Role of CCL7 in Type I Hypersensitivity Reactions in Murine Experimental Allergic Conjunctivitis

Abstract

Molecules that are necessary for ocular hypersensitivity reactions include the receptors CCR1 and CCR3; CCL7 is a ligand for these receptors. Therefore, we explored the role of CCL7 in mast cell activity and motility in vitro and investigated the requirement for CCL7 in a murine model of IgE-mediated allergic conjunctivitis. For mast cells treated with IgE and Ag, the presence of CCL7 synergistically enhanced degranulation and calcium influx. CCL7 also induced chemotaxis in mast cells. CCL7-deficient bone marrow-derived mast cells showed decreased degranulation following IgE and Ag treatment compared with wild-type bone marrow-derived mast cells, but there was no difference in degranulation when cells were activated via an IgE-independent pathway. In vivo, CCL7 was upregulated in conjunctival tissue during an OVA-induced allergic response. Notably, the early-phase clinical symptoms in the conjunctiva after OVA challenge were significantly higher in OVA-sensitized wild-type mice than in control challenged wild-type mice; the increase was suppressed in CCL7-deficient mice. In the OVA-induced allergic response, the numbers of conjunctival mast cells were lower in CCL7-deficient mice than in wild-type mice. Our results demonstrate that CCL7 is required for maximal OVA-induced ocular anaphylaxis, mast cell recruitment in vivo, and maximal FcεRI-mediated mast cell activation in vitro. A better understanding of the role of CCL7 in mediating ocular hypersensitivity reactions will provide insights into mast cell function and novel treatments for allergic ocular diseases.

FIGURE 1.

CCL-7 synergistically induces degranulation in RBL-CCR1 cells and mouse BMMCs. RBL-CCR1 cells and BMMCs were cultured or not with 25 or 100 ng/ml anti-DNP IgE for 16 h and then stimulated for 20 min with or without 10 ng/ml DNP-HSA and six concentrations of recombinant human or mouse CCL7. β-hexosaminidase activity was measured. Data are mean ± SEM and are representative of four separate experiments. *p < 0.05, **p < 0.01, ANOVA.

FIGURE 2.

CCL-7 synergistically induces a significant increase in calcium influx in mouse BMMCs. Studies were performed with cultured BMMCs loaded with Fluo-4 AM. Fluorescence was measured over time before and after treatment with DNP-HSA with and without recombinant mouse CCL7. (A) Intracellular Ca²⁺ transient release and dose-response curves to CCL7. The arrow indicates the time of addition of DNP-has, with or without CCL7. The graph represents the average fluorescence intensity from cultured BMMCs. (B) The Fluo-4 ratio represents mean fluorescence after treatment (20–300 s)/mean baseline fluorescence before treatment (0–20 s). Data are mean ± SEM. The data shown are the average of the Fluo-4 ratio of four separate experiments. These studies were performed in the absence of extracellular calcium. *p < 0.05 from four experiments.

FIGURE 3.

CCL7 induces chemotaxis of RBL-CCR1 cells and mouse BMMCs. (A) After sensitization with anti-DNP IgE, 2 × 10⁴ RBL-CCR1 cells and BMMCs in serum-free medium were transferred to the upper compartment of a Transwell chamber; the lower compartment was filled with serum-free medium containing one of six concentrations (0–100 ng/ml) of recombinant human or mouse CCL7. Cells were incubated for 3 h before quantifying the number of migrated cells. (B) After sensitization with anti-DNP IgE, RBL-CCR1 cells were stimulated with 1 ng/ml recombinant human CCL7 or were left unstimulated. Then cells were fixed and processed for polymerized actin staining with TRITC-conjugated phalloidin. Original magnification ×40. (C) Ruffling for individual cells stimulated with control or CCL7 was assessed. For each group, 100 cells were counted per condition in three separate experiments. Data are mean ± SEM. *p < 0.05, **p < 0.01, ANOVA.

FIGURE 4.

The Akt inhibitor wortmannin inhibits synergistic induction of degranulation by CCL7. (A) After sensitization, RBL-CCR1 cells were treated with wortmannin (10 μM) for 1 h before stimulation with CCL7 (10 and 100 ng/ml), with or without Ag (10 ng/ml), for 15 min. Cells were lysed, and Western blotting was performed using Abs against total Akt and phospho-Akt (Ser⁴⁷³). Data are from one representative of two separate experiments. (B) After sensitization, BMMCs were treated with wortmannin (1 or 10 μM) or left untreated and then stimulated or not for 20 min with 10 ng/ml DNP-HSA and/or recombinant mouse CCL7. β-hexosaminidase activity was measured as a reflection of degranulation. Data are mean ± SEM and are representative of three separate experiments. *p < 0.05, ANOVA.

FIGURE 5.

BMMCs cultured from CCL7^−/− mice have no obvious defects in morphology, surface markers for mast cells, or proliferation, yet they display reduced degranulation in response to Ag stimulation. (A) Mature BMMCs (>4 wk of culture) from wild-type and CCL7^−/− mice were stained with Giemsa to visualize morphology. Original magnification ×40. (B) Surface expression of FcεRI and c-kit on CCL7^−/− BMMCs cultured for 5 wk was detected by flow cytometry. (C) Cell number was determined by staining with 0.4% trypan blue and counting cells using a hemocytometer. (D) BMMCs from wild-type and CCL7^−/− mice were cultured with 100 ng/ml anti-DNP IgE for 16 h and then stimulated or not with 10 ng/ml DNP-HSA for 20 min. *p < 0.05, **p < 0.01, ANOVA.

FIGURE 6.

CCL7 is induced during anti-IgE sensitization, and its absence reduces Ag-induced Akt phosphorylation. Mature BMMCs (>4 wk of culture) from wild-type mice were sensitized or not with anti-DNP IgE (100 ng/ml), and levels of CCL7 mRNA and protein were quantified at different time points. (A) Total RNA from sensitized cells was isolated, and Ccl7 expression was examined using real-time PCR. (B) Supernatants of culture medium were collected, and protein levels were analyzed by ELISA. (C) BMMCs were stimulated or not with different concentrations of ionophore for 20 min, and β-hexosaminidase activity was determined as a measure of degranulation. Data are mean ± SEM and are representative of four separate experiments. (D) A total of 1 × 10⁶ BMMCs from wild-type and CCL7^−/− mice was incubated overnight in medium containing anti-DNP IgE (100 ng/ml) and treated with 10 ng/ml Ag for 5 min. To examine the phosphorylation of Akt, Western blotting was performed on 30 μg of total protein using Abs to total Akt, phospho-Akt (Ser⁴⁷³), and β-actin (loading control). Relative densitometric analysis is presented. Data are mean ± SEM and are representative of three separate experiments. *p < 0.05, **p < 0.01, ANOVA. ns, not significant.

FIGURE 7.

CCL7 is upregulated during the early-phase responses of EAC. (A) Time course for development of EAC. Mice were immunized i.p. with OVA and alum every other week for a total of three times, rested for 7 d, and repeatedly challenged with eye-drop administration of OVA or saline every day for a total of six times. (B) Total RNA from samples of conjunctival tissue was isolated 30 min after final challenge, and Ccl7 expression levels were examined using real-time PCR. (C) Serum of mice was collected for ELISA analysis at 3 h after the final challenge. Data are mean ± SEM for n = 5 (B) and n = 10 (C) per group and are representative of four separate experiments. (D) Conjunctival tissues were fixed, serially sectioned, and stained for CCL7 using a polyclonal Ab. Localization of CCL7 (red) in the allergen-challenged conjunctiva of wild-type mice (b), saline (control)-challenged conjunctiva of wild-type mice (a), allergen-challenged conjunctiva of wild-type mice stained using the isotype-control Ab (c), and allergen-challenged conjunctiva of CCL7^−/− mice (d) was examined using immunofluorescence. Original magnification ×10. Blue, DAPI. *p < 0.05, **p < 0.01, ANOVA.

FIGURE 8.

CCL7^−/− mice have impaired allergen-induced immediate hypersensitivity reactions. Levels of IgE Abs, but not IgG₁ Abs, are reduced in the serum of CCL7^−/− mice with experimental allergen-induced conjunctivitis. (A) Wild-type control mice and CCL7^−/− mice were sensitized and challenged with OVA or saline control. The allergen-induced clinical symptoms (clinical scores, taking into account conjunctival edema, lid edema, redness, tearing, and squinting) were analyzed 15 min after the final challenge as parameters of immediate hypersensitivity. (B) Histamine levels in the serum of the mice, obtained 3 h after the final challenge, were analyzed by ELISA. The data are representative of four separate experiments. Levels of total IgE (C), OVA-specific IgE (D), and IgG₁ (E) in the serum of saline-challenged (control) or OVA-challenged wild-type and CCL7^−/− mice were determined by ELISA. Data are mean ± SEM and are representative of four separate experiments (n = 6 per group). *p < 0.05, **p < 0.01, ANOVA. ns, not significant.

FIGURE 9.

CCL7^−/− mice display reduced mast cell recruitment in allergen-induced conjunctivitis. Conjunctival tissue of wild-type control mice and CCL7^−/− mice sensitized and challenged with OVA and saline was harvested, fixed, serially sectioned, and stained for mast cells using toluidine blue or chloroacetate esterase staining. (A) Total mast cell number was counted in toluidine blue–stained conjunctival tissue 3 h after final Ag or control challenge. (B) Chloroacetate esterase–stained conjunctival tissue from OVA-sensitized wild-type and CCL7^−/− mice was examined to detect the localization of mast cells (red) (arrows) 3 h after the final Ag challenge. Original magnification ×10. Data are mean ± SEM and are representative of four separate experiments (n = 7 per group). *p < 0.05, Student t test.

FIGURE 10.

CCL7^−/− mice display reduced numbers of mMCP-4⁺ mast cells during allergen-induced conjunctivitis. Conjunctival tissues of wild-type control mice and CCL7^−/− mice, sensitized and challenged with OVA or saline, were harvested, fixed, serially sectioned, and stained for mMCP-4, c-kit, and FcεRI. (A) Conjunctival tissue was examined to detect the localization of mMCP-4⁺ mast cells (green immunofluorescence) (arrows) 3 h after the final challenge with Ag or control in OVA-sensitized wild-type and CCL7^−/− mice. Original magnification ×20, Blue, DAPI. (B) mMCP-4⁺ mast cell number was counted in conjunctival tissues 3 h after the final challenge. Data are mean ± SEM and are representative of four separate experiments (n > 5 per group). **p < 0.01, ANOVA.

FIGURE 11.

CCL7^−/− mice display reduced expression of chemokines and cytokines in allergen-induced conjunctivitis. Wild-type and CCL7^−/− mice were challenged with Ag or saline. Total mRNA from conjunctival tissue was isolated 30 min after the final challenge, and mRNA expression levels for Ccl2, Ccl3, Ccl4, Il4, Il6, and Il13 were examined using real-time PCR. Data are mean ± SEM and are representative of four separate experiments (n = 5 per group). *p < 0.05, **p < 0.01, ANOVA. ns, not significant.