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American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1602-1609, (2002)
© 2002 American Thoracic Society

Original Article

Responses of Leukocytes to Chemokines in Whole Blood and Their Antagonism by Novel CC-Chemokine Receptor 3 Antagonists

Shannon A. Bryan, Peter J. Jose, Joanna R. Topping, Robert Wilhelm, Carol Soderberg, Denis Kertesz, Peter J. Barnes, Timothy J. Williams, Trevor T. Hansel and Ian Sabroe

National Heart and Lung Institute, and Leukocyte Biology Section, Biomedical Sciences Division, Faculty of Medicine, Imperial College School of Medicine, London, United Kingdom; and Inflammatory Diseases Unit, Roche Bioscience, Palo Alto, California

Correspondence and requests for reprints should be addressed to Ian Sabroe, MRC Clinician Scientist Fellow, University of Sheffield, Section of Functional Genomics, Division of Genomic Medicine, M Floor, Royal Hallamshire Hospital, Sheffield, S10 2JF. E-mail: i.sabroe{at}sheffield.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CC-chemokine receptor 3 (CCR3)-stimulating chemokines are likely to have important in vivo roles in the regulation of eosinophil, basophil, and potentially helper T cell type 2 and mast cell recruitment. We have developed techniques to investigate the actions of eotaxin and other chemokines on multiple leukocyte populations in whole blood, without cell purification steps that might alter leukocyte responsiveness. We have shown that the potency of eotaxin in whole blood is limited by Duffy antigen binding, which may modulate the actions of this chemokine in vivo. We have also investigated the efficacy and potency of a new panel of small molecule antagonists of CCR3 on responses of eosinophils, neutrophils, basophils, and monocytes to chemokines, using whole blood assays of shape change, chemokine receptor internalization, and CD11b upregulation. These small molecule antagonists cause selective and potent inhibition of CCR3 on eosinophils and basophils, are bioavailable in blood, and are prototypic antagonists potentially of benefit in the treatment of human allergic disease. Such whole blood methods may also be employed in the investigation of other small molecule chemokine receptor antagonists.

Key Words: antagonist • basophil • chemokine • eosinophil


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Members of the chemokine family of proteins are believed to be important in the selective recruitment of leukocytes into areas of inflammation in diseases such as asthma. In particular, the regulation of the recruitment of cells including eosinophils, basophils, and helper T cell type 2 (Th2)-type T cells is central to the processes of allergic inflammatory disease, and recruitment of all these cell types may be at least in part dependent on chemokines acting on CC-chemokine receptor 3 (CCR3) (1). The interaction between chemokines and leukocytes stimulates upregulation of adhesion molecules on the leukocyte cell surface (2, 3), and cytoskeletal changes that result in the firm arrest of the leukocytes on the endothelium and diapedesis into tissue. Once in the tissues, cells such as eosinophils and basophils may degranulate, releasing toxic granule proteins that can contribute to the tissue pathology seen in asthma (4). Chemokine-induced cytoskeletal changes in the leukocyte, mediated by polymerization of cytoplasmic monomeric actin (G-actin) to microfilamentous actin (F-actin) (5, 6), are dependent on downstream G-protein–dependent activation of signaling pathways including phospholipase C and phosphatidylinositol 3-kinase (7).

Eosinophil shape change responses to complement factor 5a (C5a) and platelet-activating factor (PAF) have been studied by laser turbimetry (8). Neutrophil shape change has also been shown to occur in response to N-formyl-methionyl-leucyl-phenylalanine (9), interleukin-8 (IL-8), and leukotriene B4 (10). Flow cytometry has also been used to detect changes in leukocyte shape (11). Using flow cytometry, we developed a method to investigate responses of leukocytes to chemokines. This method was originally developed for use with separated granulocytes and was named the gated autofluorescence forward scatter (GAFS) assay (12). Using this assay, we identified important variations in donor responses to chemokines that may have a bearing on the development of small molecule antagonists for the treatment of eosinophilic inflammation. We also showed previously unsuspected complexity in basophil chemokine responsiveness, with sequential and co-operative signaling of chemokines through receptors such as CCR2 and CCR3 on the basophil surface that may explain selective basophil recruitment in vivo (13). We also investigated the properties of a potent CCR1 and CCR3 dual antagonist by using this technique, and showed CC chemokine-induced eosinophil shape change to be inhibited by this antagonist (14).

We have adapted this method, originally designed for semipurified cell populations, to investigate the actions of chemokines in whole blood. This allowed investigation of the modulating effects of erythrocytes, other leukocytes, and plasma on chemokine responses, with no cell purification. We have combined these studies with other methodologies examining chemokine responsiveness in whole blood. We have studied the effects of a novel panel of CCR3 antagonists in whole blood assays, because CCR3 plays a central role in leukocyte trafficking in allergic disease, and therefore its antagonism is an attractive target for therapy in asthma and allergy.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Blood Preparation
The study was approved by the St. Mary's Hospital Local Research Ethics Committee. Whole blood, anticoagulated with 3.8% tri-sodium citrate, used within 10 minutes of sampling, was used in all experiments. Donors were healthy normal volunteers, atopic or asthmatic subjects on no systemic medication.

Whole Blood Gated Autofluorescence Forward Scatter Assay
The following were prepared in advance: cold ammonium chloride lysis solution (155 mM NH4Cl), assay buffer (10 mM HEPES, 10 mM glucose, 0.1% bovine serum albumin [BSA] in phosphate-buffered saline [PBS, without Ca2+ or Mg2+], pH 7.3–7.4). Chemokines were from PeproTech (London, UK). Aliquots (10 µl at 10x final whole blood concentration) of agonist or buffer were placed in 1.2-ml polypropylene cluster tubes. Whole blood (90 µl) was added, mixed, and incubated at 37° C for 4 minutes in a shaking water bath. The tubes were placed on ice and 250 µl of optimized ice-cold fixative (12) was added. After 1 minute, samples were added to NH4Cl lysis solution (1,750 µl) and left on ice for 20 minutes. After red cell lysis, samples were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA). Eosinophils were separated from neutrophils by their autofluorescence (12). A total of 250 to 500 eosinophils per sample was acquired. Samples were kept on ice before data collection.

Separated Granulocyte GAFS Assay
Cells were prepared on the basis of the method of Watts and coworkers (15). Filtered buffers were prepared: 10 mM HEPES, 10 mM glucose, and 0.1% or 0.25% BSA in PBS (without Ca2+ or Mg2+, pH 7.3–7.4). Anticoagulated whole blood (5 ml) was centrifuged at 300 x g for 10 minutes and platelet-rich plasma was removed. The cell pellet was washed in 0.1% BSA buffer, pelleted by centrifugation, reconstituted to its original volume, and layered over a discontinuous Percoll gradient (69/75%) and centrifuged at 500 x g for 30 minutes at room temperature to separate polymorphonuclear leukocytes (PMNLs) from peripheral blood mononuclear cells (PBMCs). The PMNLs were collected and washed, and red cells were lysed by hypotonic shock (12). The cells were resuspended in 0.25% BSA buffer at 5 x 106 cells/ml and incubated at 37° C for 30 minutes. After washing, the cells were resuspended in 0.25% BSA assay buffer at 3–5 x 105 cells/50 µl, and added to dilutions of eotaxin or buffer (50 µl; final volume, 100 µl). Samples were incubated for 4 minutes at 37° C, transferred onto ice, fixed with an optimized fixative, and analyzed as described above (12).

Inhibition of Eosinophil Shape Change
Anti-human CCR3 (monoclonal antibody [MAb] 7B11 [isotype IgG2a], a kind gift of P. Ponath [Leukosite, Boston, MA; now Millennium Pharmaceuticals, Boston, MA]) or small molecule antagonists were incubated with whole blood (90 µl) for 10 minutes at room temperature. The CCR3 antagonists (RO116-4875/608 [16], RO116-9132/238 [17], RO320-2947/001 [16], and RO330-0802/001 [18]) were synthesized at Roche Bioscience (Palo Alto, CA) according to procedures described in the corresponding patents (Figure 1) , reconstituted in DMSO, and stored at 10-2 M at -20° C. Serial dilutions of compounds were performed in buffer containing a constant concentration of DMSO. After incubation with antibody or antagonist, samples were stimulated with buffer or agonist as above.



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  		Figure 1. RO116-4875/608, RO116-9132/238, RO320-2947/001, and RO330-0802/001 were synthesized at Roche Bioscience (Palo Alto, CA) according to procedures described in the corresponding patents (GB2330580, GB2343894, and GB2343893-A).

 
Modulation of Chemokine-induced Cell Surface Protein Expression
Whole blood, preincubated with CCR3 antagonists for 10 minutes at room temperature, was stimulated with chemokine for 5 minutes at 37° C. Stimulation was terminated by adding ice-cold buffer (PBS, 0.25% BSA, 10 mM HEPES, pH 7.4). All processing of the samples was performed at 4° C. Samples were washed and pelleted by centrifugation. CD11b expression was measured simultaneously in neutrophils, eosinophils, and monocytes. Cell populations were identified by forward scatter/side scatter (FSC/SSC) gating and the following markers: CD11b conjugated with R-phycoerythrin (CD11b-RPE, diluted 1:10; Dako, Ely, UK), CD14 conjugated with RPE and carbocyanine 5 (CD14–RPE–Cy5, diluted 1:20; Serotec, Oxford, UK), and CD16 conjugated with fluorescein isothiocyanate (CD16–FITC, diluted 1:10; Dako) (19). After final washes, red cells were lysed, and the samples were fixed with Optilyse B (Coulter, Luton, UK) and analyzed by flow cytometry. Eosinophils were identified as CD16- granulocytes in the granulocyte FSC/SSC region. Neutrophils were identified as CD16+ granulocytes and monocytes as CD14+ (12).

To measure changes in CCR expression, stimulated blood samples were incubated with anti-human CCR3 MAb 7B11 or IgG2a control (Sigma, St. Louis, MO) for 30 minutes on ice, washed, and incubated on ice with goat anti-mouse RPE–Cy5-conjugated F(ab')2 Ab (Dako) for 30 minutes. After washing, mouse immunoglobulins (Sigma) were used in a blocking step before the samples were stained with FITC-conjugated HLA-DR MAb (clone HK14; Sigma) and RPE-conjugated CDw123 (clone 7G3; Becton Dickinson) to identify basophils. Red cells were lysed with Optilyse B. Eosinophils were identified in the granulocyte region of the FSC/SSC plot by their autofluorescence. Basophils were identified as HLA-DR-/CD123+ mononuclear cells (13).

CCR3 Receptor-binding Assay
CCR3 antagonism by small molecule inhibitors was characterized by inhibiting the binding of 125I human eotaxin (125I-eotaxin; New England Nuclear, Boston, MA) to butyrate-treated hCCR3 L1.2 transfectant cells (20). Compounds (in DMSO) were diluted with binding buffer (50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA, 0.02% sodium azide, pH 7.24). Twenty-five microliters of test solution or buffer was added to each well of a 96-well polypropylene plate, followed by 25 µl of 100 pM 125I-eotaxin and 1.5 x 105 hCCR3 L1.2 transfectant cells in 25 µl of binding buffer. After incubation for 1 hour at room temperature, the reaction was terminated by filtration through a polyethylenimine-treated Packard (Chicago, IL) Unifilter GF/C filter plate. The filters were washed four times with ice-cold wash buffer containing 10 mM HEPES and 0.5 M sodium chloride (pH 7.2) and dried at 65° C for 30 minutes. Microscint-20 scintillation fluid (25 µl/well; Packard) was added and filter radioactivity was determined with a Packard TopCount.

Inhibition of Eotaxin-mediated Chemotaxis of CCR3 L1.2 Transfectant Cells
The assay (21) was performed in a 96-well chemotaxis plate with 5-µm pore size filters (Neuroprobe, Gaithersburg, MD). Compounds were added both to the top and bottom chambers (highest final DMSO concentration, 1%) with 2 nM human eotaxin in the lower chambers in RPMI 1640 with 0.1% BSA and 10 mM HEPES. Butyrate-treated CCR3 transfectants were resuspended at 5 x 106 cells/ml in RPMI 1640 with 0.1% BSA and 10 mM HEPES, mixed with concentrations of drug identical to those used in the corresponding lower wells, and added to the top of the filter plates at 2.25 x 105 cells/well (final volume, 50 µl). The plate was incubated at 37° C. After 3 hours, the unmigrated cells were removed from the top of the filter plate, the plates were spun for 10 minutes at 750 x g, the filters were removed, and 150 µl of medium was aspirated from the plates. One hundred microliters of a solution of 0.1% Triton X-100 and 5 µM propidium iodide in H2O was added to each well to lyse the cells. Plates were stored overnight at 4° C and read with a fluorescence plate reader with excitation and emission wavelengths set at 530 and 590 nm, respectively.

Inhibition of Eotaxin-mediated Chemotaxis of Human Eosinophils
The eosinophil chemotaxis assay protocol is identical to the hCCR3 L1.2 chemotaxis assay described above with the following exceptions: Human eosinophils were used at 1.13 x 105 cells/well in RPMI with 2% fetal calf serum and 10 mM HEPES as the assay buffer. Eosinophils were isolated from acid–citrate–dextrose (ACD) anticoagulated human blood by dextran sedimentation to remove red blood cells, followed by a Ficoll–Hypaque gradient to remove PBMCs. The resulting granulocyte-enriched pellets were treated by hypotonic lysis to remove contaminating red cells, and neutrophils were depleted by negative selection with anti-CD16 beads (Miltenyi Biotech, Auburn, CA) together with an MACS Varios magnet.

Statistical Analysis
Data are presented as means ± standard error of the mean (SEM). For analysis of three of more groups in the dose–response curves, two-way analyses of variance (ANOVAs) with repeated measures (chemokine and group as effects) were performed. For analysis of two groups the paired t test was performed. A value of p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Eosinophil and Neutrophil Shape Change Responses to Various Chemokines
The potency and efficacy of eotaxin, eotaxin-2, macrophage chemoattractant protein-4 (MCP-4), macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), and IL-8 were investigated in whole blood. Eotaxin-2 was shown to be the most potent inducer of eosinophil shape change (Figure 2A) . Eotaxin was also found to be potent in causing eosinophil shape change. Responses to MCP-4 varied between donors, and whereas most chemokine response curves tended to plateau at high concentrations, the dose–response curve to MCP-4 was markedly bell shaped. Responses to MIP-1{alpha}, which acts primarily through CCR1, varied between donors. However, when compared with the shape change response seen with eotaxin or eotaxin-2, the mean shape change was negligible in the donors studied here. There was no eosinophil shape change response to IL-8; however, there was a marked neutrophil response (Figure 2B). No neutrophil responses were seen after stimulation with either eotaxin or eotaxin-2.



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  		Figure 2. Whole blood samples were incubated with buffer or chemokine as described, red cells were lysed, and eosinophil (A) and neutrophil (B) shape change was measured by flow cytometry. Responses to chemokines are shown as the percent increase in basal FSC values, and are displayed as means ± SEM; n = 4 separate donors.

 
Shape Change Responses in Duffy-Negative Volunteers
We have found that eotaxin and eotaxin-2 bind with different affinities to the promiscuous chemokine receptor known as the Duffy antigen on the red blood cell surface (J. R. Topping and P. J. Jose, unpublished data). To investigate the effects of erythrocyte Duffy antigen, we investigated eosinophil shape change responses to eotaxin and eotaxin-2 in whole blood from Duffy-negative and Duffy-positive individuals. We compared responses of cells in whole blood with those of separated granulocytes, in the absence of red blood cells. We found that responses to eotaxin in Duffy-positive individuals were significantly less than in Duffy-negative individuals (Figure 3A) . However, responses to eotaxin-2 in Duffy-negative and Duffy-positive individuals showed no significant differences. (Figure 3B). Responses to eotaxin of isolated leukocytes from Duffy-positive individuals in the absence of red cells were identical to those of Duffy-negative donor leukocytes in whole blood (Figure 3A).



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  		Figure 3. Whole blood samples from Duffy antigen-positive and -negative individuals were incubated with buffer or chemokine [eotaxin (A); eotaxin-2 (B)] as described, red cells were lysed, and eosinophil shape change was measured by flow cytometry. Responses to chemokines are shown as the percent increase in basal FSC values, and are displayed as means ± SEM; n = 3 for Duffy-negative donors, n = 4 for Duffy-positive donors. There was a significant difference in eosinophil shape change response to eotaxin between Duffy-negative individuals and Duffy-positive individuals (p < 0.001, ANOVA).

 
Inhibition of Eosinophil Shape Change by CCR3 Blockade
Initial studies in which whole blood was pretreated with the anti-CCR3 antibody 7B11 showed a dose-dependent inhibition of eosinophil responses to eotaxin in the whole blood assay of shape change (data not shown). We therefore used the whole blood GAFS assay to investigate the potency and efficacy of a novel group of small molecule antagonists of CCR3. All induced significant inhibition of responses, and RO116-9132/238 (Figure 1) was found to be the most potent inhibitor of eosinophil shape change responses, with both 100 nM and 1,000 nM RO116-9132/238 completely inhibiting eosinophil shape change to concentrations of up to 40 nM eotaxin. The remaining compounds were of lower potency (Figures 4A and 4B) .



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  		Figure 4. Whole blood was incubated with a panel of CCR3 antagonists [100 nM (A) or 1,000 nM (B)] for 10 minutes at room temperature, after which the samples were incubated at 37° C with buffer or eotaxin as described, red cells were lysed, and eosinophil shape change was measured by flow cytometry. Responses are shown as the percent increase in basal FSC values, and are displayed as means ± SEM, n = 4 separate donors. There was significant inhibition of the shape change response to 10 and 40 nM eotaxin by both 100 and 1,000 nM RO116-9132/238 (***p <= 0.001). RO116-4875/608, RO320-2947/001, and RO330-0802/001 significantly inhibited eosinophil shape change up to 10 nM eotaxin at 100 nM (p < 0.05) and 1,000 nM (p < 0.001).

 
In further experiments, various concentrations of compounds were incubated with whole blood at room temperature for 10 minutes, after which a fixed concentration of eotaxin (10 nM) was added to each sample, and the resulting shape change response measured. Again, RO116-9132/238 was shown to be the most potent inhibitor of shape change (50% inhibitory concentration [IC50] = 7 nM), with the remaining compounds showing similar inhibitory effects (IC50 = 175 to 193 nM) (Figure 5) .



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  		Figure 5. Whole blood was incubated with various concentrations of a panel of CCR3 antagonists for 10 minutes at room temperature, after which the samples were incubated at 37°C with buffer or a fixed concentration (10 nM) of eotaxin, red cells were lysed, and eosinophil shape change was measured by flow cytometry. Responses to chemokines are shown as FSC values, and are displayed as means ± SEM, n = 4 separate donors. Although all compounds tested induced significant inhibition of eosinophil shape change, RO116-9132/238 was found to be the most potent inhibitor (IC50 = 7 nM, ***p < 0.001 by ANOVA).

 
Inhibition of Chemokine-induced Eosinophil CD11b Upregulation
CD11b is a member of the ß-integrin family, whose expression on leukocytes is upregulated on chemoattractant stimulation (22). To investigate whether antagonists of CCR3 also blocked other eosinophil chemokine responses that could be measured in whole blood, we investigated the ability of RO116-9132/238 to inhibit eotaxin-induced CD11b upregulation. We used the method to simultaneously measure changes in CD11b expression on eosinophils, neutrophils, and monocytes by a combination of FSC/SSC gating and labeling with anti-CD16 and anti-CD14 MAbs as described in METHODS and (19).

Eotaxin, IL-8, and MCP-1 caused a significant increase in CD11b expression on eosinophils (p = 0.01), neutrophils (p = 0.005), and monocytes (p = 0.005), respectively (Figures 6, 7A, and 7B) . Eotaxin had no significant effect on the expression of CD11b on neutrophils. Addition of eotaxin to whole blood caused a significant increase in CD11b expression on monocytes (p = 0.03), but this was not inhibited by the CCR3 antagonist (Figure 7B). There was a small but significant upregulation of CD11b on all leukocytes stimulated with buffer alone at 37° C in all cell groups, although this was not of the same magnitude as that induced by chemokine stimulation.



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  		Figure 6. Whole blood samples were preincubated with or without RO116-9132/238 (antag) and then with buffer or 100 nM eotaxin as described, after which CD11b upregulation on CD16-negative granulocytes was measured by flow cytometry. Data are displayed as mean fluorescence ± SEM, n = 4 separate donors. There was significant upregulation of eosinophil CD11b in samples stimulated with buffer alone at 37°C compared with samples left on ice. Eotaxin induced further eosinophil CD11b upregulation and this was inhibited by 10, 100, and 1000 nM RO116-9132/238 (antag).

 


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  		Figure 7. In the same experiment as described in Figure 6, whole blood samples preincubated with or without RO116-9132/238 (antag) were incubated with buffer and IL-8 (A) or MCP-1 (B), after which CD11b expression on neutrophils (A, identified as CD16-positive granulocytes) and monocytes (B, identified by CD14 positivity) was measured by flow cytometry. Data are displayed as mean fluorescence ± SEM, n = 4 separate donors.

 
The CCR3 antagonist RO116-9132/238 inhibited eotaxin-induced eosinophil CD11b expression (Figure 6). There was no inhibition of CD11b upregulation on neutrophils stimulated with IL-8, or on monocytes stimulated with MCP-1 (Figures 7A and 7B).

Investigation of Eosinophil and Basophil CCR3 Internalization and Inhibitory Effects of CCR3 Antagonist
Exposure of CCR3 to its ligands such as eotaxin causes dose-dependent internalization of the receptor via clathrin-coated pits (23). To further characterize the CCR3 antagonists, we investigated the inhibition of eotaxin-induced CCR3 internalization simultaneously on both eosinophils and basophils in the presence and absence of CCR3 antagonists. To identify the basophil population in whole blood, the samples were stained with fluorochrome-conjugated antibodies against HLA-DR and CDw123 (13). Eosinophils were identified in the same samples by autofluorescence as previously described.

Stimulation with eotaxin resulted in a dose-dependent internalization of the CCR3 receptor on both eosinophils (100 nM eotaxin, p < 0.05) and basophils (10 nM and 100 nM eotaxin, p < 0.05) (Figures 8A and 8B) . Incubation with drug alone did not induce either eosinophil or basophil CCR3 internalization. Incubation of whole blood at 37° C with buffer or DMSO buffer solution in the absence of eotaxin did not alter eosinophil or basophil CCR3 expression. Addition of RO116-9132/238 resulted in a dose-dependent inhibition of eotaxin-induced eosinophil and basophil CCR3 internalization (Figures 8A and 8B).



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  		Figure 8. Whole blood samples were preincubated with or without RO116-9132/238 (antag) and then with buffer or eotaxin as described, after which CCR3 expression on eosinophils (A) and basophils (B) was measured by flow cytometry. Data are displayed as mean fluorescence ± SEM, n = 4 separate donors. (A) Eotaxin at 100 nM caused a significant decrease in eosinophil CCR3 expression, indicating receptor internalization (*p < 0.05, **p < 0.01). RO116-9132/238 (100 and 1000 nM antag) inhibited this internalization. (B) Incubation of whole blood with 10 and 100 nM eotaxin caused a significant decrease in basophil CCR3 expression, indicating receptor internalization (*p < 0.05, **p < 0.01). RO116-9132/238 (100 and 1000 nM antag) inhibited this internalization.

 
Inhibition of 125I-Eotaxin Binding and Chemotaxis
The four tested CCR3 small molecule antagonists all inhibited binding of 125I-eotaxin to transfectants, and also chemotaxis of transfectants and purified human eosinophils to eotaxin. These data are summarized in Table 1. The binding and chemotaxis IC50 values also showed that the quaternary salt (RO116-9132/238) demonstrated the greatest potency.


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  		TABLE 1. Inhibition of binding of 125i-labeled eotaxin by CCR3 antagonists

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Neutralization of murine chemokine ligands acting through CCR3 has been shown to be effective in decreasing eosinophil influx into the airways in models of inflammatory disease, and also in modulating Th2-type T cell recruitment to the lung and subsequent bronchial hyperreactivity (24). Depletion of eotaxin, using targeted gene disruption in mice, has also been shown to reduce eosinophil numbers in bronchoalveolar fluid 18 hours after inhaled allergen challenge when compared with eotaxin+/+ mice (although eotaxin-/- mice still mounted an eosinophil response when compared with unsensitized control mice) (25). To study the role of CCR3 in allergic inflammation, pharmacologically active monoclonal antibodies have been developed against guinea pig and mouse CCR3 (26, 27). Similarly, CCR3 ligands have a clear role in regulating eosinophil and basophil recruitment in humans (28–30), and possibly in regulating Th2-type T cell recruitment (31–33). Small molecule antagonists of CCR3 therefore have the potential to modify human allergic inflammatory disease. Data have demonstrated that a monoclonal antibody against interleukin-5 almost completely ablates the presence of eosinophils in the blood and airways of subjects with mild asthma, but in this study of a small subject population, there was no detectable effect on airway hyperresponsiveness and responses to inhaled allergen challenge (34). However, further studies are still required before the consequences of eosinophil-targeting strategies in allergic inflammatory disease can be definitively stated. In addition, the presence of CCR3 on basophils, mast cells, and Th2 cells as well as eosinophils suggests that CCR3 has an important role to play in the pathogenesis of asthma and allergy.

The function of CCR3 in whole blood has been largely unexplored, and because preparative techniques may modulate leukocyte function, we set out to study in detail the actions of CCR3 ligands in whole blood, and to investigate and characterize the properties of a new group of CCR3 small molecule antagonists. We developed a flow cytometric method to quantify the shape change responses of eosinophils when exposed to chemokines in whole blood. Eotaxin and eotaxin-2 were found to induce shape change of eosinophils but not of neutrophils. In contrast, interleukin-8 induced potent shape change responses in neutrophils but not in eosinophils. These data are in concordance with results previously obtained in separated granulocyte preparations (12, 13) and suggest that these previous studies were not confounded by preparation artifact. Whereas most chemokines tended to induce responses that plateaued at high concentrations, MCP-4 induced a markedly bell-shaped dose–response curve in eosinophils, suggesting that this chemokine signals differently than other chemokines through the CCR3, and data from CCR3 receptor truncation mutants support this (I. Sabroe and J. E. Pease, unpublished data).

The Duffy antigen on red blood cells is a nonsignaling, promiscuous receptor for many chemokines (35), and we have found that the binding of eotaxin to this receptor is higher than that of eotaxin-2 (J. R. Topping and P. J. Jose, unpublished data). Accordingly, we found that the potency of eotaxin in the whole blood eosinophil shape change assay was markedly reduced in the presence of Duffy antigen on the red blood cells, whereas that of eotaxin-2 was hardly affected.

CCR3 is a major drug target for the future treatment of allergic inflammatory disease, but to date only two groups of small molecule antagonists of CCR3 have been described in the published literature (14, 36). We investigated here the therapeutic potential of a panel of novel CCR3 antagonists, using the whole blood shape change system. Because chemokines such as eotaxin can bind to and be removed from the circulation by Duffy antigen, it is also possible that small molecule antagonists of CCR3 might bind to and be similarly removed from the accessible plasma compartment by Duffy antigen. Our data show that these compounds are still active in the circulation, and that therefore Duffy antigen binding of this group is not a major obstacle to their therapeutic use. RO116-9132/238 showed an IC50 in the assay of shape change of 7 nM, compared with an IC50 for the other three tested drugs of between 175 and 193 nM. In assays investigating the chemotactic responses of purified eosinophils, RO116-9132/238 was again the most potent with an IC50 of 0.4 nM compared with 1.8, 3.4, and 4.9 nM for the other compounds tested. On CCR3 L1.2 transfectant cells, RO116-9132/238 was again the most potent and showed an IC50 of 1 nM in assays of ligand binding and chemotaxis. The remaining three compounds were all more potent in the transfectant-based assays than in whole blood. These variations may reflect subtle differences in receptor expression and function in transfected versus native cells, for example, through posttranslational modifications such as glycosylation.

To investigate their potencies and selectivities further, we studied the effect of these small molecule CCR3 antagonists on the upregulation of leukocyte CD11b expression induced by chemokines. On the basis of studies showing that eosinophils responded to CCR3 stimulation in whole blood by the upregulation of CD11b (19), we used the technique to simultaneously investigate activities on multiple leukocyte types. We showed that CCR3 antagonists inhibited eotaxin-induced upregulation of CD11b on eosinophils. Although CD11b was upregulated on all cell groups by incubating sample with buffer at 37° C for 5 minutes (which could be expected, as CD11b is known to be sensitive and easily upregulated by sampling and processing methods [37]), the chemokines used were able to upregulate CD11b expression further. The sensitivity of leukocyte CD11b expression to upregulation in the presence of buffer alone is a further indication that techniques such as these, based in whole blood with the minimum of manipulation, are highly advantageous in the study of leukocyte chemokine responsiveness when seeking to understand the responsiveness of cells in vivo.

Interestingly, stimulation of whole blood with eotaxin caused a significant increase in monocyte CD11b expression. Monocytes are not generally held to express CCR3 in the circulation. Furthermore, apparent monocyte responses to eotaxin were not blocked by the CCR3 small molecule antagonists. Therefore, this bystander upregulation may be explained either by release of active mediators from eosinophils such as leukotrienes and PAF, or else by displacement of other chemokines by eotaxin from Duffy receptors in whole blood. These data suggest that during inflammation in vivo, there are multiple mechanisms by which specific recruiting pathways may "overspill" and activate or recruit other leukocyte types.

Interaction of chemokines with their cognate receptors typically results in receptor internalization (38). We exploited this response to develop further techniques to investigate ligand and antagonist specificity in whole blood. We showed that CCR3 antagonists were able to inhibit eotaxin-induced CCR3 internalization on both eosinophils and basophils, and showed that basophils, like eosinophils, may be responsive to CCR3-stimulating ligands in the circulation. Inhibition of receptor internalization by chemokines is likely to interfere with leukocyte chemotaxis, and indeed manipulation of receptor internalization has been identified as a potential mechanism that may be targeted by selective CCR antagonists (39).

Assays in whole blood are more convenient, quicker to use, and closer to the normal physiological state, and could be useful in the context of clinical studies to demonstrate pharmacodynamics and pharmocokinetics of new therapies. Using these systems, we have identified potentially important differences in the availability of chemokines in the circulation through their binding to Duffy antigen. The small molecule antagonists that we studied have been shown to be specific for CCR3 and to be potent inhibitors of eosinophil shape change. These compounds are therefore potential candidates for the prevention of eosinophil, basophil, Th2-type T cell, and mast cell influx into the airways.


   Acknowledgments
 
The authors thank Mrs. Jackie Turner for invaluable help in statistical analyses of the data, and Mr. Martin Redman (Immunohaematology, North London Blood Transfusion Centre, London, UK) for the phenotyping of Duffy status.

Supported by grants from the Imperial College School of Medicine (I.S.), the Wellcome Trust (P.J.J.), the National Asthma Campaign (T.J.W.), a Pfizer-sponsored studentship (J.R.T.), and the Medical Research Council (I.S.).

Received in original form November 4, 2001; accepted in final form December 26, 2001


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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