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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 420-427, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0155OC

CC Chemokine Receptor 3 Mobilizes to the Surface of Human Mast Cells and Potentiates Immunoglobulin E–Dependent Generation of Interleukin 13

Kursteen S. Price, Daniel S. Friend, Elizabeth A. Mellor, Nidia De Jesus, Gerald F. M. Watts and Joshua A. Boyce

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital; and Departments of Medicine, Pediatrics, and Pathology, Harvard Medical School, Boston, Massachusetts

Address correspondence to: Joshua A. Boyce, M.D., Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Smith Research Building, 1 Jimmy Fund Way, Boston, MA 02115. E-mail: Jboyce{at}RICS.bwh.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Eotaxins-1, -2, and -3 mediate the recruitment of blood-borne eosinophils to allergically inflamed tissues by binding CC chemokine receptor (CCR) 3. Mast cells (MCs) are resident tissue cells that also express CCR3. In the present study, we demonstrate that human (h) MCs in nasal polyps and cultured cord blood–derived hMCs express CCR3 not only on their surfaces but also in their secretory granules. Activation of hMCs mediated by the high-affinity Fc receptor for immunoglobulin (Ig)E (Fc{epsilon}RI) increased the surface presentation of CCR3 within 1 h, with a parallel decrease in intracellular CCR3 as determined by flow cytometry on saponin-permeabilized hMCs. Recombinant eotaxin-1 alone did not induce histamine release or cytokine generation, and did not significantly augment IgE-dependent histamine release by interleukin (IL)-4–primed hMCs. Nevertheless, stimulation of hMCs with eotaxin-1 2 h after Fc{epsilon}RI cross-linkage (concomitantly with maximal surface CCR3 expression) increased Fc{epsilon}RI-dependent IL-13 generation by hMCs, compared with their replicates stimulated simultaneously with both agonists. Thus, hMCs may store CCR3 and rapidly mobilize it to their surface with IgE-dependent activation, providing a novel potential mechanism for enhanced hMC effector function, including IL-13 production.

Abbreviations: CC chemokine receptor 3, CCR3 • CXC chemokine receptor 4, CXCR4 • enzyme-linked immunosorbent assay, ELISA • high-affinity Fc receptors for IgE, Fc{epsilon}RI • G-protein–coupled receptor, GPCR • Hanks' balanced salt solution containing 2% bovine serum albumin, HBA • human mast cell, hMC • human progenitor mast cell, hPrMC • immunoglobulin, Ig • interleukin, IL • mast cell, MC • median fluorescence intensity, MFI • phosphate-buffered saline, PBS • reverse transcriptase–polymerase chain reaction, RT-PCR • stem cell factor, SCF • stromal cell-derived factor, SDF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mast cells (MCs) initiate both innate and adaptive immune responses. In type I (allergic) hypersensitivity, MCs respond to cross-linkage of their high-affinity Fc receptor for immunoglobulin (Ig)E (Fc{epsilon}RI) by releasing preformed secretory granule–associated mediators, generating newly formed arachidonic acid metabolites, and secreting several cytokines and chemokines de novo (reviewed in Ref. 1). MCs constitutively reside around the blood vessels in multiple organs, including the skin and the submucosal connective tissues of the gastrointestinal, respiratory, and genitourinary tracts. In allergic rhinitis, asthma, and seasonal conjunctivitis, MCs also intercalate in mucosal epithelial surfaces (2–5), along with eosinophils, basophils, and T helper type 2 (Th2) cells. The presence of another population of MCs, localized to the bronchial smooth muscle, distinguishes the histopathology of asthma from that of eosinophilic bronchitis (6), a condition characterized by bronchial eosinophilia without bronchial hyperreactivity or airflow obstruction. Anatomically distinct subpopulations of MCs exhibit heterogeneous effector properties, including their content of proinflammatory cytokines (7), depending upon their anatomic location. As compared with other hematopoietic effector cells, however, relatively little is known about the role of chemokines and their receptors in the determination of MC tissue localization patterns, changes in these patterns in association with disease, or regional functional MC heterogeneity.

CC chemokine receptor 3 (CCR3) is a seven-transmembrane spanning G-protein–coupled receptor (GPCR) that is strongly expressed by eosinophils (8). CCR3 binds the CC chemokines eotaxin-1, -2, and -3, monocyte chemotactic proteins-2, -3, and -4, and regulated on activation, normal T cells expressed and secreted (9). Of these ligands, eotaxin-1, -2, and -3 signal exclusively through CCR3. CCR3 and its ligands are essential for the basal migration of eosinophils to the small bowel (10), and they regulate eosinophil recruitment to lung (11) and gut (12) in experimentally elicited inflammation in mice. In addition to eosinophils, human basophils (13) and some Th2 lymphocytes (14) express surface CCR3, and eotaxin-1 mediates chemotaxis of these cells in vitro. Human (h) MCs derived in vitro from cord blood mononuclear cells cultured with recombinant stem cell factor (SCF), interleukin (IL)-6, and IL-10 express modest levels of surface CCR3 and respond to eotaxin-1 with a sustained, dose-dependent calcium flux (15). hMCs purified from lung also express CCR3, and respond chemotactically to eotaxin-1 in vitro (16). However, unlike its dramatic effect on eosinophil recruitment, targeted disruption of CCR3 in mice does not attenuate the recruitment of MCs to the intestine (12) and increases the numbers of lung MCs at baseline (11). Thus, although the significance of CCR3 expression by MCs is incompletely understood, the function of this chemokine receptor for MCs differs from its obligate role in both basal and inflammation-induced eosinophil recruitment.

Immunohistochemistry performed on human nasal polyps suggested that CCR3 localizes to different compartments in hMCs and in eosinophils. We therefore undertook this study to define the localization and functions of CCR3 in hMCs. We now report that hMCs store CCR3 intracellularly, at least partly within secretory granules. Moreover, intracellular CCR3 selectively mobilizes to the surfaces of hMCs challenged with Fc{epsilon}RI cross-linking. Eotaxin-1 provides a second signal that amplifies their Fc{epsilon}RI-dependent generation of IL-13, especially when it is presented to coincide with maximal induction of surface CCR3 expression. Intracellular stores of CCR3, perhaps unique to hMCs, are a mobile pool of receptors that could amplify hMC-dependent effector responses that are prominent in Th2-polarized immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of hMCs
hMCs were derived in vitro from cord blood mononuclear cells as described previously (15). The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 at a density of 1 x 106/ml in RPMI 1640 (Gibco BRL, Gaithersburg, MD) enriched with 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO), 2 mM L-glutamine, 0.2 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 µg/ml gentamycin, and 0.1 mM nonessential amino acids, supplemented with recombinant human SCF, IL-6 (both from R&D Systems, Minneapolis, MN), and IL-10 (Endogen, Waltham, MA). Each week, medium and cytokines were replaced, and samples of 2 x 104 cultured cells were spun onto glass slides (Cytospin 2; Shandon, Pittsburgh, PA) and stained with Toluidine blue. Cultures were harvested for experiments when > 95% of the cells had become Toluidine blue–positive (usually between 6 and 9 wk of culture). Experiments involving CCR5 were performed on 4-wk-old cultures of human progenitor MCs (hPrMCs), because this marker is lost with maturation (15).

Activation of hMCs
hMCs were primed with IL-4 (10 ng/ml; Endogen) for 5 d in the presence of SCF (100 ng/ml) to maximize their expression of Fc{epsilon}RI (17). Human myeloma IgE (2 µg/ml; Chemicon International, Tecaluma, CA) was added to saturate Fc{epsilon}RI during the last 16 h. hMCs were washed and stimulated at a concentration of 5 x 105 cells/ml in medium containing SCF (100 ng/ml), either with or without a rabbit anti-human IgE polyclonal Ab (0.01–1 µg/ml; ICN Pharmaceuticals, Costa Mesa, CA). Cells were harvested for immunofluorescent microscopy and for cytofluorographic analyses at various times after activation (see below). For mediator release, samples of 105 cells were stimulated concomitantly with anti-IgE and eotaxin-1 (0.1–100 ng/ml; Peprotech, Kennelworth, NJ) or stromal cell–derived factor (SDF)-1{alpha} (0.1–100 ng/ml; Peprotech); with anti-IgE followed by eotaxin-1 or SDF-1{alpha} (10 ng/ml) at various intervals; or with each agonist alone. The reactions were terminated at 30 min for determination of percent histamine release, and at 6 h after the addition of the first agonist for cytokine analysis. Samples were frozen at -20°C until analyzed. IL-13 and IL-5 were measured with commercial enzyme-linked immunosorbent assay (ELISA; Endogen). Histamine was measured by ELISA (ICN), and percent release was calculated as described elsewhere (18). The values of the triplicate (for cytokines) or duplicate (for histamine) samples were averaged for each experiment.

Immunofluorescence
Samples of 6 x 104 cells were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) on ice for 10 min, washed, resuspended in Hanks' balanced salt solution containing 2% bovine serum albumin (HBA), and immobilized onto round 12 mm glass coverslips by cytocentrifugation. The coverslips were placed cell side up in a 24-well flat bottom plate (Corning, Corning, NY) for permeabilization with 400 µl of 100% methanol for 20 min at -20°C. The coverslips were washed with HBA and the cells were blocked for 30 min with 1% human serum in PBS, and were incubated at room temp for 1 h in a 1:500 dilution of the primary monoclonal antibody (mAb) specific for CCR3 (7B11, IgG2a; National Institutes of Health [NIH] AIDS Repository, Bethesda, MD) or an equal concentration of an isotype-matched negative control Ab (PharMingen, Burlingame, CA). The coverslips were washed three times for 5 min with HBA, incubated for 1 h with a 1:3,000 dilution of the secondary Cy3-conjugated donkey anti-mouse IgG (Jackson Immunoresearch, West Grove, PA), washed again three times, and mounted with a solution of 15% wt/vol vinol-205, 33% wt/vol glycerol, and 0.1% sodium azide in PBS. The images were analyzed qualitatively by fluorescence microscopy by two investigators blinded to the experimental conditions.

Immunohistochemical and Histochemical Analysis
Surgically excised nasal polyps were fixed for 4 h at room temperature in 4% paraformaldehyde and 0.1 M sodium phosphate, pH 7.6. The specimens were then washed twice with 2% dimethylsulfoxide in PBS and suspended overnight at 4°C in 50 mM NH4Cl. Dehydration and embedding was performed with the JB-4 kit (Polysciences Inc., Warrington, PA). After 1–2 d of hardening, 1.5-µm-thick sections were cut on a Reichert-Jung Supercut microtome (Leica, Wetzlar, Germany) with glass knives. Sections were placed on glass slides and incubated in the following sequence: 15 min at 37°C in 2 mM CaCl2 containing 0.025% pancreatic trypsin; 15 min at room temperature in PBS containing 0.05% Tween-20 and 0.1% BSA; 30 min at 37°C in PBS containing 0.05% Tween-20 and 4% normal horse serum; and overnight at 4°C in 4% normal goat serum containing 7B11 or an isotype-matched control (PharMingen) at a 1:200 dilution. The slides were washed, incubated for 40 min at room temperature in buffer containing biotin-labeled horse anti-mouse IgG, washed twice in 0.1% BSA and Tween-20 0.05% in PBS, and incubated for 40 min at room temperature in Vectastain ABC-AP reagent (Vector Laboratories, Inc., Burlingame, CA). The slides were then incubated for 15 min in the dark at room temperature in an alkaline phosphatase substrate solution, counterstained with Gill's hematoxylin in 20% ethylene glycol, and mounted with coverslips using ImmunoMount (Shandon).

Flow Cytometry
hMCs were fixed in 5% paraformaldehyde in PBS for 5 min to preserve their surface presentation of chemokine receptors (19). In some of the experiments in which activation-dependent changes in CCR3 distribution were studied, the hMCs were stimulated in the presence of brefeldin A (Sigma), which disrupts the transport of nascent proteins from the Golgi apparatus (20). To partition chemokine receptors to intracellular and extracellular compartments, we optimized a saponification procedure for the simultaneous depletion of surface chemokine receptor stores and preservation of intracellular stores using immature, 4-wk-old human mast cell progenitors (hPrMCs). Unlike mature cultured hMCs, hPrMCs express CCR5, which resides exclusively on the cell surface, as well as CCR3 and CXCR4 in both intracellular and surface stores (15,19). As anticipated, abundant CCR5 and CCR3 signals were present in these immature hPrMCs after fixation and staining with the anti-CCR5 and anti-CCR3 mAbs (19). Saponin treatment at 0.1 and 1.0% depleted the CCR5 signal nearly completely, while revealing progressive increases in CCR3 staining (identical results for two experiments, data not shown). Thus, in all subsequent experiments for the selective cytofluorographic visualization of the intracellular pools of chemokine receptors, hMCs were incubated for 1 h in PBS containing 3% BSA and 1% saponin. Samples of 1 x 105 hMCs were then suspended in cold Hanks' balanced salt solution containing 2% FBS and 0.1% human serum (FACS buffer), and incubated on ice with 7B11, a mouse anti-human CXCR4 mAb (12G5; NIH AIDS Repository), mouse anti-human CCR5 (IgG2b; PharMingen), or isotype-matched control Abs at 1:200 dilutions for 45 min. The hMCs were washed in cold FACS buffer, resuspended, and incubated again at 4°C with fluorescein isothiocyanate–conjugated sheep anti-mouse IgG (Jackson Immunoresearch). The tracings were analyzed with FACSort (Becton Dickinson, Oxnard, CA) as previously described (15). The net median fluorescence intensity (MFI) for each receptor was calculated by subtracting the median fluorescence signal elicited by the negative control Ab from that elicited by the Ab of interest. Percent changes in net MFI were then calculated to quantitate activation and time-dependent alterations in receptor expression.

Immunogold Electron Microscopy
hMCs were fixed at room temperature with 2% paraformaldehyde and 0.2% glutaraldehyde (Polysciences) in 60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2 (pH 6.9) for 2 h. The fixed cells were then processed for ultrathin cryosectioning as previously described (22). Cryosections (60–70 nm thick) were obtained with a Reichert-FC S Ultracut microtome and were incubated for 30 min at room temperature with a range of dilutions of a rabbit polyclonal anti-human CCR3 Ab (provided by Dr. Bruce Daugherty, Merck, Kennelworth, N.J.) in 1% BSA, or an anti-CCR3 mAb, 7B11, or with a control Ab for 30 min and washed. Optimal concentrations at which background staining was minimal were 1:250 and 1:5000 for the polyclonal and monoclonal Abs, respectively. As specificity controls, additional sections were incubated with nonspecific mouse IgG2a or with rabbit IgG (PharMingen). For sections treated with 7B11 or control IgG2a, incubation was performed with rabbit anti-mouse Ig Ab (1:200 dilution) (Dako, Carpenteria, CA) for a further 30 min. The sections were then washed and incubated with protein A-gold (10 nm; Electron Microscopy Laboratory, Utrecht University, Utrecht, The Netherlands) for 20 min. The labeled sections were viewed with a JEOL 100CX electron microscope at 80 kV.

Reverse Transcriptase–Polymerase Chain Reaction
Samples of total RNA were harvested from hMCs 4 h after cell stimulation. The samples were randomly primed and subjected to reverse transcriptase–polymerase chain reaction (RT-PCR) according to the protocol provided by the manufacturer (Clontech, Carlsbad, CA). PCR (30 cycles) with IL-13– and G3PDH-specific primers was performed as previously described (18). Analysis of the PCR products on ethidium bromide–stained 1.5% agarose gels revealed the predicted 600-base pair band corresponding to IL-13 mRNA and the 983-base pair G3PDH band. The densities of the bands were measured with an automated laser spot densitometer (Chemi-imager 4400; Alpha Innotech Corp, San Leandro, CA), and the relative intensity of the IL-13 signal was calculated as a percentage of the corresponding G3PDH sample for each experiment.

Statistics
Except where indicated otherwise, the results are expressed as the mean ± SEM. Differences between groups were analyzed with the Student's t test. P values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Microscopic Localization of CCR3 in hMCs
Immunocytochemistry with the CCR3-specific mAb, 7B11, elicited a diffuse cytoplasmic staining pattern in nasal polyp–associated hMCs (Figures 1B and 1D), identified by the intense chloroacetate esterase–staining cells in adjacent sections (Figure 1A). In the same specimens, polyp-associated eosinophils, identified by red staining with hematoxylin azure-2 eosin (not shown), stained at the cell perimeter; this pattern is consistent with a dominant surface distribution (Figure 1D). Immobilized cultured hMCs also showed a diffuse pattern of CCR3 immunostaining (Figure 1E). Higher magnification images of both the polyp-associated hMCs and the cultured hMCs (insets, Figure 1D and 1E, respectively) showed a granular pattern of staining.



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  		Figure 1. Immunostaining for CCR3 in hMCs. Low magnification image of a section from a surgically excised nasal polyp stained with chloroacetate esterase to localize hMCs (A, arrows). An adjacent section stained with the anti-human CCR3 mAb, 7B11, shows intense staining of multiple cells, including hMCs (B, arrows). Higher magnification image (D) shows CCR3 immunoreactivity concentrated at the rim of eosinophils (arrow), but in a diffuse, granular pattern in hMCs (inset). Immunostaining for CCR3 in cultured hMCs (E) reveals a diffuse pattern with granule-like accentuation (inset). No reactivity was noted either in the polyps (C) or on the cultured hMCs (F) with an irrelevant IgG control. The results in A–D are from one experiment representative of two performed with polyps from different donors. The images in E and F are from one experiment representative of three.

 
To obtain clearer definition of the intracellular location of CCR3 in cultured hMCs, immunogold for CCR3 was performed with two different Abs. Gold particles localized predominantly to secretory granules (Figure 2A), mostly in the granule matrices (Figure 2B), and to a lesser extent in the granule membranes (Figure 2D). Many of the CCR3-positive granules had a lucent separation between the granule matrix and membrane, suggesting partial cell activation (23) by the cytoprotective SCF that was maintained throughout the experiments. Some particles localized to surface microplicae (Figure 2C). Comparable results were obtained with both Abs. No staining occurred with nonspecific rabbit IgG (Figures 2E and 2F) or with mouse IgG2a (not shown).



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  		Figure 2. Immunogold transmission electron microscopy for CCR3. A 1:250 dilution of a specific rabbit anti-human CCR3 polyclonal Ab (A–D) or an equal concentration of a rabbit IgG (E, F) were used to stain frozen thin sections through a pellet of cultured hMCs. Low magnification images show staining of the hMCs with the anti-CCR3 Ab (A), but not with the control IgG (E). Higher magnification images show CCR3 localization to granule matrices (B) and membranes (D), with staining of some surface microplicae (C). Neither granules (F) nor microplicae (not shown) stained with the negative control IgG. Similar results were obtained with the mAb 7B11 (not shown). Scale bars are equal to 1 µM.

 
Activation-Dependent Changes in CCR3 Distribution
Cytofluorographic and immunofluorescent analyses were used to follow changes in localization of CCR3 in response to Fc{epsilon}RI cross-linkage in IL-4–primed, passively sensitized hMCs. Neither priming nor sensitization of hMCs altered their baseline expression of CCR3 or its distribution (not shown). SCF (100 ng/ml) was maintained under all conditions to sustain viability. Primed hMCs expressed low levels of surface CCR3 (net MFI 4.7 ± 0.8 arbitrary units, mean ± SEM, n = 5), which did not change with sham treatment over the course of these experiments (as shown for one donor, Figure 3A). Staining for CCR3 in permeabilized hMCs was consistently greater than in the nonpermeabilized samples (11.8 ± 2.0 net MFI units, n = 3, P = 0.007 compared with surface staining).



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  		Figure 3. Effect of activation on CCR3 localization in cultured hMCs. Flow cytometry performed on primed, passively sensitized hMCs with the CCR3-specific mAb 7B11 (A, left panels) or the CXCR4-specific mAb 12G5 (A, right panels) 2 h after the addition of a sham control (top row), anti-IgE (middle row), or anti-IgE plus eotaxin-1 (bottom row). Cytofluorographic signals obtained on nonpermeabilized and permeabilized hMCs reflect the results of one experiment representative of three performed in the case of CCR3, and two in the case of CXCR4. Quantitative data are presented in the text. (B) Time-dependent changes in net median fluorescence intensity corresponding to surface CCR3 expression in activated hMCs relative to nonactivated controls. Results are the mean ± SEM from 3–6 experiments per time point. (C) Immunofluorescence for CCR3 in sham-treated (middle panel) and anti-IgE-treated (left panel) hMCs. A control IgG2a showed no immunoreactivity (right panel). Arrows indicate rim pattern of staining in the activated hMCs relative to the granular staining of the nonactivated controls. Results in C are from a single experiment representative of three performed.

 
Fc{epsilon}RI cross-linkage resulted in an initial {bsim} 25% decrease in surface-associated CCR3 at 15 min relative to the sham-treated conditions (Figure 3B), followed by a sharp increase in surface staining for CCR3 that peaked at 2 h (205 ± 81% increase over sham-treated controls, n = 6, P = 0.02), and then declined slightly by 6 h to levels that were still more than twice the levels expressed by sham-treated hMCs (Figure 3B). The activation-dependent increase in surface CCR3 at 2 h was unaffected by brefeldin A treatment of the hMCs (from 4.6 ± 1.3 net MFI units for the sham-treated samples to 8.3 ± 2.5 and 9.3 ± 2.0 net MFI units for the samples stimulated with anti-IgE in the absence or presence of brefeldin A, respectively, mean ± SEM, n = 3). Brefeldin A treatment nevertheless completely ablated IL-13 and IL-5 generation (n = 2, data not shown), thereby confirming its activity. Intracellular CCR3 decreased in the anti-IgE–stimulated hMCs at 2 h (77 ± 19% decrease compared with sham-treated hMCs, mean ± SEM, n = 3, as shown for one experiment; Figure 3A) concomitant with maximal levels of surface CCR3. Simultaneous stimulation of the hMCs with anti-IgE and eotaxin-1 (10 ng/ml) attenuated both the activation-induced increase in surface-associated CCR3 (by 55 ± 30%, n = 3) and the decrease in intracellular CCR3 (by 77 ± 18%, n = 3, as shown for one experiment; Figure 3A). Intracellular CCR3 remained abundant and unchanged in the sham-treated controls (n = 3, as shown for one donor; Figure 3A).

In contrast to the activation-dependent changes in CCR3, the distribution of CXCR4 (another chemokine receptor stored intracellularly by hMCs [21]) was relatively unchanged by IgE-dependent activation in these experiments (16 ± 37% increase and 5 ± 11% decrease, respectively, in surface and intracellular CXCR4, mean ± 1/2 range, n = 2, one experiment displayed; Figure 3A). Fc{epsilon}RI-mediated changes in the cytofluorographic distribution of CCR3 were accompanied by enhancement of CCR3 immunofluorescence at the cell membrane in the activated hMCs, contrasting sharply with the granule-type signals in the sham-treated controls (n = 3, as shown for a single donor; Figure 3C). No staining was observed with a mouse IgG2a control Ab (Figure 3C). Therefore, CCR3 and CXCR4 may be stored by hMCs in different intracellular compartments, their respective intracellular trafficking may be controlled by different mechanisms, or their mobilization occurs with very different kinetics.

Effect of CCR3 Ligation on hMC Activation Responses
Primed, sensitized hMCs stimulated either with recombinant eotaxin-1 or with SDF-1{alpha} (0.1–100 ng/ml each) did not undergo exocytosis, generate cytokines, or produce lipid mediators (n = 3, data not shown). When provided concomitantly with anti-IgE at various doses, eotaxin-1 (10 ng/ml) did not significantly affect Fc{epsilon}RI-dependent generation of IL-13 or IL-5, and did not enhance histamine release (n = 5, data not shown). However, hMCs stimulated with eotaxin-1 at 1, 2, and 3 h after Fc{epsilon}RI cross-linkage (so as to coincide with maximal CCR3 membrane presentation) generated increased amounts of IL-13. The additive effect of eotaxin-1 was maximal and statistically significant at 2 h, coincident with the highest levels of surface CCR3 expression (471 ± 132 versus 783 ± 42 pg/106 hMCs, net increase of 312 ± 96 pg/106 hMCs, P = 0.02, n = 3; Figure 4). SDF-1{alpha} added at 2 h did not further enhance IL-13 generation over its provision concomitantly with Fc{epsilon}RI cross-linkage (n = 2, data not shown). Eotaxin-1 costimulation at 2 h did not significantly augment the steady-state levels of IL-13 mRNA expression at 4 h after Fc{epsilon}RI cross-linking, as determined by semiquantitative RT-PCR (relative intensity 35 ± 3% versus 45 ± 6% of the G3PDH signal for the anti-IgE and anti-IgE plus eotaxin-1 groups, respectively, n = 3, data not shown).



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  		Figure 4. Costimulatory effect of eotaxin-1 on Fc{epsilon}RI-dependent IL-13 generation by hMCs. Primed, sensitized hMCs were activated with Fc{epsilon}RI cross-linkage and stimulated with eotaxin-1 (10 ng/ml) at various intervals afterward. The supernatants were collected 6 h after the addition of anti-IgE. The results are the mean ± SEM of the net eotaxin-1–mediated increases in IL-13 production four experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CCR3 is confined to the cell surface of unstimulated human peripheral blood eosinophils (24), where it functions in their eotaxin-mediated recruitment. In contrast, three lines of evidence in our study indicate that hMCs constitutively express CCR3 in a predominantly intracellular distribution. First, immunocytochemistry with the CCR3-specific mAb, 7B11, elicited a diffuse cytoplasmic staining pattern in nasal polyp-associated hMCs (Figures 1B and 1D). This pattern was also observed in cultured hMCs (Figure 1E). In both instances, higher magnification images suggested a granule-type distribution (Figures 1D and 1E, insets). This staining pattern contrasted with that of the polyp-associated eosinophils in the same specimens, which stained at the cell perimeter, consistent with a dominant surface distribution (Figure 1D). Second, immunogold electron microscopy localized CCR3 predominantly in secretory granule matrices (Figures 2A and 2B), with some staining of granule membranes (Figure 2D) and surface microplicae (Figure 2C). Some background staining was observed as well. Two different CCR3-specific Abs from different species yielded similar results. Third, flow cytometry performed on samples permeabilized with saponin under conditions optimized to remove most surface chemokine receptors revealed intracellular staining for CCR3 in nonstimulated hMCs (Figure 3A). Together with a previous report localizing the IL-8 receptor CXCR2 to the secretory granules of skin hMCs (25), these findings suggest that hMCs are unique among hematopoietic cells for their storage of some chemokine receptors in their secretory organelles.

Activation of lung hMCs ex vivo by cross-linkage of Fc{epsilon}RI fuses secretory granule membranes with plasma membranes (26). Because CCR3 localized partly to secretory granules in cultured hMCs (Figures 2A, 2B, and 2D), we hypothesized that IgE-dependent exocytosis might mobilize intracellular CCR3 to the hMC surface. For these experiments, conditions were optimized for the selective cytofluorographic visualization of intracellular chemokine receptors. Saponin at 0.1 and 1.0% markedly decreased the cytofluorographic signal for CCR5 (a receptor expressed exclusively on the surfaces of hPrMCs and not on mature hMCs), while enhancing the signal for CCR3 in a concentration-dependent fashion. The selective depletion of surface chemokine receptor staining could reflect the known disruptive effects of saponin on plasma membrane cholesterol, which is critical for maintaining the stability and conformation of several GPCRs (27). hMCs were then primed for 5 d with IL-4 (10 ng/ml) and passively sensitized overnight to maximize their surface expression of Fc{epsilon}RI (28). Surface CCR3 was assessed in activated hMCs and sham-treated controls at various intervals after stimulation. The low levels of surface CCR3, which did not change with sham treatment over the course of these experiments (as shown for one donor; Figure 3A), are consistent with the results of our previous studies (15).

Fc{epsilon}RI cross-linkage produced an initial slight decrease in surface-associated CCR3 at 15 min, followed by a sharp increase that peaked at 2 h and was maintained above the baseline for at least 6 h. That the activation-dependent increase at least partly reflected mobilization of the intracellular stores is supported by a concomitant > 50% decrease in intracellular staining over the same interval (as shown for one experiment; Figure 3A). The increase in CCR3 could reflect mobilization from granules. Some CCR3 was seen in the granule membranes of the unstimulated hMCs (Figure 2D), which fuse with the plasma membrane after Fc{epsilon}RI cross-linkage (26). However, the peak increment of surface CCR3 expression occurred 2 h after activation (Figure 3B), well beyond the time required for MC granule membrane fusion with plasma membrane. Inasmuch as most of the CCR3 localized to the granule matrix (Figure 2B), it is possible that granule-associated CCR3 could be exocytosed in a complex with lipids, or in a soluble form, before its incorporation with the plasma membrane. Although the increment in CCR3 could have originated partly from an additional, non–granule-associated intracellular store, the fact that the increment was resistant to brefeldin A treatment (which completely abolished de novo cytokine production) makes this possibility less likely. The preservation of intracellular CCR3 in hMCs concomitantly treated with anti-IgE and eotaxin-1, and the lack of Fc{epsilon}RI-mediated increase in surface CCR3 in the same samples (Figure 3A), likely reflects ligand-induced CCR3 internalization, as previously described for eosinophils (24), and further validates the efficacy of the saponification procedure for the selective cytofluorographic visualization of intracellular CCR3 stores. The cytofluorographic changes in the distribution of CCR3 were accompanied by a shift in the enhancement of CCR3 immunofluorescence from the granules to the cell membrane in the activated hMCs (as shown for a single donor; Figure 3C). Taken together, these observations support the concept that hMCs can mobilize their intracellular stores of CCR3 for presentation at the cell surface to mediate effector function(s) after cell activation. The fact that the distribution of CXCR4, another receptor reported to localize intracellularly in hMCs (21), was relatively unchanged by IgE-dependent activation in these experiments indicates that hMCs may store CCR3 and CXCR4 in different intracellular compartments, may control their respective intracellular trafficking patterns by different mechanisms, or may mobilize them with very different kinetics.

After Fc{epsilon}RI cross-linkage, hMCs generate several cytokines, including IL-13, an effector of tissue fibrosis, goblet cell metaplasia, and bronchial hyperreactivity (29, 30), and IL-5, the major mediator of eosinophilia (31). Eotaxin-1 induces a sustained rise in intracellular calcium in cultured hMCs (15), which is required for calcineurin-dependent cytokine transcription in other cell types (32), and potentiates the IgE-dependent generation of IL-4 by human basophils (33), suggesting a potential costimulatory activity for cytokine generation in cells bearing both Fc{epsilon}RI and CCR3. In our study, neither eotaxin-1 alone nor the CXCR4 ligand, SDF-1{alpha}, induced exocytosis or cytokine generation from primed, sensitized hMCs, and neither chemokine amplified Fc{epsilon}RI-dependent histamine release. Furthermore, although there was a trend toward eotaxin-1–induced amplification of Fc{epsilon}RI-dependent IL-13 generation at all doses of anti-IgE, it did not reach significance. However, delaying eotaxin-1 stimulation until 2-h after Fc{epsilon}RI cross-linkage further amplified IL-13 generation. Indeed, the 2 h point, at which the additive effect of eotaxin-1 was maximal and significant (Figure 4), coincided temporally with the highest levels of surface CCR3 expression (Figure 3B). The enhanced costimulatory effect of eotaxin-1 for IL-13 generation likely relates to increased membrane presentation of CCR3, rather than an to undefined priming effect for GPCR-mediated signal transduction, because SDF-1{alpha} added at 2 h (when surface CXCR4 levels were unaffected) did not enhance IL-13 generation over its provision concomitantly with Fc{epsilon}RI cross-linkage.

In a recent study, mice with a targeted disruption of CCR3 showed an unexpected increase in tracheal intraepithelial MCs, both at baseline and especially after allergen challenge, when compared with wild-type controls (11). A separate study failed to show any differences in the ability of CCR3-/- and CCR3+/+ mice to mount a reactive intraepithelial jejunal MC hyperplasia in response to experimental helminth infection (12). Because eosinophil recruitment was profoundly impaired in both studies, it is clear that CCR3 has distinct functions for these two allergic effector cells and may have organ-specific effects on MC distribution. CCR3-bearing human lung MCs do migrate ex vivo in response to eotaxin-1 (16), and thus it is tempting to speculate that CCR3 is important for the egress of MCs from the mucosal intraepithelial compartment of the lung, rather than for their recruitment to this compartment, during allergen exposure. If so, IgE-dependent activation of MCs in the bronchial mucosa could facilitate their surface expression of CCR3 and thus favor their eotaxin-mediated egress, while simultaneously potentiating subepithelial fibrosis, mucous gland hyperplasia, and IgE synthesis through their production of IL-13. Our study identifies CCR3 in the hMC secretory granule, defines its mobilization to the cell surface with Fc{epsilon}RI cross-linkage, and recognizes its co-stimulatory function for cytokine production by hMCs. These observations expand the possible functions of CCR3 and its ligands, which are prominent in allergen-associated pulmonary inflammation (34), and suggest that treatment strategies that target CCR3 could alter both MC distribution and function as well as eosinophil trafficking.


   Acknowledgments
 
This work was supported by National Institutes of Health grants AI-48802, AI-52353, AI-31599 and HL-36110, by a grant from the Charles Dana Foundation, by a grant from the Vinik Family Fund for Research in Allergic Diseases, and by a grant from the Hyde and Watson Foundation. K.S.P. is the recipient of grants from Glaxo Smith Kline and from the American Academy of Allergy, Asthma, and Immunology. The authors wish to acknowledge Dr. Nancy Kedersha for her expert input in the use of immunofluorescence microscopy.

Received in original form August 15, 2002

Received in final form November 1, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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