Ablation of type I hypersensitivity in experimental allergic conjunctivitis by eotaxin-1/CCR3 blockade

Abstract

The immune response is regulated, in part, by effector cells whose activation requires multiple signals. For example, T cells require signals emanating from the T cell antigen receptor and co-stimulatory molecules for full activation. Here, we present evidence indicating that IgE-mediated hypersensitivity reactions in vivo also require cognate signals to activate mast cells. Immediate hypersensitivity reactions in the conjunctiva are ablated in mice deficient in eotaxin-1, despite normal numbers of tissue mast cells and levels of IgE. To further define the co-stimulatory signals mediated by chemokine receptor 3 (CCR3), an eotaxin-1 receptor, effects of CCR3 blockade were tested with an allergic conjunctivitis model and in ex vivo isolated connective tissue-type mast cells. Our results show that CCR3 blockade significantly suppresses allergen-mediated hypersensitivity reactions as well as IgE-mediated mast cell degranulation. We propose that a co-stimulatory axis by CCR3, mainly stimulated by eotaxin-1, is pivotal in mast cell-mediated hypersensitivity reactions.

Introduction

Allergic diseases affect approximately one-third of the population and constitute one of the major health care problems in the Western world (1). IgE-mediated immediate hypersensitivity reactions (e.g. asthma, conjunctivitis, rhinitis and dermatitis) can affect any mucosal tissue and can have serious consequences (e.g. systemic anaphylaxis). Costs associated with allergic diseases dominate public health budgets. Moreover, current treatments do not provide complete solutions because of their insufficient effectiveness, poor persistence of effects or unwanted side effects. A comprehensive understanding of the cellular and molecular events underlying the pathogenesis of allergic disease could greatly facilitate improvement of current treatment strategies and development of innovative therapeutics.

The allergic events following allergen exposure in a sensitized individual take place in two phases: an acute phase, occurring within minutes, and a late phase, spanning from 6 to 72 h after exposure. The acute-phase reaction stems primarily from the release of preformed and inducible mediators of immediate hypersensitivity following the cross-linking of allergen-specific IgE bound to the surface of resident mast cells (2). In addition to histamine and mast cell tryptases, the sulfidopeptide leukotrienes LTC4, LTD4 and LTE4 contribute to clinical symptoms associated with the acute-phase reaction (3, 4). The late-phase reaction, characterized by the infiltration of inflamed tissue with eosinophils, basophils, T cells, neutrophils and macrophages, occurs in most cases of allergic inflammation, and there is a general correlation between the degree of cellular infiltration and the severity of disease. Indeed, products from infiltrating cells are known to damage the epithelium, promote bronchial hyper-responsiveness (5), conjunctival irritation and mast cell/basophil secretion (6).

The discovery of eotaxin-1 and other CC chemokines has helped to explain the molecular basis of chemoattraction in the late-phase reaction (7–11). Chemokines are a large group of cytokines that promote leukocyte trafficking into inflammatory sites, regulate hematopoeisis and activate leukocyte function. The CC chemokines such as eotaxin-1 (CCL11), eotaxin-2 (CCL24), RANTES (CCL5), monocyte chemoattractant protein-3 (MCP-3, CCL7), MCP-4 (CCL13) and macrophage inflammatory protein-1 alpha (MIP-1α, CCL3) have been implicated in driving the late-phase reaction (12, 13). Recently, eotaxin-1 and eotaxin-2 have been shown to play distinctive roles in eosinophilic lung inflammation. Eotaxin-2, induced by IL-13, was shown to be a pivotal mediator of lung eosinophilia, whereas eotaxin-1 appeared to serve a regulatory function; both chemokines exerted their effects via chemokine receptor 3 (CCR3) (14). Interestingly, these CCR3 ligands appear to exert unique roles depending on target organs or tissue structure (15). We have recently reported that MIP-1α serves as a co-stimulatory signal for mast cell degranulation and is a critical acute-phase mediator (16–18). Still, little information has been obtained about the roles of CC chemokines in early-phase reactions, in contrast to the progress made in dissecting the roles of particular chemokines in late-phase reactions (19).

Previously, Romagnani et al. have suggested that eotaxin-1 serves as a differentiation or homing factor for connective tissue-type mast cells; indeed, the authors propose that mast cells differentiate into their connective tissue type under the influence of fibroblast-derived factors, including stem cell factor (SCF) and eotaxin-1 (20–24). This theory is supported by the selective expression of CCR3 on this category of mast cells and by the effects of CCR3 on expression of specific proteases. We have demonstrated that pharmacological inhibition of CCR3 almost completely suppresses mast cell-mediated immediate hypersensitivity in allergen-sensitized mice, as evaluated by clinical scores, Evans Blue dye extravasation and conjunctival mast cell degranulation (25). This finding further suggests a role for eotaxin-1–CCR3 signaling in mast cell function.

Although eotaxin-1 is recognized as a homeostatic factor for mast cells, its potential involvement in mast cell activation remains controversial, as CCR3-bearing mast cells are refractory to eotaxin-1-induced degranulation. Additionally, in vitro-established, CCR3-transfected mast cell lines such as RBL-2H3 or MC-9 have notoriously scarce expression of CCR3 on their cell surface. These cell properties hamper detailed analysis of molecular events downstream of the eotaxin-1 and IgE-mediated signals in mast cells. Even with approaches employing in vitro-differentiated mast cells taken from bone marrow, heterogeneity of mast cell lineage, which can vary depending on the differentiation regimen or mucosal environment, complicates proper interpretation of the eotaxin-1 impact on the physiological process of allergen-stimulated mast cell degranulation in vivo. To overcome shortcomings of analyses using established cell lines or conventional mast cell preparations such as bone marrow-derived mast cells, we isolated and examined properties of mature connective tissue mast cells.

In this paper, we describe experiments that show the crucial role of the eosinophilic CC chemokine eotaxin-1 as a signal for mast cell priming. Eotaxin-1 was rapidly induced after allergen challenge, and this expression was required for optimal mast cell degranulation. Mice deficient in eotaxin-1 exhibited greatly diminished clinical symptoms in the acute-phase reaction. This ablation of the immediate hypersensitivity reaction was not due to decreased numbers of resident mast cells or impaired IgE/IgG₁ priming; rather, it appeared to be caused by a failure of mast cell degranulation in situ. Passive sensitization experiments using ex vivo mast cells demonstrated that neutralization of eotaxin-1 in sensitized mast cells inhibits mast cell degranulation. Elevated expression of CCR3 on isolated mast cells from the conjunctiva or the skin was confined to high affinity IgE receptor (FcεRI)^high subset. Functionally, CCR3 blockade by mAb or specific CCR3 antagonist significantly suppressed IgE-mediated degranulation of isolated mast cells. We propose that the involvement of CCR3 in mast cell activation by eotaxin-1 is a new critical component of the mechanism of action of the acute-phase reaction in ocular allergy.

Materials and methods

Animals

BALB/c, SWR/J and 129/SvEv mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Eotaxin-1-deficient mice were maintained inbred on either 129/SvEv or BALB/c backgrounds (26). Control wild-type mice were age and sex matched and maintained under identical conditions. The present study conformed to all regulations for laboratory animal research outlined by the Animal Welfare Act, NIH guidelines and the Association for Research in Vision and Ophthalmology statement regarding the experimental use of animals and was approved by the Home Office (London, UK).

Induction of allergic inflammation in the conjunctiva

129/SvEv mice were sensitized using a protocol based on those that we have previously reported (18, 25, 27, 28). For repeated sensitization, mice were injected intra-peritoneally with 1 mg of aluminum hydroxide conjugated with Fel d1 extract (2000 AU per mouse; ALK Laboratories, Hørsholm, Denmark) on days 1, 14 and 24. Concomitantly, aluminum hydroxide-conjugated Fel d1 extract (1000 AU μl⁻¹, 25 μg per eye) was topically administered onto the eye on days 1, 2, 3, 7 and 14. Thereafter, mice were topically challenged once per week with Fel d1 extract without alum (1000 AU μl⁻¹). Eight weeks after the initial sensitization, affinity-purified Fel d1 was instilled onto the eyes (0.5 mg ml⁻¹, 10 μl per eye) for three consecutive days for the final challenge (days 56–57). Control mice were mock sensitized in a similar manner using saline and challenged using antigen solution. The specificity of the responses was confirmed by challenging sensitized mice with irrelevant antigens. After the final challenge, the clinical responses were recorded within the first 30 min and graded using the criteria described in our previous reports, with modifications detailed here (29). The symptoms were evaluated in a double-blinded fashion and graded 0–4 by an ophthalmologist unaware of the identity of each mouse using defined criteria (Supplementary Table 1, available at International Immunology Online). The cumulative clinical score was calculated as the sum of the scores of each of these four parameters (0–16).

For evaluation of the effector-phase contribution to allergen-induced clinical symptoms, a single exposure protocol was used (28). For active immunization, mice were injected with a suspension of 50 μg of ragweed pollen (ICN, Aurora, OH, USA) and 1 mg of aluminum hydroxide (Sigma, St Louis, MO, USA) into the left hind footpad under anesthesia. On day 22, conjunctivitis was induced by topical application of 1.5 mg ragweed suspended in 10 μl of PBS. For passive immunization, mice were intravenously injected with monoclonal ragweed-specific IgE (3.5 μg per mouse) (30) and challenged with the ragweed suspension on the following day.

Control mice were mock sensitized and challenged identically with ragweed suspension. For CCR3 blockade of the effector phase, anti-CCR3 antibody (clone 83103, R&D Systems, Minneapolis, MN, USA) was intravenously administered (on days 21 and 22, total of 120 μg per mouse) before allergen challenge.

For pharmacological inhibition of CCR3, the specific CCR3 antagonist W-56750, [4-(3-aminophenyl)thiazol-2-ylthio]-N-[1-(3,4-dichlorobenzyl)piperidin-4-yl] acetamide (Mitsubishi Tanabe Pharma Co.) was used (25). W-56750, a benzylpiperidine compound, inhibits eotaxin-induced intracellular calcium influx in human eosinophils with an IC₅₀ of 3.8 nM. W-56750 antagonizes not only human CCR3 but also murine CCR3 with 50% inhibitory concentration (IC₅₀) values of ∼235 nM (H. Higashi, M. Murata and S. Takeda, unpublished data). W-56750 has no affinity for other GPCRs, including CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, PAF and LTD₄ (H. Higashi, M. Murata and S. Takeda, unpublished data).

For histological evaluation of immediate hypersensitivity reactions, mice were sacrificed and then tissues were collected, fixed in 4% PFA and embedded in Historesin (Leica Instruments GmbH). Serial sagittal sections (3 μm in thickness) were stained with toluidine blue, Giemsa or hematoxylin and eosin. Three consecutive conjunctival tissue sections from each eye were examined, and mast cells and other inflammatory cells were counted under a 200× field microscope by an independent scientist in a double-blinded fashion.

Immunohistochemistry

Serial frozen sections (10 μm in thickness) were stained for CCR3 and FcεRI using monoclonal anti-CCR3 antibody (R&D Systems) and FITC-conjugated anti-FcεRI α subunit (eBiosciences, San Diego, CA, USA). CCR3 was labeled with Alexa Fluor 555 using Tyramide Signal Amplification Kit (Invitrogen, Carlsbad, CA, USA). For mouse mast cell protease-5 (mMCP-5) and mMCP-6 staining, eyes perfusion fixed using 4% PFA were embedded in paraffin and serially sectioned. After antigen retrieval by microwave treatment, serial sections were stained using polyclonal antibodies to mMCP-5 and mMCP-6 (generous gifts from M. F. Gurish, Brigham and Women's Hospital). The positive signals were detected using Alexa Fluor 488-conjugated anti-IgG antibody.

Real-time reverse transcription–PCR

Total conjunctival RNA or isolated mast cell RNA was reverse transcribed using superscript III (Invitrogen). The cDNAs were amplified and quantified by LightCycler (Roche, Mannheim, Germany) using QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA, USA). Primers for IL-3R were 5′-GAAGGGCAGGGACATCTTT and 5′-AGAGGGAGAGCGACTGGAAT; for CCL2, 5′-CATCCACGTGTTGGCTCA and 5′-GATCATCTTGCTGGTGAATGAGT; for CCL3, 5′-TGCCCTTGCTGTTCTTCTCT and 5′-GTGGAATCTTCCGGCTGTAG; for CCL4, 5′-GCCCTCTCTCTCCTCTTGCT and 5′-GGAGGGTCAGAGCCCATT; for CXCL2, 5′-AAAATCATCCAAAAGATACTGAACAA and 5′-CTTTGGTTCTTCCGTTGAGG; for RANTES, 5′-GCAAGTGCTCCAATCTTGCA and 5′-ATGTATTCTTGAACCCACTTCTTCTC; for CCL7, 5′-TTCTGTGCCTGCTGCTCATA and 5′-TTGACATAGCAGCATGTGGAT; for CCL15, 5′-GCCCTCTCTCTCCTCTTGCT and 5′-GGAGGGTCAGAGCCCATT; for CCL24, 5′-GCAGCATCTGTCCCAAGG and 5′-GCAGCTTGGGGTCAGTACA and for CCL26, 5′-CACCAGTGACGGTGTGATCT and 5′-CAAATGGTTCCTGGTGTTCA. For quantification of mMCP-5, mMCP-6 and FcεRI α subunit, QuantiTect Primer Assay (Qiagen) was used. The specificity of the primers was verified by agarose gel electrophoresis and melting curve analysis. To ensure equal loading and amplification, all products were normalized to reduced form of guanosine adenine dinucleotide phosphate transcript as an internal control, and relative copy numbers of the products were calculated.

In situ hybridization with anti-sense eotaxin-1 RNA probes

To analyze conjunctival expression of eotaxin-1, we conducted in situ hybridization of eotaxin-1 on frozen sections as previously described (18, 26). Briefly, the full-length cDNA of murine eotaxin-1 in pBluescript SK+ was linearized, and anti-sense and sense RNA probes were generated using T7 and T3 RNA polymerase, respectively (Promega Corporation, Madison, WI, USA). The ³⁵S-radiolabeled probes were reduced to 200–300 ribonucleotides by alkaline hydrolysis. After hybridization, slides were washed at 65°C and autoradiographed for 4 days to 5 weeks at 4°C. Slides were then counterstained by hematoxylin and eosin or Giemsa staining. We established the specificity of the hybridization using a sense probe.

In vitro passive sensitization assay using isolated conjunctival tissue

Conjunctival tissue was collected surgically from naive mice under an operating microscope and sensitized by incubation with anti-2,4-dinitrophenol (anti-DNP)–keyhole lympet hemocyanin IgE (SPE7, 50 ng ml⁻¹; Sigma) in HBSS overnight at 4°C. After being washed three times, the conjunctival tissue was challenged in vitro with DNP-albumin (1 mg ml⁻¹) at 37°C. Supernatants were collected 45 min after challenge and assayed for histamine using ELISA (IBL Immuno-Biological Laboratories). For eotaxin-1 supplementation, eotaxin-1 was added to media 15 min after challenge. The conjunctival samples were processed so that mast cells could be identified by metachromatic staining. Toluidine blue staining showed the same mast cell number and degranulation in the conjunctiva as did Giemsa staining. Mast cell degranulation after in vitro challenge was confirmed by morphological analysis to correlate with histamine release.

ELISA

For evaluation of released eotaxin-1 protein levels, eyes with attached eyelids were collected and analyzed using an in vitro passive sensitization assay. After in vitro challenge with DNP-conjugated albumin, supernatants were measured for released eotaxin-1 using a commercial ELISA Kit (R&D Systems). The sensitivity of the eotaxin-1 ELISA was <0.4 pg ml⁻¹. The inhibitory monoclonal anti-eotaxin-1 antibody (R&D Systems) did not affect measurement of the recombinant eotaxin-1 standard (our unpublished observation).

For measurement of serum IgE, IgG₁ or IgG_2a levels, mice were bled and sera collected after final antigen challenge. Serum ELISA of IgE, IgG₁ or IgG_2a was performed using the Opt EIA mouse IgE set or biotin-conjugated anti-mouse IgG₁ or IgG_2a antibodies (BD Biosciences, Franklin Lakes, NJ, USA). Antigen-specific ELISA was performed using plates coated with Fel d1 instead of capture antibodies.

Subconjunctival injection of eotaxin-1

Five microliters of recombinant murine eotaxin-1 (10 ng ml⁻¹ in 0.1% BSA/PBS) was subconjunctivally injected into naive mice using a 30-gage needle attached to a Hamilton syringe (18). The LPS levels determined by the limulus amebocyte lysate method were <0.1 ng mg⁻¹. At the indicated time point after injection, the mice were sacrificed and the conjunctival samples prepared for morphological analysis.

Isolation and FACS analysis of mast cells from the conjunctiva and the skin

Conjunctival mast cells were isolated from mouse conjunctiva using an enzymatic digestion protocol, as described in a previous report (28), and connective tissue mast cells were isolated from mouse skin. Briefly, conjunctiva and skin of 8- to 12-week old SWR/J mice were enzymatically digested in RPMI1640 medium supplemented with 10% fetal bovine serum, 1.5 mg ml⁻¹ collagenase (Nitta Gelatin, Osaka, Japan), 0.5 mg ml⁻¹ hyaluronidase (Sigma) and 0.5 mg ml⁻¹ DNase I (Sigma) for 2 h at 37°C. Dispersed cells were layered onto isotonic Percoll density medium (density 1.041, Amersham Pharmacia Biotech, Piscataway, NJ, USA) and centrifuged at 800 × g for 20 min. The cell pellet at the bottom was cultured in RPMI1640 with murine recombinant IL-3 (10 ng ml⁻¹, Peprotech, Rocky Hill, NJ, USA), recombinant SCF (10 ng ml⁻¹, Peprotech) and 5% serum for 1–7 days. Lineage marker-positive cells and plasmacytoid dendritic cells were depleted after labeling with rat anti-CD4, rat anti-CD8a, rat anti-CD11b, rat anti-B220 (all from eBiosciences) and rat anti-plasmacytoid dendritic cell antigen 1 (PDCA-1) (Miltenyi Biotec, Auburn, CA, USA) using anti-rat Ig κ chain antibody-conjugated microbeads (BD Biosciences). For isolation of CCR3⁺ mast cells, the isolated lineage marker-negative cells were labeled with PE-conjugated anti-CCR3 (clone 83101, R&D Systems). CCR3-positive fractions were positively isolated using anti-PE antibody-conjugated microbeads (BD Biosciences).

For FACS analysis, cultured mast cells were blocked by anti-CD32 and subjected to immunofluorescence staining. Analysis was conducted using the FACS Calibur flow cytometer (BD Biosciences). PE-conjugated anti-CCR3 antibody (R&D Systems), biotin-conjugated anti-c-Kit antibody (2B8, eBiosciences), PE-conjugated anti-c-Kit antibody (eBiosciences), FITC-conjugated-anti-FcεRI α subunit (MAR-1, eBiosciences) and peridinin-chlorophyll-protein complex (PerCP)-conjugated streptavidin (BD Biosciences) were used for staining. FITC-conjugated IgG (eBiosciences), PE-conjugated IgG (eBiosciences), PerCP-conjugated IgG (BD Biosciences) and biotin-conjugated IgG (eBiosciences) were used for isotype control staining.

β-Hexosaminidase assay

Isolated mast cells were sensitized by incubation with anti-DNP-IgE (SPE7) in RPMI1640 containing 10 ng ml⁻¹ of murine recombinant IL-3, 10 ng ml⁻¹ of recombinant SCF and 5% murine serum. The cells were then washed with HBSS containing 10 ng ml⁻¹ of murine recombinant IL-3, 10 ng ml⁻¹ of recombinant SCF, 0.04% BSA and 10 mM HEPES. Re-suspended cells at 2–8 × 10⁴ cells per 100 μl were transferred into triplicate wells of a 96-well U-bottom plate and allowed to equilibrate at 37°C for 10 min prior to addition of DNP-albumin (Sigma) or compound 48/80 (Sigma). After 45 min, the plate was centrifuged at 290 × g for 5 min at 4°C. β-Hexosaminidase activity of the culture supernatant was determined using a previously reported protocol (16). Fifty-microliter aliquots of the supernatant were placed into wells of another 96-well plate together with 100 μl of 2.5 mM p-nitrophenyl-N-acetyl β-D glucosaminide (Sigma) solubilized in 0.04 M citrate buffer adjusted with disodium phosphate to pH 4.5. After incubation at 37°C for 90 min, the reactions were terminated by addition of 50 μl of 0.4 M glycin adjusted with sodium hydroxide to pH 10.7. The colored product was measured at a wavelength of 405 nm with a reference filter of 570 nm. Relative β-hexosaminidase release was defined as the activity in the supernatant of tested cells divided by the activity in the positive control cell supernatant, multiplied by 100. Compound 48/80 stimulus was used for assay control.

Statistical analysis

Data were summarized as mean ± standard error of the mean. Statistical analyses were performed using the unpaired Student's t-test (two tailed), Mann–Whitney U-test or analysis of variance (ANOVA), as appropriate.

Results

Localization of eotaxin-1 and CCR3 expression in the conjunctiva

Since eotaxin-1, a known regulator of mast cell activity, was detected in the conjunctival transcriptome of allergic hypersensitivity responses (25), we chose to further examine the mechanism of action for this cytokine using a murine experimental allergic conjunctivitis model. In this model, the immediate hypersensitivity reaction, manifested as clinical symptoms such as conjunctival edema, is a direct parameter of mast cell degranulation in the challenged conjunctiva (18, 25, 28). We first determined the location of the cells producing eotaxin-1 after allergen challenge and their spatial relationship to conjunctival mast cells. Conjunctival tissue harbors large numbers of mast cells, particularly in the substantia propria of the eyelid root (18). In situ hybridization analysis was performed on conjunctival serial sections from allergen-challenged animals (Fig. 1). The eotaxin-1-positive signals were localized mainly to mononuclear cells and fibroblasts in the substantia propria, particularly in the eyelid root, which is in close proximity to the resident mast cells. During the acute-phase reaction, the resident mononuclear cells and fibroblasts in this region were the predominant sources of eotaxin-1. PBS-treated mice showed low constitutive eotaxin-1 expression.

Fig. 1.

Localization of eotaxin-1-expressing cells in the allergen-challenged conjunctiva. (A) Localization of eotaxin-1 mRNA-positive cells in the allergen-challenged conjunctivas from sensitized mice. Eotaxin-1 mRNA-positive cells are observed in the forniceal conjunctiva. Slides were exposed for 1 week, developed and counterstained with hematoxylin and eosin. (B) Eotaxin-1-positive cells (*) from mock (PBS)-immunized mice, exposed for 1 week. (C) Schematic description of conjunctival eotaxin-1-positive cells (red dots) in the allergen-challenged conjunctiva. Ep, conjunctival epithelium.

We next examined spatial expression of chemokine receptors in the conjunctiva. We found that conjunctival mast cells express the eotaxin-1 receptor CCR3 (Fig. 2A). Interestingly, CCR3-positive cells in the conjunctiva of naive mice were mainly mast cells, suggesting that eotaxin-1 stimulates mast cells directly, rather than indirectly via stimulation of bystander cells. This observation, together with the localization of eotaxin-1 expression to cells in close proximity to mast cells, suggests that the eotaxin-1 secreted by mononuclear cells and fibroblasts following allergen exposure might bind directly to nearby CCR3-bearing mast cells.

Fig. 2.

Mast cell expression of CCR3. (A) Expression of CCR3 in conjunctival mast cells. Conjunctival mast cells from a naive mouse stain positive for CCR3 and FcεRI α subunit. Merging demonstrates co-localization of CCR3 and FcεRI α subunit (right panel). Scale = 50 μm. (B and C) Expression of CCR3 on connective tissue-type mast cells. Analysis by FACS on isolated mast cells from the conjunctiva (B) and ear skin (C). The PE-gated CCR3⁺ population expressed FcεRI α subunit (B and C, left panel). Mature mast cells expressing both FcεRI α subunit and c-Kit were strongly positive for CCR3 (B and C, right panels). (D) Expression of CCR3 and FcεRI α subunit on conjunctival mast cells.

To further confirm CCR3 expression on conjunctival mast cells, the mast cells were isolated using enzymatic techniques and analyzed for FcεRI expression using FACS. FcεRI is a tetrameric receptor composed of α, β and γ subunits. The α subunit directly binds to IgE and inducible expression of FcεRI is determined by α subunit expression. To examine whether CCR3 expression is related to mast cell maturation, surface expression of FcεRI α subunit and c-Kit was analyzed together with CCR3 expression. The mature mast cell fraction, expressing both FcεRI α subunit and c-Kit, was strongly positive for CCR3 (Fig. 2B, right panel). The majority of cells in the FcεRI α subunit^high fraction expressed CCR3 (Fig. 2B, left panel). Notably, surface CCR3 on mast cells appeared to be co-expressed with the FcεRI α subunit (Fig. 2A, B and D).

Since conjunctival mast cells are connective type, this observation suggests that co-expression of CCR3 and FcεRI α subunit might be a characteristic feature of the mature connective tissue-type mast cells. To test this hypothesis, we analyzed skin-derived mast cells. FACS analysis confirmed strong expression of CCR3 on FcεRI α subunit⁺ c-Kit⁺ mature mast cells (Fig. 2C, right panel).

Effects of eotaxin-1 injection on conjunctival eosinophils and mast cells

In vitro studies using human mast cell progenitors (22) and mature lung mast cells (20) have shown that eotaxin-1 plays roles in mast cell differentiation and homing. It is not known whether these roles are distinct or whether eotaxin-1 function depends on maturity or heterogeneity of mast cells in the tissue of origin. Therefore, we performed an in vivo experiment to test whether eotaxin-1 modulates mast cell function. We injected recombinant eotaxin-1 beneath the conjunctiva of naive mice. This technique allowed us to evaluate the eotaxin-1 effect on mast cells before commencement of the FcεRI priming effect and without indirect bystander effects initiated by TCR activation. We analyzed the morphology of resident mast cells by Giemsa and hematoxylin and eosin staining at 1 and 4 h after injection. As expected, eotaxin-1 injection (10 ng ml⁻¹) induced rapid recruitment of eosinophils in as little as 1 h (eosinophil count after eotaxin-1 injection: 57.6 ± 17.0 and 7.7 ± 2.5 eosinophils per field of view after injection with eotaxin-1 and PBS, respectively; P < 0.05). Eosinophil recruitment increased over the first 4 h after injection. Mast cells, in contrast, did not show any sign of recruitment into the conjunctiva (mast cell count at 4 h after injection of eotaxin-1: 24 ± 4 cells per field of view, PBS: 27 ± 3 cells per field of view; non-significant, NS). Neither increased degranulation nor morphological changes were observed (degranulated mast cell count after injection of eotaxin-1: 3.5 ± 0.9 cells per field of view, PBS: 2.4 ± 1.1 cells per field of view; NS). Our data indicate that conjunctival CCR3-bearing mast cells do not respond to eotaxin-1 by eliciting a chemotactic response. Therefore, we hypothesized that eotaxin-1 may instead serve as a co-stimulatory signal for an undefined activation cascade.

Impairment of mast cell degranulation and immediate hypersensitivity in experimental allergic conjunctivitis by eotaxin-1 deficiency

To test the above hypothesis, we analyzed allergen-induced mast cell-related symptoms and degranulation in eotaxin-1-deficient mice. Conjunctival mast cell degranulation can be evaluated using clinical parameters in murine experimental allergic conjunctivitis (18, 28). Whereas wild-type mice (129/SvEv) developed clear parameters of acute inflammation during the sensitization period, eotaxin-1-deficient mice failed to show clinical symptoms of immediate hypersensitivity. Remarkably, eotaxin-1-deficient mice were almost devoid of acute inflammation upon final challenges (Fig. 3A). Every symptom of clinical disease was strongly inhibited. We also evaluated mast cell degranulation in the challenged conjunctivas of wild-type and eotaxin-1-deficient mice. Since mast cell activation drives the acute-phase reaction (18, 28), these data suggest that eotaxin-1 deficiency does indeed affect mast cell activation in vivo. Consistent with the suppression of clinical symptoms, mast cell degranulation was significantly inhibited in eotaxin-1-deficient mice (Fig. 3A). Total conjunctival mast cell numbers were exactly the same between wild-type and eotaxin-1-deficient mice (Fig. 3B). When we assessed the kinetics of clinical symptoms in eotaxin-1-deficient mice, we observed no development of symptoms of acute inflammation after repeated challenges through days 56–58.

Fig. 3.

Impairment of allergen-induced immediate hypersensitivity reaction and mast cell degranulation in eotaxin-1-deficient mice. Eotaxin-1-deficient mice and wild-type control mice were sensitized and challenged by Fel d1 on days 56, 57 and 58. The allergen-induced clinical symptoms (clinical scores) and degranulated conjunctival mast cell count were analyzed as parameters of immediate hypersensitivity. (A) The immediate hypersensitivity reaction was abolished in eotaxin-1-deficient (−/−) mice. Kinetics of scores is shown through allergen challenges (days 56–57). N = 10 per group, *P < 0.001. Degranulation of mast cells was significantly impaired in eotaxin-1-deficient mice (right panel). *P < 0.05 (B) Total mast cell counts were not affected by eotaxin-1 deficiency (left panel). *P < 0.05, **P < 0.01. (C) Allergen-specific serum Igs (IgE, IgG₁ and IgG_2a) (N = 13 per group). Levels of allergen-specific Igs (IgE, IgG₁ and IgG_2a) in the mock-immunized mice were below detection limits (D. Miyazaki, unpublished data). Values are expressed as mean ± standard error of the mean.

Elevated levels of allergen-specific IgE, which is required for mast cell activation, are caused by a complex series of cellular and molecular events summarized as the inductive-phase events. Proper antigen processing and presentation, B cell maturation and class switching driven by T_h2 cells are required for Ig priming. Our analysis of IgE, IgG₁ and IgG_2a revealed that there is no antibody deficit in eotaxin-1-deficient mice (Fig. 3C). To the contrary, production of allergen-specific IgE was increased in these mice, although not to a significant level (P = 0.06).

To examine whether impairment of the immediate hypersensitivity reaction in eotaxin-1-deficient mice was an outcome of a direct impact on mast cell physiology and independent of inductive-phase phenomena, eotaxin-1-deficient mice were further evaluated using a passive sensitization protocol. Wild-type mice sensitized by intravenous injection of ragweed-specific IgE showed significant clinical symptoms on allergen exposure (Fig. 4A). In contrast, clinical scores of eotaxin-1-deficient mice were significantly depressed, again confirming a requirement for eotaxin-1 in the immediate hypersensitivity reaction. Since CC chemokine receptors on mast cells are candidate receptors for mast cell activation, levels of representative CC chemokines were examined. Real-time PCR analysis of RNA isolated from the conjunctivas of allergen-challenged eotaxin-1-deficient mice revealed significant reduction of MIP-1α, RANTES and MIP-1β expression (Fig. 4B).

Fig. 4.

Impairment of allergen-induced passive anaphylactic reaction and local chemokine production in wild-type and eotaxin-1-deficient mice. (A) Eotaxin-1-deficient mice and wild-type control mice were sensitized by intravenous injection of ragweed-specific IgE and challenged by ragweed instillation. The allergen-induced clinical symptoms (clinical scores) were significantly suppressed in eotaxin-1-deficient (−/−) mice. (B) Profiles of chemokine induction in eotaxin-1-deficient eye homogenates 2 h after ragweed instillation by real-time RT–PCR. Eotaxin-1 deficiency caused significant reduction of CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (RANTES) (C) Eotaxin-1-deficient conjunctivas showed reduced expression of mMCP-5 and mMCP-6, but this reduction was not significant. Expression of FcεRI α subunit was not altered in naive eotaxin-1-deficient mice but, was, significantly decreased in allergen-exposed, eotaxin-1-deficient mice (N = 5 per group). *P < 0.05. Values are expressed as mean ± standard error of the mean. (D) Immunohistochemical localization of mMCP-5⁺ mast cells in the conjunctiva of eotaxin-1-deficient and wild-type mice. The superficial conjunctival area of naive mice without allergen exposure showed mMCP-5⁺ mast cells (arrow) in eotaxin-1-deficient mice that were altered in distribution compared with wild type, but not significantly reduced in number. Scale = 200 μm.

We also evaluated whether eotaxin-1 deficiency might affect the responsiveness of conjunctival mast cells. In the mouse, 12 different proteases are stored in mast cell secretory granules. Since distinct functions have been recognized for these enzymes, profiling of mast cell proteases can be used to characterize effector functions. We examined transcript levels of mast cell-specific proteases to understand the possible alteration of effector function in the passively sensitized mice. Conjunctival mast cells are connective tissue type, which express tryptase mMCP-6 as well as chymase mMCP-5 (31, 32). Real-time reverse transcription (RT)–PCR analysis showed that conjunctiva from eotaxin-1-deficient mice displays some decrease in mMCP-5 and mMCP-6, though the difference was not significant compared with expression in wild-type mice (Fig. 4C). We next measured FcεRI transcript levels to determine whether eotaxin-1 deficiency might cause impairment of mast cell activation. It is known that α subunit expression represents mast cell activation and that this subunit is selectively expressed in mast cells or basophils. Real-time RT–PCR analysis showed no deficit in α subunit level in eotaxin-1-deficient mice, though the level was decreased after allergen exposure (Fig. 4C). This indicates that eotaxin-1 deficiency does not affect constitutive levels of FcεRI expression in conjunctival mast cells, since eotaxin-1-deficient mice harbor equivalent numbers of conjunctival mast cells (Fig. 3B), and basophils are rarely observed in murine conjunctiva.

Next, we used immunohistochemistry to examine distribution of sub-populations in conjunctival mast cells of naive animals. In the superficial conjunctival area, mMCP-5-expressing mast cell number appeared somewhat reduced, although not significantly so (Fig. 4D, arrow). Geographical distribution of tryptase mMCP-6-positive mast cells was not significantly altered in eotaxin-1-deficient mice compared with the wild type (D. Miyazaki and M. Ohbayashi, unpublished data). Altogether, eotaxin-1 deficiency did not markedly affect conjunctival mast cell sub-populations.

Requirement of eotaxin-1 for histamine release from sensitized conjunctiva

To examine whether the impaired mast cell function is a direct outcome of eotaxin-1 insufficiency, we first used the in vitro histamine release assay with isolated conjunctiva. We tested whether blockade of eotaxin-1 by mAb treatment could suppress IgE-dependent mast cell activation. Upon IgE stimulation, sensitized conjunctival mast cells promptly released histamine. When eotaxin-1 was blocked by mAb treatment, histamine release was significantly suppressed to 38% of that released by cells treated with control IgG (Fig. 5A). This suppression was reversed by providing an excess of recombinant eotaxin-1 (1 ng ml⁻¹) during in vitro challenge (Fig. 5A). To assess whether the histamine release is related to CCR3 activation, we evaluated effects of a CCR3 antagonist on mast cell degranulation. We selected W-56750 as an inhibitor because of its high selectivity for CCR3. When W-56750 was added to the media of allergen-stimulated conjunctivas, allergen-induced histamine release was suppressed to 42% of that released by control cells, and this inhibition was reversed by adding excess recombinant eotaxin-1 during in vitro challenge (D. Miyazaki, unpublished data) (25, 33).

Fig. 5.

Effects of eotaxin-1 neutralization on allergen-induced histamine release and eotaxin-1 secretion. (A) The experimental design involved a passive sensitization assay using isolated conjunctival tissue from naive mice (SWR/J). The left panel shows histamine release from the in vitro-sensitized tissue challenged with DNP-albumin. Treatment with a neutralizing antibody to eotaxin-1 (2 μg ml⁻¹) significantly suppressed allergen-induced histamine release of in vitro-sensitized conjunctival mast cells. Excess recombinant eotaxin-1 (r-eotaxin-1, 1 ng ml⁻¹) negated the effect of eotaxin-1 neutralization. The right panel shows eotaxin-1 secretion from the in vitro-sensitized tissue challenged with DNP-albumin. Neutralization of eotaxin-1 had no effect on its secretion. N = 7 per group. *P < 0.05, **P < 0.01. (B) Effects of eotaxin-1 on mast cell histamine release. Passive sensitization using isolated tissue from naive mice (eotaxin-1-deficient mice on BALB/c background or wild-type mice) was used. Addition of recombinant eotaxin-1 during the challenge phase stimulated allergen-induced histamine release from eotaxin-1-deficient mast cells in a dose-dependent fashion. Supplementation without allergen stimulus did not affect histamine release in either eotaxin-1-deficient or wild-type mice. N = 4 per group. *P < 0.05, **P < 0.01.

Taken together, the above findings support the idea that eotaxin-1, secreted on allergen stimulus, directly activates mast cells. To confirm that eotaxin-1 is indeed secreted upon allergen stimulus, we measured eotaxin-1 levels in the conjunctival histamine release assay. Eotaxin-1 was released into the reaction media within 90 min after allergen stimulus, and its levels increased 4-fold over those in the mock-challenged control (DNP-albumin challenged: 23.7 ± 3.3 pg per eye, mock challenged: 5.6 ± 1.4 pg per eye, P < 0.005; Fig. 5A). Interestingly, suppression of histamine release by eotaxin-1 blockade was independent of secreted eotaxin-1 levels (Fig. 5A).

Next, we examined whether eotaxin-1 had any direct stimulatory function on degranulation using the in vitro histamine release assay. Consistent with previous studies (20), supplementation of eotaxin-1 to wild-type conjunctiva had no stimulatory effect on allergen-induced histamine release (Fig. 5B). Eotaxin-1 therefore does not directly stimulate mast cell degranulation but might constitute an amplifying link in the FcεRI-mediated cascade required for mast cell activity. To investigate this hypothesis, we tested whether supplementing eotaxin-1 in eotaxin-1-deficient mice might result in mast cell activation. Indeed, conjunctival mast cells from eotaxin-1-deficient mice exhibited a dramatic increase in histamine release following allergen challenge, in an eotaxin-1 dose-dependent manner (Fig. 5B). Wild-type mice, on the other hand, showed no significant increase in histamine release following treatment with eotaxin-1 (Fig. 5B).

Impaired mast cell degranulation by CCR3 inhibition

To discriminate between differentiation and activation of mast cells, we examined two different culture conditions in the presence or absence of CCR3 blockade. Isolated lin⁻ PDCA-1⁻ connective tissue-type mast cells from the conjunctiva were cultured in IL-3/SCF (10 ng ml⁻¹)-containing media with or without W-56750 (100 nM) for 1 week to induce terminal differentiation. In the absence of W-56750, semi-adherent colonies of mast cells with large and granule-rich appearances were formed. To examine the effects of CCR3 blockade on mast cell differentiation, we analyzed connective tissue-type mast cell-related transcripts for mMCP-5, mMCP-6 and FcεRI α subunit. When lin⁻ PDCA-1⁻ connective tissue-type mast cells were cultured with W-56750 to impair CCR3 signaling, levels of connective tissue-type mast cell-related transcripts, including mMCP-5, mMCP-6 and FcεRI α subunit, were significantly reduced (P < 0.01, Fig. 6C). IL-3 receptor α chain level was not changed (Fig. 6C). These data support the hypothesis that CCR3 activation acts also as a differentiation stimulus for connective tissue-type mast cells.

Fig. 6.

Suppression of mast cell degranulation by impaired CCR3 activation. (A) Semi-mature connective tissue-type mast cells cultured with W-56750 (100 nM, open circle, open triangle and open square) and mature connective tissue-type mast cells (cultured for 1 week without W-56750, filled circle, both from the skin) were tested for DNP-albumin-induced degranulation by measurement of β-hexosaminidase release. The connective tissue-type mast cells were sensitized with anti-DNP-IgE and stimulated by DNP-albumin with or without eotaxin-1 (5 ng ml⁻¹) and/or W-56750 (20 nM). Semi-mature mast cells failed to respond to allergen stimulus without eotaxin-1 (open circle). The response was restored by addition of eotaxin-1 (5 ng ml⁻¹; open square) and inhibited by addition of W-56750 (20 nM; open triangle). Mature connective tissue-type mast cells, cultured without W-56750, vigorously responded to DNP-albumin (filled circle). (B) Anti-DNP-IgE-sensitized mature connective tissue-type mast cells were stimulated by DNP-albumin with anti-CCR3 antibody or isotype control. Anti-CCR3 antibody treatment suppressed β-hexosaminidase release in a dose-dependent manner; release was restored by addition of eotaxin-1 (5 ng ml⁻¹). (C) Impaired induction of mast cell-restricted proteases and FcεRI by CCR3 blockade. Isolated lin⁻ PDCA-1⁻ conjunctival mast cells were cultured with or without W-56750 (100 nM) for 1 week and assayed for transcripts of mMCP-5, mMCP-6 and FcεRI α subunit and IL-3 receptor α chain. Induction of mMCP-5, mMCP-6 and FcεRI α subunit was significantly impaired with W-56750 treatment. (D) Intrinsic CCR3 ligands (RANTES, CCL7, eotaxin-1, CCL15, CCL24 and CCL26) of connective tissue-type mast cells, with or without CCR3 blockade, were evaluated by RT–real-time PCR. Connective tissue-type mast cells were stimulated with phorbol myristate acetate (10 ng ml⁻¹) and ionomycin (1 μM) and were evaluated for CCR3 ligand transcripts. Of these CCR3 ligands, CCL7 was predominantly expressed in both conditions. No appreciable suppression of CCR3 ligands was observed in W-56750-treated cells (400 nM). Figures are representative of two repeated experiments. Connective tissue-type mast cells from the conjunctiva showed similar results. *P < 0.05, **P < 0.01. N = 3 per group. Values are expressed as mean ± standard error of the mean.

Next, we attempted to identify the exact mast cell maturation stage stimulated by CCR3 in allergic responses. Reactivity of mast cells isolated from the skin and conjunctiva was measured by allergen-induced β-hexosaminidase release. Reactivity of the presumable semi-mature mast cells was evaluated after complete removal of supplemented W-56750 during differentiation blockade. As expected, eotaxin-1 in the absence of allergen exposure did not stimulate degranulation in either mast cell differentiation stage (Fig. 6A). This is consistent with previous reports and with our organ culture experiments (25) (Fig. 5B). We further examined whether CCR3 exerts its effects when mast cells are activated by allergen and IgE. Allergen stimulus failed to provoke β-hexosaminidase release from semi-mature mast cells (mast cells cultured with W-56750, Fig. 6A). When CCR3 was partially blocked using a sub-optimal dose of W-56750 (20 nM) with eotaxin-1 supplementation (5 ng ml⁻¹), allergen-induced β-hexosaminidase release was partially restored (Fig. 6A).

Next, fully mature connective tissue-type mast cells, cultured without blockade of CCR3, were tested for allergen-induced responses. These mast cells displayed a more vigorous response compared with the semi-mature mast cells (Fig. 6A). To assess the requirement of CCR3 stimulation for mature mast cell responses, fully mature mast cells were challenged with allergen in the presence of anti-CCR3 antibody or control antibody. Addition of anti-CCR3 significantly suppressed β-hexosaminidase release in a dose-dependent manner (Fig. 6B). This suppressive effect of CCR3 blockade was also observed with W-56750 treatment and was confirmed using conjunctiva-derived mast cells (D. Miyazaki, unpublished data).

To examine whether CCR3-mediated stimulatory effects have any relationship with mast cell-derived CCR3 ligands, connective tissue-type mast cells, cultured with or without W-56750, were examined for induction of CCR3 ligands after activation. We detected CCL7, CCL15 and eotaxin-1 transcripts in phorbol myristate acetate/ionomycin-activated cells (Fig. 6D), but appreciable suppression was not observed in W-56750-treated cells. This further confirms that suppressive effects of W-56750 on mast cells are independent of intrinsic CCR3 ligand induction.

Taken together, these data suggest that eotaxin-1/CCR3 axis serves both as a default master switch for IgE-mediated degranulation and as a regulator of degranulation levels.

Discussion

Our observations clearly illustrate important aspects of eotaxin-1 activity. First, eotaxin-1 is a critical mediator for IgE-mediated mast cell activation and provides the igniting signal for ocular allergic reactions. This complementary signal to IgE cross-linking appears not to require de novo colonization of inflammatory cell subsets. Moreover, an important aspect of our findings is a pivotal role of CCR3 in activation of mature connective tissue-type mast cells in the ocular tissue.

Our results indicate that eotaxin-1 is not required for the inductive phase of mucosal immunity but is an important effector-phase mediator. The eotaxin-1/CCR3 signal contributes to eosinophil homing or allergen-dependent chemoattraction in disease models, without affecting homing of mast cells. Furthermore, eotaxin-1/CCR3 is required for physiologically relevant levels of mast cell activation in vivo. The profound decrease in mast cell degranulation and the near loss of clinical symptoms in the acute-phase reaction in mice deficient in eotaxin-1, as well as after CCR3 blockade, indicate that eotaxin-1/CCR3 is a biologically relevant mast cell activation signal in the conjunctiva. Moreover, skin-derived connective tissue-type mast cells utilized CCR3 input for allergic responses as well, although relevant CCR3 ligands in the skin remain unidentified.

Mast cell progenitors, derived from pluripotent precursor cells, exit the bone marrow and circulate through the systemic bloodstream. Their fate is determined by their homing signal and their location of residence in situ. CCR3 was proposed to be an attribute surface molecule in connective tissue-type mast cells, since eotaxin-1 in combination with SCF promotes differentiation of fetal mast cell progenitors into connective tissue-type mast cells (34). In contrast to these earlier observations, when an allergic airway model was evaluated for impact of CCR3 deficiency, no appreciable defect in mast cell activity was observed (35). Increased numbers of intra-epithelial mast cells were, however, found in the trachea (35). In the skin inflammation model, disruption of CCR3 did not alter mast cell numbers or degranulation (36). Findings from these elegant studies are in marked contrast to our observations. We suspect that this is due to heterogeneity of mast cells (mucosal or connective tissue type) depending on the tissue of residence, presumably dictated by both expression of chemokine receptors and availability of specific ligands from the surrounding tissue (37). In this scenario, ablation of CCR3 may instead alter the repertoire of chemokine receptors, resulting in a switch in the default receptor. Indeed, we also observed expression of CCR1, CXCR3 and other candidate receptors on connective tissue-type mast cells (25, 37).

Our observation that the CCR3 signal is necessary for connective tissue-type mast cell activation may indicate that CCR3 is the preferred receptor in the environment of the eye. Thus, tissue specificity may dictate mast cell functions.

Chemokine stimulation and mast cell degranulation are controlled by receptor-mediated signaling and regulation of receptor expression. Our current understanding is that CCR3 activates Gi protein to release the FcεRI α subunit, which then couples to phosphatidylinositol 3-kinase γ and amplifies the allergen-stimulated Ca²⁺ influx (38). These findings agree with our recent observation of co-stimulatory, CCR1-mediated signaling by MIP-1α (16, 18) and support our present observation that eotaxin-1-mediated signaling is an important stimulus for mast cell activation. A second aspect of eotaxin-1/CCR3 signaling is regulation of the surface expression of CCR3. CCR3 is regulated in both transcriptional and post-transcriptional manners, a combination of regulatory mechanisms commonly observed for G-protein-coupled receptors. Upon ligand binding, CCR3 undergoes desensitization via either receptor internalization or receptor degradation (39, 40). This might explain the poor response of eotaxin-1-sufficient mast cells to a CCR3 stimulus, which can be provided by fibroblasts or mononuclear cells in the surrounding tissue environment.

To search for other candidate molecules of CCR3 ligands to conjunctival immediate hypersensitivity reaction, we previously conducted whole-genome scanning of immediate early transcripts using allergic conjunctivitis model after CCR3 antagonist (W-56750) treatment (25, 37). Of all the differentially expressed transcripts identified by ANOVA analysis (P < 0.01), eotaxin-1 ranked in the top 20 and was the sole CCR3 ligand in the group. Another CCR3 ligand, CCL7, was detected in conjunctival mast cell transcripts (Fig. 6); however, no statistical difference in expression was observed. This suggests that the contribution of CCL7 is negligible in allergic conjunctivitis. An additional ligand of CCR3, eotaxin-3, was recently shown to be pivotal in eosinophilic esophagitis by using the whole-genome scanning approach (41). Taken together, these data support the idea that differential usage of CCR3 ligands is dependent on the tissue location.

We previously proposed that MIP-1α is an essential co-stimulatory signal for mast cells (18). However, we consider that defective activation of mast cells by eotaxin-1/CCR3 is not secondary to action of MIP-1α. Previous analysis of chemokine expression profiles in MIP-1α-deficient mice showed suppressed expression of RANTES and MIP-2 and elevated expression of MIP-1β and TCA-3 (18). The eotaxin-1-deficient mice in the current study showed a profile of chemokine expression clearly distinct from that of MIP-1α-deficient mice (Fig. 4). To confirm that the reduction in MIP-1α expression was caused by reduced eotaxin-1–CCR3 signaling, we administered a CCR3 antagonist, W-56750, to allergen-stimulated wild-type mice (25). CCR3 antagonism resulted in both reduced MIP-1α expression and significantly suppressed mast cell degranulation (D. Miyazaki, unpublished data). Reduced MIP-1α expression may therefore be a general indicator of suppressed mast cell function.

Our data support the emerging view that antagonizing the eotaxin-1–CCR3 interaction or signaling from CCR3 has potential as a treatment for ocular allergy. Ongoing clinical trials have been devised to modulate this axis via utilization of CCR3 antagonists or humanized monoclonal eotaxin-1 antibody (25, 33, 42). We believe that mast cell-mediated pathological processes in connective tissue are under control of the eotaxin-1/CCR3 axis and expect to observe significant suppression of the late-phase reaction by these treatments in phase II trials. To summarize, we propose that CCR3 in the ocular tissue regulates IgE-induced degranulation and presumably maturation. We believe that these findings may provide in-depth understanding of various mast cell-mediated allergic diseases, leading to discovery of novel types of anti-allergy therapy.

Supplementary data

Supplementary table is available at International Immunology Online.

Funding

National Institutes of Health Grants (R01 AI42242 to M.E.R., R01 AI45898 to M.E.R., P01 HL-076383-01 to M.E.R.).

Glossary

Abbreviations

ANOVA

analysis of variance

CCR3

chemokine receptor 3

DNP

2,4-dinitrophenol

FcεRI

high affinity IgE receptor

IC₅₀

50% inhibitory concentration

MCP-3

monocyte chemoattractant protein-3

MIP-1α

macrophage inflammatory protein-1 alpha

mMCP-5

mouse mast cell protease-5

NS

non-significant

PDCA-1

plasmacytoid dendritic cell antigen 1

PerCP

peridinin-chlorophyll-protein complex

RT

reverse transcription

SCF

stem cell factor