, University of Maryland and Baltimore Veterans Administration Medical Center, Baltimore, MD 21201; {dagger} Department of Cardiothoracic Surgery, Vanderbilt University and Nashville Veterans Administration Medical Center, Nashville, TN 37232; {ddagger} Merck Research Laboratories, Rahway, NJ 07065; and § Division of Infectious Disease, Department of Medicine, Vanderbilt University and Nashville Veterans Administration Medical Center, Nashville, TN 37232


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Pharmacologic antagonism of CCR5, a chemokine receptor expressed on macrophages and activated T cells, is an effective antiviral therapy in patients with macrophage-tropic HIV infection, but its efficacy in modulating inflammation and immunity is only just beginning to be investigated. In this regard, the recruitment of CCR5-bearing cells into clinical allografts is a hallmark of acute rejection and may anticipate chronic rejection, whereas conventionally immunosuppressed renal transplant patients homozygous for a nonfunctional {Delta}32 CCR5 receptor rarely exhibit late graft loss. Therefore, we explored the effects of a potent, highly selective CCR5 antagonist, Merck’s compound 167 (CMPD 167), in an established cynomolgus monkey cardiac allograft model. Although perioperative stress responses (fever, diminished activity) and the recruitment of CCR5-bearing leukocytes into the graft were markedly attenuated, anti-CCR5 monotherapy only marginally prolonged allograft survival. In contrast, relative to cyclosporine A monotherapy, CMPD 167 with cyclosporine A delayed alloantibody production, suppressed cardiac allograft vasculopathy, and tended to further prolong graft survival. CCR5 therefore represents an attractive therapeutic target for attenuating postsurgical stress responses and favorably modulating pathogenic alloimmunity in primates, including man.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Until we learn how to achieve tolerance, progress in heart transplantation will depend on better control of pathogenic influences on the donor heart. Chemotactic cytokines or "chemokines" play a critical role in activation of innate immunity (1), ischemia/reperfusion injury (2), and induction of adaptive immune responses (3). Because each of these pathways is associated with important injury in transplanted organs, chemokines and their receptors are attractive candidates for new interventions to protect allografts from immunologic injury and for induction of tolerance (4). The current inflammation paradigm holds that chemokine receptors afford homing and activation signals critical to the migration and function of lymphohemopoietic cells in a variety of circumstances, including embryonic development, growth, and inflammation (1, 5, 6). Despite promiscuity among the many possible chemokine ligand-receptor pairs and "cross-talk" between different receptors on individual cells, a few specific chemokine receptors, particularly CCR1, CCR2, CCR5, and CXCR3, have emerged in murine models as promising targets to prevent allograft injury and to attenuate innate immune vascular injury (7, 8, 9, 10, 11).

CCR5 is best known as a major coreceptor for macrophage-tropic human immunodeficiency viruses. Its natural ligands include RANTES (CCL5), MIP-1{alpha} and MIP-1beta (CCL3 and 4), and MCP-2 (CCL8). CCR5 is expressed on the surfaces of resident tissue monocytes and dendritic cells and on activated T cells, macrophages, and NK cells in both lymphoid and nonlymphoid tissues (5, 6). Human peripheral blood monocytes and T cells exhibit low levels of CCR5, but expression is increased several weeks after in vitro stimulation or in vivo in the setting of established Th1-type inflammation (8), and particularly on monocytes and activated/memory Th1 lymphocytes. Because these cells are capable of entering sites of inflammation and executing pathogenic effector functions, blockade of CCR5 might be expected to prevent injury to the graft by CCR5+ helper or effector T cell subsets or by other important immunocyte populations (dendritic cells, macrophages) that express CCR5.

The absence of functional CCR5 is well tolerated in humans homozygotic for the CCR5{Delta}32 mutation (12), suggesting that "normal CCR5 function is well compensated or redundant" (5). Similarly, CCR5 knockouts and mice treated with CCR5 inhibitors exhibit only subtly diminished stress response to endotoxin and preserved or enhanced Th1 and Th2 responses (13). Together, these data suggest that CCR5 inhibition is not likely to be associated with other clinically important immune deficits (in contrast to CCR2; for example, see Ref. 14). Based on promising rodent transplant data (10, 15, 16, 17, 18) and these clinical considerations, this study was undertaken to investigate the role of CCR5 in primate cardiac allograft rejection using compound 167 (CMPD 167 (N-[(1R,3S,4S)-3-((4-(3-benzyl-1-ethylpyrazol-5-yl)piperidin-1-yl)methyl)-4-(3-fluorophenyl) cyclopentan-1-yl]-N-methyl-D-valine).4 This compound is an allosteric inhibitor of CCR5 that has similar affinity for the G protein-coupled and uncoupled states of the receptor (M. S. Springer and P. E. Finke, manuscript in preparation). Consistent with this profile, CMPD 167 blocks the binding of all known ligands of CCR5, including chemokines, R-5 tropic gp120 envelope proteins (including YU2), and nonpeptidyl antagonist ligands with similar affinities ranging from 0.05 to 2.5 nM. In addition, it binds with high affinity to the CCR5 receptor expressed on human PBMCs. The compound has similar affinities for the human, chimpanzee, rhesus monkey, and cynomolgus monkey, suppresses chemokine ligand-induced CCR5–mediated functions (G protein-dependent calcium signaling, chemotaxis, and MAPK (ERK1/2) activation) over a broad range of physiologically achievable serum concentrations, and prevents infection by SIV in macaques (19, 20). Our initial observations in this regard have also led to additional preliminary efforts to elucidate a potential role for CCR5 in the nociceptive and febrile response to surgical stress in the cynomolgus monkey heart transplant model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
CMPD 167

Merck’s CMPD 167 is a lead compound selected for exquisitely specific high-affinity binding to human and cynomolgus monkey CCR5 (21, 22). The structure, as well as the pharmacokinetic properties of this proprietary acidic cyclopentyl derivative in preclinical species, has been published (World Intellectual Property Organization patent WO 00/76972, December, 2000; A1; 69). CMPD 167 was administered in a twice daily (bid; bis in die) dosing paradigm via a tether jacket system implanted on the day of transplant to maintain trough levels of >100 nM, its IC90 value in whole monkey blood.

Cynomolgus monkeys

Wild-caught cynomolgus monkeys (Macaca fascicularis) were obtained from Coulston Foundation (Alamogordo, NM) and Charles River Laboratories. All procedures were approved by the institutional animal care and use committees of Vanderbilt University (Nashville, TN), University of Maryland (Baltimore, MD), and Merck and were conducted in compliance with National Institutes of Health guidelines for the care and use of laboratory animals (23). Males weighing 3.8 to 4.3 kg, were selected as organ recipients of AB blood type-compatible, MHC class II-mismatched (stimulation index: mean 7.7, median 6.2, range 3.1–26) donor hearts (24, 25). Donor-recipient pairs were determined so as to maximize MLR response within groups of blood type-compatible animals.

Surgical procedures

All recipient animals underwent heterotopic intraabdominal cardiac allograft transplantation as described previously (26, 27). Graft function (electrocardiogram; left ventricular pressure) and core temperature were assessed by telemetry (D70-PCTP, Data Sciences International; implanted at the time of transplantation) 2 to 3 times daily until graft demise. When clinical condition allowed, open cardiac biopsies were performed by protocol on postoperative days 4, 7, 14, and 28 and monthly thereafter until graft failure. Additional protocol biopsies were performed at predetermined intervals between days 3 and 5 after transplant in monotherapy and vehicle-treated animals. An additional biopsy was performed whenever an examiner appreciated decreased graft contractility (intraventricular pressure wave form damping), diminished graft heart rate (<150), or fever (temperature >38°C after day 3), which are typical early signs suggestive of graft rejection. Graft failure was defined as loss of telemetric, palpable, and visible graft activity. Failed grafts were explanted promptly and examined histologically.

Experimental groups and dosing regimens

Five cardiac allograft recipients were treated with CMPD 167 monotherapy at 5 (n = 3) or 10 (n = 2) mg/kg bid i.v. In three contemporaneous animals a similar volume of drug vehicle was administered IV bid, and two animals received no treatment. In an additional group of five animals, cyclosporine (CsA) was added to CMPD 167 at 10 (n = 2 (animals M376 and M347) or 15 (n = 3) mg/kg bid. CsA was dosed once daily at 12.5 mg/kg on the day of transplant, 10 mg/kg for 7–10 days, and 5 mg/kg/day for 3–14 days followed by 2.5–5 mg/kg/day thereafter until graft loss and was dose adjusted as needed to maintain subtherapeutic levels of 150–200 ng/ml. A reference group of three concurrent animals and three historical animals received tapered monotherapy with CsA dosed as described above.

In one historical and all concurrent animals, clinically diagnosed and pathologically confirmed acute rejection episodes occurring on or before day 32 were treated with methyl prednisolone (40 mg/kg once followed by 20 mg/kg daily for two days). One combined CMPD and CsA-treated animal (M292) with early recurrence of rejection on day 22 after treatment with steroids on day 14 was treated additionally with three daily doses of antithymocyte globulin (10 mg/kg). One monotherapy animal (M627) was treated with steroids for biopsy-proven acute rejection on day 8 and an additional subtherapeutic tapered dose of CsA for 46 days (see below). A contemporaneous subset of a large group of animals treated with a CD154-blocking Ab provided reference values for recipient postoperative body temperature and cytokine measurements (28).

Immunohistochemistry

Heart explants and biopsies of heart and skin were rapidly dissected and immediately fixed in a Nakane fixative (29) and additional unfixed pieces were immediately frozen in RNase-free OCT compound (TissueTek). After 4 h at 4°C, Nakane-fixed tissues were gradually infused with 20% sucrose plus 5% glycerol overnight, snap frozen in OCT-filled cryomolds, and stored at –80°C.

Double-label immunofluorescence microscopy with Abs specific for CCR5, CD3, or CD68 was used to characterize the leukocyte-infiltrating, acutely rejecting (days 3, 4, 5, & 7) cynomolgus cardiac allografts using 5-µm frozen sections blocked serially with 5% donkey serum, clarified nonfat dried milk, and Fc blocker (Accurate Chemical). Sections were labeled for 1 h with affinity-purified primary Abs or appropriate IgG controls (2–5 µg/ml). Slides were stained with the following Abs: rabbit anti-human CCR5 (Merck in-house R4627, which is reactive with the C terminus of the receptor and whose interaction is not perturbed by CMPD 167 binding), mouse anti-human CD68 mAb (clone KP1; DakoCytomation), rat anti-human CD3 (clone CD3-12; Serotec), rabbit anti-human RANTES (Santa Cruz Biotechnology), and goat-anti-human P-selectin (Santa Cruz Biotechnology). All nonimmune IgG controls were obtained from Jackson ImmunoResearch Laboratories. Slides were washed and incubated with affinity purified F(ab')2 anti-goat, anti-mouse, or anti-rabbit donkey IgGs conjugated to either Cy3 (red fluorescence) or Cy5 (green fluorescence) (5 µg/ml, 30 min.) obtained from Jackson ImmunoResearch Laboratories. Coverslips were mounted on the slides with Vectashield plus 4',6'-diamidino-2-phenylindole nuclear stain (Vector Laboratories).

Quantitation of cell infiltrates

Sections were photographed and analyzed with an Everest imaging system from Intelligent Imaging Innovations equipped with an Axioplan 2 microscope (Carl Zeiss). Single- or double-labeled monocytes/macrophages and T cells were counted microscopically for each allograft section in a blinded fashion. The sectioned tissue areas were measured planimetrically and used to calculate the numbers of infiltrating leukocytes per square millimeter of allograft.

Human monocyte isolation and culture

Monocytes were prepared from monocyte enriched leukopacks (Biological Specialties) using a Ficoll density gradient with subsequent sheep RBC rosetting to remove T cells. Isolated monocytes were suspended and stored overnight at 4°C before culture. Monocytes at 2 x 106 cells/ml in RPMI 1640 supplemented with 12% FBS were cultured for 24 h at 37°C with 5% CO2 in suspension using Teflon jars to enhance CCR5 expression. The resulting cell population was homogeneous and CD14+CCR1+CCR5+. Subsequently, 106 cells per well were seeded into 6-well plates and allowed to attach for >2 h.

CCR5-mediated cytokine release from human macrophages

To assess the role of chemokine-induced CCR5-mediated elaboration of the proinflammatory cytokines IL-1, IL-6, and TNF by cultured human macrophages, following the replating procedure the cells were preincubated with either DMSO (vehicle control) or CMPD 167 at various concentrations for 1 h, after which either the control medium or the CCR5 agonist/ligand RANTES (PeproTech) was added to a final concentration of 250 nM at 37°C. Because CCR1 also ligates RANTES and is expressed on cultured human macrophages, pretreatment with a CCR1-specific inhibitor (Merck 940 at 300 nM) was included to ascertain specificity. After incubating the cells at 37°C for 24 h, total RNA was isolated by chloroform extraction from cells lysed in 1 ml of Ultraspec RNA isolation system buffer (Biotecx Laboratories) and purified on an RNase mini column (Qiagen, Valencia, CA); genomic DNA contamination was eliminated by applying an RNase-free DNase kit (Qiagen) on the column. mRNA expression was determined by real-time quantitative PCR (TaqMan) on an ABI PRISM 7700 sequence detector system using cDNA prepared from 1 µg of RNA samples using reagents and protocols provided by Applied Biosystems. The relative mRNA fold induction of cytokine gene expression was calculated relative to day 2 nonstimulated, nontreated control macrophages (i.e., day 2 control; fold induction = 1) normalized against beta-actin.

Rejection scoring and cardiac allograft vasculopathy (CAV) quantification

Classification of cellular infiltrates was done according to International Society of Health and Lung Transplantation (ISHLT) criteria for acute allograft rejection (30, 31). Three independent evaluators (T.Z., R.N.P., and B.-N.H.N.), each blinded to treatment group, evaluated a minimum of 25 and up to 50 epicardial arteries and intramyocardial arterioles by morphology on H&E and elastin stain in multiple levels from each explant. In biopsies, 2–5 arterioles were scored. CAV was graded as follows: grade 0, normal arterial morphology; grade 1, activated endothelial cells with enlarged nuclei and/or adherent leukocytes and without luminal narrowing (<10%); grade 2, distinct neointimal thickening and luminal narrowing <50%; grade 3, extensive neointimal proliferation with >50% luminal occlusion (for illustrative histology see Fig. 9a) Individual CAV scores were tabulated for each explant, and mean and median scores were calculated. After unblinding, the median of the three final CAV measurements was accepted as the score for that sample. In four instances where discrepancies occurred (scoring range between observers of >0.5), the score was adjudicated by pathologic review (B.-N.H.N. and R.N.P.).


Figure 9
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  		FIGURE 9. a, Representative grading of CAV. A, native heart (animal M282, H&E staining, original magnification x200, CAV grade 0) shows an essentially normal vessel with flat endothelial cells without subendothelial intimal thickening and with preserved muscular medium (arrow). B, CAV grade 1 (animal M1494 at day 38, H&E staining, original magnification x400) is characterized by activated endothelial cells with enlarged nuclei and adherent leukocytes but minimal expansion. C, CAV grade 2 (animal M1494 at day 38, H&E staining, original magnification x150) with distinct neointimal thickening but <50% luminal narrowing. D, CAV grade 3 (animal M1494 day 38, H&E staining, original magnification x350) is characterized by extensive neointimal proliferation with >50% luminal occlusion. b, Effect of CCR5 blockade on CAV. E, representative vessel from a cardiac allograft treated with CsA monotherapy (animal M1454 on day 49, H&E staining, original magnification x150) shows grade 3 CAV with severe intimal thickening (double-headed arrow) and significant cellular infiltrates. F, In contrast, combined CCR5 antagonist and CsA therapy (animal M287 on day 34, H&E staining, original magnification x150) is associated with acute rejection (with perivascular lymphocytic infiltrates, open arrow), but markedly less neointimal thickening and fibrosis characteristic of CAV (arrowhead). c, CAV on scheduled biopsies and at graft explant. The animals treated with subtherapeutic CsA are shown in black and those treated with CCR5 antagonist plus subtherapeutic CsA are shown in red. The addition of a CCR5 antagonist delayed the onset of CAV.

 
Alloantibody assay

Recipient serum specimens were collected weekly during the first month, biweekly for 2 months, and monthly thereafter until graft rejection and stored at –80°C until use. Alloantibodies were measured retrospectively by flow cytometry using frozen donor splenocytes as described previously (32). Results were expressed as the percentage of positive T cells (defined as CD3+DR–) as compared with recipient pretransplant serum. Donor serum and archived historical samples were used as internal negative and positive controls, respectively. Two animals treated with CsA were not evaluated because viable donor cells were not available.

Statistical analysis

Unless otherwise mentioned, all data are presented as means and SD for all variables. Continuous variables were checked for normality by plotting histograms. Variables that were not normally distributed were analyzed using the Kruskal-Wallis test and the Mann-Whitney U test. Those that were normally distributed were assessed with a one-way ANOVA and Student’s t test. A {chi}2 test for trend was used for scoring variables and their changes over time. Survival statistics were calculated by the Kaplan-Meier method using the log-rank test for significance analysis. Correlations between parameters were studied using the Pearson or Spearman correlation test. p values of <0.05 were considered statistically significant. All tests were two tailed. All statistical analyses were performed on a personal computer with the statistical package SPSS for Windows (version 13.0) except for the power analyses, which were performed using the shareware PS: Power and Sample Size Calculations, version 2.1 (W. D. Dupont and W. D. Plummer, Vanderbilt University).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CCR5 inhibition blunts the febrile response

The average temperature over the first 3 days after surgery in animals treated with CMPD 167 (37.3 ± 0.5°C, n = 5) was unexpectedly >1°C lower than in vehicle-treated or untreated animals (38.5 ± 0.4°C, n = 5, p = 0.0047; Fig. 1) or animals treated with a mAb directed at CD154 (IDEC-131, 38.4 ± 0.7, n = 4, p < 0.05; data not shown), and 0.3°C below CsA-treated animals (n = 5, p = NS). As independently documented by the veterinary animal care technicians and veterinarians, animals treated with CMPD 167 were more active and resilient following multiple surgical procedures within the first 2 wk (transplant, protocol biopsies on days 4 and 7, and graft explant) than were animals in any other treatment group.


Figure 1
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  		FIGURE 1. Individual body temperature after heterotopic allo-heart transplant. A single line represents body temperature of an individual animal. A, Green lines depict vehicle control animals (n = 5) and blue lines show CMPD 167 monotherapy animals (n = 5). For the first 3 days after transplant the mean body temperature is significantly lower in CMPD 167-treated animals. B, Black lines depict CsA monotherapy animals (n = 3), red lines show combined CsA and CMPD 167 (CsA + CMPD 167)-treated animals (n = 5). CsA treatment lowers the mean body temperature by ~0.5°C and may attenuate the febrile response in this model.

 
Inhibition of CCR5 could potentially blunt fever by interfering with the proximal triggers of innate immune activation in the wound or the revascularized graft by inhibiting the release of associated proinflammatory cytokines or by modulating the response of constitutively CCR5+ cells to these cytokines. To explore whether CCR5 ligands are expressed locally in the wound and thus might initiate the fever consistently seen in these animals, we evaluated RANTES expression at the surgical site. As shown in Fig. 2, RANTES expression is prominent in the skin incision 90 min after wounding. Similar to inflamed and atherosclerotic endothelia (33), RANTES is associated with aggregated CD62P+ platelets that appear sequestered at the edge of the surgical wound as they are in the rejecting graft. Intense RANTES expression, presumably arising from platelets surrounding skin incisions shortly after wounding and on the vascular endothelia of rejecting cardiac allografts (Fig. 3), strongly implies that RANTES plays a role in leukocyte chemotaxis in these settings.


Figure 2
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  		FIGURE 2. Activated platelets in a surgical skin wound are a source of RANTES at the time of incision. Cryosections of vehicle-treated cynomolgus monkey skin adjacent to the wound were obtained 1.5 h after incision and double labeled for RANTES and P-selectin (platelets). A, Green fluorescence exhibits focal RANTES labeling (arrowheads). B, Red fluorescence shows the corresponding distribution of P-selectin at matching arrowheads. C, Merged image demonstrates that RANTES is localized in platelet aggregates (corresponding arrowheads). Bar, 50 µm.

 

Figure 3
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  		FIGURE 3. Vascular endothelium shows prominent RANTES expression in a 6-day rejecting cynomolgus monkey heart allograft. A, Depicts RANTES (red fluorescence) localized on the cardiac vascular endothelium (arrowheads). B, IgG control displays very low background staining (4',6'-diamidino-2-phenylindole counterstain). Bar, 50 µm.

 
We next assessed the effect of CMPD 167 on macrophage cytokine elaboration during in vitro stimulation of CCR5+CCR1+ cultured human monocytes with RANTES and found that expression of IL1beta and IL6 (but not TNF-{alpha} or IFN-{gamma}) mRNA was inhibited by a CCR5-dependent, CCR1-independent mechanism (Fig. 4). Because suppression of fever was an unanticipated finding in our preliminary studies, serum samples for reference levels of inflammatory markers were not systematically obtained at clinically relevant intervals (2–6 and 12–36 h after surgery) in most CMPD 167 monotherapy-, vehicle-, or CsA monotherapy-treated animals. However, initial findings from the blood of a small subset of the experimental subjects reported here indicate that IL1beta, IL6, IL8, C-reactive protein, and soluble CD154 elaborations on day 1 after transplant or at the time of graft demise are highly variable between individual animals (data not shown) and suggest that a very large number of observations (>10–25 per group) would be required to determine whether serum cytokine levels are significantly different between groups of CMPD-167-treated animals and relevant reference populations. Sample sizes of this magnitude are impractical in this model.


Figure 4
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  		FIGURE 4. Effect of CMPD 167 on macrophage cytokine elaboration. Expression of IL1beta and IL6 (but not TNF-{alpha} or IFN-{gamma}) mRNA was inhibited (Inh.) by CMPD 167 but not by a selective CCR1 inhibitor. Thus elaboration of IL1beta and IL6 by macrophages in response to RANTES occurs via a CCR5-dependent, CCR1-independent mechanism, whereas TNF-{alpha} and IFN-{gamma} are little affected by RANTES or a blockade of either of these receptors.

 
CCR5 modulates graft leukocyte infiltration

We defined the kinetics of leukocyte infiltration into cardiac allografts and studied the efficacy of CMPD 167 to block this immigration. Intense multifocal perivascular and interstitial infiltration of leukocytes (lymphocytes, macrophages, and monocytes) into the graft reliably occurs by days 4–7 in untreated animals (Fig. 5). The greatest CCR5 expression was observed on CD68+ macrophages (45%) with a minority of graft-infiltrating CCR5+ T cells (15%) (Fig. 5 and Table I). These data suggest a prominent role for CCR5+CD68+ leukocytes in cardiac allograft rejection. Histopathological analysis of rejecting control biopsies shows a prominent leukocytic infiltrate consisting of macrophages and lymphocytes without polymorphonuclear cells or dendritic cells (Fig. 6A). In animals treated with CMPD 167, infiltration of CCR5+, CD68+, and CD3+ leukocytes was inhibited in association with CCR5 blockade on days 4 and 7 and at rejection (Figs. 6 and 7) relative to vehicle-treated control animals at similar intervals. Importantly, infiltration by CD3+ cells was also inhibited relative to vehicle-treated controls on days 4 and 7 (Fig. 7C) despite the absence of detectable CCR5 expression on 85% of T cells found in untreated grafts.


Figure 5
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  		FIGURE 5. CCR5+ macrophages and CCR5+ T cells are present in a rejecting a cynomolgus monkey heart allograft (day 7). Cryosections of a heart allograft biopsy were double labeled with Abs against CCR5, CD68, and CD3 as described in Materials and Methods. A–C, Fusiform-shaped macrophages were intensely positive for CCR5 (A, red fluorescence shown by arrowheads) and coexpressed CD68 (B and C, green fluorescence shown by matching arrowheads). D–F, CCR5 (red fluorescence) was also expressed by some CD3+ T cells (matching arrowheads in D–F), but most of the CD3+ T cells (green fluorescence) were CCR5 negative (F). Corresponding arrows in D–F depict additional CCR5+ macrophages. Bar, 50 µm.

 

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  		Table I. Expression of CCR5 in CD68+ macrophages and CD3+ T cells in rejecting cynomolgus monkey heart allografts (7 days)a

 

Figure 6
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  		FIGURE 6. Monotherapy with CCR5 antagonist CMPD 167 (5 mg/kg bid) inhibits the influx of CCR5+ macrophages into a monkey heart allograft (4 days). A, H&E-stained section of a control rejecting heart biopsy depicting a myofiber (M) surrounded by numerous infiltrating leukocytes. Many macrophages (arrows) and lymphocytes (arrowheads) are present, but polymorphonuclear cells and dendritic cells are not evident. Bar, 15 µm. B and C, Biopsy cryosections were stained with Abs to CCR5 (red fluorescence) and CD68 (green fluorescence). B, Control biopsy displays several inflammatory foci with prominent CCR5+CD68+ macrophages (arrowheads) and large cord-like aggregates of CCR5+ leukocytes invading cardiac muscle fibers (arrows). C, CMPD 167-treated allograft biopsy exhibits a greatly reduced influx of CCR5+ leukocytes (arrowheads) and unlabeled leukocytes (arrow); CD68+ cells are conspicuously absent. Bar (for B and C), 50 µm.

 

Figure 7
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  		FIGURE 7. CCR5 blockade with CMPD 167 inhibits the influx of CCR5+ (A), CD68+ (B), and CD3+ (C) leukocytes in cynomolgus monkey heart allografts during the entire acute rejection period. Frozen sections of heart biopsies (1-mm diameter) were immunostained and the tissue area was measured planimetrically. Labeled leukocytes were counted microscopically for the entire section and expressed as the number of labeled cells per square millimeter of heart tissue. For vehicle controls ({square}), each biopsy was obtained from a separate monkey (except for 30 min where n = 3 or 4), while the counts for CMPD 167-treated animals ({blacksquare}) are the means derived from two or three monkeys.

 
Anti-CCR5 monotherapy prolongs allograft survival

Five cynomolgus monkeys treated with CMPD 167 monotherapy at 5 (n = 3) or 10 mg/kg/bid (n = 2) exhibited significantly prolonged mean graft survival with monotherapy (8.1 days ± 0.4 SEM, chemotaxis index 7.4–8.9), relative to 6.4 ± 0.2 (confidence interval, 6.1–6.6, p = 0.002) for five vehicle-treated or untreated animals (Fig. 8, top panel). Rejected grafts in all groups exhibited a typical intense myocardial infiltrate rich in lymphocytes, monocytes, and macrophages, including CCR5+ cells, although (as noted above) the proportion of CCR5+ cells was lower in CMPD 167-treated animals at rejection than in vehicle-treated reference animals. Thus, CCR5+ cells are capable of graft infiltration during CCR5 inhibition as used here, and acute allograft rejection can occur despite apparently efficient CCR5 antagonism.


Figure 8
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  		FIGURE 8. Graft survival after heterotopic allo-heart transplantation. Representation of graft survival and description of histological analysis of cardiac allografts treated with increasing doses of CCR5 blockade (top panel) or CsA alone or combined with a high dose of CCR5 blockade (bottom panel). Color coding corresponds to treatment groups. Bar length corresponds to graft survival time. The codified histological findings for graft biopsy and graft explant tissues are depicted inside and outside the histogram bars, respectively. Multiple sections representative of each tissue were examined by H&E stain and scored as follows: grade 1A/B, focal perivascular or interstitial lymphocytic inflammation, sometimes diffuse (1B); grade 2, focal mononuclear infiltrate spread into the interstitium; grade 3, multifocal (3A) or diffuse (3B) lymphocytic infiltrate with myocyte damage; grade 4, perivascular and interstitial inflammatory infiltrate with extensive myocyte necrosis. X, clinical signs of rejection but no tissue biopsy; *, rejection treatment with 3 days of steroids; CAV, intimal proliferative lesions typical of CAV. Upper panel shows significantly longer primary graft survival for CCR5-monotherapy (blue, n = 5)-treated animals compared with vehicle controls (green, n = 5) (p = 0.002). Lower panel shows primary and/or secondary graft survival related to acute rejection from CsA-monotherapy treated animals vs combination CsA and CPMD 167 (CsA + CPMD 167) therapy. Although the survival difference is statistically significant (p = 0.04), most animals in the combined CsA and CPMD 167 group but only one animal in the CsA group were treated with steroids for symptomatic acute rejection occurring within the first 32 days after transplant.

 
In one case (animal M627), the rejecting CMPD 167 monotherapy graft was successfully treated with "rescue" steroids initiated on day 8. The addition of cyclosporine from day 8 to 54 was associated with survival for 85 days, 31 days after cyclosporine withdrawal.

Synergy studies: acute cellular rejection

Twelve animals were treated with CsA dosed to achieve subtherapeutic CsA levels (100–300 ng/ml in our cynomolgus monkey heart model) 10–14 days after transplant, 11 as primary therapy and one after acute rejection treatment on day 8. Mean time to the first symptomatic acute rejection episode was 30 ± 7 days (days 11, 15, 21, 39, 46, and 49) with subtherapeutic CsA compared with >20 ± 6 days (days 7, 14, 16, >21, and 44) with CsA plus CMPD 167 (p = 0.32) (Fig. 8, bottom panel). Two early acute rejection episodes in the CsA group (days 15 and 21) were not treated with steroids and were associated with graft failure as expected. Acute rejection episodes occurring in two CsA plus CMPD 167 animals within the first month were successfully treated with steroids. One CsA monotherapy was rejected on day 13 despite steroid treatment on day 11, and one CsA plus CMPD 167 animal exhibited recurrent rejection on day 25 (M292), 1 wk after steroid therapy, that responded to antithymocyte globulin. Two animals treated using combined CMPD 167 and CsA were sacrificed on days 21 (as per the IACUC protocol for >10% weight loss despite normal activity and food intake) and 35 (aspiration) with normal graft function without exhibiting acute rejection. Overall, spontaneously resolving asymptomatic (biopsies with ISHLT score ≥1) or symptomatic (fever, bradycardia, or diminished graft contractility) acute rejection episodes occurred at similar intervals and with similar frequencies in both groups.

Effect of CCR5 antagonism on CAV

CAV scores from day 0–28 in the CsA group (1.6 ± 0.6, n = 7) were significantly higher than with combined CMPD and CsA (0.4 ± 0.3, n = 11, p = 0.002) (Fig. 9C). Upon histology at explant, although all rejected grafts had moderate to severe cellular infiltrates (ISHLT grades 3A to 4), only mild to moderate or mild CAV was observed with combined CMPD and CsA, even in the three longest-surviving grafts (animal M347, CAV score 1.1; M292, CAV score 1.5; 627, CAV score 0.8 on day 54) as illustrated in Fig. 9C. This result stands in distinct contrast to grafts surviving for similar intervals in animals treated with CsA, with or without steroid rescue therapy, in which CAV is relatively severe (>2) in most animals.

Effect of CCR5 antagonism on anti-donor Ab

We reasoned that CCR5 might attenuate CAV by inhibiting adaptive immunity to alloantigens and by delaying elaboration of the alloantibody. Thus, anti-donor Ab levels were measured serially in three vehicle-treated animals, three animals treated with CsA monotherapy, five animals treated with CMPD 167 monotherapy, five animals with CsA plus CMPD 167, and animal M627 (delayed addition of CsA after steroid rescue). Animals treated with CsA alone or with vehicle exhibited high levels of antidonor IgM and IgG Ab within 3 wk (Fig. 10). In contrast, anti-donor Ab was absent or present at low titers at 3 wk in animals treated with CMPD 167 alone. Alloantibody elaboration and class switching (from IgM to IgG) was significantly delayed with combined CsA and CMPD 167 relative to CsA alone.


Figure 10
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  		FIGURE 10. Effect of CCR5 blockade on the production of anti-donor Abs. Recipient Ab binding on donor spleen T cells was revealed by flow cytometry using secondary Abs reactive with monkey IgM (left panels) or IgG (right panels) as described in Materials and Methods. A, Representative flow profiles are depicted for the day of transplant (dotted line) and at the graft explant for recipients treated with CsA alone (solid line, animal M634 at day 14) or CsA combined with CCR5 blockade (filled histogram, animal M347 at day 44). B, Production of anti-donor Abs is summarized for monkey allograft recipients treated with subtherapeutic CsA alone (top panels), CCR5 blockade alone (middle panels), or both therapies (bottom panels). In the bottom panels, one animal treated with CCR5 blockade monotherapy (M627) was rescued with steroids and therapeutic CsA at day 12 and is shown by a dotted line. Antidonor Abs were expressed as the percentage of positive cells at respective time points after subtracting the day 0 (day of transplant) baseline value. Ab levels are depicted from day 0 until graft explant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CCR5 inhibition using CMPD 167 monotherapy modestly but significantly delayed the onset of acute cellular rejection and prolonged graft survival, confirming in primates observations initially made in rodents (10, 17, 18, 34, 35). However, the effect of delaying infiltration of CCR5+ cells and attenuating acute allograft injury, while qualitatively similar to what is seen rodents (10, 18), is quantitatively much less efficacious in protecting the allograft from lethal injury. Although incomplete receptor blockade by CMPD 167 cannot be excluded based on our study, in our estimation it is unlikely. Trough drug levels in treated animals were consistently at or above the in vitro whole blood IC90, and infiltration of the allograft by CCR5+ cells was significantly inhibited even at the time of graft failure. We conclude that, as expected, other chemokines and receptors can mediate graft infiltration in primates despite CCR5 blockade to a degree that is clinically more important than in rodents. We infer that CCR5 blockade as monotherapy or with subtherapeutic conventional immunosuppression is unlikely to attenuate acute rejection in humans.

Although CCR5 was expressed in 45% of graft-infiltrating monocytes and macrophages but was only detected in 15% of graft-associated T cells, the influx of T cells was significantly inhibited in CMPD-treated animals. Whether CCR5 blockade attenuates acute cellular rejection kinetics by a direct effect on CCR5+ monocytes or by indirect effects on T cells that enter the graft remains to be determined. Despite its salutary effect on early monocyte infiltration, the early febrile response, and late CAV, CCR5 inhibition in conjunction with CsA is associated with an incidence of steroid-responsive acute rejection at least as high as that in the CsA-treated reference group, although allograft survival is prolonged. Alloantibody elaboration is delayed, an observation anticipated by some rodent studies (36), but not prevented. Our data leave open the possibility that a CCR5 blockade exerts competing effects on alloimmunity, both delaying infiltration of pathogenic CCR5+ (and CCR5–) immunocytes into the graft and interfering with the emergence or function of regulatory pathways, because CCR5 is expressed on regulatory T cells and plasmacytoid dendritic cells (37, 38, 39, 40). If so, clinical application could be targeted to the first days of the transplant episode to inhibit surgical stress responses and, after stable graft function is established, to suppress CAV after donor-specific T regulatory cells have become established.

That CCR5 modulates inflammation (fever) and malaise occurring in the context of surgical stress is a novel observation with potentially important implications for the clinical care of patients undergoing cardiac surgery or with infection or trauma. The mechanisms causing fever remain incompletely understood (41, 42), complicating our efforts to explain our observations. The current paradigm holds that fever is mediated centrally by hypothalamic PGE1 elaboration. Because CMPD 167 poorly penetrates the CNS, we presume that this agent does not directly modulate prostanoid-driven signaling in the brain. Increased activity and appetite on the first days after surgery in CMPD 167-treated monkeys are consistent with an anti-nociceptive effect on CCR5+ postganglionic neurons, but this mechanism, if operative, would not likely directly account for the antipyrexial effect associated with CCR5 antagonism. CsA treatment partially attenuated the febrile response, an effect perhaps ascribable to the blunting of cyclophilin-mediated, NFAT- and/or NF-{kappa}B-dependent cytokine pathways. In contrast, CsA monotherapy had no effect on appetite or malaise, suggesting that these features of the CCR5 blockade phenotype are distinct from cyclophilin-driven innate immune responses and perhaps specific to this pathway.

To explore how CCR5 might modulate inflammation, we reasoned that peripheral events elicited by wounding might trigger the elaboration of CCR5-dependent humoral mediators of inflammation pivotal to fever. In support of this notion, we find platelet-associated RANTES in surgical wounds and show that RANTES amplifies IL-1 and IL-6 mRNA in cultured monocytes by a CCR5-dependent mechanism. Based on these observations, we posit that CCR5 blockade attenuates fever by inhibiting the amplification of platelet-derived RANTES-driven inflammation in the wound, which in turn modulates the elaboration of cytokines by resident CCR5+ tissue monocytes and dendritic cells or blood monocytoid populations activated by coagulation and the associated inflammation in the wound.

Our observations and derivative hypotheses build upon the evolving paradigm linking innate immunity to the classical alloantigen-driven pathogenic adaptive immune response to an allograft. In this study we demonstrate that CCR5 plays an important role in innate immune responses to surgical stress following transplantation and in CAV. Innate immunity is increasingly recognized as an important modulator of adaptive responses to donor Ags through "danger" signals (43, 44, 45, 46, 47, 48) and in vascular disease (35, 49, 50, 51, 52). Whether this effect on innate immunity accounts in part or wholly for the delay in Ab elaboration and, conversely, how Ag presentation and lymphocyte responses are affected by CCR5 blockade are important remaining questions and the focus of ongoing work. As the broad array of reagents needed to perform informative studies becomes available for the cynomolgus monkey, comprehensive and pathway-specific gene expression profiling in tissues archived from these studies should shed additional light on the mechanisms involved.

We conclude that CCR5 antagonism modulates perioperative inflammation, delays graft infiltration by monocytes/macrophages and T cells, and slightly prolongs acute cardiac allograft survival in cynomolgus monkeys. Combined with CsA, CMPD 167 delays (but does not prevent) alloantibody elaboration and confers significant protection from CAV relative to CsA monotherapy. Attenuation of CAV despite failure to prevent intense cellular infiltrates, alloantibody elaboration, and myocyte injury suggests that CCR5 plays an important role in vascular remodeling downstream from alloimmune injury. The number of observations to date is relatively small, and ongoing work with this and other CCR5 antagonists will be required to confirm the specific role of this promising therapeutic target in allograft injury and to determine how CCR5 affects innate and adaptive immunity (6, 38, 39, 40). If efficacy in safely inhibiting chronic rejection is confirmed, CCR5 inhibition could substantially improve long-term outcomes after transplantation of the heart and other organs and address a pressing and currently unmet clinical need.


   Acknowledgments
 
We acknowledge the important contributions of James Atkinson for grading cardiac rejection scores and Qi Feng, Amal Laaris, Athba Hammed, Hui Zhou, Chris Avon, and Nitin Sangrampurkar for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was presented in part orally and in abstract form at the American Transplant Congress and the International Society for Heart and Lung Transplantation annual meetings 2002–2006. This work was supported by a Merck Research Laboratory sponsored research agreement and gift, National Institutes of Health Grant U01 AI 066719, and an American Heart Association Award (0455722U) (to R.N.P.); National Institutes of Health Grant F32 HL079818 and Thoracic Surgery Foundation for Research and Education resident research awards (to B.-N.H.N.); Deutsche Forschungsgemeinschaft research award (to C.S.); and an Other Tobacco Related Diseases research grant from the Maryland Cigarette Restitution Fund Program. Back

2 C.S. and R.N.P. contributed equally to the first authorship of this study. C.S. and B.-N.H.N. were primarily responsible for the conduct of the in vivo studies, and C.S. made the cardinal observation regarding the effect of CCR5 inhibition on the febrile response. In conjunction with J.A.D. and I.I.S., R.N.P. was primarily responsible for study design and manuscript authorship. Back

3 Address correspondence and reprint requests to Dr. Richard N. Pierson III, Division of Cardiac Surgery, University of Maryland, 22 South Greene Street, Room N4W94, Baltimore, MD 21201. E-mail address: rpierson{at}smail.umaryland.edu Back

4 Abbreviations used in this paper: CMPD 167, compound 167 (N-[(1R,3S,4S)-3-((4-(3-benzyl-1-ethylpyrazol-5-yl)piperidin-1-yl)methyl)-4-(3-fluorophenyl)cyclopentan-1-yl]-N-methyl-D-valine; bid, twice daily (bis in die); CAV, cardio allograft vasculopathy; CsA, cyclosporine; ISHLT, International Society of Heart and Lung Transplantation. Back

Received for publication June 15, 2006. Accepted for publication June 1, 2007.


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 Materials and Methods
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