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Originally published online as doi:10.1189/jlb.0904511 on January 26, 2005

Published online before print January 26, 2005
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(Journal of Leukocyte Biology. 2005;77:759-766.)
© 2005 by Society for Leukocyte Biology

Differential expression of CCR3 and CXCR3 by human lung and bone marrow-derived mast cells: implications for tissue mast cell migration

Christopher E. Brightling*,1, Davinder Kaur*, Patrick Berger{dagger}, Angela J. Morgan*, Andrew J. Wardlaw* and Peter Bradding*

* Institute for Lung Health, Department of Infection, Immunity and Inflammation, Leicester-Warwick Medical School and University Hospitals of Leicester, United Kingdom; and
{dagger} Laboratoire de Physiologie Cellulaire Respiratoire, INSERM E356, University Victor Segalen Bordeaux 2, France

1 Correspondence: Department of Respiratory Medicine, University Hospitals of Leicester, Groby Road, Leicester, LE3 9QP, UK. E-mail: ceb17{at}le.ac.uk


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ABSTRACT
 
The selective microlocalization of mast cells within specific airway structures, such as the airway smooth muscle and submucosal glands, in asthma is important in the pathophysiology of inflammatory lung disease. Chemokines are likely candidates mediating mast cell migration into these tissue compartments. In this study, we have defined the chemokine receptor profile of human lung mast cells (HLMC) compared with mast cells derived from human bone marrow (BM) and the human mast cell line HMC-1. CXC chemokine receptor 3 (CXCR3) was the most highly expressed chemokine receptor on ex vivo HLMC analyzed by flow cytometry, and CXCR3 expression by mast cells in the bronchial mucosa was confirmed by immuno-histochemistry. CXCR3 was functional, inducing a rise in cytosolic-free Ca2+, actin reorganization, and chemotaxis in response to the CXC ligands CXCL9, -10, and -11. CXCR3 activation did not induce degranulation or cytokine synthesis. In addition, more than 10% of ex vivo HLMC expressed CC chemokine receptor 3, CXCR1, and CXCR4. It is interesting that CXCR3 was not expressed by human BM-derived mast cells, suggesting its expression is induced during tissue maturation. As CXCR3 ligands are elevated in many pulmonary diseases, CXCR3 may be important for determining the anatomical microlocalization of mast cells within the human lung.

Key Words: chemotaxis • HMC-1 • chemokine receptors • CXCL10 • CXCR1 • CXCR4


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INTRODUCTION
 
The mast cell plays a central role in allergic disease and innate immunity [1 2 3 ]. Mast cells are widely distributed throughout the body in connective tissue and at mucosal surfaces. There is increasing evidence that the microlocalization of mast cells within specific tissue structures may be particularly important in the pathogenesis of disease. For example, in asthma, through their selective localization within the airway smooth muscle bundles [4 ] and submucosal glands [5 ], mast cells are likely to contribute to the disordered airway physiology that characterizes this disease [3 ].

A key question is how then do mast cells accumulate at these specific tissue sites. Mast cells originate from CD34+ progenitor cells in the bone marrow (BM), circulate as undifferentiated mononuclear cells in the peripheral circulation, and subsequently, following migration into tissue, mature under the influence of locally derived growth factors and cytokines, including stem cell factor (SCF) [6 ]. It is important that mature mast cells themselves can migrate within tissue [7 8 9 ]. It is likely that this selective mast cell recruitment will require a chemotactic signal arising from the tissue. The C-C and C-X-C chemokines, in particular, are attractive candidates as mast cell chemoattractants. These ubiquitous, structurally related peptides mediate the chemotaxis of many cell types [10 ]. Cultured human mast cell (HMC) precursors express functional CXC chemokine receptor 2 (CXCR2), CXCR4, CC chemokine receptor 3 (CCR3), and CCR5 [11 , 12 ]. Thus, ligands for these receptors may act in recruitment of mast cell progenitors to specific tissue sites. Hitherto, CCR3 is the only functional chemokine receptor reported to be expressed by tissue HMC [13 ], so the chemokine receptor expression of tissue mast cells awaits full characterization.

In this study, our aim was to define the chemokine receptor expression profile of human lung mast cells (HLMC); to compare this with HMC in long-term culture (LT-HLMC), BM-derived mast cells (BMMC), and the HMC-1; and to investigate the function of highly expressed, novel HLMC chemokine receptors.


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MATERIALS AND METHODS
 
Mast cells
HLMC were obtained from normal lung obtained at surgery for carcinoma by positive selection using anti-CD117-coated immunomagnetic beads (Dynal collection kit, Dynal, Oslo, Norway) as described previously [14 ]. A typical lung sample provided a yield of 0.1–0.5 x 106 HLMC/g lung of >98% purity. Cells were cultured in Dulbecco’s modified Eagle’s medium and 10% fetal bovine serum, supplemented with SCF (100 ng/ml), interleukin (IL)-10 (10 ng/ml), and IL-6 (50 ng/ml; R&D Systems, Abingdon, Oxfordshire, UK), and were used for experiments within 48 h of isolation (HLMC) or maintained in LT-HLMC for 4 weeks prior to experiments [15 ]. Progenitor cells from BM were obtained from the femur at hip surgery and were grown over 8–12 weeks in the same supplemented medium as for LT-HLMC. The BMMC were confirmed to be mast cells with expression of tryptase and CD117. The HMC-1 cell line was a generous gift from Dr. Joseph Butterfield (Mayo Clinic, Rochester, MN). HMC-1 cells were maintained in Iscove’s modified Dulbecco’s medium as described previously [16 ].

Bronchial tissue samples
Large airway tissue from lung resection material was immediately transferred into ice-cooled acetone containing the protease inhibitors iodoacetamide (20 mM) and phenylmethylsulfonyl fluoride (2 mM) for fixation, stored at –20°C for 24 h, and then processed into the water-soluble resin, glycolmethacrylate (GMA; Polysciences, Northampton, UK), for embedding [17 ].

Chemokine receptor expression
Flow cytometry
For surface chemokine receptor expression, HLMC, LT-HLMC, and BMMC were resuspended in phosphate-buffered saline containing 0.5% bovine serum albumin at a concentration of 1 x 106cells/ml. Nonspecific antibody binding was blocked using mouse immunoglobulin G (IgG; Sigma Chemical Co., Poole, UK). Mast cells were stained with a directly conjugated monoclonal antibody (mAb) against CD117-phycoerythrin (PE; Dako, Ely, Cambridgeshire, UK) and antibodies to the following chemokine receptors: mouse mAb CCR1, -2, -4, -5, and -6, CXCR1, -2, -3, -4, -5, and -7 (R&D Systems), and CCR3, -7, -9, and -10 (gift from Millenium, Cambridge, MA); rabbit polyclonal antibodies CCR8 (AMS Biotechnology, Abingdon, Oxfordshire, UK) and CX3CR1 (Chemicon, Hampshire, UK). These were indirectly labeled with fluorescein isothiocyanate (FITC), and appropriate isotype controls were performed (mouse mAb IgG1, IgG2a, IgG2b, or mAb rabbit IgG, Dako, UK). HLMC and BMMC were gated for CD117 and were analyzed by two-color flow cytometry on a FACScan (Becton Dickinson, Oxford, UK). HMC-1 cells were labeled with chemokine receptors or appropriate controls alone and analyzed by single-color flow cytometry. To assess coexpression of chemokine receptors, HLMC were dual-stained with anti-CXCR3 mAb, indirectly labeled with FITC and preconjugated anti-CCR3-phycoerythrin (PE), -CXCR1-PE, or -CXCR4-PE mAb (R&D Systems), and then analyzed by two-color flow cytometry.

Immunohistochemistry
The technique of immunostaining, applied to GMA-embedded tissue, has been described previously [17 ]. To assess the colocalization of chemokine receptors with mast cells in the bronchial submucosa in large airway tissue, sequential sections were stained with mAb (Dako, UK) to tryptase, as described previously [18], the chemokine receptors of interest, and appropriate isotype controls.

Imunofluorescence
HLMC (105 cells/well) were seeded onto fibronectin-coated chamber slides and cultured for 24 h. The HLMC were double-stained for mast cell tryptase and the chemokine receptor of interest. First, the cells were labeled with the chemokine receptor mAb, indirectly labeled with FITC, and then stained with tryptase-biotin (Chemicon), indirectly labeled with Texas Red streptavidin (Vector Lab Inc., Burlingame, CA). Cells were counterstained with 4',6-diamidino-2-phenylindole (Sigma Chemical Co.). Appropriate isotype controls were performed.

Functional assessment of chemokine receptors
Calcium imaging
Changes in the cytosolic-free Ca2+ concentration ([Ca2+]i) in response to chemokine receptor activation by known ligands was measured by ratiometric Ca2+ imaging on FURA-2-loaded cells using Openlab software (Improvision, Coventry, UK) [14 ]. This was converted to [Ca2+]i using a commercially available calibration kit (Molecular Probes, Junction City, OR).

Actin reorganization
After chemokine activation, mast cells were stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC; Sigma Chemical Co.), which binds to intracellular polymeric F-actin, and staining was assessed by immunofluorescence and flow cytometry.

Chemotaxis assays
Chemotaxis assays were performed using Transwells with fibronectin-coated inserts with a pore size of 8 µm (Becton Dickinson). We placed 1 x 105 mast cells in 100 µl culture medium supplemented with 10% fetal calf serum into the top well and 450 µl recombinant chemokine or appropriate negative control at varying concentrations in the bottom well. After incubating the cells for 3 h at 37°C, we counted the number of mast cells that migrated into the bottom well using Kimura stain in a haemocytometer. To confirm that chemotaxis was mediated via the activation of the chemokine receptor, HLMC and HMC-1 cells were preincubated with the receptor-blocking antibodies or isotype control (R&D Systems). Checkerboard analysis was used to distinguish chemotactic from chemokinetic activity.

Mediator release by mast cells following chemokine receptor activation
We placed 0.5 x 106 HLMC and HMC-1 in 250 µl into the wells of a 96-well plate. The HLMC were presensitized with human myeloma IgE, 2.5 µg/ml for 1 h, and washed to remove excess IgE. The HLMC were stimulated with goat polyclonal anti-human IgE (1:1000 dilution; Sigma Chemical Co.) or chemokine receptor ligand (200 ng/ml), and the HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 5 ng/ml) and calcium ionophore (A23187, 250 ng/ml) or chemokine receptor ligand (100 ng/ml) for 2 h and 24 h with unstimulated controls. The cell-free supernatants were stored at –80°C for later mediator measurement. The concentration of histamine was measured by radioenzymatic assay in the 2-h samples, and IL-13 was measured by enzyme-linked immunosorbent assay (Caltag-Medsystems, Silverstone, UK) in the 24-h samples.

Statistical analysis
Mast cell chemokine receptor expression is presented as the mean (SEM) percentage of cells that expressed the receptors compared with the relevant isotype control. Comparisons between the proportions of chemokine receptors expressed by each different type of mast cell were made by ANOVA. The number of cells in the bronchial biopsies that expressed chemokine receptors was described as the median [interquartile range (IQR)], and the proportion of these cells that colocalized to mast cells was described as the mean (SEM). The [Ca2+]i is expressed as the mean (SEM). The migration of mast cells is expressed as the fold difference in migration compared with control and is described as the mean (SEM). Nonparametric data were analyzed using the Mann-Whitney test and parametric data by using t-tests or by ANOVA across more than two groups. A value of P< 0.05 was taken as being statistically significant.


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RESULTS
 
CXCR3 is the most highly expressed HLMC chemokine receptor
We examined the expression of all known human chemokine receptors on HLMC derived from lung resection specimens (n=6) using flow cytometry. These HLMC expressed a number of chemokine receptors, namely CCR1, -3, -4, and -7 and CXCR1, -3, -4, and -6 as shown (Fig. 1A and 1B ). Those expressed on >10% of cells were CCR3, CXCR1, CXCR3, and CXCR4. CXCR3 was the most highly expressed receptor on HLMC (32±11% of cells). The mean (SEM) proportion (%) of CCR3, CXCR1, and CXCR4+ cells, which coexpressed CXCR3, was 100 (0), 96 (3), and 88 (2), respectively (n=4; Fig. 1C ). The expression of CXCR3 by isolated HLMC was also demonstrated by immunofluorescence (Fig. 2A 2B 2C 2D ). In the bronchial submucosa, the median (IQR) number of tryptase+ mast cells was 32 (10) cells/mm2, and the number of CXCR3+ cells was 58 (18) cells/mm2. By colocalization, we found that 47 (4)% of the mast cells were CXCR3+, and 30 (5)% of the CXCR3+ cells were mast cells (Fig. 2E and 2F) , confirming that in vivo HLMC express CXCR3.



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  		Figure 1. HLMC chemokine receptor expression. (A) Representative dot plots of all of the chemokine receptors that were significantly expressed by HLMC. (B) Bar graph of mean ± SEM proportion of HLMC expressing each chemokine receptor (n=6). Solid bars correspond to chemokine receptors that were significantly expressed by HLMC compared with control (P<0.05), whereas open bars were chemokine receptors not significantly expressed by HLMC. (C) Representative dot plots for coexpression of CCR3, CXCR1, and CXCR4 with CXCR3 by HLMC.



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  		Figure 2. CXCR3 expression by HLMC and mast cells in the bronchial submucosa. HLMC CXCR3 expression was confirmed by immunofluorescence, as illustrated by (A) isotype control, (B) green CXCR3+ cells, (C) red tryptase+ cells, and (D) double-stained cells with blue nuclear counterstain. Bronchial tissue was cut at 2 µm to enable colocalization of tryptase and CXCR3 expression by the same cell on sequential sections. Representative photomicrographs are shown in (E) the tryptase-positive mast cells and in (F) the corresponding cells stained for CXCR3, highlighted by arrows.

Differential expression of CXCR3 and CCR3 by HLMC and BMMC
We compared the chemokine receptor expression of HLMC and LT-HLMC, BMMC, and HMC-1. The proportion of each type of mast cell that expressed the chemokine receptors that were significantly expressed by HLMC is as shown (Table 1 ). The chemokine receptors that were not significantly expressed by HLMC were also not highly expressed by the other mast cell types (data not shown). The HLMC demonstrated increased expression of CCR3 [95% confidence interval (CI) 1, 18; P=0.015] and CXCR3 (95% CI 3, 59; P=0.038) compared with BMMC, suggesting differential expression of these chemokine receptors between progenitor BMMC and mature tissue mast cells.


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  		Table 1. Mean (SEM) Proportion (%) of Mast Cells That Express the Chemokine Receptors

CXCR3 receptors on HLMC are functional and mediate chemotaxis
The above data demonstrate that CXCR3 is the chemokine receptor most commonly expressed by HLMC and HMC-1 cells. Whether this receptor is functional is unknown, whereas the other highly expressed chemokine receptors CCR3, CXCR1, and CXCR4 have been previously reported as mediating HMC chemotaxis [12 , 13 , 19 ]. Therefore, we further examined the function of CXCR3 in isolated HLMC. There was a significant increase in [Ca2+]i in HLMC following incubation with all three known CXCR3 ligands (P<0.001): mean ± SEM increase for CXC chemokine ligand 10 [CXCL10; interferon (IFN)-inducible protein 10 (IP-10)] 194 ± 11 nM, CXCL11 [IFN-inducible T cell-{alpha} chemoattractant (ITAC)] 207 ± 14 nM, and CXCL9 [monokine induced by IFN-{gamma} (Mig)] 213 ± 11 nM. The increase was similar for each ligand (comparison between ligands, ANOVA, P=0.52). An example of the transient increase in [Ca2+]i following CXCL10 (IP-10) activation is shown in Figure 3A and 3B . Ligand activation of the CXCR3 receptor resulted in actin reorganization with a 1.4-fold increase in the median fluorescence following phalloidin-TRITC staining (n=9; 95% CI 1.1–2.0, P=0.03), as illustrated in Figure 3C and 3D . There were no differences between the ligands. Similarly, the [Ca2+]i increased significantly in HMC-1 cells following incubation with CXCL10 (IP-10) 88 ± 11 nM, CXCL11 (ITAC) 85 ± 5 nM, and CXCL9 (Mig) 51 ± 6 nM (P<0.001).



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  		Figure 3. Functional assessment of CXCR3 on HLMC. (A) Example of increased [Ca2+]i following CXCL10 (IP-10) activation, as illustrated by increased intracellular green fluorescence by FURA-2-loaded HLMC in the right panel compared with the left. (B) Corresponding graph showing transient rise in [Ca2+]i after activation by CXCL10 (arrow represents point at which CXCL10 added to cells). (C) Actin reorganization in HLMC demonstrated by increased phalloidin-TRITC immunofluorescence before (left panel) and 5 min after activation (right panel) by CXCL10. (D) Representative histogram of flow cytometry of HLMC phalloidin-TRITC following CXCL10 activation, illustrating increased phalloidin-TRITC immunofluorescence.

CXCL10 (IP-10) was chemotactic for HLMC (n=6) in a concentration-dependent manner, statistically significant chemotaxis occurred from 25 ng/ml, and the maximal response was 200 ng/ml (2.5-fold migration above medium alone as control, 95% CI 1.5–3.5; P=0.014; Fig. 4A ). CXCL10 (IP-10) was also chemotactic for HMC-1 (n=3) in a concentration-dependent manner with maximal response at 100 ng/ml (3.3-fold migration above medium alone, 95% CI 2.2, 4.3; P=0.011). Checkerboard analysis confirmed that HMC-1 and HLMC migration toward recombinant CXCL10 (IP-10) was a result of chemotaxis rather than chemokinesis and that chemotaxis was inhibited by blocking CXCR3 (n=4) with a specific blocking antibody (Fig. 4B) . We also compared HLMC (n=3) migration toward CC chemokine ligand 11 (CCL11), CXCL8, CXCL10, and CXCL12 (Fig. 4C) .



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  		Figure 4. CXCL10 (IP-10) was chemotactic for HLMC. (A) Concentration response curve of HLMC chemotaxis to CXCL10 (IP-10; n=6). *, P< 0.05, significant chemotaxis toward CXCL10 (IP-10) compared with medium alone (control). (B) HLMC (n=6) and HMC-1 cells (n=3) were analyzed for chemotactic and chemokinetic activity toward CXCL10 (IP-10; solid bars) at a concentration of 200 ng/ml for HLMC and 100 ng/ml for HMC-1 cells. The migration toward CXCL10 (IP-10) was assessed by preincubating mast cells with a specific CXCR3-blocking mAb (hatched bars, HLMC, n=4). Addition of chemokine to the upper (where cells were loaded) or lower chamber was as indicated. CXCL10 (IP-10) was chemotactic but not chemokinetic, and migration was blocked by CXCR3-blocking mAb. Data shown represent means ± SEM. *, P < 0.05. (C) HLMC (n=3) chemotaxis toward CXCL10, CCL11, CXCL12, and CXCL8 at a concentration of 10 ng/ml and 100 ng/ml. *, P < 0.05, chemotaxis toward chemokine compared with control medium alone.

CXCR3 activation by CXCL10 did not alter the basal release of histamine by HLMC (n=5) and did not increase the release of IL-13 by HMC-1 or HLMC (n=3; Fig. 5A and 5B ).



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  		Figure 5. Histamine and IL-13 secretion after CXCR3 activation. (A) Histamine concentration in cell-free supernatant from HLMC (n=5), unstimulated and stimulated with IgE/anti-IgE or CXCL10 (100 ng/ml). *, P < 0.05. (B) IL-13 concentration in cell-free supernatants from HLMC (n=3) and HMC-1 cells (n=3; 2x106cells/ml) cultured for 24 h and unstimulated or stimulated with CXCL10, 100 ng/ml, anti-IgE, or PMA and ionomycin. Data shown represent means ± SEM. *, P < 0.05.


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DISCUSSION
 
In this report, we identify for the first time that CXCR3 is the most abundantly expressed, functional chemokine receptor on HLMC. In contrast, CXCR3 expression by BMMC was low. These observations suggest that CXCR3 activation may facilitate the migration of mast cells within tissue and therefore, may be important in the microlocalization of mast cells within specific tissue structures.

Until now, the profile of chemokine receptor expression by HLMC has been described poorly. CCR3 is the only reported HLMC chemokine receptor [13 , 20 ], although a number of chemokine receptors have been identified on human cord blood-derived mast cells or HMC-1 cells, namely CCR1, -2, -3, -4, and -5 and CXCR1, -2, and -4 [11 , 12 , 19 , 21 22 23 ]. Our investigation into the pattern of chemokine receptor expression on ex vivo HLMC, using fluorescein-activated cell sorter analysis, confirmed the expression of CCR3 and for the first time, also demonstrated expression of CCR1, CCR4, CCR7, CXCR1, CXCR3, CXCR4, and CXCR6. CXCR3 was the chemokine receptor most highly expressed and was also clearly expressed by mast cells present within the bronchial submucosa. It is important that we have demonstrated for the first time that the CXCR3 expressed on the HMC is functional, as the CXCR3 ligands CXCL10 (IP-10), CXCL11 (ITAC), and CXCL9 (Mig) induced a transient rise in [Ca2+]i, cytoskeletal reorganization, and chemotaxis.

The ligands for CXCR3 are IFN-{gamma}-inducible chemokines and therefore are regarded as markers of T helper cell type 1 (Th1) activity [24 ]. CXCR3 ligands have been implicated in the pathogenesis of a number of inflammatory diseases, such as rheumatoid arthritis [25 ], post-lung transplant bronchiolitis obliterans syndrome [26 ], and sarcoidosis [27 ], which are all characterized by increased numbers of tissue mast cells [25 , 28 , 29 ]. However, increased expression of CXCL10 (IP-10) is not restricted to Th1-mediated disease but is also a feature of Th2-mediated allergic disease. Thus, CXCL10 (IP-10) is overexpressed in bronchial biopsies from stable asthmatics [27 ] compared with normal controls, and the concentration of CXCL10 (IP-10) is markedly elevated in bronchoalveolar lavage fluid in asthmatics following allergen challenge [30 ]. There are numerous sources of CXCR3 ligands, including structural cells such as endothelial cells [31 ], nerve cells [32 ], and smooth muscle [33 ] and epithelial cells [34 ]. This wide array of potential cellular sources of CXCR3 ligands may be of particular importance for the microanatomical distribution of mast cells within tissue. The differential localization of mast cells within particular tissue structures is an important feature in a number of diseases, such as the colocation of mast cells with nerves in the skin in the development of the symptom of itch [35 ] and the localization of mast cells within the airway smooth muscle bundles and mucosal glands in asthma [4 , 5 ]. As we have shown that CXCR3 is highly expressed by lung mast cells and is functional, and the concentration of CXCR3 ligands are elevated in pulmonary disease, the CXCR3/CXCR3 ligand axis may play a critical role in determining the distribution of mast cells within the human lung.

In addition to defining the chemokine receptor profile of HLMC, our study allowed us to compare the expression of chemokine receptors by human BMMC, HMC-1 cells, fresh HLMC, and LT-HLMC culture. It is known that chemokine receptor expression by cord blood-derived mast cells in vitro changes with maturation and that in particular, CCR3 expression is maintained in mature mast cells [11 ]. Indeed, we found that CCR3 expression by HLMC was increased compared with BMMC, HMC-1 cells, and cultured LT-HLMC, suggesting that the increased CCR3 expression in mature HLMC is dependent on specific factors within the local microenvironment. CXCR3 was highly expressed by HLMC and cultured LT-HLMC but not by BMMC, suggesting that like CCR3, CXCR3 expression is up-regulated once mast cells mature within tissue. The recent finding that mast cells in the synovium of patients with rheumatoid disease express CXCR3 supports this view [25 ]. However, in contrast to CCR3, HLMC expression of CXCR3 persisted even when the cells were maintained in LT-HLMC culture, suggesting that once CXCR3 expression is established, it is independent of the tissue microenvironment.

Although CXCR3 was the chemokine receptor most highly expressed, HLMC also expressed the chemokine receptors CCR3, CXCR1, and CXCR4 in more than 10% of cells. CCL11 (eotaxin) mediates HLMC chemotaxis [13 ], and the ligands for CXCR1 and CXCR4, namely CXCL8 (IL-8) and CXCL12 (stromal cell-derived factor-1{alpha}), respectively, are chemotactic for cord blood-derived mast cells [12 , 19 ]. Indeed, we confirmed that CCL11 and CXCL12 were chemotactic for HLMC. Therefore, the mechanisms of migration of mature tissue mast cells are likely to be complex, in particular, because of interactions between chemokines themselves. For example, CXCL10 (IP-10) is a natural antagonist for CCL11 (eotaxin). Thus, the relative importance of mast cell migration mediated by CXCR3 compared with other chemokines and other chemoattractants [36 ] in controlling the localization of mast cells within tissue structures needs to be explored fully.

In summary, we have described a specific chemokine receptor profile for HLMC and have found that CXCR3 was the most highly expressed and functional chemokine receptor, suggesting that CXCR3 ligands may contribute to the microlocalization of mast cells within tissue. Specifically targeting CXCR3-mediated mast cell migration may provide a novel, effective treatment for many inflammatory diseases.


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ACKNOWLEDGEMENTS
 
This work was supported by Asthma UK, DoH UK Clinician Scientist Scheme. We are grateful to Millenium for kindly providing some of the chemokine receptor antibodies and to Dr. S. Saha and Ms. A. Sutcliffe for technical support.

Received September 13, 2004; revised December 10, 2004; accepted December 23, 2004.


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REFERENCES
 
    1
  1. Williams, C., Galli, S. (2000) The diverse potential effector and immunoregulatory roles of mast cells in allergic disease J. Allergy Clin. Immunol. 105,847-859[CrossRef][Medline]
    2. 2
  2. Marshall, J. S., Jawdat, D. M. (2004) Mast cells in innate immunity J. Allergy Clin. Immunol. 114,21-27[CrossRef][Medline]
    3. 3
  3. Brightling, C. E., Bradding, P., Pavord, I. D., Wardlaw, A. J. (2003) New insights into the role of the mast cell in asthma Clin. Exp. Allergy 33,550-556[CrossRef][Medline]
    4. 4
  4. Brightling, C. E., Bradding, P., Symon, F. A., Holgate, S. T., Wardlaw, A. J., Pavord, I. D. (2002) Mast-cell infiltration of airway smooth muscle in asthma N. Engl. J. Med. 346,1699-1705[Abstract/Free Full Text]
    5. 5
  5. Carroll, N. G., Mutavdzic, S., James, A. L. (2002) Increased mast cells and neutrophils in submucosal mucous glands and mucus plugging in patients with asthma Thorax 57,677-682[Abstract/Free Full Text]
    6. 6
  6. Austen, K. F., Boyce, J. A. (2001) Mast cell lineage development and phenotypic regulation Leuk. Res. 25,511-518[CrossRef][Medline]
    7. 7
  7. Wang, H. W., Tedla, N., Lloyd, A. R., Wakefield, D., McNeil, P. H. (1998) Mast cell activation and migration to lymph nodes during induction of an immune response in mice J. Clin. Invest. 102,1617-1626[Medline]
    8. 8
  8. Turner, C. R., Kolbe, J., Spannhake, E. W. (1988) Rapid increase in mast cell numbers in canine central and peripheral airways J. Appl. Physiol. 65,445-451[Abstract/Free Full Text]
    9. 9
  9. Montefort, S., Gratziou, C., Goulding, D., Polosa, R., Haskard, D. O., Howarth, P. H., Holgate, S. T., Carroll, M. P. (1994) Bronchial biopsy evidence for leukocyte infiltration and upregulation of leukocyte-enothelial cell adhesion molecules 6 hours after local allergen challenge of sensitized asthmatic airways J. Clin. Invest. 93,1411-1421
    10. 10
  10. Baggiolini, M. (1998) Chemokines and leukocyte traffic Nature 392,565-568[CrossRef][Medline]
    11. 11
  11. Ochi, H., Hirani, W. M., Yuan, Q., Friend, D. S., Austen, K. F., Boyce, J. A. (1999) T helper cell type 2 cytokine-mediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro J. Exp. Med. 190,267-280[Abstract/Free Full Text]
    12. 12
  12. Juremalm, M., Hjertson, M., Olsson, N., Harvima, I., Nilsson, K., Nilsson, G. (2000) The chemokine receptor CXCR4 is expressed within the mast cell lineage, and its ligand stromal cell-derived factor-1{alpha} acts as a mast cell chemotaxin Eur. J. Immunol. 30,3614-3622[CrossRef][Medline]
    13. 13
  13. Romagnani, P., de Paulis, A., Beltrame, C., Annunziato, F., Dente, V., Maggi, E., Romagnani, S., Marone, G. (1999) Tryptase-chymase double-positive human mast cells express the eotaxin receptor CCR3 and are attracted by CCR3-binding chemokines Am. J. Pathol. 155,1195-1204[Abstract/Free Full Text]
    14. 14
  14. Sanmugalingam, D., Wardlaw, A. J., Bradding, P. (2000) Adhesion of human lung mast cells to bronchial epithelium: evidence for a novel carbohydrate-mediated mechanism J. Leukoc. Biol. 68,38-46[Abstract/Free Full Text]
    15. 15
  15. Duffy, S. M., Lawley, W. J., Kaur, D., Yang, W., Bradding, P. (2003) Inhibition of human mast cell proliferation and survival by tamoxifen in association with ion channel modulation J. Allergy Clin. Immunol. 112,965-972[CrossRef][Medline]
    16. 16
  16. Duffy, S. M., Leyland, M. L., Conley, E. C., Bradding, P. (2001) Voltage-dependent and calcium-activated ion channels in the human mast cell line HMC-1 J. Leukoc. Biol. 70,233-240[Abstract/Free Full Text]
    17. 17
  17. Britten, K. M., Howarth, P. H., Roche, W. R. (1993) Immunohistochemistry on resin sections: a comparison of resin-embedding techniques for small mucosal biopsies Biotech. Histochem. 68,271-280[Medline]
    18. 18
  18. Bradding, P., Feather, I. H., Howarth, P. H., Mueller, R., Roberts, J. A., Britten, K., Bews, J. P., Hunt, T. C., Okayama, Y., Heusser, C. H. (1992) Interleukin 4 is localized to and released by human mast cells J. Exp. Med. 176,1381-1386[Abstract/Free Full Text]
    19. 19
  19. Inamura, H., Kurosawa, M., Okano, A., Kayaba, H., Majima, M. (2002) Expression of the interleukin-8 receptors CXCR1 and CXCR2 on cord-blood-derived cultured human mast cells Int. Arch. Allergy Immunol. 128,142-150[CrossRef][Medline]
    20. 20
  20. de Paulis, A., Annunziato, F., Di Gioia, L., Romagnani, S., Carfora, M., Beltrame, C., Marone, G., Romagnani, P. (2001) Expression of the chemokine receptor CCR3 on human mast cells Int. Arch. Allergy Immunol. 124,146-150[CrossRef][Medline]
    21. 21
  21. Juremalm, M., Olsson, N., Nilsson, G. (2002) Selective CCL5/RANTES-induced mast cell migration through interactions with chemokine receptors CCR1 and CCR4 Biochem. Biophys. Res. Commun. 297,480-485[CrossRef][Medline]
    22. 22
  22. Nilsson, G., Mikovits, J. A., Metcalfe, D. D., Taub, D. D. (1999) Mast cell migratory response to interleukin-8 is mediated through interaction with chemokine receptor CXCR2/interleukin-8RB Blood 93,2791-2797[Abstract/Free Full Text]
    23. 23
  23. Lippert, U., Artuc, M., Grutzkau, A., Moller, A., Kenderessy-Szabo, A., Schadendorf, D., Norgauer, J., Hartmann, K., Schweitzer-Stenner, R., Zuberbier, T., Henz, B. M., Kruger-Krasagakes, S. (1998) Expression and functional activity of the IL-8 receptor type CXCR1 and CXCR2 on human mast cells J. Immunol. 161,2600-2608[Abstract/Free Full Text]
    24. 24
  24. Kaplan, A. P. (2001) Chemokines, chemokine receptors and allergy Int. Arch. Allergy Immunol. 124,423-431[CrossRef][Medline]
    25. 25
  25. Ruschpler, P., Lorenz, P., Eichler, W., Koczan, D., Hanel, C., Scholz, R., Melzer, C., Thiesen, H. J., Stiehl, P. (2003) High CXCR3 expression in synovial mast cells associated with CXCL9 and CXCL10 expression in inflammatory synovial tissues of patients with rheumatoid arthritis Arthritis Res. Ther. 5,R241-R252[CrossRef][Medline]
    26. 26
  26. Belperio, J. A., Keane, M. P., Burdick, M. D., Lynch, J. P., III, Zisman, D. A., Xue, Y. Y., Li, K., Ardehali, A., Ross, D. J., Strieter, R. M. (2003) Role of CXCL9/CXCR3 chemokine biology during pathogenesis of acute lung allograft rejection J. Immunol. 171,4844-4852[Abstract/Free Full Text]
    27. 27
  27. Miotto, D., Christodoulopoulos, P., Olivenstein, R., Taha, R., Cameron, L., Tsicopoulos, A., Tonnel, A. B., Fahy, O., Lafitte, J. J., Luster, A. D., Wallaert, B., Mapp, C. E., Hamid, Q. (2001) Expression of IFN-{gamma}-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in T(H)1- and T(H)2-mediated lung diseases J. Allergy Clin. Immunol. 107,664-670[CrossRef][Medline]
    28. 28
  28. Yousem, S. A. (1997) The potential role of mast cells in lung allograft rejection Hum. Pathol. 28,179-182[CrossRef][Medline]
    29. 29
  29. Wardlaw, A. J., Cromwell, O., Celestino, D., Fitzharris, P., Geddes, D. M., Collins, J. V., Kay, A. B. (1986) Morphological and secretory properties of bronchoalveolar lavage mast cells in respiratory diseases Clin. Allergy 16,163-173[CrossRef][Medline]
    30. 30
  30. Pilette, C., Francis, J. N., Till, S. J., Durham, S. R. (2004) CCR4 ligands are up-regulated in the airways of atopic asthmatics after segmental allergen challenge Eur. Respir. J. 23,876-884[Abstract/Free Full Text]
    31. 31
  31. Ishiguro, N., Takada, A., Yoshioka, M., Ma, X., Kikuta, H., Kida, H., Kobayashi, K. (2004) Induction of interferon-inducible protein-10 and monokine induced by interferon-{gamma} from human endothelial cells infected with Influenza A virus Arch. Virol. 149,17-34[CrossRef][Medline]
    32. 32
  32. Fujioka, T., Purev, E., Rostami, A. (1999) Chemokine mRNA expression in the cauda equina of Lewis rats with experimental allergic neuritis J. Neuroimmunol. 97,51-59[CrossRef][Medline]
    33. 33
  33. Hardaker, E. L., Bacon, A. M., Carlson, K. A. R. E., Roshak, A. K., Foley, J. J., Schmidt, D. B., Buckley, P. T., Comegys, M. E. G. H., Panettieri, R. A., Jr, Sarau, H. M., Belmonte, K. E. (2004) Regulation of TNF-{{alpha}}- and IFN-{{gamma}}-induced CXCL10 expression: participation of the airway smooth muscle in the pulmonary inflammatory response in chronic obstructive pulmonary disease FASEB J. 18,191-193[Abstract/Free Full Text]
    34. 34
  34. Sauty, A., Dziejman, M., Taha, R. A., Iarossi, A. S., Neote, K., Garcia-Zepeda, E. A., Hamid, Q., Luster, A. D. (1999) The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells J. Immunol. 162,3549-3558[Abstract/Free Full Text]
    35. 35
  35. Jarvikallio, A., Harvima, I. T., Naukkarinen, A. (2003) Mast cells, nerves and neuropeptides in atopic dermatitis and nummular eczema Arch. Dermatol. Res. 295,2-7[Medline]
    36. 36
  36. Berger, P., Girodet, P. O., Begueret, H., Ousova, O., Perng, D. W., Marthan, R., Walls, A. F., Tunon de Lara, J. M. (2003) Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis FASEB J. 17,2139-2141[Abstract/Free Full Text]



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