CCR3 is essential for skin eosinophilia and airway hyperresponsiveness in a murine model of allergic skin inflammation

Abstract

The CC chemokine receptor 3 (CCR3) is expressed by eosinophils, mast cells, and Th2 cells. We used CCR3(-/-) mice to assess the role of CCR3 in a murine model of allergic skin inflammation induced by repeated epicutaneous sensitization with ovalbumin (OVA), and characterized by eosinophil skin infiltration, local expression of Th2 cytokines, and airway hyperresponsiveness (AHR) to inhaled antigen. Eosinophils and the eosinophil product major basic protein were absent from the skin of sham and OVA-sensitized CCR3(-/-) mice. Mast cell numbers and expression of IL-4 mRNA were normal in skin of CCR3(-/-) mice, suggesting that CCR3 is not important for infiltration of the skin by mast cells and Th2 cells. CCR3(-/-) mice produced normal levels of OVA-specific IgE, and their splenocytes secreted normal amounts of IL-4 and IL-5 following in vitro stimulation with OVA, indicating effective generation of systemic Th2 helper responses. Recruitment of eosinophils to lung parenchyma and bronchoalveolar lavage (BAL) fluid was severely impaired in CCR3(-/-) mice, which failed to develop AHR to methacholine following antigen inhalation. These results suggest that CCR3 plays an essential role in eosinophil recruitment to the skin and the lung and in the development of AHR.

Figure 1

Eosinophils are virtually absent in OVA-sensitized skin sites of CCR3^–/– mice. (a) Eosinophils/HPF in skin sites of CCR3^–/– mice and WT controls sensitized with either OVA or saline. The bars represent the mean (n = 6–7 animals per group). *P < 0.01. (b) Immunoperoxidase MBP staining of sensitized skin sites from CCR3^–/– mice and WT controls. MBP stains brown.

Figure 2

Total mononuclear cell counts in CCR3^–/– mice are normal (a). CCR3^–/– mice have normal expression of IL-4 (b) and IFN-γ (c) mRNA in OVA-sensitized skin. Cytokine mRNA levels were normalized to β2-microglobulin. The bars represent the mean (n = 6 animals per group). *P < 0.05; **P < 0.01.

Figure 3

IL-4 (a) and IL-5 (b) in supernatants of splenocytes from CCR3^–/– mice and WT controls sensitized with either OVA or saline following in vitro stimulation with OVA. The bars represent the mean (n = 4–6 animals per group). CCR3^–/– mice mount normal serum OVA-specific IgE (c) Ab responses. The bars represent the mean (n = 6–7 animals per group). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

CCR3^–/– mice epicutaneously sensitized with OVA do not develop BAL or lung tissue eosinophilia following inhalation challenge with allergen. (a) Eosinophils, neutrophils, and lymphocytes in BAL fluid were counted. The bars represent the mean (n = 6–7 animals per group). *P < 0.01. (b) Lung sections taken 24 hours after a single exposure of inhaled 1% OVA and stained with Congo red dye, which stains eosinophils orange (arrows). (c) Trachae sections taken 24 hours after a single exposure of inhaled 1% OVA and stained with a solution containing naphthol AS-D chloroacetate to identify mast cells (arrows).

Figure 5

CCR3^–/– mice epicutaneously sensitized with OVA do not develop AHR to Mch following inhalation challenge with allergen. AHR was measured by whole body plethysmography in conscious CCR3^–/– mice and WT controls following inhalation challenge with OVA. Penh, an index of airway obstruction, was calculated from the box pressure/time wave after aerosolization of increasing doses of Mch. Numerically higher values of Penh are indicative of increased airway obstruction. Data represents mean Penh values ± SEM (n ≥ 6 mice).

Figure 6

The Th2 cytokine is expressed normally in lungs of epicutaneously sensitized CCR3^–/– mice following allergen inhalation challenge. IL-4 mRNA levels were normalized to β2-microglobulin. The bars represent the mean (n = 5 animals per group). *P < 0.05; **P < 0.01.