CCR2 membrane binding assay

The radio-ligand binding assay was done using scintillation proximity assay (SPA) technology as previously described with some modifications [11]. Membranes (6 μg per assay point) from Ba/F3 cells transfected with mouse CCR2), and wheat germ agglutinin-coated SPA beads (450 μg per point; Amersham, Arlington Heights, IL), were pre-incubated for 30 min at room temperature in mouse plasma (C57BL/6 mouse, anticoagulant - sodium citrate; Bioreclamation, Inc., Hicksville, NY) containing 10 mM HEPES, pH 7.2. Then, prebound to WGA-SPA beads membranes were incubated at room temperature for 6 h with the indicated concentrations of CCR2 antagonists (in mouse plasma, 1% DMSO, final) and 0.2 nM ¹²⁵I-rhCCL2 in mouse plasma (S.A. 2200 Ci/mmol, PerkinElmer Life and Analytical Science, Boston, MA). Binding was measured using a 1450 Microbeta Trilux counter (Wallac, Gaithersburg, MD). Binding constants (IC₅₀ and slope) were calculated using GraphPad Prism software (GraphPad Software, Inc., La Jolla CA).

CCR2 chemotaxis assay

Chemotaxis experiments were performed as described previously with some modifications [11]. WeHi-274.1 cells (ATCC) were resuspended at 4 × 10⁶ cells/ml in mouse plasma (C57BL/6 mouse, anticoagulant - sodium citrate; Bioreclamation, Inc.) containing 10 mM HEPES, pH 7.2. Various concentrations of rmCCL2, diluted in mouse plasma, were dispensed at 30 μl/well into disposable microchemotaxis plates (ChemoTx 101-5sp; Neuro Probe, Gaithersburg, MD), which were then covered with a filter. Cells were pre-incubated at 37°C with various concentrations of MK0812 in a CO₂ incubator for 30 min, and then 25-μl cell aliquots were applied to each spot on the filter. After incubation for 2 h at 37°C in a 5% CO₂ incubator, the filters were removed. Migrated cells in the bottom wells were transferred to a Microlite luminometer plate (Thermo Fisher Scientific, Waltham, MA), and assayed using CellTiter-Glo kit according to the manufacturer's instructions (Promega, Madison, WI). The area under the curve (AUC) and inhibition constants (IC₅₀ and slope) were calculated using GraphPad Prism software.

Chemokine-induced monocyte migration in vivo

Recombinant mouse CCL2 and CCL3 (R & D Systems, Minneapolis, MN) were dissolved in PBS and injected i.v. in the lateral tail vein of BALB/c mice, CCR2^-/- mice or CCR2^+/+ littermate controls. 30 min later, chemokine-injected and control animals were euthanized, blood was collected into EDTA-coated tubes. Plasma was collected after centrifugation and frozen at -20°C until used for measurements of CCL2, CCL7 or CXCL12 by ELISA (R & D Systems). Where indicated, animals were dosed with the CXCR4 antagonist AMD3100 (Sigma-Aldrich, St. Louis, MO) subcutaneously 1 h prior to dosing with MK0812 by oral gavage (p.o.). In all studies involving MK0812, the compound was dosed as a solution in 0.4% hydroxypropyl methylcellulose (MC) via oral gavage 1 h prior to CCL2 or CCL3 administration.

Blood cell differential counts and flow cytometry

White blood cell differential counts to quantify total leukocytes, lymphocytes, monocytes, neutrophils, basophils and eosinophils were performed using mouse whole blood collected into EDTA-coated tubes. Blood samples were analyzed using the ADVIA 120 Hematology System (Bayer HealthCare, LLC, Tarrytown, New York) according to the manufacturer's instructions for analysis of mouse blood.

To measure the number of CD11b⁺Ly6C^hi monocytes, blood was collected from euthanized mice into EDTA-coated tubes. Blood was aliquoted into 96-well V-bottom plates. To block nonspecific binding, normal rat serum was added to a final concentration of 20% and incubated for 10 min at room temperature. Blood was then incubated with PerCP-Cy5.5-anti-CD11b, PE-antiLy6G, FITC-anti-Ly6C and/or anti-CXCR4 antibodies or isotype controls (BD Biosciences, Franklin Lakes, NJ) for 20 min at RT. The cells were pelleted by centrifugation and erythrocytes were lysed using FACS Lysing Solution (BD Biosciences) according to the manufacturer's instructions. Cells were washed and analyzed on a FACS Calibur instrument (BD Biosciences).

Experimental autoimmune encephalomyelitis

EAE was induced in C57BL/6 female mice at 10-12 weeks of age. Mice were injected with 200 ng pertussis toxin (Sigma-Aldrich, St. Louis, MO) i.v. on days 0 and 2. On day 1, mice were immunized with 50 μg MOG_35-55 peptide (Princeton Biomolecules, Lanhorne, PA) emulsified with 300 μg heat-killed Mycobacterium tuberculosis (Difco, Detroit, MI) and injected in two 50 μl s.c. injections over the flanks. Beginning on day 5 post immunization, animals were dosed with FTY720 (1 mg/kg, q.d.) or MK0812 (10 or 30 mg/kg, b.i.d.) by oral gavage. Both compounds were dissolved in 0.4% hydroxypropyl methylcellulose (MC). Control animals were dosed b.i.d with MC. Animals were scored for clinical symptoms daily using the following scoring system: 0, no signs of disease; 1, tail paralysis; 2, tail paralysis and hind limb weakness; 3, hind limb and tail paralysis; 4, hindlimb and forelimb paralysis; 5, moribund or dead. Mice that reached a score of 4 that did not improve within 24 h were euthanized and assigned a score of 5 for humane reasons.

Analysis of central nervous system (CNS) infiltrates

To assess the infiltration of leukocytes into CNS tissues in the EAE model, naïve mice or mice with EAE that had been dosed with either MC (the vehicle) or MK0812 (30 mg/kg, b.i.d.) were euthanized and then perfused with 50 ml cold PBS containing 2% glucose and heparin (2.1 U/ml). The spinal cord and brains were dissected and mononuclear cells isolated using a Neural Tissue Dissociation Kit (Papain) (Miltenyi Biotec, Inc., Auburn, CA) according to the manufacturer's instructions. Isolated mononuclear cells were then stained for flow cytometric analysis using fluorochrome-conjugated anti-CD45, anti-CD11b, anti-CD3, anti-Ly6G, anti-Ly6C antibodies (BD Biosciences), washed and analyzed on a FACS Calibur instrument (BD Biosciences).

The CCR2 antagonist MK0812 blocks CCR2 function in vitro

Having determined that this CCL2-induced monocyte migration in vivo was CCR2 mediated, we used this system to characterize the effects of the small molecule CCR2 antagonist MK0812, a highly potent and selective reagent [12,13]. We first confirmed the potency of this compound against CCR2 using a membrane binding assay. MK0812 inhibited the binding of ¹²⁵I-labeled recombinant human CCL2 to CCR2-containing mouse cell membranes with an IC₅₀ of 19 nM in the presence of 97% mouse plasma, accounting for any shift in potency due to protein content in vivo (Figure ​(Figure3A).3A). Similarly, MK0812 potently inhibited CCL2-mediated chemotaxis of WEHI-274.1 cells with an IC₅₀ = 5 nM in the presence of 99% mouse plasma (Figure 3B, C). Having defined the high potency of this molecule against mouse CCR2, we sought to characterize its effects on monocyte migration in vivo.

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Figure 3

MK0812 inhibits binding to CCR2 and CCL2-mediated chemotaxis in vitro. A, Inhibition of binding of ¹²⁵I-rhCCL2 to membranes from Ba/F3 cells transfected with mouse CCR2 was tested using the indicated concentrations of MK0812 or unlabeled rmCCL2. B, The inhibition of WeHi-274.1 cell chemotaxis was tested with the indicated concentrations of MK0812. C, The area under the curves from the data depicted in (B) were calculated and plotted against the concentration of CCL2 to determine an IC₅₀ = 5 nM for MK0812. The data presented are the mean ± S.E.M. and are representative of three independent experiments.

Pharmacokinetic analysis of MK0812

We next evaluated the pharmacokinetic behavior of MK0812 in vivo. Mice were dosed with MK0812 at 50 mg/kg, p.o. and plasma was collected 1, 4, 8, or 16 h later to determine the plasma exposure over time. The compound displayed a T_1/2 = 1.6 h, with C_max = 27 μM and an AUC = 104 μM·h (data not shown). Thus, 16 h after a 50 mg/kg dose, the plasma concentration of MK0812 was 50 nM, or ten-fold higher than the IC₅₀ for inhibition of chemotaxis in the presence of 99% mouse plasma in vitro. Substantially lower doses would therefore be required for complete inhibition of CCR2 activity in acute in vivo assays of 2-3 h duration, as described below.

Pharmacodynamic effects of the CCR2 antagonist MK0812

We next sought to assess the effect of MK0812 on CCR2-dependent monocyte migration in vivo. As shown above, intravenous injection of a 300 ng bolus of CCL2 led to a clear increase in the number of blood CD11b⁺Ly6C^hi monocytes (Figure ​(Figure4A).4A). Oral administration of MK0812 1 h prior to the CCL2 challenge dose-dependently and completely inhibited this response. We determined the concentration of MK0812 present in the plasma at the time of blood collection in order to define the pharmacokinetic - pharmacodynamic relationship for this compound. We observed a linear relationship between the administered dose of MK0812 and the levels of the compound present in the plasma. The most complete inhibition of the CCL2-driven increase in monocyte number was observed at doses that achieved plasma concentrations of MK0812 above the in vitro IC₅₀ for this compound (Figure ​(Figure4B).4B). Thus, the in vivo effects of MK0812 align well with the predicted potency based on the in vitro characterization of the compound.

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Figure 4

MK0812 inhibits CCL2-mediated monocyte migration in vivo. A, BALB/c mice were administered the indicated dosage of vehicle (MC) or MK0812 by oral gavage. 1 h later, the mice were injected with PBS or 300 ng CCL2 i.v. 30 min later, peripheral blood CD11b⁺Ly6C^hi monocyte numbers were determined as in Figure 2. B, The plasma concentrations of MK0812 were determined from the animals used in (A) and plotted versus the dosage of MK0812 administered. The IC₅₀ for inhibition of CCL-2 mediated chemotaxis (see Figure 3) is indicated by a dashed line. The data presented are the mean ± S.E.M. of six mice per group and are representative of three independent experiments. **p < 0.01 compared to MC/CCL2 group.

In the course of this experiment, we noted that the circulating pool of CD11b⁺Ly6C^hi monocytes decreased in animals as a consequence of dosing with MK0812 (Figure ​(Figure4A).4A). This finding is likely a reflection of the role of CCR2 in the homeostatic process of monocyte recruitment from the bone marrow to the peripheral blood. In order to extend the characterization of the effects of CCR2 antagonist administration, we performed additional analyses of the blood from animals dosed with MK0812. As shown above, oral administration of MK0812 led to a dose-dependent reduction of up to 75% of circulating CD11b⁺Ly6C^hi monocytes (Figure ​(Figure5A).5A). In addition, the levels of CCL2 and CCL7, two important ligands for CCR2 implicated in recruitment from the bone marrow, were significantly elevated in mice dosed with MK0812 (Figure 5B, C). This effect was selective for CCR2 ligands, since no change was observed in plasma levels of a selection of other chemokines, including CCL3, CCL4, CXCL1, CXCL2, and CXCL12 (data not shown). Further, we repeated these studies using the structurally distinct CCR2 antagonist INCB3344 and observed similar pharmacodynamic effects on blood Ly6C^hi monocyte number and circulating CCL2 and CCL7 levels (data not shown). In addition, elevated levels of CCL2 and CCL7, but not other chemokines, was observed in the plasma of CCR2^-/- mice relative to CCR2^+/+ littermates, indicating that these changes in CCR2 ligand levels were general phenomena of CCR2 antagonism and absence of CCR2 expression (data not shown).

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Figure 5

Pharmacodynamic effects of MK0812 in vivo. BALB/c mice were administered the indicated dosage of vehicle (MC) or MK0812 by oral gavage. 2 h later, peripheral blood CD11b⁺Ly6C^hi monocyte numbers were determined as in Figure 2 (A) and plasma concentrations of CCL2 (B) and CCL7 (C) were determined. ***p < 0.001 and **p < 0.01 compared to MC group. D, The effect of MK0812 (3 mg/kg, p.o.) or vehicle (MC) on CCL2 and CCL3 mediated changes in CD11b⁺Ly6C^hi monocyte numbers were evaluated as in Figure 4. **p < 0.01 compared to PBS group. E, BALB/c mice were dosed with vehicle (MC) or MK0812 (30 mg/kg, p.o.) and 2 h later, blood was collected and analyzed for white blood cell counts using an ADVIA system to quantify total leukocytes, lymphocytes, monocytes, neutrophils, basophils and eosinophils. *p < 0.05 compared to MC group. The data presented are the mean ± S.E.M. of six mice per group and are representative of three independent experiments.

While CCR2 antagonism in vivo rendered CD11b⁺Ly6C^hi monocytes non-responsive to CCL2, we sought to determine if this monocyte population remained responsive to other chemotactic signals. We determined that intravenous administration of the CCR1 ligand macrophage inflammatory protein-1α (CCL3) into naive mice caused a recruitment of monocytes into the peripheral blood similar to that observed with CCL2 (Figure ​(Figure5D).5D). This result suggests that both CCR1 and CCR2 ligands can mediate the direct recruitment of monocytes from the bone marrow to the peripheral blood. Notably, while administration of MK0812 paralyzed the ability of this monocyte population to response to CCL2, responsiveness to CCL3 remained intact, such that CCL3 induced a 2.5-3-fold increase in Ly6C^hi monocyte number both in the presence and absence of MK0812 (Figure ​(Figure5D).5D). As discussed later, this finding may have important implications for the use of CCR2 antagonists to treat human disease. Finally, administration of MK0812 to mice caused a selective decrease in the circulating monocyte population, as white blood cell counts for the other major blood cell populations remained unchanged (Figure ​(Figure5E5E).

Role for CXCR4 in the modulation of peripheral monocyte number by MK0812

The reduction in circulating CD11b⁺Ly6C^hi monocytes that occurs after CCR2 antagonist treatment could be due to a recycling of monocytes from the blood back to the bone marrow or could simply reflect the further migration of monocytes into tissues where differentiation to macrophage and dendritic cell subsets occurs. One potential recycling mechanism could involve the stromal cell-derived factor-1 (CXCL12) - mediated retention of leukocytes mediated through CXCR4 [8,14]. We first examined the expression of CXCR4 on Ly6G^-CD11b⁺Ly6C^hi monocytes in the blood and bone marrow of naïve mice. While CXCR4 was clearly detectable on 34% of bone marrow Ly6C^hi monocytes, we did not detect expression on the corresponding blood monocyte population (Figure ​(Figure6A6A).

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Figure 6

The pharmacodynamic effect of MK0812 on monocytes is mediated by CXCR4. A, Blood and bone marrow were isolated from naïve BALB/c mice and stained for expression of CD11b, Ly6G, Ly6C and CXCR4. Neutrophils were excluded by gating on the Ly6G^- population and monocytes were gated upon based on CD11b⁺Ly6C^hi expression. The expression of CXCR4 on the gated CD11b⁺Ly6C^hi monocytes is indicated. B, BALB/c mice were administered the indicated dosage of vehicle (MC) or AMD3100 s.c. 1 h later, mice were dosed p.o. with MK0812 or vehicle. 30 min later, peripheral blood CD11b⁺Ly6C^hi monocyte numbers were determined as in Figure 2. Plasma concentrations of CCL2 and CXCL12 were determined from the same mice. ***p < 0.001, *p < 0.05 compared to the Vehicle group. C, The effect of AMD3100 was assessed on Ly6G^-CD11b⁺Ly6C^hi monocytes and Ly6G⁺Ly6C⁺ neutrophils. **p < 0.01 as shown. The data presented are the mean ± S.E.M. of six mice per group and are representative of three independent experiments.

To assess the role of CXCR4 on the decrease in blood monocytes upon CCR2 antagonism, we treated animals with the selective CXCR4 antagonist AMD3100 prior to administration of MK0812. As shown above, in animals pretreated with vehicle alone, MK0812 caused a significant decrease in CD11b⁺Ly6C^hi blood monocytes (Figure ​(Figure6B).6B). Pretreatment with AMD3100 significantly and dose-dependently counteracted the effect of the CCR2 antagonist. These data suggest that the reduced peripheral monocyte number in CCR2 antagonist treatment is largely due to recycling of this population back into the bone marrow via CXCR4. In all animals dosed with the CCR2 antagonist, there was a clear elevation in plasma CCL2 levels, suggestive of comparable CCR2 receptor coverage in all groups (Figure ​(Figure6B).6B). An elevation in plasma CXCL12 levels was detected in animals treated with the highest dose of AMD3100, consistent with substantial coverage of CXCR4 at the 10 mg/kg dose (Figure ​(Figure6B).6B). Notably, this dose of AMD3100 also had the greatest impact on monocyte levels in the plasma.

To rule out the possibility that CXCR4 antagonism alone could cause an elevation in peripheral monocyte number independent of CCR2 antagonism, we examined whether mice dosed with AMD3100 alone affected peripheral monocyte and neutrophil populations. At the highest dose tolerable to mice, 30 mg/kg, AMD3100 had only a modest effect on the peripheral Ly6C^hi monocyte number relative to vehicle treated animals, while a clear reversal of the effects of MK0812 were again observed (Figure ​(Figure6C).6C). As expected, CXCR4 antagonism alone did lead to a substantial increase in peripheral blood Ly6G⁺Ly6C⁺ neutrophils, an effect that was observed to be independent of CCR2 antagonism (Figure ​(Figure6C).6C). Thus, CXCR4-mediated retention of neutrophils in the bone marrow is a mechanism to limit neutrophil egress from the bone marrow under homeostatic conditions.