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The Journal of Immunology, 2000, 165: 7224-7233.
Copyright © 2000 by The American Association of Immunologists

Basophil Responses to Chemokines Are Regulated by Both Sequential and Cooperative Receptor Signaling1

Akos Heinemann2,*, Adele Hartnell*, Victoria E. L. Stubbs*, Kazuki Murakami*, Dulce Soler{dagger}, Gregory LaRosa{dagger}, Philip W. Askenase3,*, Timothy J. Williams* and Ian Sabroe4,*

* Leukocyte Biology Section, Biomedical Sciences Division, Imperial College School of Medicine, South Kensington, London, United Kingdom; and {dagger} Millennium Pharmaceutical Inc., Cambridge, MA 002139


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate human basophil responses to chemokines, we have developed a sensitive assay that uses flow cytometry to measure leukocyte shape change as a marker of cell responsiveness. PBMC were isolated from the blood of volunteers. Basophils were identified as a single population of cells that stained positive for IL-3R{alpha} (CDw123) and negative for HLA-DR, and their increase in forward scatter (as a result of cell shape change) in response to chemokines was measured. Shape change responses of basophils to chemokines were highly reproducible, with a rank order of potency: monocyte chemoattractant protein (MCP) 4 (peak at <1 nM) >= eotaxin-2 = eotaxin-3 >= eotaxin > MCP-1 = MCP-3 > macrophage-inflammatory protein-1{alpha} > RANTES = MCP-2 = IL-8. The CCR4-selective ligand macrophage-derived chemokine did not elicit a response at concentrations up to 10 nM. Blocking mAbs to CCR2 and CCR3 demonstrated that responses to higher concentrations (>10 nM) of MCP-1 were mediated by CCR3 rather than CCR2, whereas MCP-4 exhibited a biphasic response consistent with sequential activation of CCR3 at lower concentrations and CCR2 at 10 nM MCP-4 and above. In contrast, responses to MCP-3 were blocked only in the presence of both mAbs, but not after pretreatment with either anti-CCR2 or anti-CCR3 mAb alone. These patterns of receptor usage were different from those seen for eosinophils and monocytes. We suggest that cooperation between CCRs might be a mechanism for preferential recruitment of basophils, as occurs in tissue hypersensitivity responses in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Eosinophil and basophil granulocytes are important effector cells in allergy and host defense responses, particularly to parasitic infections such as helminths (1, 2) and ectoparasitic ticks (3). These cells are directed by chemotactic factors, chemokines particularly, to sites of inflammation where they release proinflammatory mediators and toxic granule products. Although exceeded in numbers by eosinophils, basophils are a constituent of the cellular infiltrate in bronchial tissue of atopic asthmatic patients (4, 5) and are further increased after allergen challenge (6). More prominently, basophils are a major constituent of the cellular infiltrate in the cutaneous late-phase reaction to allergens (6, 7) and dominate some T cell-mediated delayed-type hypersensitivity responses, such as cutaneous basophil hypersensitivity (8), contact dermatitis (9, 10), and late-phase IgG1- (11, 12), IgE- (13), and other T cell-mediated responses (14, 15).

Basophils respond to many CC chemokines, including eotaxin (16, 17), eotaxin-2 (18), eotaxin-3 (19), RANTES (16, 20, 21), monocyte chemoattractant protein (MCP)5-1 (22), MCP-2 (23), MCP-3 (24), and MCP-4 (16, 25, 26). Besides CCR3, which is believed to mediate principally cell recruitment and chemotaxis (as opposed to degranulation) (16, 17, 27), basophils also express other CCRs, including CCR1 and CCR2. Although the role of CCR1 is still unclear, CCR2 is strongly linked to histamine release and leukotriene secretion (16, 22). Major phenotypic similarities with respect to the expression of chemokine receptors (CCR3 in particular) and adhesion molecules (28) by eosinophils and basophils suggest common pathways of recruitment. However, although there is growing evidence for a crucial role of CCR3 in allergy-related tissue eosinophilia (29, 30, 31, 32, 33), the magnitude and kinetics of basophil influx after allergen challenge parallel neither eosinophil recruitment (6, 8, 9, 10, 11, 13, 14, 15, 32), nor generation of eotaxin or eotaxin-2 at the inflammatory site (6, 32). Thus, the existence of basophil-specific chemoattractants or mechanisms of recruitment need to be given serious consideration.

Having encountered a chemotactic factor in vivo, leukocytes immediately begin to rearrange their cytoskeleton and change their shape to facilitate their attachment to microvascular endothelial cells (34, 35, 36, 37, 38, 39). Such responses can be detected by flow cytometry as changes in either forward or right-angle light scattering of the cells (40, 41). On the basis of this observation, we developed a simple flow cytometric assay, the gated autofluorescence forward scatter (GAFS) assay, to measure chemokine responses of human eosinophils in a mixed population of polymorphonuclear leukocytes (PMNL) (42).

Because basophils make up <1% of leukocytes in the blood of healthy donors and are hence difficult to obtain in quantities relevant for experimental studies, current knowledge on the regulation of basophil migration is still limited. In the present study, we have adapted the GAFS assay to measure responses of human basophils to chemokines in a mixed population of PBMC. We have used this basophil-gated forward scatter (BGFS) assay to dissect the roles of CCRs, particularly CCR2 and CCR3, on basophils, and demonstrate the ability of these chemokine receptors to cooperate, suggesting a possible mechanism for the specific regulation of basophil recruitment in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

BSA, glucose, mouse IgG1 (clone MOPC 21) and IgG2a (clone UPC 10) control Abs, and FITC-conjugated anti-human HLA-DR mAb (clone HK14) were obtained from Sigma (Poole, U.K.). PBS (with and without Ca2+ and Mg2+) and HEPES were purchased from Life Technologies (Paisley, U.K.). FITC-conjugated F(ab')2 goat anti-mouse Ig and R-PE-labeled anti-human CD16 mAb were supplied by Dako (Cambridge, U.K.). FITC-conjugated anti-human CD14 was obtained from Autogen Bioclear (Calne, U.K.). Dextran T-500 and Percoll were purchased from Amersham Pharmacia Biotech (St. Albans, U.K.). Human recombinant eotaxin-2, RANTES, macrophage-inflammatory protein (MIP)-1{alpha}, MCP-1, MCP-2, MCP-3, MCP-4, and MDC were purchased from PeproTech (London, U.K.). R-PE-conjugated anti-human CDw123 mAb (clone 7G3), FACS-Flow, and Cellfix were obtained from Becton Dickinson (Mountain View, CA). Mouse anti-human CCR3 mAb LS-7B11 (isotype IgG2a) (43), CCR2 mAb LS-132.1D9 (isotype IgG2a), CCR1 mAb LS-2D4 (isotype IgG1), CCR4 mAbs LS-1G1 and LS-2B10 (44), and recombinant human eotaxin and synthetic eotaxin-3 were produced by LeukoSite/Millennium Pharmaceuticals (Cambridge, MA). Recombinant human IL-8 was a generous gift from J. White (SmithKline Beecham, King of Prussia, PA). Stock solutions of chemokines (10 µM) were prepared in PBS containing 0.1% BSA. Ten millimolar PBS containing 10 mM HEPES, 10 mM glucose, and 0.1% BSA (pH 7.2–7.4) was used as assay buffer. Staining buffer consisted of Ca2+/Mg2+-free PBS with 0.5% BSA and 0.1% azide.

Cell preparation

Volunteer blood donors were healthy normal subjects or atopics, as defined by a history of asthma, eczema, or hay fever, and symptoms on exposure to common aeroallergens, including pollens and house dust mites. The donors were taking no systemic medication. Blood was sampled according to a local ethics committee-approved protocol and was prepared as previously described (45). Briefly, platelet-rich plasma was removed by centrifugation of citrated whole blood, after which the erythrocytes were removed by dextran sedimentation. PMNL (containing neutrophils and eosinophils) were then separated from PBMC over a discontinuous plasma/Percoll gradient. Any erythrocyte contamination of the PBMC or PMNL pellet was removed by hypotonic shock lysis (46). To identify basophils, PBMC (1 x 108/ml) were stained with R-PE-labeled anti-human CDw123 mAb at 2 µg/ml and FITC-labeled anti-human HLA-DR mAb at 0.6 µg/ml for 10 min at room temperature and washed before use. Similarly, monocytes were distinguished by labeling of PBMC with FITC-conjugated anti-human CD14 mAb at 1/100 dilution (42).

In some experiments, purified basophils were prepared according to a modification of the technique of Tsang et al. (47). Citrated blood was centrifuged as above to remove platelets and plasma. The erythrocyte/buffy coat pellet was diluted with PBS and layered over Histopaque 1077 (Sigma) and centrifuged at 400 x g for 20 min. The resulting PBMC layer was incubated with the StemSep basophil purification Ab cocktail (StemCell Technologies, Vancouver, British Columbia, Canada) and colloidal magnetic particles, and unlabeled basophils were separated from other cell types by passage through a magnetic field according to the manufacturer’s instructions (StemCell Technologies). Cell purity was assessed by staining of an aliquot with anti-CDw123 and anti-HLA-DR mAbs and was found to be routinely >93% basophils, with a cell yield of 8.5 x 104 to 5 x 105/donor (from 80 ml of citrated blood). Any contaminating cells had forward light scatter (FSC)/side light scatter (SSC) characteristics of lymphocytes.

Measurement of leukocyte shape changes using flow cytometry

Agonist-induced shape change was measured using a modification of the protocol described previously (42). The cells were resuspended in assay buffer containing Ca2+ and Mg2+ and left for a short period (5–10 min at room temperature) to allow equilibration of intracellular and extracellular calcium. In experiments to investigate receptor usage by chemokines, this incubation period was also used to treat cells with buffer, anti-CCR2 mAb LS-132.1D9, or the anti-CCR3 mAb LS-7B11. Aliquots of cells (5 x 105 PMNL or PBMC) were then mixed with the respective agonist or buffer in 1.2-ml polypropylene cluster tubes (Costar, Cambridge, MA) in a final volume of 100 µl, and the tubes were placed in a 37°C shaking water bath for 4 min (basophils and eosinophils) or 10 min (monocytes). In experiments investigating the kinetics of basophil shape change responses, PBMC were incubated with buffer or eotaxin for the time points indicated before fixation and analysis. After these incubation times, which had been determined to be optimal in preliminary experiments (see Results) or in a previous study (42), the cells were transferred to an ice-water bath, and 250 µl of ice-cold fixative (1x Cellfix diluted 1/4 in FACS-Flow) was added to terminate the reaction and maintain the change in cell shape until analysis. The samples were then analyzed immediately on a FACScalibur flow cytometer (Becton Dickinson).

Basophils were identified as a single population of cells that stained positive for CDw123 (FL-2 fluorescence channel) and negative for HLA-DR (FL-1 channel; Fig. 1Go) (48, 49, 50). Anti-CD14-labeled monocytes were detected by their increased fluorescence on the FL-1 channel. Eosinophils were distinguished from neutrophils by their higher autofluorescence on the FL-2 channel (42). FSC, SSC, FL-1, and FL-2 data were acquired, and acquisition was terminated after 500 (eosinophils and basophils) or 1000 (monocytes) target events. Shape change of leukocytes was quantified as percentage of cells showing an increase in FSC above baseline (for detailed description, see Results).



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  		FIGURE 1. The BGFS assay. Flow cytometric detection of basophil shape change in a mixed PBMC population stained with anti-human HLA-DR and CDw123 mAbs. A, A representative dot plot of CDw123 (FL-2, 585 nm) vs HLA-DR (FL-1, 530 nm). Basophils are detected as highly positive for CDw123 but negative for HLA-DR, labeled as R1, and selectively displayed on a FSC vs SSC dot plot in B and C. In a sample of buffer-treated cells, a region (R2) was defined such that it contained 20% of basophils with the highest FSC values (see Materials and Methods and Results) (B). Upon stimulation with a chemokine, a concentration-dependent increase in FSC could be observed which was quantified as percent of cells in R2 (C). The current example shows a typical response to 1 nM eotaxin where R2 contains 76% of basophils.

 
Shape change of purified basophils was determined as above, using 1.7–10 x 103 cells per point, except that the purified basophils did not require labeling with anti-CDw123 and anti-HLA-DR mAbs.

Immunofluorescence flow cytometry

To investigate the expression of CCR1, CCR2, CCR3, and CCR4 on leukocytes, PMNL or PBMC were suspended in staining buffer at 5 x 106 cells/ml. Samples were incubated on ice for 30 min with 10 µg/ml rabbit IgG to saturate Fc{gamma} receptors and then with 10 µg/ml anti-CCR1 mAb LS-2D4, 10 µg/ml anti-CCR2 mAb LS-132.1D9, 3 µg/ml anti-CCR3 mAb LS-7B11, 10 µg/ml anti-CCR4 mAbs 1G1 or 2B10, or 0.6 µg/ml HLA-DR mAb. Isotype-matched mouse IgG1 and IgG2a mAbs were used to determine nonspecific binding. The cells were washed and resuspended in staining buffer containing FITC-conjugated F(ab')2 goat anti-mouse Ig on ice for 30 min and then washed again. To saturate free binding sites of the polyclonal goat anti-mouse Ab, the cells were incubated with a mixture of mouse IgG1 and IgG2a. Leukocytes were then counterstained with R-PE-conjugated anti-CD16 (to distinguish neutrophils from eosinophils in PMNL populations) or CDw123 (2 µg/ml, to identify basophils in PBMC populations) for 20 min. After a final wash, the samples were resuspended in fixative and held on ice until analysis.

To allow for a direct comparison of receptor expression, PBMC and PMNL were analyzed using the same FL-1 setting. Neutrophils were discriminated from eosinophils by their high expression of CD16. Monocytes were identified and gated on a FSC/SSC plot. Basophils were distinguished by their high CDw123 staining. Contaminating dendritic cells did not interfere with the measurement because they either showed the same level of staining (CCR3 and control mAbs) or were totally separated from basophils such that two distinct populations could be observed on a FL-1/FL-2 plot. The characteristics of these populations were also confirmed by anti-HLA-DR staining (0.6 µg/ml). In all instances, receptor density was recorded on the FL-1 channel and represented as mean fluorescence intensity, and nonspecific binding was determined by the relevant control Abs.

Statistical analysis

Data are shown as means ± SEM. Statistical evaluation was performed using the Wilcoxon signed rank test or ANOVA and Bonferroni’s post test where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Discrimination of basophils and measurement of shape change in PBMC using flow cytometry

Fig. 1GoA shows a fluorescence dot plot of PBMC stained with anti-HLA-DR-FITC (FL-1) and anti-CDw123-PE (FL-2). Basophils formed a well-separated population that stained highly positive for CDw123 but negative for HLA-DR and accounted for 0.5–3% of PBMC. Another, smaller population of CDw123 bright cells was also present among the PBMC, but separated clearly from basophils due to positive staining for HLA-DR. This population has previously been identified as monocyte-derived dendritic cells (49). When the CDw123+/HLA-DR–cells were gated (R1) and displayed selectively on a FSC vs SSC dot plot, they again formed a homogenous population (Fig. 1Go, B and C). Stimulation with chemokine caused an increase in forward scatter of basophils (Fig. 1GoC, 1 nM eotaxin), as compared with buffer (Fig. 1GoB). We quantified this effect as percentage of cells in a higher FSC region, defined as a region that contained only 20% of cells with high FSC values in a control sample (Fig. 1GoB). When subsequent samples of PBMC from the same donor were exposed to a chemokine, e.g., eotaxin, a concentration-dependent increase in the number of basophils in the high forward scatter region could be observed (Figs. 1GoC and 3). At maximal stimulation, up to 80% of basophils were present in this region. Staining with the anti-CDw123 mAb, directed against the {alpha} subunit of the IL-3 receptor, did not interfere with the IL-3 receptor functionally because basophils labeled in this manner responded to pM concentrations of IL-3 (data not shown).

Comparison of chemokine receptor density on basophils, eosinophils, and monocytes

The expression levels of CCR1, CCR2, and CCR3 on leukocytes are shown in Fig. 2Go. Of these receptors, only very low levels of CCR1, were found on neutrophils. Basophils and eosinophils predominantly expressed CCR3, whereas CCR2 was dominant on monocytes. CCR1 was expressed on all the cell types investigated, its density being three times higher on monocytes than eosinophils and basophils. The levels of CCR1 and CCR3 were similar in eosinophils and basophils, but, importantly, basophils were also positive for CCR2, which was absent on eosinophils. Despite the original cloning of the CCR4 receptor from a basophilic cell line, we were unable to detect any expression of this receptor on peripheral blood basophils using specific anti-CCR4 mAbs (data not shown).



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  		FIGURE 2. Expression levels of CCR1, CCR2, and CCR3 on eosinophils, neutrophils, basophils, and monocytes. PMNL or PBMC populations were stained with anti-human CCR1 mAb LS-2D4, CCR2 mAb LS-132.1D9, CCR3 mAb LS-7B11, or isotype-matched control mAbs followed by FITC-conjugated goat anti-mouse Ig. Receptor expression was determined as an increase in fluorescence above the level observed with the relevant control mAb. Data are shown as mean ± SEM with n = 4, each using cells from different donors.

 
Basophil chemokine responsiveness

Basophil responsiveness to a range of chemokines was investigated using the BGFS assay. The order of potency among chemokines was MCP-4 (peak at < 1 nM) >= eotaxin-2 = eotaxin-3 >= eotaxin > MCP-1 > MIP-1{alpha} > RANTES = IL-8 (Figs. 3Go, A and B). In contrast, the CCR4 agonist MDC was completely ineffective up to 10 nM. Maximal responses were highest with eotaxin, eotaxin-2, eotaxin-3, and RANTES, followed by MCP-4 and MCP-1 (Fig. 3Go, A and B). The efficacy of MIP-1{alpha} and IL-8 was only ~50% that of eotaxin (Fig. 3GoB). Optimal responses to chemokines were seen after 4 min of stimulation. When basophils were exposed to eotaxin for longer times, the dose-response curves tended to become more bell shaped with an apparently reduced maximum efficacy (Fig. 3GoC).



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  		FIGURE 3. Basophil shape change in response to chemokines. Basophils in mixed PBMC populations were stimulated for 4 min at 37°C with buffer or chemokine, fixed, and their shape change was measured by flow cytometry. The curves show the concentration-dependent increase of basophils in the stimulated region R2 (see Fig. 1Go). The rank order of chemokines tested was MCP-4 >= eotaxin-2 = eotaxin-3 >= eotaxin > MCP-1 > MIP-1{alpha} > RANTES = IL-8 >> MDC, suggesting that CCR3 and CCR2 ligands are most effective at inducing basophil shape change followed by CCR1 and CXCR1/2, whereas CCR4 is ineffective. The potency of RANTES, MIP-1{alpha}, and IL-8 were significantly less than the potency of eotaxin and MCP-4 (p < 0.05). C, The dose-response curves of basophils to eotaxin when stimulated with chemokine for 4, 8, or 12 min at 37°C before fixation and analysis as described above. Data are shown as mean ± SEM with n = 4–9, each using cells from different donors.

 
Comparison of basophil, eosinophil and monocyte shape change in response to MCP-1, MCP-2, MCP-3, and MCP-4

Parallel experiments determined the responses of monocytes, basophils and eosinophils. MCP-1 was most potent on monocytes (Fig. 4GoA), showing a bell-shaped dose-response curve with a threshold concentration below 0.1 nM and responses that peaked at 1 nM. On basophils, MCP-1 was significantly less potent, but, after its peak at 3 nM, maximal effectiveness was maintained up to 100 nM (Fig. 4GoA). In contrast, MCP-1 did not elicit a response in eosinophils at 10 nM and below, but induced marked shape change at 30–100 nM, which corresponded to a 30- to 100-fold reduced sensitivity of eosinophils compared with basophils (Fig. 4GoA). MCP-2 was generally less potent than the other MCPs at inducing shape change in all three cell types, but, of the three, basophils were most sensitive to this chemokine (Fig. 4GoB). MCP-3 induced marked shape change in all three cell types, with eosinophils being only slightly less sensitive than monocytes and basophils (Fig. 4GoC).



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  		FIGURE 4. Comparison of the ability of the MCPs to induce shape change in eosinophils, basophils, and monocytes. Mixed PBMC populations (labeled with mAbs to identify basophils and monocytes) and PMNL populations (identifying eosinophils by autofluorescence) were stimulated in parallel experiments with MCP-1 (A), MCP-2 (B), MCP-3 (C), or MCP-4 (D) and the resulting leukocyte shape change was measured as described. Although MCP-1 was most potent in monocytes, basophils were most sensitive to MCP-2, MCP-3, and MCP-4. Monocytes and eosinophils exhibited desensitization to MCP-1 and MCP-4 at higher concentrations, respectively, whereas responses were maintained in basophils at supramaximal concentrations. Data are shown as mean ± SEM with n = 5–6, each using cells from different donors. *, p < 0.05 significant difference between basophil shape change responses and those of other cell types (eosinophil or monocytes).

 
MCP-4 caused little shape change in monocytes at concentrations <10 nM (Fig. 4GoD). In contrast, MCP-4 was more potent on basophils and eosinophils (Fig. 4GoD). However, the dose-response curve to MCP-4 in eosinophils was bell shaped, so that supramaximal concentrations of MCP-4 were ineffective, whereas there was a small trough followed by a further increase in efficacy at high concentrations of MCP-4 in basophils (Fig. 4GoD).

Involvement of CCR2 and CCR3 in eosinophil and basophil responses

The dose-response curve to eotaxin was shifted to the right by a factor of 100 for eosinophils and basophils in the presence of 16 µg/ml anti-CCR3 mAb LS-7B11, whereas the anti-CCR2 mAb LS-132.1D9 (Fig. 5Go, A and B) and the respective control Abs (data not shown) were without effect. Similarly, basophil responses to eotaxin-3 were abolished in the presence of anti-CCR3 (20.7 ± 1.3%, 70.8 ± 1.8%, and 23.7 ± 3.7% basophils responding to control, 10 nM eotaxin-3, and 10 nM eotaxin-3 plus anti-CCR3 mAb, respectively; n = 4).



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  		FIGURE 5. Receptor usage of chemokines in leukocytes. Mixed leukocyte populations were incubated with buffer, control Abs, or anti-chemokine receptor mAbs (anti-human CCR2 mAb LS-132.1D9 and CCR3 mAb LS-7B11) as indicated for 5 min at room temperature. The leukocyte populations were treated with chemokine (eotaxin (A and B) and MCP-1 (C and D)) or buffer as indicated for 4 min at 37°C, fixed, and their shape change (eosinophils (A and C) and basophils (B and D)) was measured as described. The dose-response curve to eotaxin was left unaltered by anti-CCR2 mAb but shifted to the right by anti-CCR3 mAb by a factor of 100 both in eosinophils (A) and basophils (B) as compared with buffer. Similarly, eosinophil responses to MCP-1 were abolished by anti-CCR3 mAb but left unaltered by anti-CCR2 mAb (C). In contrast, anti-CCR2 mAb shifted the dose-response curve to MCP-1 to the right by a factor of 100 in basophils, whereas anti-CCR3 mAb was ineffective against low concentrations but inhibited high concentrations of MCP-1 (D). Data are shown as mean ± SEM with n = 5–6. each using cells from different donors. *, p < 0.05 significant suppression of leukocyte chemokine responsiveness by pretreatment with anti-chemokine receptor mAbs.

 
Eosinophil shape change in response to high-dose MCP-1 was likewise inhibited by anti-CCR3 but was left unaltered by anti-CCR2 (Fig. 5GoC). In contrast, the MCP-1 dose-response curve in basophils was markedly shifted to the right by anti-CCR2, but was not modified by anti-CCR3 up to the maximal-effective concentration of MCP-1 (Fig. 5GoD). Basophil shape change to supramaximal concentrations of MCP-1, however, was clearly inhibited by anti-CCR3 and almost completely abolished by the combination of anti-CCR2 and anti-CCR3 (Fig. 5GoD). As a consequence, in the presence of anti-CCR2 mAb the MCP-1 dose response-curve in basophils was identical with that in eosinophils (Fig. 5Go, D and C), whereas in the presence of anti-CCR3 mAb it resembled the bell-shaped response-curve as seen in monocytes (Figs. 5GoD and 4A).

In basophils, responses to MCP-2 over the range 0–10 nM were not reduced by anti-CCR2 mAb but were almost completely abolished by anti-CCR3 mAb (Fig. 6GoA).



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  		FIGURE 6. Effect of the anti-human CCR2 mAb LS-132.1D9 and CCR3 mAb LS-7B11 on shape change responses to MCP-2 (A), MCP-3 (B), and MCP-4 (C) in basophils. Basophils were pretreated for 5 min at room temperature with control or blocking anti-chemokine receptor mAbs before stimulation with chemokine at 37°C and measurement of shape change was as previously described. MCP-2 responses were left unaltered by anti-CCR2 mAb LS-132.1D9 but markedly inhibited by anti-CCR3 mAb LS-7B11 (A). Shape change induced by MCP-3 was largely unaffected by either mAb, but attenuated by a combination of anti-CCR2 mAb plus anti-CCR3 mAb (B). MCP-4 responses were shifted to the right by a factor of 100 by anti-CCR3 mAb, whereas anti-CCR2 mAb only significantly blocked high concentrations of MCP-4 (D). Data are shown as mean ± SEM with n = 5–8, each using cells from different donors. *, p < 0.05 significant suppression of basophil chemokine responsiveness by pretreatment with anti-chemokine receptor mAbs.

 
Anti-CCR2 or anti-CCR3, alone, had a minimal effect on basophil responses to MCP-3, whereas the combination of anti-CCR2 plus anti-CCR3 markedly depressed these responses (Fig. 6GoB).

Anti-CCR3 mAb inhibited responses to low concentrations of MCP-4 in basophils, but was ineffective against high concentrations (Fig. 6GoC). Conversely, anti-CCR2 mAb had a minimal inhibitory effect on responses to low MCP-4 concentrations, but significantly reduced the response to high concentrations of MCP-4 (Fig. 6GoC). The combination of anti-CCR2 plus anti-CCR3 was slightly more effective that any of the mAbs alone, but still could not abolish responses to high concentrations of MCP-4.

Effect of CCR2 stimulation on CCR3 responsiveness

To investigate whether stimulation of CCR2 could enhance the response of basophils to CCR3 agonists, basophils were stimulated with increasing concentrations of eotaxin in the absence or presence of a low concentration of MCP-1 (0.3 nM), with both chemokines being added to the cells simultaneously. Stimulation with 0.3 nM MCP-1 caused a small basophil response. Stimulation of basophils with 0.3 nM MCP-1 in combination with increasing concentrations of eotaxin resulted in a left-shift of the dose-response curve to eotaxin, such that the threshold concentration and EC50 for eotaxin were decreased by a factor of 3 compared with the response to eotaxin alone (Fig. 7Go). Maximal responses to eotaxin were not affected by MCP-1, indicating that only sensitivity to, but not effectiveness of, CCR3 activation was enhanced (Fig. 7Go).



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  		FIGURE 7. Synergistic effect of simultaneous activation of CCR2 and CCR3 in basophils. Basophils in mixed PBMC populations were stimulated with eotaxin, either alone or in combination with a fixed concentration of MCP-1 (0.3 nM). A low level of CCR2 activation by 0.3 nM MCP-1 enhanced the responsiveness of basophils to CCR3 stimulation, and shifted the concentration-response curve to eotaxin leftward by a factor of three. *, The point at which significant enhancement of eotaxin signaling by coincubation with MCP-1 was first apparent (p < 0.05). Data are shown as mean ± SEM with n = 5, each using cells from different donors.

 
Responses of purified basophils

To investigate whether the patterns of responsiveness observed above were directly due to agonist stimulation of basophils or dependent upon indirect stimulation of basophils via other cell types such as monocytes, experiments were undertaken using highly purified basophils prepared by negative magnetic selection. Fig. 8Go shows that purified basophils exhibited similar dose-response relationships to the basophils in mixed cell populations above, after stimulation with eotaxin, MCP-1, or MCP-3. In two of these experiments, basophil responses to eotaxin, MCP-1, -2, -3, -4, and MIP-1{alpha} were compared in purified and mixed cell populations from the same donor and found to be identical (data not shown). These highly purified basophils did not require labeling with anti-CDw123 and anti-HLA-DR, thus also demonstrating that these labeling steps did not affect basophil responsiveness. Fig. 8Go also shows that, in purified basophil populations, similar patterns of chemokine receptor usage by eotaxin (CCR3 only), MCP-1 (sequential dependence upon CCR2 and CCR3), and MCP-3 (dependence on more than one receptor) were observed to those described above.



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  		FIGURE 8. Chemokine responsiveness of purified basophils. Pure basophil populations were prepared by negative magnetic selection, and their shape change responses to chemokines were measured as for the mixed cell populations above (except that labeling with anti-CDw123/HLA-DR to identify basophils was not required). A, The responses of basophils to eotaxin, MCP-1, and MCP-3. B, The responses of purified basophils to fixed concentrations of these chemokines after treatment with a control IgG Ab, or blocking mAbs to CCR2 or CCR3 alone or in combination, as per Figs. 5Go and 6Go above. Significant inhibition of responses by pretreatment with blocking Abs is indicated by *, p < 0.05 and **, p < 0.01. Data shown are the mean ± SEM of cells from four different donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using a novel assay of basophil responses, the BGFS assay, we have shown here that basophils integrate chemokine signals in a manner that has not been described previously. These findings may provide mechanisms that could account for selective basophil recruitment in vivo.

Previously, we have used a flow cytometric assay of leukocyte shape change (the GAFS assay) to measure responses of eosinophils, neutrophils, and monocytes to chemokines in vitro (42). This sensitive assay is capable of measuring chemokine responsiveness of individual cell types in mixed cell populations. Basophils have been historically difficult to study because of their low incidence in the circulation. We therefore modified the GAFS assay to measure basophil responses in mixed PBMC preparations, without the need for further leukocyte purification. We identified basophils using an anti-IL-3R{alpha} (CDw123) mAb (staining predominantly basophils and monocyte-derived dendritic cells), in conjunction with an anti-HLA-DR mAb (which labeled the dendritic cells and distinguished them from basophils).

We found that the kinetics of basophil chemokine responsiveness were similar to those of eosinophils, being detectable within 1 min and reaching a maximum response by 4 min. After longer periods of stimulation, the dose-response curves to eotaxin tended to become more bell shaped with an apparent reduction in efficacy (Fig. 3Go). Eosinophil responses to MCP-4 are similarly time dependent, although, in contrast to basophils, eosinophil responses to eotaxin are preserved over time (42). Basophil shape change was induced by many chemokines, with rank order of potency of MCP-4 >= eotaxin-2 = eotaxin-3 >= eotaxin > MCP-1 = MCP-3 > MIP-1{alpha} > RANTES = MCP-2 = IL-8. Similar patterns of chemokine responsiveness were observed in highly purified basophil populations, demonstrating that these rapid responses to chemokines were not dependent on other cell types (e.g., monocytes), nor were they modulated by the anti-CDw123/HLA-DR labeling step, which was not required for the purified cells.

The major human eosinophil chemokine receptor is CCR3, with lower levels of CCR1 expression (42, 51). As we show here, monocytes principally express CCR2, relatively high levels of CCR1, and no detectable CCR3. Basophils show a different pattern of CC chemokine receptor expression, with high levels of CCR3, intermediate levels of CCR2, and low levels of CCR1. The ability of the various chemokines tested to cause basophil shape change reflected these patterns of CCR expression. MCP-4, eotaxin, and eotaxin-2 were the most potent stimulators of basophil shape change. A recently described eotaxin functional homologue, eotaxin-3, also showed similar potency to eotaxin and eotaxin-2. Basophil shape change induced by eotaxin-3 was completely inhibited by a blocking anti-CCR3 Ab.

MCP-1 and MIP-1{alpha} were less potent inducers of basophil shape change than eotaxin, in keeping with the lower levels of expression of their principle receptors, CCR2 and CCR1, respectively.

IL-8 has been shown previously to cause basophil histamine release through specific IL-8 receptors (52), and also caused shape change in basophils. Both IL-8 and MIP-1{alpha} were less efficacious than eotaxin, MCP-1, and MCP-4, causing responses in <50% of the cells at the concentrations tested. The FSC changes of basophils in response to chemokine stimulation had some appearances of being "all or nothing," with discrete movement of increasing numbers of cells into the higher FSC region upon exposure to increasing concentrations of chemokine (Fig. 1Go). Thus, the lower efficacy of MIP-1{alpha} may be due to the lower level of basophil CCR1 expression (Fig. 2Go), with some cells not expressing sufficient levels of coupled receptors to be able to respond to ligand. We have previously found eosinophil shape change induced by chemokines to be pertussis toxin sensitive (I. Sabroe, unpublished data). Other studies have shown basophil responses to chemokines including RANTES, MCP-1, and IL-8 to be similarly sensitive to pertussis toxin (22, 52, 53, 54).

MDC, whose receptor CCR4 was originally described in a basophil-derived cell line (55), failed to elicit any response, and we were unable to detect expression of CCR4 on peripheral blood basophils using specific mAbs. Curiously, at higher concentrations, MDC appeared to cause a reduction in the baseline numbers of basophils that were in the high FSC gate, an effect not seen with other chemokines. This may reflect basophil responses mediated by another MDC receptor, the existence of which has been speculated in other work (56).

The MCP group of chemokines, comprising MCP-1, -2, -3, and -4, all show varying selectivities for CCR2 and CCR3. We postulated that the differing expression patterns of these receptors on eosinophils (CCR3 but no CCR2), monocytes (CCR2 but no CCR3), and basophils (both receptors) may result in leukocyte-specific patterns of chemokine responsiveness. Our data indicated that basophils respond differently to MCPs, when compared with eosinophils and monocytes, and uncovered two patterns of integrated basophil chemokine responses.

Sequential receptor activation

MCP-1 induced a bell-shaped dose-response curve in monocytes (Fig. 4GoA), suggesting desensitization of responses at higher concentrations, and this signaling was completely blocked by an anti-CCR2 mAb (data not shown). Eosinophils only responded to MCP-1 at high concentrations, in a CCR3-dependent manner (Fig. 5GoC). Responses of eosinophils to high concentrations of MCP-1 using assays of intracellular calcium flux were previously observed when eotaxin was first described (57). Consistent with this, one study showed that MCP-1 bound to CCR3, but that the affinity of MCP-1 for CCR3 was much lower than that of eotaxin (51). In both that study and work of other groups, CCR3 transfectants were not responsive to MCP-1 (51, 58), suggesting subtle differences in the coupling of these receptors in transfectants compared with native eosinophils. Basophils, both purified and in mixed PBMC populations, showed responses to MCP-1 across a broad concentration range, with signaling being mediated exclusively by CCR2 at low concentrations and predominantly by CCR3 at high concentrations (Figs. 5GoD and 8). These data suggest that in the presence of high concentrations of MCP-1 in vivo, CCR2 may be desensitized, and basophil and eosinophil recruitment may be favored over monocyte recruitment.

Leukocyte responses to MCP-4 showed an inverse pattern. At low concentrations, basophil responses to MCP-4, like eosinophil responses (42), were exclusively mediated via CCR3 (Fig. 6GoC), but, at high MCP-4 concentrations, eosinophils showed a bell-shaped dose-response curve that was not seen in basophils (Fig. 4GoD). In eosinophils, this bell-shaped dose-response curve of MCP-4 signaling is different to the sigmoidal dose-response curve of eotaxin and suggests different mechanisms of MCP-4 and eotaxin signaling via CCR3 (42). The maintained responses of basophils to higher concentrations of MCP-4 correlated with an increasing monocyte response to MCP-4, likely to be mediated by CCR2. The combination of anti-CCR3 and anti-CCR2 mAbs was unable to completely block the responses of basophils to high concentrations of MCP-4. These data are consistent with either the involvement of a third receptor (e.g., CCR1) in MCP-4 signaling, or more likely reflect the inability of the anti-CCR3 Ab to fully block the activity of a high-affinity ligand at high concentration, consistent with the data shown in Fig. 5GoA. Thus, where high concentrations of MCP-4 are present in vivo, CCR3 may be desensitized and basophil and monocyte recruitment may be favored over eosinophils. These data are consistent with recent observations from Ying et al. (32), who demonstrated that basophil recruitment in the late phase allergic response in human skin, although not clearly correlated with expression of a single chemokine, paralleled more closely MCP-4 kinetics. In contrast, eosinophil recruitment occurred earlier (peaking at 6 h) compared with basophils (peaking at 24 h), and correlated well with eotaxin generation.

Both MCP-1 (Figs. 4GoA and 5D) and MCP-4 (Figs. 4GoD and 6C) appear to exhibit two optimal concentrations for shape change of basophils, with an early peak response at 1 nM, a small dip in response at 10 nM corresponding with a switch in receptor usage, and a subsequent further response at 100 nM. At these higher concentrations of chemokines, almost all of the basophils have changed shape and thus further cooperation between CCR2 and CCR3 cannot be demonstrated. It is interesting to note that a recent paper described two optimal concentrations of NAP-2 in neutrophil chemotaxis, mediated by the sequential activation of CXCR2 and CXCR1 (59), presumably through regulation of receptor desensitization and expression (46).

Cooperation of chemokine receptor signaling

MCP-2 proved to be a weak stimulator of leukocyte shape change, but was a more potent stimulator of basophil than eosinophil responses (Fig. 4GoB). Blocking mAbs demonstrated that basophil responses to MCP-2 were predominantly mediated by CCR3 (Fig. 6GoA). However, an element of cooperative receptor signaling between basophil CCR3 and another receptor not present on eosinophils may explain the higher sensitivity of basophils to MCP-2. In keeping with this, another study has shown that MCP-2 interacts with both the receptors for RANTES and MCP-1 on basophils (23). To investigate this further, we measured basophil CCR3-mediated responses to eotaxin in the presence of low levels of CCR2 stimulation by MCP-1. Basophil responses to eotaxin were significantly enhanced in the presence of a low concentration of MCP-1, suggesting that cooperation between CCR2 and CCR3 may be important in responses of basophils in vivo.

An alternative pattern of receptor cooperativity was seen in basophil responses to MCP-3. This chemokine showed potent stimulation both of eosinophils and monocytes. As therefore anticipated, MCP-3 was a potent stimulator of basophil shape change. Blockade of basophil CCR2 or CCR3 alone had no significant effect on the response to MCP-3, whereas both mAbs in combination caused a significant reduction in basophil MCP-3 responses (Fig. 6Go). The failure of these Abs to block MCP-3 responses completely when employed together suggests that a component of basophil MCP-3 responsiveness is mediated by a third receptor, most likely CCR1. These data are in keeping with previous observations that basophil calcium responses to MCP-3 could only be desensitized by RANTES and MCP-1 in combination, but not by either of these chemokines singly (24). However, our data suggest that CCR2 and CCR3 may cooperate to induce basophil activation when stimulated together, so it was surprising that no reduction in MCP-3 response occurred with these mAbs used singly. Thus, these data may be explained by maximal cooperativity being reached when any two of CCR1, 2, and 3 are stimulated, but we were unable to test this hypothesis further because the available anti-CCR1 mAb does not cause effective receptor blockade in the shape change assay (42). Combined blockade of CCR2 and CCR3 was less effective at inhibiting MCP-3-induced shape change in purified basophil populations (Fig. 8Go), which could be due to the functional up-regulation of CCR1 during the additional handling steps required to produce these highly purified cells. An alternative explanation might rest in receptor heterodimerization. In opioid receptors, heterodimerization of two different G protein-coupled receptors results in altered patterns of ligand binding and receptor signaling (60). The CCR2 G protein-coupled receptor forms homodimers that regulate responsiveness to MCP-1 (61) and thus it is possible that a component of the basophil response to MCP-3 is regulated through the formation of CC chemokine receptor heterodimers.

Taken together, our data demonstrate differential responses to CC chemokines between basophils and eosinophils, regulated by induction of sequential or cooperative receptor signaling. Eosinophils dominate helminth responses (1) and allergen late phase responses (6, 32), whereas basophils dominate cutaneous basophil reactions (8, 11, 12, 13, 14, 15) and tick (3), as well as contact dermatitis responses (9, 10). Differential basophil signaling via sequential and cooperatively acting chemokines could be a mechanism explaining these in vivo differences.


   Acknowledgments
 
We thank Dr. Peter Jose and Professor Bernhard A. Peskar for helpful discussions.


   Footnotes
 
1 This work was supported by the Austrian Science Foundation (J1700-MED) (to A. Heinemann), the Wellcome Trust (to A. Hartnell), the National Asthma Campaign (to V.E.L.S. and T.J.W.), the National Institutes of Health (AI-43371 and HL-56389) (to P.W.A.), the Welfide Corporation (to K.M.), and Imperial College School of Medicine (to I.S.). Back

2 Current address: Department of Experimental and Clinical Pharmacology, Karl-Franzens-University, Universitätsplatz 4, A-8010 Graz, Austria. Back

3 Current address: Section of Allergy and Clinical Immunology, Yale University School of Medicine, New Haven, CT 06510. Back

4 Address correspondence and reprint requests to Dr. Ian Sabroe, Leukocyte Biology Section, Biomedical Sciences Division, Imperial College School of Medicine, South Kensington, London SW7 2AZ, U.K. Back

5 Abbreviations used in this paper: MCP, monocyte chemotactic protein; MIP-1{alpha}, macrophage inflammatory protein-1{alpha}; PMNL, polymorphonuclear leukocytes (comprising eosinophils and neutrophils); BGFS, basophil-gated forward scatter assay; FSC, forward scatter; SSC, side scatter. Back

Received for publication May 9, 2000. Accepted for publication September 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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