The Journal of Allergy and Clinical Immunology
Volume 111, Issue 1 , Pages 97-105, January 2003

Receptor internalization is required for eotaxin-induced responses in human eosinophils☆☆☆

Division of Allergy and Immunology, Department of Pediatrics, Children's Hospital Medical Center, Cincinnati. Cincinnati, Ohio

Received 9 August 2002; received in revised form 17 September 2002; accepted 25 September 2002.

Article Outline

Abstract 

Background: CC chemokine receptor 3 (CCR3) is a major chemokine receptor involved in regulating eosinophil trafficking, and therefore the elucidation of ligand-induced CCR3 events has important implications in understanding the biologic and pathologic properties of eosinophils. After ligand binding to CCR3, cellular signals include stimulatory (ie, calcium mobilization, actin polymerization, shape change, and chemotaxis) and inhibitory (ie, desensitization of the receptor) events. We have previously demonstrated that CCR3 undergoes rapid and prolonged ligand-induced internalization. Objective: Here we explore the role of internalization in downstream cellular processes, including shape change, actin polymerization, calcium mobilization, and desensitization. Methods: Peripheral blood–derived human eosinophils were pretreated with 2 mechanistically distinct inhibitors of internalization, sucrose and phenylarsine oxide, and functional responses were monitored. Results: We first demonstrate that ligand-induced internalization is required for chemokine-induced eosinophil shape change. To define which signaling components upstream of eosinophil shape change required internalization, we next studied the role of internalization in calcium mobilization and actin polymerization. Sucrose and phenylarsine oxide pretreatment inhibited actin polymerization, implicating receptor internalization in this early response. In contrast, calcium mobilization was not inhibited by blockade of internalization. Finally, we were interested in testing the role of internalization in receptor desensitization. We first demonstrated that preincubation with eotaxin induced a dose-dependent desensitization in eotaxin-induced eosinophil transepithelial migration. However, this phenomenon was not inhibited by blockade of internalization. Conclusion: These results establish that CCR3 internalization is critically involved in select eosinophil functional responses (ie, cellular shape change and actin polymerization) but not desensitization and calcium mobilization. (J Allergy Clin Immunol 2003;111:97-105.)

Keywords:  Allergy, chemokines, eosinophils

Abbreviations:  CCR3 , CC chemokine receptor 3, FSC , Forward scatter, GPCR , G protein-coupled receptor, MCP , Monocyte chemoattractant protein, PAO , Phenylarsine oxide

 

CC chemokine receptor 3 (CCR3), a 7-transmembrane-spanning G protein–coupled genetically polymorphic receptor, is a major chemokine receptor expressed on allergic inflammatory cells, especially eosinophils.1, 2, 3, 4, 5, 6 CCR3 binds multiple ligands, including RANTES (CCL51), monocyte chemoattractant protein [MCP] 3 (CCL7), MCP-2 (CCL8), MCP-4 (CCL13), and macrophage inflammatory protein 5 (CCL15, HCC-2, and leukotactin), as well as the CCR3-exclusive ligands designated eotaxin 1, 2, and 3 (CCL11, CCL24, and CCL26, respectively). Sites of allergic inflammation are characterized by increased expression of CCR3 ligands and accumulation of CCR3+ inflammatory cells (eg, eosinophils, basophils, and mast cells).1, 2, 3, 4, 5, 6 Thus understanding the biochemical events associated with CCR3 signaling is of critical importance.

Binding of chemotactic factors to G protein–coupled receptors (GPCRs) on the cell surface of granulocytes induces a variety of responses, including secondary granule release, cellular migration, and phagocytosis.7, 8 After chemokine exposure, stimulation of GPCRs causes a transient increase in intracellular calcium that activates several signaling pathways, including actin polymerization in eosinophils.9 With the activation of the intracellular motile apparatus, cells undergo shape change, followed by acquisition of a polarized morphology and subsequent migration. After activation, chemoattractant receptors undergo desensitization and recycling. These processes can serve as important mechanisms by which leukocytes maintain their ability to sense a chemoattractant gradient during an inflammatory response.10

After engagement of ligand, GPCRs can be internalized.11, 12 We have previously shown that CCR3 undergoes rapid and prolonged ligand-induced internalization into the early endosome compartment, as demonstrated by colocalization with the transferrin receptor with confocal microscopy.13 Subsequently, the receptor traffics to the cell surface membrane; alternatively, the receptor can be targeted for degradation through a lysosomal pathway. The functional importance of CCR3 internalization has not been examined. Previous studies have shown that the specific function of GPCR internalization varies considerably between distinct receptors. For example, internalization has been implicated for resensitization and activation of the mitogen-activated protein kinase pathway but not for desensitization of the β2-adrenergic receptor.14, 15, 16, 17, 18 In contrast, internalization of CXCR2 is required for chemotaxis, but not for mitogen-activated protein kinase activation.19 This highlights the importance of determining the role of internalization for each GPCR.

In this report our goal was to determine the importance of plasma membrane receptor internalization for CCR3-mediated events in human eosinophils. We demonstrate that internalization is critical for eosinophil shape change and actin polymerization after activation with a CCR3 ligand but is not required for receptor desensitization and calcium mobilization. These results establish a novel functional role for receptor internalization in chemokine-signaling events in human eosinophils.

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Methods 

Eosinophil purification 

Eosinophils were purified from healthy or mildly atopic volunteers by means of negative immunomagnetic selection on the basis of the method of Hansel et al.20 Briefly, granulocytes were isolated from heparin-anticoagulated whole blood by means of dextran sedimentation, Percoll centrifugation, and hypotonic lysis of red blood cells. Cells were resuspended in HBSS (Gibco BRL) with 2% FCS and incubated with 0.75 μL/106 cells anti-CD16–conjugated microbeads (MACS, Miltenyi Biotech Inc) for 30 minutes at 4°C. The cell suspension was then applied into a CS MACS column, and negative populations were collected through a magnetic field. The isolates routinely contained greater than 95% eosinophils with viability of greater than 95%, as assessed by means of Trypan blue exclusion. For shape-change experiments, the entire granulocyte fraction (comprised of 5%-30% eosinophils, depending on the donor) was used. Each experiment was repeated multiple times (indicated in the figure legends and text) with eosinophils from separate donors.

Intracellular [ca2+] measurement 

Cells (2 × 106/mL) were loaded with 5 μmol/L Fura-2 AM (Mo-lecular Probes) in HBSS with 1% FCS at 37°C in the dark. After 2 washes in flux buffer (145 mmol/L NaCl, 4 mmol/L KCl, 1 mmol/L NaHPO4, 0.8 mmol/L MgCl2, 1.8 mmol/L CaCl2, 25 mmol/L HEPES, and 22 mmol/L glucose, pH 7.6), cells were resuspended at 2 × 106 cells/mL and maintained on ice. Cells were subsequently stimulated with chemokine in a cuvette with a continuously stirring magnetic bar by using a RatioMaster fluorimeter (Photon Technology, Inc). Data were recorded as the relative ratio of fluorescence emitted at 510 nm after excitation at 340 and 380 nm (y axis) over time (x axis). The agent was added to cells for the last 5 minutes of loading to assess the role of phenylarsine oxide (PAO; Sigma). Cells were resuspended in flux buffer with 0.3 mol/L sucrose for 20 minutes at 35°C and stimulated as above to assess the effect of sucrose on calcium mobilization.

Transmigration assay 

Eosinophil transmigration through a monolayer of respiratory epithelial cells was performed as previously described.21, 22 Briefly, A549 cells (human respiratory epithelial cells, ATCC) were grown as monolayers in tissue-culture flasks in Dulbecco's modified Eagle's medium (Gibco BRL) supplemented with 10% FCS, penicillin, and streptomycin. Cell monolayers were trypsinized, centrifuged at low speed, and resuspended in fresh medium before culture on permeable filters (polycarbonate filters with 3-μm pores) in Transwell tissue-culture plates (Corning Costar Corp). Cells (1.5 × 105) in 100-μL volumes were grown to confluence on the upper surface of the filters for 2 days, and TNF-α (10 ng/mL) was added for the final 18 hours. Monolayer integrity was assessed by means of microscopic analysis with toluidine blue staining. On the day of the assay, 6 to 10 × 105 eosinophils in HBSS plus 0.5% BSA (low endotoxin, Sigma) were placed in the upper chamber, and the chemokine (in HBSS plus 0.5% BSA) was placed in the lower chamber. After incubation, cells in the lower chamber were combined with cells washed from the bottom of the transwell (with 0.5 mL of HBSS plus 10 mmol/L EDTA) and counted by means of flow cytometry, as previously described.23 Briefly, relative cell counts were obtained by acquiring events for 60 seconds. Alternatively, cells were counted with a hemocytometer.

Shape-change assay 

Eotaxin-induced eosinophil shape change was assessed by using gated autofluorescence/forward scatter (FSC).24 This assay uses differential autofluorescence to identify leukocyte types and changes in FSC to measure shape change in response to agonist. Briefly, granulocytes were isolated by means of dextran sedimentation, Percoll gradient centrifugation, and hypotonic red blood cell lysis and were preincubated for 30 minutes at 37°C in shape-change buffer (PBS with 0.9 mmol/L CaCl2, 0.5 mmol/L MgCl2, 10 mmol/L glucose, 10 mmol/L HEPES, and 0.1% BSA). After a wash in the same buffer, 1 × 106 cells in 200 μL were pretreated with sucrose (0.3 mol/L, 20 minutes), PAO (0.8 μmol/L, 5 minutes), or pervanadate (250 μmol/L, 5 minutes) when chemokine was added for an additional 6 minutes. Cells were incubated at 37°C in a shaking water bath, and the reaction was stopped by placing cells on ice and fixing with 600 μL of cold 4% paraformaldehyde in shape-change buffer. Cells were further analyzed by means of flow cytometry on the FACScalibur flow cytometer (Beckton Dickinson). Eosinophils were distinguished by their high autofluorescence in the FL2 channel (585 nm), and the FSC of 5000 cells was acquired. The percentage change in cell shape was calculated as follows:

100 × {[FSC (Chemokine) − FSC (Media)]/FSC (Media)}.

Pretreatment with inhibitors did not significantly alter basal FSC. The effectiveness of pervanadate to increase tyrosine phosphorylation of cellular proteins was assessed by means of Western blotting, as described previously.13 Briefly, whole-cell lysates were prepared from eosinophils, and 50 μg was separated by means of electrophoresis and transferred to a nitrocellulose membrane. The presence of phosphorylated tyrosines was assessed by using the PY20 antibody (Transduction Laboratories).

Actin polymerization assay 

Eotaxin-induced actin polymerization was assessed with nitrobenzoxadiazole-phallacidin (Molecular Probes). This fluorescence method has been shown to correlate well with biochemical measurements of F-actin.25, 26 Purified eosinophils were resuspended at 106 cells/mL in PBS and stimulated with eotaxin at 37°C for indicated amounts of time. After stimulation, cells were fixed in 3.7% formaldehyde for 60 minutes. Lysophosphatidylcholine (100 μg/mL, Sigma) and 3.3 × 10−7 mol/L nitrobenzoxadiazole-phallacidin were added to the cells and incubated for 1 hour in the dark. Cells were analyzed on a FACScalibur with a linear fluorescence channel (FL1), where the fluorescence is proportional to F-actin content. Relative F-actin content is expressed as the ratio of the mean channel fluorescence between eotaxin- and media-stimulated cells. The percentage of inhibition of the maximal response is calculated for relative F-actin content for sucrose- or PAO-treated cells compared with that seen in untreated cells.

Flow cytometry 

CCR3 internalization was monitored as previously reported.13 Briefly, cells (5 × 105) were washed with FACS buffer (2% BSA and 0.1% Na azide in PBS) and incubated with 0.5 μg of anti-hCCR-3 antibody (clone 7B11, kindly provided by Dr Paul Ponath, LeukoSite, Cambridge, Mass, or clone 61828.111, R&D Systems) or the respective isotype-matched control (Pharmingen) for 30 minutes at 4°C. The binding of both primary antibodies was shown not to be inhibited by ligand binding to the receptor (data not shown).13 After 2 washes in FACS buffer, cells were incubated with 0.5 μg of FITC-conjugated isotype-specific secondary antibody (Pharmingen) for 30 minutes at 4°C in the dark. After 2 washes, labeled cells were subjected to flow cytometry on a FACScan flow cytometer and analyzed by using the CELLQuest software (Becton Dickinson). Inhibition of internalization of surface CCR3 was assayed by incubating cells at 37°C for indicated lengths of time with PAO and sucrose, followed by eotaxin (Peprotech). After chemokine exposure, cells were immediately placed on ice, washed, and stained as described above. In select experiments cells were treated with biotinylated eotaxin (R&D Systems), and its presence on the cell surface was assessed by staining with streptavidin-FITC. As a negative control, biotinylated soybean trypsin inhibitor was used.

Statistical analysis 

Results are expressed as means and SDs of replicate samples, and statistical significance was calculated by using the Student t test.

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Results 

Role of internalization in eotaxin-induced eosinophil shape change 

We have previously shown that CCR3 undergoes rapid ligand-induced internalization.13, 21 The functional importance of this receptor-trafficking event is largely unknown. We hypothesized that internalization is required for downstream functional responses after CCR3 stimulation. Initially, we examined the role of internalization in chemokine-induced eosinophil shape change.24 In this assay granulocytes were exposed to eotaxin for a brief period of time, and the change in shape of eosinophils (gated as highly autofluorescent cells) was monitored by FSC on a flow cytometer (Fig 1, A and B ).

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  • Fig. 1. 

    Internalization is required for eosinophil shape change induced by eotaxin. In A eosinophils were distinguished in the granulocyte population by their high autofluorescence in FL2. Cells in gate R2 were monitored for changes in FSC in response to chemokine (B) . Cells treated with media alone are shown as the shaded histogram , and cells treated with 10 ng/mL eotaxin (E) are shown as the open histogram . In C and D granulocytes were pretreated with PAO (squares ; Fig 1, C ), sucrose (squares ; Fig 1, D ), or media alone (circles) , followed by treatment with the indicated doses of eotaxin. Fixed cells were analyzed by means of flow cytometry for change in FSC indicative of cell shape change. Percentage change is calculated relative to the FSC of cells not treated with chemokine. The viability of the cells was greater than 95%, as determined by means of Trypan blue staining, and cells treated with sucrose and PAO are still functionally active in that they mobilize calcium (Fig 3). In E and F the effect of PAO and sucrose pretreatment on ligand-induced internalization is shown. Eosinophils were pretreated with 0.8 μmol/L PAO for 5 minutes (Fig 1, E ) or 0.3 mol/L sucrose for 20 minutes (S ; Fig 1, F ), and 100 ng/mL eotaxin (E) was added for an additional 15 minutes. FACS analysis was performed to assess surface expression of CCR3 on cells. The shaded histogram is the isotype-matched control, and the open histogram represents cells without any pretreatment. The dashed line represents cells pretreated with eotaxin alone, and the dotted line represents cells treated with eotaxin in the presence of PAO or sucrose, respectively. Representative experiments of 4 similar experiments are shown.

As shown in Fig 1, C and D (media), eotaxin induced a reproducible increase in eosinophil shape change; a significant effect was seen at 1 ng/mL, and a plateau was seen between 10 and 100 ng/mL. To assess the role of receptor internalization in this functional process, we used 2 well-established inhibitors of internalization: PAO and hypertonic sucrose. PAO and sucrose both inhibit the clathrin-coated pit pathway involved in receptor internalization, although by different mechanisms.14, 27, 28, 29, 30, 31, 32, 33 Pretreatment of eosinophils with either of the inhibitors blocked eotaxin-induced CCR3 internalization (Fig 1, E and F ). PAO was effective at a range of doses from 0.8 to 80 μmol/L; we chose to use the lowest effective dose (0.8 μmol/L) for subsequent experiments. Sucrose was used at 0.3 mol/L because this dose was both effective (Fig 1, F ) and nontoxic to cells (as assessed by Trypan blue staining). When cells were pretreated with PAO or sucrose, basal shape change was not affected (data not shown); however, eotaxin-induced shape change was inhibited (Fig 1, C and D ). After pooling experiments from multiple donors, PAO and sucrose inhibited shape change by 116% ± 15% (P = .009) and 82% ± 31% (P = .02), respectively. Cells were also treated with the tyrosine phosphatase inhibitor pervanadate (250 μmol/L) before and during eotaxin treatment to rule out the possibility that the action of PAO was related to its ability to inhibit tyrosine phosphatases. However, this did not inhibit eosinophil shape change, although it did increase the tyrosine phosphorylation of cellular proteins (data not shown). Taken together, these data demonstrate that receptor internalization is required for eotaxin-induced shape change in eosinophils.

Role of internalization in actin polymerization 

We were next interested in deciphering which cellular responses that precede cellular shape change might be affected by internalization. Previous studies in neutrophils have shown that cell shape change correlates very closely with total F-actin content change during actin polymerization.34, 35 We thus hypothesized that internalization would be required for chemokine-induced actin polymerization and, consequently, cell shape change. As seen in Fig 2, eotaxin (10 ng/mL) induces a rapid increase in the F-actin content in eosinophils.

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  • Fig. 2. 

    The effect of inhibiting receptor internalization on chemokine-induced actin polymerization. Eosinophils were pretreated with 0.8 μmol/L PAO for 5 minutes (squares) , 0.3 mol/L sucrose for 20 minutes (triangles) , or media alone (circles) and then treated with eotaxin (10 ng/mL) for the indicated period of time. Cells were fixed and stained with nitrobenzoxadiazole-phallacidin. Relative F-actin content is expressed as the ratio of the mean channel fluorescence between eotaxin- and media-stimulated cells. A representative experiment of 4 similar experiments is shown.

Eosinophils were pretreated with PAO and sucrose to assess the role of internalization in chemokine-induced actin polymerization. In Fig 2, a representative experiment, inhibition of internalization is shown to reduce eotaxin-induced actin polymerization. Pooling experiments from multiple donors, PAO and sucrose inhibited actin polymerization by 84% ± 52% (n = 6 experiments with 3 separate donors, P = .01) and 72% ± 16% (n = 4 experiments with 2 separate donors, P = .03), respectively. Thus internalization is critically involved in chemokine-induced actin polymerization.

Role of ccr3 internalization in eotaxin-induced calcium mobilization 

To determine the role of CCR3 internalization in another rapid functional response, we assessed the increase of intracellular calcium in response to eotaxin. Previous studies have shown that actin polymerization in human eosinophils depends on intracellular calcium mobilization.9 As seen in Fig 3 (top panels), eosinophils responded with a robust calcium increase on stimulation with eotaxin (250 ng/mL).

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  • Fig. 3. 

    Receptor internalization is not required for calcium mobilization. Calcium mobilization in response to 250 ng/mL eotaxin was monitored in Fura-2 AM–loaded eosinophils (A and B , top panels ). Cells were also pretreated with 0.8 μmol/L PAO for 5 minutes (Fig 3, A , bottom panel ) or 0.3 mol/L sucrose for 20 minutes (Fig 3, B , bottom panel ). Representative experiments are shown (n = 4).

When the cells were pretreated with PAO for 5 minutes, they still responded to eotaxin with a rapid increase in intracellular calcium (Fig 3, A , bottom panel). Similarly, cells pretreated with sucrose had a normal calcium mobilization when treated with eotaxin (Fig 3, B , bottom panel). Over a range of doses (10-250 ng/mL) of eotaxin, there was no significant decrease in calcium mobilization after sucrose or PAO pretreatment (data not shown). These data indicate that internalization is not required for eotaxin-induced calcium mobilization.

Role of internalization in receptor desensitization 

We were next interested in testing the role of ligand-induced internalization in an inhibitory response, such as receptor desensitization. We first demonstrated that eotaxin induced cellular desensitization in a functional response. Eosinophils were incubated with or without eotaxin (100 and 500 ng/mL) for 15 minutes, and after removal of the ligand, transmigration through a monolayer of epithelial cells in response to eotaxin (100 ng/mL) in the lower chamber was monitored after 1 hour. As shown in Fig 4, A , eotaxin pretreatment inhibited subsequent CCR3-mediated transmigration in a dose-dependent manner, indicating functional desensitization.

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  • Fig. 4. 

    Effect of inhibiting receptor internalization on CCR3 desensitization. In A cells were pretreated with the indicated amounts of eotaxin for 15 minutes. Transmigration through an epithelial monolayer for 1 hour in response to 100 ng/mL eotaxin after removal of unbound ligand is shown. The line represents baseline transmigration to media alone. The data are expressed as means ± SD of duplicate measurements, and a representative experiment is shown (n = 3). In B eosinophils were pretreated with 100 ng/mL eotaxin, washed to remove ligand, and then allowed to transmigrate in response to the indicated amount of eotaxin. The solid line represents control cells, and the dashed line represents eotaxin-pretreated cells. In C and D cells were pretreated with 0.3 mol/L sucrose for 20 minutes and 500 ng/mL eotaxin for additional 15 minutes as noted. After ligand removal, transmigration in response to media alone (gray bar) or 100 ng/mL eotaxin (black bar) for 15 minutes is shown (C) . The data are expressed as means ± SD of duplicate measurements, and a representative experiment (n = 2) is shown. In D , after ligand removal, cells were allowed to rest for 15 minutes, and CCR3 expression was analyzed by means of flow cytometry. The shaded histogram is the isotype-matched control, and the open histogram represents cells without any pretreatment. The dashed line represents cells pretreated with eotaxin alone (E) , and the dotted line represents cells treated with eotaxin in the presence of sucrose (E + S) .

For example, eosinophils pretreated with 100 and 500 ng/mL eotaxin exhibited 42.8% (n = 2) and 68.4% (n = 3) inhibition in transmigration, respectively. The desensitization was also examined by demonstrating reduced responsiveness of eotaxin-pretreated eosinophils to different eotaxin concentration gradients during the transmigration assay (Fig 4, B ). Nonpretreated cells displayed a typical bell-shaped curve, with peak transmigration seen at 100 ng/mL eotaxin and high-dose inhibition visible at 500 ng/mL. Eotaxin-pretreated cells also displayed a bell-shaped curve, with the maximal chemotactic response at 100 ng/mL. However, this response was significantly reduced in comparison with that seen in nonpretreated cells. Together, these results indicate that pretreatment of eosinophils with eotaxin effectively desensitizes subsequent CCR3-mediated responses.

Eosinophils were pretreated with eotaxin in the presence or absence of sucrose to test the hypothesis that internalization was required for desensitization (Fig 4, C ). After removal of sucrose and eotaxin, cells transmigrated in response to eotaxin (100 ng/mL) through a layer of respiratory epithelial cells. Cells pretreated with eotaxin in the presence of sucrose still underwent desensitization compared with non–sucrose-treated cells. Cells were pretreated with sucrose and eotaxin to ensure that CCR3 internalization did not occur once sucrose was removed, as above. After removal of sucrose and eotaxin by washing, cells were allowed to rest for the duration of the transmigration in Fig 4, C . At the end of the incubation period, the location of CCR3 was assessed by means of flow cytometry. As shown in Fig 4, D , CCR3 internalization remained significantly inhibited after sucrose removal. Additionally, it remained possible that inhibition of internalization by pretreatment of cells with sucrose caused retention of the receptor-ligand complex on the cell surface, and thus the subsequent treatment with li-gand might have been unable to cause transmigration because of steric inhibition by ligand already present rather than because of desensitization. Cells were pretreated as above with biotinylated eotaxin that was capable of inducing receptor internalization in the absence of sucrose to investigate this possibility (data not shown). After washing to remove sucrose and ligand, the presence of biotinylated eotaxin was assessed by staining cells with streptavidin-FITC. These experiments indicated that washing effectively removed sucrose and ligand, reinforcing that the assay was assessing desensitization rather than epitope blockade by eotaxin (data not shown). Collectively, these data indicate that internalization is not required for desensitization of CCR3-mediated responses.

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Discussion 

CCR3 is rapidly internalized into the early endosome compartment after agonist stimulation in a manner similar to that observed for several other GPCRs13, 36; however, the functional significance of this event has not been previously elucidated. In this article we report several novel findings concerning the function of internalization in eosinophils. First, we report that ligand-induced CCR3 internalization is inhibited by PAO and sucrose. These 2 distinct well-established inhibitors of internalization were used because they block receptor internalization by different biochemical mechanisms. PAO inhibits receptor endocytosis by forming stable ring structures with vicinal sulfhydryl groups on plasma membrane proteins.27 In contrast, hypertonic sucrose inhibits the proper formation of clathrin-coated pits at the plasma membrane surface.28, 29 Notably, PAO induced dose-dependent inhibition of internalization and was effective at doses comparable with those reported in other studies.31, 32, 33 Second, we report that PAO and sucrose both blocked eotaxin-induced actin polymerization and cellular shape change (but not calcium mobilization). It is notable that these 2 mechanistically distinct inhibitors had comparable blocking effects on eosinophil function, suggesting that inhibition of receptor internalization, rather than other effects of these agents, was responsible for blocking actin polymerization and cellular shape change. Although PAO also blocks tyrosine phosphatases, control experiments of eotaxin-induced eosinophil shape change in the presence of sodium orthovanadate demonstrated that altering the levels of tyrosine phosphorylation in eosinophils did not block eosinophil shape change. It remained possible that hypertonic sucrose was affecting receptor-ligand interactions on the cell surface, resulting in inhibition of subsequent events. However, cells pretreated with sucrose still elicited calcium transients in response to eotaxin, indicating that the sucrose-treated cells were fully functional in select responses. Third, we report that eotaxin-induced cellular desensitization was not blocked by inhibition of receptor internalization. These later experiments were based on our novel desensitization assay by using epithelial transmigration after brief exposure to eotaxin; previous studies have demonstrated eotaxin-induced desensitization by examination of calcium mobilization responses.37

Our findings are consistent with the results of Yang et al,19 who have demonstrated that internalization of CXCR2 is required for chemotaxis of CXCR2-transfected cells. However, other studies have shown that the chemotaxis caused by N-formyl-methionyl-leucyl-phenylalanine and MCP-1 in cells transfected with their respective receptors is not dependent on receptor internalization.38, 39 Although Yang et al19 have used dominant-negative mutants of dynamin to inhibit internalization of CXCR2, the other studies examined the consequences of mutating all serine and threonine residues in the carboxy terminus of receptors themselves. It remains possible that the effect of dominant-negative mutants of dynamin is due to some alternative function of dynamin. For instance, dynamin is also involved in internalization of caveolae.40, 41 Alternatively, specific GPCRs might have different requirements for chemotactic responses. Importantly, our study demonstrates a role of internalization in primary cells rather than chemokine receptor–transfected cells. We speculate that internalization has a role in chemotaxis by allowing the receptor to traffic to the appropriate intracellular location, where it can interact with signaling molecules. Recently, mitogen-activated protein kinases have been shown to be phosphorylated and activated within 1 minute after exposure of eosinophils to CCR3 ligands.42, 43 This activation is required for eotaxin-induced eosinophil chemotaxis, actin polymerization, and degranulation.42, 43 Additionally, the src family kinases Hck and c-Fgr, which have been shown to be important for CXCR2-mediated degranulation in neutrophils, have been shown to associate with CCR3 after eotaxin stimulation.44 Previous studies have shown that shape change correlates closely with actin polymerization and that mobilization of calcium is required for chemokine-induced actin polymerization in eosinophils.9, 34, 35 Our study demonstrates that internalization is required for actin polymerization but not for calcium mobilization. Calcium mobilization and actin polymerization are rapid responses that peak within 10 seconds after stimulation (Fig. 2, Fig. 3). Future studies will be aimed at better defining the timing of receptor internalization, which was not possible with the flow cytometry method used in our study.

In summary, we report several novel findings concerning CCR3-mediated signaling in eosinophils. First, we report that ligand-induced CCR3 internalization is inhibited by PAO and sucrose. Second, we demonstrate that the induction of select eotaxin-induced functional responses (shape change and actin polymerization) are dependent on receptor internalization. Finally, we report that in addition to stimulating eosinophils to undergo migration, long-term treatment with eotaxin actually blocks transmigration to subsequent stimuli. However, this desensitization is not dependent on receptor internalization. Collectively, our study is the first to define a functional role for plasma membrane protein internalization in eotaxin-induced events in eosinophils. These results have implications for therapeutic intervention in allergic diseases because blockade of CCR3 internalization is likely to have some protective consequences on pathologic responses mediated by eosinophils.

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Acknowledgements 

We thank Patricia Fulkerson, Laura Kindinger, and Jessica Kavanaugh for excellent technical assistance. We also thank Drs Gurjit Hershey, David Williams, and Fred Finkelman for critical discussions and Andrea Lippelman for editorial assistance.

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References 

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☆ Supported in part by the American Heart Association Scientist Development Grant (N.Z.), National Institutes of Health grants R01 AI42242-05 (M.E.R.) and R01 AI45898-03 (M.E.R.), the Human Frontier Science Program (M.E.R), International Life Sciences Institute (M.E.R.), and Burroughs Wellcome Fund (M.E.R.). NZ is a Parker B. Francis fellow.

☆☆ Reprint requests: Marc E. Rothenberg, MD, PhD, Division of Allergy and Immunology, Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229.

PII: S0091-6749(02)91514-9

doi:10.1067/mai.2003.3

The Journal of Allergy and Clinical Immunology
Volume 111, Issue 1 , Pages 97-105, January 2003

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