SEARCH

SEARCH BY CITATION

Keywords:

  • Chemokines;
  • Chemotaxis;
  • Eosinophils;
  • Human

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Epithelial cells play an important role in orchestrating mucosal immune responses. In allergic-type inflammation, epithelial cells control the recruitment of eosinophils into the mucosa. Th2-type cytokine-driven release of eosinophil-active chemokines from epithelial cells directs eosinophil migration into the mucosal epithelium. CCR3, the main eosinophil chemokine receptor, regulates this process; however, the respective contribution of individual CCR3 ligands in eosinophil transepithelial migration is less well understood. Using an in vitro transepithelial chemotaxis system, we found that eotaxin-3 produced by IL-4-stimulated airway epithelial cells and CCR3 on eosinophils exclusively mediate eosinophil transepithelial migration. Eotaxin-3 protein levels were also increased in the nasal mucosal epithelium recovered from allergic patients as compared to non-allergic patients. Surprisingly, eotaxin-3 in IL-4-stimulated airway epithelial cells was predominantly cell surface bound, and the cell surface form was critical for eosinophil transepithelial migration. Eotaxin-3 cell surface association was partially glycosaminoglycan (GAG) dependent, but was completely protein dependent, suggesting that eotaxin-3 associates with both GAG and cell surface proteins. We thus provide evidence that cell surface-associated eotaxin-3 is the critical IL-4-dependent chemotactic signal mediating eosinophil transepithelial migration in the setting of allergic inflammation.

Abbreviations:
APC:

allophycocyanin

GAG:

glycosaminoglycan

MCP:

monocyte chemotactic protein

NHBEC:

normal human bronchial epithelial cells

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mucosal epithelial cells actively participate in orchestrating the mucosal immune responses by producing biologically active cytokines and chemokines. Allergic asthma is a chronic inflammatory disease of the airways, characterized by airway eosinophilia, hyper-responsiveness and mucus hyper-secretion 1. CD4+ Th2 cells are thought to drive this response by producing IL-4, IL-13 and IL-5 24.

Chemokines are a superfamily of 8–10-kDa secreted basic proteins that induce leukocyte chemotaxis by binding to G protein-coupled receptors on target cells 5. The key eosinophil chemokine receptor is CCR3 6, and the major CCR3 ligands include eotaxin-1, -2, -3 (CCL11, CCL24 and CCL26, respectively), and monocyte chemotactic protein (MCP)-4 (CCL13) 712. It is believed that CD4+ Th2 cell-derived IL-4 and IL-13 induce the expression of STAT6-dependent chemokines in the lung that recruit eosinophils into the airways.

Eosinophil transepithelial migration is one of the key steps in allergic airway inflammation, leading to migration of eosinophils into the airways and mucosal damage 13. Understanding the mechanisms mediating eosinophil transepithelial migration is critical in gaining insight into the mechanisms of allergic airway inflammation. It remains unclear which eosinophil-active chemoattractant is important in eosinophil transepithelial migration. Airway epithelial cells are known to produce a variety of eosinophil-active chemokines under different cytokine stimulations. While the Th2 cytokines IL-4 and IL-13 have been shown to stimulate low levels of eotaxin-1 production from epithelial cells and fibroblasts 1416, pro-inflammatory cytokines, such as TNF-α, IFN-γ and IL-1, appear to be the most potent inducers of eotaxin-1 production in human lung epithelial cells 17, 18. Eotaxin-1-deficient mice are able to develop airway eosinophilia and airway hyper-responsiveness in a model of experimental asthma 19, 20, implying that other eosinophil-active chemoattractants are also involved in eosinophil recruitment into the airways. Th2 cytokines differentially regulate eotaxin-2 expression in monocytes and macrophages 21. Recent studies of eotaxin-2-deficient mice have revealed an eotaxin-2-dependent mechanism in IL-13-induced airway eosinophilia 22. Airway epithelial cells can produce eotaxin-3 following Th2 cytokine stimulation 2325. However, the relative contribution of individual eosinophil-active chemokines in eosinophil transepithelial migration has never been reported.

Functionally active eotaxin-1 and eotaxin-2 have been identified in both human and mouse. However, the functional orthologues for human eotaxin-3 and MCP-4 have not been identified in the mouse, and the mouse eotaxin-3 gene is likely a pseudogene 22. Therefore, eotaxin-3 gene regulation and function cannot be studied in the mouse and must be studied in humans. In the current study, we found that eotaxin-3 was the predominant CCR3 ligand induced in airway epithelial cells following Th2 cytokine stimulation. Eotaxin-3 expression was also increased in the mucosal epithelium of nasal biopsies recovered from allergic patients as compared to non-allergic patients. Eotaxin-3 was transcriptionally regulated in a STAT6-dependent manner, and the eotaxin-3 protein was predominantly cell surface associated. This cell surface-associated eotaxin-3 was the functionally active eosinophil chemokine that mediated eosinophil transepithelial migration.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Eotaxin-3-CCR3 is critical in mediating eosinophil transepithelial migration

To determine which eosinophil-active chemoattractant is involved in eosinophil transepithelial migration, we performed in vitro eosinophil transmigration assays through IL-4-stimulated airway epithelial cells (BEAS-2B cells, a cell line derived from human bronchial epithelial cells). BEAS-2B cells, grown to a confluent monolayer on filter membranes, were stimulated with IL-4 (50 ng/mL) for 18–20 h and washed twice prior to the chemotaxis assays. Eosinophils were freshly isolated from healthy human volunteers and placed in the top compartment of the chemotaxis chamber. A 2.8-fold increase in eosinophil transmigration was observed when BEAS-2B cells were grown on the bottom of the filter membranes and stimulated with IL-4 as compared to unstimulated cells (p <0.001) (Fig. 1A, left panel). Interestingly, this increase was not observed when BEAS-2B cells were grown on the top of the filter membranes and stimulated with IL-4 (p = 0.26) (Fig. 1A, right panel), suggesting that IL-4-stimulated BEAS-2B cells produce biologically active chemoattractant(s) in a polar fashion (baso-laterally) to mediate eosinophil transepithelial migration. In parallel experiments, using untreated BEAS-2B cell monolayers grown on either side of the filter membranes, eosinophil transmigration was observed when exogenous eotaxin-3 or eotaxin-1 was present in the bottom of the chemotaxis chambers (Fig. 1B), suggesting that airway epithelial cells do not act as a simple mechanical barrier but rather actively participate in eosinophil transepithelial migration.

thumbnail image

Figure 1. Eosinophil transepithelial migration. (A) Migration through IL-4-stimulated BEAS-2B cells. BEAS-2B cells, grown on opposite sides of filter membranes as shown in the diagrams, were stimulated with IL-4. Human eosinophils were added to the top of the chamber. The y axis represents the chemotactic index (mean ± SD from three independent experiments with triplicate samples for each data point). Open and filled symbols represent sham-treated and IL-4-treated BEAS-2B cells, respectively. (B) Migration through unstimulated BEAS-2B cells with exogenous chemokines. BEAS-2B cells were grown on the opposite sides of the filter membranes, as shown in the schematic diagrams. Exogenous eotaxin-3 or eotaxin-1 was added to the bottom of the chambers. As a control, bare filters were used without BEAS-2B cells. Human eosinophils were added to the top of the chambers. The y axis represents the chemotactic index (mean ± SD from three independent experiments with six samples for each data point in each experiment), and the x axis represents chemokine concentrations. Open and filled symbols represent bare filter and BEAS-2B cell monolayer, respectively. * indicates p value <0.001 for statistically significant difference, and N.S. indicates non-significant difference.

Download figure to PowerPoint

To delineate individual chemokines involved in eosinophil transmigration through IL-4-stimulated BEAS-2B cells, we performed a series of antibody blocking experiments. BEAS-2B cell monolayers, grown on the bottom of the filter membranes, were stimulated with IL-4 and washed twice prior to the chemotaxis assays. Addition of an anti-human CCR3 mAb completely abolished eosinophil transmigration in a dose-dependent fashion (Fig. 2A). Similarly, anti-human eotaxin-3 mAb, but not anti-human eotaxin-1 mAb or isotype-matched control antibody, completely inhibited eosinophil transmigration (Fig. 2B), indicating that eosinophil transepithelial migration is exclusively mediated by eotaxin-3 and CCR3. Specificity of the anti-eotaxin-3 mAb was studied in separate experiments using bare filters with exogenous eotaxin-3 or eotaxin-1 present in the bottom chambers (Fig. 2C). Anti-eotaxin-3 mAb specifically inhibited eosinophil chemotaxis towards eotaxin-3, but had no effect on eotaxin-1-mediated eosinophil chemotaxis (Fig. 2C). Taken together, these data demonstrate that the interaction of eotaxin-3, produced by IL-4-stimulated BEAS-2B cells, and CCR3 on eosinophils mediates eosinophil transepithelial migration. Furthermore, eotaxin-3 produced by IL-4-stimulated BEAS-2B cells is likely attached to the monolayer, because the BEAS-2B cell monolayer was washed after IL-4 stimulation prior to addition of eosinophils.

thumbnail image

Figure 2. CCR3 and eotaxin-3 mediate eosinophil transepithelial migration in IL-4-stimulated airway epithelial cells. (A) Effect of CCR3 mAb inhibition. BEAS-2B cells on the filter membrane were treated with IL-4 or were left untreated (sham). Anti-CCR3 mAb or control mAb was added at the indicated concentrations. Eosinophils were pre-incubated with mAb before adding to the top of the chemotaxis chambers. Open symbol represents sham-treated cells; filled symbols represent IL-4-treated cells incubated with anti-CCR3 mAb; and hatched symbols represent IL-4-treated cells incubated with control mAb. (B) Effect of anti-eotaxin-3 mAb. BEAS-2B cells on filter membrane were treated with IL-4 or left untreated (sham). After washing off the IL-4-containing medium or sham medium, BEAS-2B cells were pre-incubated with anti-eotaxin-3, anti-eotaxin-1 or control mAb. Eosinophils were added to the top of the chemotaxis chamber with the presence of antibodies on both sides of the membrane. Open symbol represents sham-treated cells; filled symbols represent IL-4-treated cells with anti-eotaxin-3 mAb; hatched symbols represent IL-4-treated cells with control mAb; and dotted symbols represent IL-4-treated cells with anti-eotaxin-1 mAb. (C) Eosinopil chemotaxis to exogenous eotaxin-3 or eotaxin-1 in the presence of blocking mAb. Human eotaxin-3 or eotaxin-1 was added to the bottom chambers at the indicated concentrations. Anti-eotaxin-3 or -eotaxin-1 or control mAb was present in both sides of the chamber. Open symbol represents sham-treated cells; filled symbols represent chemotaxis to eotaxin-3 in the presence of anti-eotaxin-3 mAb; gray symbols represent chemotaxis to eotaxin-1 in the presence of anti-eotaxin-3 mAb; and hatched symbols represent chemotaxis to eotaxin-3 in the presence of control mAb. (D) Eotaxin-3 binding to unstimulated BEAS-2B cells induces eosinophil transepithelial migration. BEAS-2B cells were grown on the bottom of the filter membrane and incubated with exogenous eotaxin-1, -2, -3 at the basal surface. After washing off unbound chemokines, eosinophils were added to the top chamber. As a control, chemokines were incubated with the bare filter membrane and washed off before adding eosinophils. The y axis represents the chemotactic index (mean ± SD from three independent experiments with triplicate samples for each data point in each experiment) for all figures. * indicates p value <0.001 and ** indicates p value <0.005 for statistically significant differences.

Download figure to PowerPoint

To study the ability of CCR3 ligands to adhere to BEAS-2B cells, we added human eotaxin-1, -2, and -3 to unstimulated BEAS-2B cells and studied their ability to mediate eosinophil transepithelial migration. Human eotaxin-1, -2, and -3 were added individually to the basal surface of untreated BEAS-2B cell monolayers grown on the bottom of the filter membranes, and washed off after a 3-h incubation prior to the eosinophil transmigration assay. In control experiments, individual chemokines were incubated with the bare filters for 3 h and washed off before adding eosinophils. Addition of eotaxin-3 uniquely induced a dose-dependent and statistically significant increase in eosinophil transmigration, whereas eotaxin-1 and eotaxin-2 had no effect (Fig. 2D). Addition of the chemokines to the bare filter membranes did not induce significant eosinophil chemotaxis for any ligands tested (Fig. 2D). These results demonstrate a unique feature of eotaxin-3 in cell surface interaction in airway epithelial cells as compared to eotaxin-1 and eotaxin-2. Cell-bound eotaxin-3 in untreated bronchial epithelial cells is functionally active in mediating eosinophil transepithelial migration, further supporting our finding that the biologically active form of eotaxin-3 produced by IL-4-stimulated BEAS-2B cells is attached to the cell surface.

Eotaxin-3 is the predominant eosinophil-active chemokine in IL-4-stimulated epithelial cells

To determine the differential expression of eosinophil-active chemokines in airway epithelial cells, we treated human airway epithelial cells with a panel of cytokines and measured mRNA levels of eotaxin-1, -2, -3 and MCP-4 using Northern blot analysis. We used BEAS-2B and A549 cell lines, primary airway epithelial cells [normal human bronchial epithelial cells (NHBEC)] and, for comparison, normal human umbilical vein endothelial cells (HUVEC). Eotaxin-3 mRNA was markedly induced in all cells tested following stimulation with the Th2 cytokines IL-4 and IL-13 (Fig. 3). In contrast, IL-4 and IL-13 did not induce detectable mRNA expression of other eosinophil-active chemoattractants, including eotaxin-1, -2 and MCP-4. As we have previously reported, and as was subsequently confirmed by others 7, 8, 17, 26, only the inflammatory cytokines TNF-α and IFN-γ induced significant eotaxin-1 and MCP-4 mRNA expression in the airway epithelial cells (Fig. 3). These data indicate that eotaxin-3 is the predominant CCR3 ligand induced in airway epithelial cells following stimulation with the Th2 cytokines IL-4 or IL-13.

thumbnail image

Figure 3. Northern blot analysis of eosinophil-active chemokine expression in airway epithelial cells. Total RNA was isolated from A549 cells, BEAS-2B cells, NHBEC and HUVEC stimulated with cytokines as indicated, fractionated on a 1.2% agarose gel containing 0.2 M formaldehyde and probed with DNA probes for human eotaxin-1, -2, -3 and MCP-4.

Download figure to PowerPoint

To determine if any of the CCR3 ligands are transcriptionally regulated by IL-4 and IL-13, we molecularly engineered ∼1 kb 5′ flanking DNA of the genes for human eotaxin-1, -2, -3 and MCP-4 into a firefly luciferase reporter construct. BEAS-2B cells were transfected with these promoter-reporter constructs, followed by stimulation with different cytokines. Promoter activity in the transfected cells was determined by measuring luciferase activity. Consistent with a previous report 27, we found a small twofold induction of eotaxin-1 promoter activity following IL-4 or IL-13 stimulation (Fig. 4A). There was no synergistic or additive effect of IL-4 and TNF-α for induction of eotaxin-1 promoter activity (Fig. 4A). In striking contrast, eotaxin-3 promoter activity was markedly induced 15-fold following stimulation with either IL-4 or IL-13 (p <0.005) (Fig. 4A). Addition of TNF-α had no additive effect on eotaxin-3 promoter activity. Cytokine treatment did not induce eotaxin-2 or MCP-4 promoter activities (Fig. 4A). Taken together, these data demonstrate that in bronchial epithelial cells, IL-4 and IL-13 strongly and selectively induce eotaxin-3 promoter activity (15-fold), a much lower level of the eotaxin-1 promoter activity (twofold), and have no effect on eotaxin-2 or MCP-4 promoter activities.

thumbnail image

Figure 4. Promoter activity of human eotaxin-1, -2, -3 and MCP-4. (A) Activation of chemokine promoter-reporter constructs with Th2- and Th1-type cytokines. Schematic diagrams depict the DNA constructs containing ∼1 kb promoter sequences for eotaxin-1, -2, -3 and MCP-4, the luciferase reporter (LUC), and the putative STAT6 and NF-κB binding sites. The promoter-reporter constructs were transfected into BEAS-2B cells. Following stimulation with cytokines, cells were lysed and luciferase activity was measured. Results were presented as fold induction (mean ± SD) compared with untreated controls from three independent experiments with duplicate samples for every data point in each experiment. (B) Activity of eotaxin-3 promoter STAT6 mutants. A schematic presentation of STAT6 binding site mutants is shown on the left side. BEAS-2B cells were transfected with the promoter-luciferase reporter constructs and stimulated with IL-4. M1 and M2 represent the distal and proximal STAT6 binding site mutants, respectively. The promoter activity was presented as fold induction of luciferase activity (mean ± SD) compared with untreated controls from three independent experiments with duplicate samples for each data point in each experiment. The open symbols represent no cytokine stimulation and the filled symbols represent cells stimulated with IL-4. * indicates p value <0.005, ** indicates p value <0.05 for statistically significant differences, and N.S. indicates non-significant difference.

Download figure to PowerPoint

Since STAT6 is recognized as a master regulator of allergic inflammation by controlling Th2 cell generation and trafficking, we decided to study the role of STAT6 in IL-4-induced eotaxin-3 transcription. There are two putative STAT6 binding sites in the human eotaxin-3 promoter region between positions –622 and –613 (distal) and –18 and –9 (proximal) (Fig. 4B, diagram). We engineered two mutant constructs whereby the first two nucleotides in the STAT6 binding site TT were changed to AG individually for the two STAT6 sites (Fig. 4B, diagram). While the distal STAT6 mutant M1 had no effect on eotaxin-3 promoter activity in BEAS-2B cells following IL-4 stimulation (p = 0.98), the eotaxin-3 promoter activity was completely abolished in the proximal STAT6 mutant (M2) (p <0.05) (Fig. 4B). These results demonstrate that the proximal STAT6 site is essential for eotaxin-3 promoter activity in bronchial epithelial cells stimulated with Th2-type cytokines.

Eotaxin-3 protein in the nasal mucosal epithelium of allergic human subjects

To extend our study to an in vivo setting, we examined eotaxin-3 immunoreactivity in the nasal epithelium of allergic and non-allergic human subjects using immunohistochemistry. To do so, we obtained middle turbinate mucosal biopsies from allergic and non-allergic human subjects (three patients in each group). The three allergic patients were skin test positive to perennial allergens (house dust mite). Frozen tissue sections were prepared from individual patients and stained with anti-eotaxin-3 mAb or isotype-matched control mouse IgG. In all three allergic patients, there was increased eotaxin-3 staining in the mucosal epithelium, as compared to non-allergic individuals. Fig. 5 shows staining results of one allergic patient and one non-allergic patient from each group. The increased eotaxin-3 expression was seen as the brown color staining around the mucosal epithelial cells, as compared to control antibody staining (Fig. 5, top left panel vs. top right panel). In non-allergic patients, there was weak eotaxin-3 staining in the mucosal epithelium, as compared to control antibody staining (Fig. 5, bottom left panel vs. bottom right panel).

thumbnail image

Figure 5. Immunohistochemistry staining of eotaxin-3 in nasal biopsies from allergic and non-allergic human subjects. Frozen biopsy specimens of the middle turbinate of an allergic patient (top panels) and a non-allergic patient (bottom panels) were stained with mouse anti-human eotaxin-3 mAb (left panels) and control mouse IgG (right panels). The brown color staining around the epithelial cells with anti-eotaxin-3 mAb indicates positive staining. Results represent one allergic patient and one non-allergic patient from each group with three patients per group.

Download figure to PowerPoint

Eotaxin-3 is attached to cell surface in IL-4-stimulated airway epithelial cells

To confirm that eotaxin-3 protein was produced in bronchial epithelial cells stimulated with Th2 cytokines, we first performed ELISA analysis of BEAS-2B cell culture supernatants following stimulation with IL-4 or IL-13. To our surprise, we found minimally detectable eotaxin-3 in the supernatant of IL-4-stimulated cells (data not shown). However, the eotaxin-3 protein was readily detectable by Western blot analysis in cell lysates of IL-4-stimulated BEAS-2B cells (Fig. 6A), implying that the eotaxin-3 protein was produced but either retained intracellularly or attached to the cell surface. To address this, we performed immunocytochemistry analysis on non-fixed and non-permeabilized BEAS-2B cells following IL-4 stimulation and found that eotaxin-3 protein was clearly visualized on the cell surface (Fig. 6B). These data demonstrate that eotaxin-3, a secreted chemokine with no transmembrane domain, is produced by IL-4-stimulated BEAS-2B cells and is efficiently retained on the cell surface. This is consistent with our finding that cell surface-attached eotaxin-3 in IL-4-stimulated BEAS-2B cells mediates eosinophil transepithelial migration.

thumbnail image

Figure 6. Analysis of eotaxin-3 protein in IL-4-stimulated airway epithelial cells. (A) Western blot analysis of eotaxin-3 protein in BEAS-2B cells. Total ERK (1 and 2) was used as a control for equal protein loading. (B) Immunofluorescence staining of eotaxin-3 on the surface of non-fixed, non-permeabilized BEAS-2B cells following stimulation with IL-4. Representative arrows indicate regions of intense staining consistent with surface distribution.

Download figure to PowerPoint

Eotaxin-3 cell surface association in airway epithelial cells is sensitive to heparin and salt

To study the mechanism of eotaxin-3 cell surface association in BEAS-2B cells, we first studied the effect of heparin and salt on this interaction. BEAS-2B cells were grown on tissue culture plates and stimulated with IL-4 (50 ng/mL) or IL-13 (50 ng/mL) for 18–20 h. After removing the IL-4-containing supernatant, BEAS-2B cells were washed and further incubated with fresh medium in the presence or absence of heparin (50 µg/mL). The cell supernatants were collected and the eotaxin-3 released into the supernatant was measured by ELISA. In BEAS-2B cells stimulated with either IL-4 or IL-13, there was a 6–7-fold increase in eotaxin-3 in the supernatants after incubation with heparin, as compared to no heparin treatment (Fig. 7A, top panel), indicating that the eotaxin-3 protein was released from the cell surface. Our kinetic studies showed that maximum release of surface-bound eotaxin-3 by heparin was achieved after 4 h of incubation at 37°C (data not shown). The small increase of eotaxin-3 in the supernatant after incubation with heparin in BEAS-2B cells following stimulation with TNF-α and IFN-γ is likely nonspecific because this increase is not statistically different from that with sham-treated BEAS-2B cells (Fig. 7A). These results are consistent with our Northern blot analysis (Fig. 3). Similar results were obtained in primary airway epithelial cells (NHBEC) (Fig. 7A, middle panel), suggesting that the eotaxin-3 cell surface association is a common feature in airway epithelial cells, and not a cell line-dependent phenomenon. As a comparison, we performed the same experiments in human endothelial cells. In HUVEC, IL-4 and IL-13 were potent stimuli for eotaxin-3 production, and eotaxin-3 was actively secreted into the cell supernatant (Fig. 7A, bottom panel). There was also a cell membrane-bound fraction of eotaxin-3 in HUVEC, which was releasable by heparin (Fig. 7A, bottom panel). Thus, in airway epithelial cells, eotaxin-3 is exclusively cell surface associated, whereas in vascular endothelial cells it is both secreted and cell surface bound.

thumbnail image

Figure 7. Release of cell-bound eotaxin-3 by heparin or salt. (A) Effect of heparin on cell-bound eotaxin-3 in BEAS-2B cells, NHBEC and HUVEC. Cells were either sham-treated or treated with cytokines. The cytokine-treated cell supernatants (open symbols) were collected, and the cells were further incubated with fresh medium alone (gray symbols) or fresh medium containing heparin (50 µg/mL) (filled symbols) for 4 h at 37°C. Eotaxin-3 in the supernatants before and after heparin treatment was measured by ELISA and is shown in the y axis. Data represent means ± SD from three independent experiments with triplicate samples for each data point in each experiment. (B) Effect of heparin and NaCl on cell-bound eotaxin-3 in BEAS-2B cells. BEAS-2B cells were either sham-treated or treated with IL-4. After collecting the cell supernatants for ELISA analysis, the cells were further incubated for 4 h with either fresh medium alone or fresh medium containing heparin or NaCl at the indicated concentrations. Eotaxin-3 in the supernatants was measured by ELISA and is shown in the y axis. Data represent means ± SD from three independent experiments with triplicate samples for each data point in each experiment. (C) Immunocytochemistry staining of cell surface bound eotaxin-3 in IL-4-stimulated BEAS-2B cells before and after treatment with heparin (50 µg/mL). Representative arrows indicate areas of intense staining consistent with surface distribution prior to heparin treatment, and significantly reduced staining of the same areas after heparin treatment. * indicates p value <0.001 for statistically significant difference, and N.S. indicates non-significant difference.

Download figure to PowerPoint

The surface-bound eotaxin-3 in IL-4-stimulated BEAS-2B cells was also sensitive to sodium chloride treatment, which released the surface-bound eotaxin-3 in a concentration-dependent manner (Fig. 7B). Surface staining of eotaxin-3 in IL-4-stimulated BEAS-2B cells was markedly decreased after incubation with heparin (Fig. 7C), which further supports the conclusion that the increased eotaxin-3 detected in the cell supernatant after heparin treatment was due to a release of cell surface-bound eotaxin-3.

Glycosaminoglycans participate in eotaxin-3 cell surface association

To gain insight into the molecular mechanism of eotaxin-3 cell surface association, we studied cell surface interaction of exogenous eotaxin-3 in wild-type Chinese hamster ovary cells (CHO K1) and glycosaminoglycan (GAG)-deficient CHO cells (CHO 745) using a FACS-based assay. Both CHO K1 and CHO 745 cells were first incubated with biotinylated eotaxin-3. After washing off unbound eotaxin-3, cells were incubated with allophycocyanin (APC)-conjugated streptavidin, and cell surface-bound eotaxin-3 was measured by FACS. Binding of eotaxin-3 to GAG-deficient CHO 745 cells was only 34% of eotaxin-3 binding to wild-type CHO K1 cells (Fig. 8A, B, and D). As a control, we performed similar studies using the chemokine IP-10 (CXCL10), since it has been well established that cell surface binding of IP-10 is mediated by GAG. Binding of IP-10 to GAG-deficient CHO 745 cells was only 16% of the binding of IP-10 to wild-type CHO K1 cells. Thus, GAG account for 68% of the binding for eotaxin-3 and 86% of the binding for IP-10 (p = 0.014). These results indicate that while GAG significantly contribute to eotaxin-3 cell surface association, there is also evidence for a GAG-independent component in eotaxin-3 cell surface binding.

thumbnail image

Figure 8. Cell surface binding of eotaxin-3 and IP-10 to CHO cells and BEAS-2B cells. (A) FACS histograms of cell surface binding of eotaxin-3 and IP-10. Wild-type CHO K1 cells and GAG-deficient CHO 745 cells were incubated with biotinylated eotaxin-3 or IP-10 separately, followed by APC-conjugated streptavidin. The three panels show binding of eotaxin-3 (top) and IP-10 (middle) to CHO cells, and binding of eotaxin-3 to BEAS-2B cells pretreated with pronase (lower). The x axis represents relative MFI and the y axis represents cell numbers. In the top and middle panels, dark and light lines represent wild-type and GAG-deficient CHO cells, respectively, and filled lines represent controls without chemokines. In the lower panel, the dark and light lines represent untreated and pronase-treated BEAS-2B cells, respectively, and the filled lines represent untreated controls without eotaxin-3. (B) Binding of eotaxin-3 and IP-10 to wild-type CHO K1 cells (open symbol) and GAG-deficient CHO 745 cells (filled symbol). (C) Binding of eotaxin-3 and IP-10 to BEAS-2B cells pretreated with glycosidases (upper panel) or pronase (lower panel). The open symbols represent untreated cells and the filled symbols represent either glycosidase-treated BEAS-2B cells (top panel) or pronase-treated BEAS-2B cells (lower panel). The y axis represents MFI for both (B) and (C). Data represent means from one representative experiment with triplicate samples for each data point of three independent experiments for both (B) and (C). (D) Percent of chemokine binding in GAG-deficient CHO 745 cells compared to wild-type CHO K1 cells. (E) Percent of chemokine binding in BEAS-2B cells pretreated with glycosidases (open symbol) or pronase (filled symbol) compared to untreated cells. Data represent means ± SD from three independent experiments with triplicate samples for each data point in each experiment for both (D) and (E). * indicates p value <0.05 for statistically significant difference.

Download figure to PowerPoint

To further explore the contribution of GAG to eotaxin-3 cell surface binding as well as explore the possibility that there is a protein component to eotaxin-3 cell surface binding, we treated BEAS-2B cells with glycosidases and pronase and assessed eotaxin-3 binding. Cell surface binding of eotaxin-3 was readily detectable in untreated BEAS-2B cells by FACS using biotinylated eotaxin-3 and APC-streptavidin (Fig. 8A, bottom panel). Pretreating BEAS-2B cells with glycosidases to remove cell surface GAG resulted in 58% eotaxin-3 binding, as compared to untreated BEAS-2B cells, indicating the important role of GAG in eotaxin-3 cell surface association (Fig. 8C, E). In agreement with the CHO 745 results, binding of IP-10 to glycosidase-treated BEAS-2B cells was only 36% of untreated BEAS-2B cells. Thus, GAG account for 42% of the binding for eotaxin-3 and 64% of the binding for IP-10 in BEAS-2B cells (p = 0.026), suggesting that IP-10 binding to epithelial cells is more sensitive to glycosidase treatment than eotaxin-3. However, both eotaxin-3 and IP-10 cell surface binding was equally and completely sensitive to pronase treatment, with only 10% and 12% binding compared to binding of the chemokines to untreated BEAS-2B cells, respectively (Fig. 8A, C, and E). Taken together, our data indicate that GAG contribute to eotaxin-3 cell surface association, but that there is also a GAG-independent protein component that is also involved in eotaxin-3 cell surface binding.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

One of the hallmarks of allergic airway inflammation is airway eosinophilia. Understanding the mechanisms governing eosinophil transepithelial migration will improve our understanding of allergic inflammation, since migration of blood-borne eosinophils into the airways leads to mucosal damage 13. Here, we found that eotaxin-3 was the only CCR3 ligand markedly induced by IL-4 and IL-13 in airway epithelial cells and was unique among CCR3 ligands in that it was found exclusively bound to the cell surface of airway epithelial cells. Interaction of epithelial-bound eotaxin-3 with CCR3 on eosinophils is an important mechanism in eosinophil transepithelial migration.

In a recent study by Blanchard et al.28, eotaxin-3 was found highly induced in patients with eosinophilic esophagitis, an allergic intestinal disease. Esophageal eotaxin-3 mRNA and protein levels strongly correlated with tissue eosinophilia 28. These results further strengthen our finding that eotaxin-3 plays a crucial role in eosinophil transepithelial migration and tissue eosinophilia during allergic mucosal inflammation.

Our immunohistochemistry results of increased eotaxin-3 expression in the nasal epithelium of allergic human subjects extends our in vitro studies to the in vivo setting, and are consistent with studies by Berkman et al.29 and Komiya et al.23 in asthmatic subjects. Berkman and colleagues found that eotaxin-3 was markedly up-regulated in patients with asthma 24 h after allergen challenge as demonstrated by PCR analysis of bronchial biopsies 29. Using immunohistochemistry, Komiya and colleagues demonstrated eotaxin-3 staining in bronchial epithelium isolated from asthmatic patients 23. Taken together, these results demonstrate that eotaxin-3 production is not only increased in respiratory epithelial cells following stimulation with Th2 cytokines in vitro, but it is also increased in the respiratory epithelium of allergic individuals. Consistent with the findings of Komiya et al. in bronchial epithelial cells 23, there was low-level eotaxin-3 expression in the nasal epithelium of non-allergic patients, which likely represents basal expression of eotaxin-3 in the airway epithelium.

In human vascular endothelial cells, IL-4 stimulation induces both secreted and cell surface-bound eotaxin-3 30. However, only cell-bound eotaxin-3 was critical for shear-dependent eosinophil transendothelial migration 30. Eotaxin-3 therefore is unique among the CCR3 ligands in that the functionally active form mediating eosinophil transmigration in both epithelial and endothelial cells is cell surface immobilized.

The literature on secreted eotaxin-3 by IL-4-stimulated airway epithelial cells is contradictory 2325. While both Yamamoto et al.24 and Heiman et al.25 reported ∼200 pg/mL eotaxin-3 in the cell supernatant of IL-4-stimulated airway epithelial cells, Komiya et al.23 found that the level of eotaxin-3 in IL-4-stimulated BEAS-2B cells was much higher ∼25 ng/mL. Our results are consistent with the findings by Yamamoto et al.24 and Heiman et al.25. This discrepancy may reside in different cell culture conditions. For example, presence of heparin in culture medium will increase the fraction of eotaxin-3 in the cell supernatants, as we have clearly demonstrated (Fig. 7). The secreted eotaxin-3 in the supernatant is too little to have any significant function in our assay system of eosinophil transepithelial migration.

Although the eotaxin-3 cell surface association has been previously recognized in endothelial cells 30, the molecular basis of this interaction has not been established. We found that in epithelial cells, cell surface GAG play an important, but not exclusive, role in eotaxin-3 cell surface association (Fig. 8). Chemokines have been shown to bind GAG both in vitro and in vivo31, and this interaction has important biological functions for in vivo leukocyte trafficking 3234. Enhancement of local chemokine concentration is greatly facilitated by chemokine-GAG interactions, providing a guiding signal for cell migration and trafficking during inflammation. Cell surface binding of IP-10 appears to be predominantly mediated by GAG. However, we found that there are both GAG-dependent and GAG-independent binding mediating eotaxin-3 cell surface association in airway epithelial cells, and both components are present in unstimulated epithelial cells. This mechanistic diversity in chemokine-cell surface interaction is supported by a recent report by Halden et al.35, who found that IL-8 binds to both glycosylated and non-glycosylated syndecan-2 on human endothelial cells, indicating a direct protein-protein contact in chemokine-proteoglycan interaction.

We found that there was more residual binding of eotaxin-3 and IP-10 to glycosidase-treated BEAS-2B cells than to GAG-deficient CHO 745 cells (Fig. 8D, E). This may result from incomplete digestion of glycosidases or differences in the GAG-independent binding sites between CHO and BEAS-2B cells. Nevertheless, there was significantly more binding of eotaxin-3 than IP-10 to both GAG-deficient CHO 745 cells and glycosidase-treated BEAS-2B cells, indicating that eotaxin-3 has a larger GAG-independent cell surface binding component as compared to IP-10. Furthermore, cell surface binding of both eotaxin-3 and IP-10 to BEAS-2B cells was equally and completely sensitive to pronase pretreatment, suggesting that the non-GAG cell binding sites of eotaxin-3 may be mediated by the protein component or non-GAG sugar moieties of cell surface proteoglycans, or by entirely different cell surface proteins. It has been shown that BEAS-2B cells express low levels of CCR3 receptor on their surface 36. However, we found that addition of an anti-CCR3 blocking mAb to BEAS-2B cells during IL-4 stimulation did not alter the eotaxin-3 surface binding (data not shown), which argues against a significant role of CCR3 in eotaxin-3 cell surface association in airway epithelial cells.

Regulation of eotaxin-3 promoter activity in airway epithelial cells has not been previously reported, and we provide evidence that STAT6 is critical for eotaxin-3 promoter activity in airway epithelial cells. This is of particular importance in allergic airway inflammation, because STAT6 plays a pivotal role in Th2 responses and is essential in airway inflammation in mice 3740. This STAT6-dependent regulation of eotaxin-3 expression likely has broader implications, since STAT6 also mediates the activation of eotaxin-3 gene expression in human dermal fibroblasts 41. Thus, modulation of eosinophil recruitment by targeting STAT6-dependent eotaxin-3 expression may offer a novel therapeutic approach for both allergic airway disease and atopic dermatitis.

Overall, our findings establish that eotaxin-3 is the predominant CCR3 ligand markedly induced in epithelial cells following Th2 stimulation and its expression is dependent on the transcription factor STAT6. The eotaxin-3 protein is predominantly cell surface bound in epithelial cells via cell surface GAG and protein-protein interactions, and the membrane-immobilized eotaxin-3 is the functionally active CCR3 ligand mediating eosinophil transepithelial migration. Our results provide insight into understanding the molecular mechanism of allergic airway inflammation and offer novel targets for therapeutic interventions in asthma and related allergic diseases.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Cells

The BEAS-2B cell line was derived from human bronchial epithelial cells transformed with a hybrid adeno-SV40 virus, and the A549 cell line was derived from a human lung adenocarcinoma with the alveolar cell type II phenotype. Both were maintained in F12 medium. NHBEC and HUVEC were purchased from Clonetics (San Diego, CA). The NHBEC were cultured in BEBM®-Bronchial Epithelial Medium supplemented with the BulletKit® (both from Cambrex Bio Science Walkersville, Inc., Walkersville, MD). The HUVEC were cultured using the Umbilical Vein Endothelial Cell Systems (Cambrex). Both wild-type CHO (CHO K1) and GAG-deficient CHO (CHO 745) cells were purchased from American Type Culture Collection (Manassas, VA) and maintained in complete HAM medium (Gibco) according to the manufacturer's instructions. All media were supplemented with 10% FCS, 50 U/mL penicillin, 50 µg/mL streptomycin, and 2 mM L-glutamine.

Isolation of eosinophils from peripheral human blood

Eosinophils were isolated from peripheral human blood of six healthy volunteers according to a previously published method 42. The protocol for obtaining human peripheral blood was reviewed and approved by Human Research Committee at Massachusetts General Hospital. The purity of isolated eosinophils was greater that 97% in all experiments.

Northern blot analysis

RNA from cultured cells was isolated using RNA-Stat 60 (Tel Test-B, Inc., Friendswood, TX). Total RNA (15 µg) was fractionated on a 1.2% agarose gel containing 0.2 M formaldehyde and transferred to GeneScreen membranes and hybridized with [32P]-dCTP Klenow-labeled random-primed probes for human eotaxin-1, -2, -3 and MCP-4.

Cloning of promoter reporter constructs

DNA fragments containing the human eotaxin-1, -2, -3 and MCP-4 promoters (∼1 kb) were amplified from genomic DNA using PCR primers. The PCR fragments were digested with Kpn I and Xho I and cloned into pGL-2 basic vector. The eotaxin-3 promoter proximal and distal STAT6 binding site mutants were engineered using the Transformer Site-Directed Mutagenesis Kit (Clontech, Palo Alto, CA). The primer 5′-TAACTTAGTTCAGAGCACTGGAATATTAC-3′ was used for the distal STAT6 binding site mutation (M1) and the primer 5′-CAACCACAGAAAGCTCTGGAATTG-3′ was used for the proximal STAT6 binding site mutation (M2). All the plasmids were analyzed by digestion with restriction endonucleases and DNA sequencing for sequence confirmation. Constructs for transient transfection were prepared using the Qiagen Plasmid Maxi kit (Qiagen, Santa Clarita, CA).

Promoter activity assay

BEAS-2B cells were transiently transfected with promoter-reporter construct using lipofectin. For the cytokine stimulation experiments, the indicated concentrations of cytokines were added to transfected cells for 18 h. Cells were then harvested and cell lysates were analyzed for luciferase activity using the Promega Luciferase assay system.

Chemotaxis assay

Chemotaxis assays of human eosinophils were performed in 96-well Neuroprobe chemotaxis chambers with 5 µM pore size polycarbonate membranes (Neuroprobe, Gaithersburg, MD), following the manufacturer's directions. The membrane, which has 96 pre-marked circles to perfectly match the 96 wells of the bottom plastic plate, was placed directly on top of the 96-well plate provided by the manufacturer. Eosinophils (25,000 cells) were placed onto the top of the membrane in 25 µL RPMI containing 1% BSA (Sigma). The chambers were then incubated at 37°C for 1 h. For the transmigration assay of human eosinophils through BEAS-2B cells, 2 days prior to the chemotaxis assay, 2 × 104 BEAS-2B cells in 50 µL medium were plated onto each spot of a 96-well Neuroprobe chemotaxis membrane and incubated at 37°C (each spot has been circled with black ink by the manufacturer). At 18 h prior to the chemotaxis assay, the BEAS-2B cells were stimulated with IL-4 (50 ng/mL) in 50 µL at 37°C. The confluence of BEAS-2B cell monolayers was evaluated by Diff-Quick staining after the completion of each experiment and was >95%. Prior to adding eosinophils to the top of the chamber, the BEAS-2B cells were washed twice with RPMI containing 1% BSA. For preparing the BEAS-2B cell monolayers when grown on top of the filter membrane, we added BEAS-2B cells to the top of each spot of the filter membrane. When grown on the bottom of the filter membrane, we first flipped the filter membrane (bottom up) and placed it on top of the 96-well plastic chamber. We then added BEAS-2B cells to each spot. For chemotaxis assays, we aspirated the residual volume from each spot, washed each spot twice with fresh chemotaxis medium (RPMI with 1% BSA) and flipped the filter membrane to the original orientation. In this manner, the BEAS-2B cell monolayer was on the bottom of the filter membrane with the basal surface face up in contact with the added eosinophils. In the experiments with addition of exogenous chemokines, individual chemokines (eotaxin-1, -2, and -3; all from PeproTech, Rocky Hill, NJ) were added to the basal surface of the cells for 3 h and washed twice prior to the chemotaxis assay.

Migrated eosinophils in the bottom chambers were enumerated by manual counting of the entire well in a random fashion using an Olympus microscope with a counting grid attached to the eyepiece. The chemotactic index was calculated as previously described 43, by dividing the numbers of eosinophils migrated in experimental wells by the numbers of eosinophils migrated in sham-treated wells. Assay results were presented as means ± SD of three independent experiments. There were at least three wells for each individual data point in each experiment to minimize intra-well variability. For antibody blocking experiments, anti-human CCR3 mAb (rat IgG2a, clone 61828.111, catalog no. MAB155; R&D Systems, Minneapolis, MN), anti-human eotaxin-3 mAb (mouse IgG1; PeproTech), anti-human eotaxin-1 mAb (mouse IgG1; PeproTech), and control antibodies were added to both the top and bottom of the chambers at the appropriate concentrations for the entire 1-h assay.

Western blot analysis

BEAS-2B cells, following stimulation with IL-4 (50 ng/mL) for 18–20 h, were lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, and 1% Triton-X 100) supplemented with protease inhibitors (0.1 µg/mL pepstatin A, 0.03 mM leupeptin, 145 mM benzamidine, 0.37 µg/mL aprotinin, and 1 mM PMSF; all from Sigma). To analyze eotaxin-3 protein expression, 150 µg of total protein of each sample was boiled for 5 min in Tricine Sample buffer (Bio-Rad) and 4 M urea, and fractionated on a 12% peptide Criterion Precast Gel (Bio-Rad) run in electrophoresis buffer containing 0.1 M Tris, 0.1 M Tricine, and 3.5 mM SDS. Proteins were transferred onto a PVDF membrane (NEN, Boston, MA) and eotaxin-3 protein was identified by sequentially incubating the membrane with a 1 : 10,000 dilution of an affinity-purified polyclonal goat anti-human eotaxin-3 antibody (PeproTech) followed by a 1 : 3000 dilution of an HRP-conjugated rabbit anti-goat Ig antibody (Bio-Rad), and developed using an ECL kit (Amersham Pharmacia, Piscataway, NJ). Total ERK protein was used as a control for equal protein loading.

ELISA analysis

Eotaxin-3 was measured by ELISA according to the manufacturer's instructions (R&D Systems) with separate standard curves in the presence or absence of heparin (50 µg/mL).

Immunofluorescent staining of adherent BEAS-2B cells

BEAS-2B cells were plated on glass coverslips and stimulated with IL-4 (50 ng/mL) for 18 h. The cells on the coverslips were placed on ice for the entire staining process. After rinsing with ice-cold PBS, the cells were incubated with PBS containing 1% FCS for 30 min to block nonspecific antibody binding. Without fixation or permeabilization, the cells were further incubated for 45 min with PBS containing 1% FCS and 10 µg/mL mouse mAb against human eotaxin-3 (PeproTech), or 10 µg/mL mouse IgG1 isotype-matched control mAb. The cells were then washed with cold PBS and incubated with FITC-conjugated goat anti-mouse IgG (1 : 100 dilution) for 30 min. After staining, the cells were washed with cold PBS, fixed in 2% paraformaldehyde for 10 min on ice, mounted with Vectashield mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA) and analyzed with a Nikon fluorescent microscope.

Immunohistochemistry staining of eotaxin-3 in nasal biopsies of human subjects

After receiving informed consent, six healthy subjects underwent allergy skin testing with a panel of aeroallergens, including trees, grasses, weeds, house dust mite, cat, dog and fungi. Three subjects were non-allergic and three were allergic to perennial allergens (house dust mite). Each subject also underwent a superficial (2–3 mm) biopsy of the middle turbinate. The protocol for obtaining human tissue biopsies was reviewed and approved by Human Research Committee at Massachusetts General Hospital. The biopsy specimens were frozen in OCT compound (Ames Company, Elkhart, IN) and stored at –80°C until use. Frozen tissue sections (4 µm thick) were prepared, fixed in acetone, and blocked with horse serum. The slides were stained with mouse anti-human eotaxin-3 mAb (Pepro Tech) or control mouse IgG, treated with hydrogen peroxide and then incubated with biotinylated horse anti-mouse IgG. The slides were further incubated with ABC reagent and developed in AEC (Aldrich). The photographs were taken using a Nikon microscope with a 20× objective.

Binding of eotaxin-3 and IP-10 to BEAS-2B and CHO cells

Eotaxin-3 (PeproTech) and IP-10 (CXCL10) 44 were biotinylated with EZ-Link Sulfo-NHS-Biotin (N-hydroxysulfosuccinimidobiotin) (Pierce) at a 1 : 5 molar ratio in 50 mM sodium phosphate buffer at pH 6.5 for IP-10; pH 7.5 for eotaxin-3. After incubation on ice for 3 h, the reaction was quenched by the addition of 50 mM Tris, pH 7.0. Adherent BEAS-2B cells and CHO cells were harvested from tissue culture flasks using non-enzymatic Cell Dissociation Solution (Sigma). For glycosidase treatment, 5 × 105 BEAS-2B cells were treated in suspension (100 µL of complete medium) with a combination of heparinase I (20 U), heparinase III (5 U) and chondroitinase ABC (5 U) (all from Sigma) at 37°C for 2 h. For pronase treatment, 1.25 × 106 BEAS-2B cells were treated in suspension (1 mL serum-free medium) with pronase (Sigma) at 20 µg/mL for 20 min at 37°C. Cells were then washed and resuspended in complete medium at 1 × 106/mL. Biotinylated eotaxin-3 or IP-10 was added at a concentration of 5000 ng/mL to 150 µL of cells and the mixture was incubated at 37°C for 1 h. After washing with cold PBS, cells were stained with APC-conjugated streptavidin for 20 min at 4°C and washed again. Chemokine cell surface binding was measured by flow cytometry using a FACScan machine (BD). The percent binding of chemokine in GAG-deficient CHO 745 cells or glycosidase- and pronase-treated BEAS-2B cells was determined by dividing the mean fluorescent intensity (MFI) of CHO 745 or treated BEAS-2B cells with the MFI of wild-type CHO K1 cells or untreated BEAS-2B cells, respectively. Results represent means ± SD from three independent experiments.

Statistical analysis

Student's t-test (unpaired, two-tailed) was used to calculate significance levels for all measurements. p <0.05 was considered to be statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

This work was supported by the Janeway Award from Children's Hospital Boston (Q.Y.), by the Dana Foundation (A.D.L.), and by National Institutes of Health grants K08-DK68085 to Q.Y. and R01-AI40618 to A.D.L. The authors would like to thank Nicole L. Brousaides and Jeiti Sun for their excellent technical assistance.

  • 1

    WILEY-VCH

  • 2

    WILEY-VCH

  • 3

    WILEY-VCH

  • 4

    WILEY-VCH

  • 5

    WILEY-VCH

  • 6

    WILEY-VCH

  • 7

    WILEY-VCH

  • 8

    WILEY-VCH

  • 1
    Wills-Karp, M., Immunologic basis of antigen-induced airway hyperresponsiveness. Annu. Rev. Immunol. 1999. 17: 255281.
  • 2
    Del Prete, G. F., De Carli, M., D'Elios, M. M., Maestrelli, P., Ricci, M., Fabbri, L. and Romagnani, S., Allergen exposure induces the activation of allergen-specific Th2 cells in the airway mucosa of patients with allergic respiratory disorders. Eur. J. Immunol. 1993. 23: 14451449.
  • 3
    Robinson, D. S., Hamid, Q., Ying, S., Tsicopoulos, A., Barkans, J., Bentley, A. M., Corrigan, C. et al., Predominant Th2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 1992. 326: 298304.
  • 4
    Walker, C., Bode, E., Boer, L., Hansel, T. T., Blaser, K. and Virchow, J. C., Jr., Allergic and nonallergic asthmatics have distinct patterns of T-cell activation and cytokine production in peripheral blood and bronchoalveolar lavage. Am. Rev. Respir. Dis. 1992. 146: 109115.
  • 5
    Luster, A. D., Chemokines – chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 1998. 338: 436445.
  • 6
    Heath, H., Qin, S., Rao, P., Wu, L., LaRosa, G., Kassam, N., Ponath, P. D. and Mackay, C. R., Chemokine receptor usage by human eosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody. J. Clin. Invest. 1997. 99: 178184.
  • 7
    Garcia-Zepeda, E. A., Rothenberg, M. E., Ownbey, R. T., Celestin, J., Leder, P. and Luster, A. D., Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nat. Med. 1996. 2: 449456.
  • 8
    Garcia-Zepeda, E. A., Combadiere, C., Rothenberg, M. E., Sarafi, M. N., Lavigne, F., Hamid, Q., Murphy, P. M. and Luster, A. D., Human monocyte chemoattractant protein (MCP)-4 is a novel CC chemokine with activities on monocytes, eosinophils, and basophils induced in allergic and nonallergic inflammation that signals through the CC chemokine receptors (CCR)-2 and -3. J. Immunol. 1996. 157: 56135626.
  • 9
    Kitaura, M., Nakajima, T., Imai, T., Harada, S., Combadiere, C., Tiffany, H. L., Murphy, P. M. and Yoshie, O., Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. J. Biol. Chem. 1996. 271: 77257730.
  • 10
    Forssmann, U., Uguccioni, M., Loetscher, P., Dahinden, C. A., Langen, H., Thelen, M. and Baggiolini, M., Eotaxin-2, a novel CC chemokine that is selective for the chemokine receptor CCR3, and acts like eotaxin on human eosinophil and basophil leukocytes. J. Exp. Med. 1997. 185: 21712176.
  • 11
    Kitaura, M., Suzuki, N., Imai, T., Takagi, S., Suzuki, R., Nakajima, T., Hirai, K. et al., Molecular cloning of a novel human CC chemokine (eotaxin-3) that is a functional ligand of CC chemokine receptor 3. J. Biol. Chem. 1999. 274: 2797527980.
  • 12
    Shinkai, A., Yoshisue, H., Koike, M., Shoji, E., Nakagawa, S., Saito, A., Takeda, T. et al., A novel human CC chemokine, eotaxin-3, which is expressed in IL-4-stimulated vascular endothelial cells, exhibits potent activity toward eosinophils. J. Immunol. 1999. 163: 16021610.
  • 13
    Teran, L. M., CCL chemokines and asthma. Immunol. Today 2000. 21: 235242.
  • 14
    Wenzel, S. E., Trudeau, J. B., Barnes, S., Zhou, X., Cundall, M., Westcott, J. Y., McCord, K. and Chu, H. W., TGF-beta and IL-13 synergistically increase eotaxin-1 production in human airway fibroblasts. J. Immunol. 2002. 169: 46134619.
  • 15
    Sato, E., Nelson, D. K., Koyama, S., Hoyt, J. C. and Robbins, R. A., Inflammatory cytokines modulate eotaxin release by human lung fibroblast cell line. Exp. Lung Res. 2001. 27: 173183.
  • 16
    Nakamura, H., Luster, A. D., Tateno, H., Jedrzkiewicz, S., Tamura, G., Haley, K. J., Garcia-Zepeda, E. A. et al., IL-4 differentially regulates eotaxin and MCP-4 in lung epithelium and circulating mononuclear cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 2001. 281: L1288L1302.
  • 17
    Lilly, C. M., Nakamura, H., Kesselman, H., Nagler-Anderson, C., Asano, K., Garcia-Zepeda, E. A., Rothenberg, M. E. et al., Expression of eotaxin by human lung epithelial cells: Induction by cytokines and inhibition by glucocorticoids. J. Clin. Invest. 1997. 99: 17671773.
  • 18
    Fujisawa, T., Kato, Y., Atsuta, J., Terada, A., Iguchi, K., Kamiya, H., Yamada, H. et al., Chemokine production by the BEAS-2B human bronchial epithelial cells: Differential regulation of eotaxin, IL-8, and RANTES by Th2- and Th1-derived cytokines. J. Allergy Clin. Immunol. 2000. 105: 126133.
  • 19
    Rothenberg, M. E., MacLean, J. A., Pearlman, E., Luster, A. D. and Leder, P., Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue eosinophilia. J. Exp. Med. 1997. 185: 785790.
  • 20
    Tomkinson, A., Duez, C., Cieslewicz, G. and Gelfand, E. W., Eotaxin-1-deficient mice develop airway eosinophilia and airway hyperresponsiveness. Int. Arch. Allergy Immunol. 2001. 126: 119125.
  • 21
    Watanabe, K., Jose, P. J. and Rankin, S. M., Eotaxin-2 generation is differentially regulated by lipopolysaccharide and IL-4 in monocytes and macrophages. J. Immunol. 2002. 168: 19111918.
  • 22
    Pope, S. M., Fulkerson, P. C., Blanchard, C., Akei, H. S., Nikolaidis, N. M., Zimmermann, N., Molkentin, J. D. and Rothenberg, M. E., Identification of a cooperative mechanism involving interleukin-13 and eotaxin-2 in experimental allergic lung inflammation. J. Biol. Chem. 2005. 280: 1395213961.
  • 23
    Komiya, A., Nagase, H., Yamada, H., Sekiya, T., Yamaguchi, M., Sano, Y., Hanai, N. et al., Concerted expression of eotaxin-1, eotaxin-2, and eotaxin-3 in human bronchial epithelial cells. Cell. Immunol. 2003. 225: 91100.
  • 24
    Yamamoto, S., Kobayashi, I., Tsuji, K., Nishi, N., Muro, E., Miyazaki, M., Zaitsu, M. et al., Upregulation of interleukin-4 receptor by interferon-gamma: Enhanced interleukin-4-induced eotaxin-3 production in airway epithelium. Am. J. Respir. Cell. Mol. Biol. 2004. 31: 456462.
  • 25
    Heiman, A. S., Abonyo, B. O., Darling-Reed, S. F. and Alexander, M. S., Cytokine-stimulated human lung alveolar epithelial cells release eotaxin-2 (CCL24) and eotaxin-3 (CCL26). J. Interferon Cytokine Res. 2005. 25: 8291.
  • 26
    Lamkhioued, B., Garcia-Zepeda, E. A., Abi-Younes, S., Nakamura, H., Jedrzkiewicz, S., Wagner, L., Renzi, P. M. et al., Monocyte chemoattractant protein (MCP)-4 expression in the airways of patients with asthma. Induction in epithelial cells and mononuclear cells by proinflammatory cytokines. Am. J. Respir. Crit. Care Med. 2000. 162: 723732.
  • 27
    Matsukura, S., Stellato, C., Plitt, J. R., Bickel, C., Miura, K., Georas, S. N., Casolaro, V. and Schleimer, R. P., Activation of eotaxin gene transcription by NF-kappa B and STAT6 in human airway epithelial cells. J. Immunol. 1999. 163: 68766883.
  • 28
    Blanchard, C., Wang, N., Stringer, K. F., Mishra, A., Fulkerson, P. C., Abonia, J. P., Jameson, S. C. et al., Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis. J. Clin. Invest. 2006. 116: 536547.
  • 29
    Berkman, N., Ohnona, S., Chung, F. K. and Breuer, R., Eotaxin-3 but not eotaxin gene expression is upregulated in asthmatics 24 hours after allergen challenge. Am. J. Respir. Cell. Mol. Biol. 2001. 24: 682687.
  • 30
    Cuvelier, S. L. and Patel, K. D., Shear-dependent eosinophil transmigration on interleukin 4-stimulated endothelial cells: A role for endothelium-associated eotaxin-3. J. Exp. Med. 2001. 194: 16991709.
  • 31
    Handel, T. M., Johnson, Z., Crown, S. E., Lau, E. K. and Proudfoot, A. E., Regulation of protein function by glycosaminoglycans – as exemplified by chemokines. Annu. Rev. Biochem. 2005. 74: 385410.
  • 32
    Proudfoot, A. E., Handel, T. M., Johnson, Z., Lau, E. K., LiWang, P., Clark-Lewis, I., Borlat, F. et al., Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc. Natl. Acad. Sci. USA 2003. 100: 18851890.
  • 33
    Johnson, Z., Kosco-Vilbois, M. H., Herren, S., Cirillo, R., Muzio, V., Zaratin, P., Carbonatto, M. et al., Interference with heparin binding and oligomerization creates a novel anti-inflammatory strategy targeting the chemokine system. J. Immunol. 2004. 173: 57765785.
  • 34
    Wang, L., Fuster, M., Sriramarao, P. and Esko, J. D., Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses. Nat. Immunol. 2005. 6: 902910.
  • 35
    Halden, Y., Rek, A., Atzenhofer, W., Szilak, L., Wabnig, A. and Kungl, A. J., Interleukin-8 binds to syndecan-2 on human endothelial cells. Biochem. J. 2004. 377: 533538.
  • 36
    Stellato, C., Brummet, M. E., Plitt, J. R., Shahabuddin, S., Baroody, F. M., Liu, M. C., Ponath, P. D. and Beck, L. A., Expression of the C-C chemokine receptor CCR3 in human airway epithelial cells. J. Immunol. 2001. 166: 14571461.
  • 37
    Kaplan, M. H., Schindler, U., Smiley, S. T. and Grusby, M. J., STAT6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 1996. 4: 313319.
  • 38
    Akimoto, T., Numata, F., Tamura, M., Takata, Y., Higashida, N., Takashi, T., Takeda, K. and Akira, S., Abrogation of bronchial eosinophilic inflammation and airway hyperreactivity in signal transducers and activators of transcription (STAT)6-deficient mice. J. Exp. Med. 1998. 187: 15371542.
  • 39
    Tomkinson, A., Kanehiro, A., Rabinovitch, N., Joetham, A., Cieslewicz, G. and Gelfand, E. W., The failure of STAT6-deficient mice to develop airway eosinophilia and airway hyperresponsiveness is overcome by interleukin-5. Am. J. Respir. Crit. Care Med. 1999. 160: 12831291.
  • 40
    Mathew, A., MacLean, J. A., DeHaan, E., Tager, A. M., Green, F. H. and Luster, A. D., Signal transducer and activator of transcription 6 controls chemokine production and T helper cell type 2 cell trafficking in allergic pulmonary inflammation. J. Exp. Med. 2001. 193: 10871096.
  • 41
    Hoeck, J. and Woisetschlager, M., Activation of eotaxin-3/CCLl26 gene expression in human dermal fibroblasts is mediated by STAT6. J. Immunol. 2001. 167: 32163222.
  • 42
    Yuan, Q., Austen, K. F., Friend, D. S., Heidtman, M. and Boyce, J. A., Human peripheral blood eosinophils express a functional c-kit receptor for stem cell factor that stimulates very late antigen 4 (VLA-4)-mediated cell adhesion to fibronectin and vascular cell adhesion molecule 1 (VCAM-1). J. Exp. Med. 1997. 186: 313323.
  • 43
    Bromley, S. K., Thomas, S. Y. and Luster, A. D., Chemokine receptor CCR7 guides T cell exit from peripheral tissues and entry into afferent lymphatics. Nat. Immunol. 2005. 6: 895901.
  • 44
    Campanella, G. S., Lee, E. M., Sun, J. and Luster, A. D., CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10). J. Biol. Chem. 2003. 278: 1706617074.