The prostaglandin D₂ receptor CRTH2 is important for allergic skin inflammation after epicutaneous antigen challenge

Abstract

Background: Cutaneous prostaglandin (PG) D₂ levels increase after scratching. Chemoattractant receptor-homologous molecule expressed on receptor on T(H)2 cells (CRTH2) mediates chemotaxis to PGD₂ and is expressed on T(H)2 cells and eosinophils, which infiltrate skin lesions in patients with atopic dermatitis.

Objective: We sought to examine the role of CRTH2 in a murine model of atopic dermatitis.

Methods: CRTH2(-/-) mice and wild-type control animals were epicutaneously sensitized by means of repeated application of ovalbumin (OVA) to tape-stripped skin for 7 weeks and then challenged by means of OVA application to tape-stripped previously unsensitized skin for 1 week. Skin histology was assessed by means of hematoxylin and eosin staining and immunohistochemistry. Cytokine mRNA expression was examined by means of quantitative RT-PCR. Levels of PGD₂, antibody, and cytokines were measured by means of ELISA.

Results: PGD₂ levels significantly increased in skin 24 hours after tape stripping, although not in skin subjected to repeated sensitization with OVA. Allergic skin inflammation developed normally at sites of chronic epicutaneous sensitization with OVA in CRTH2(-/-) mice but was severely impaired in previously unsensitized skin challenged with OVA, as evidenced by significantly decreased skin infiltration with eosinophils and CD4(+) cells and impaired T(H)2 cytokine mRNA expression. Impaired skin inflammation at sites of acute OVA challenge in CRTH2(-/-) mice was not due to an impaired systemic response to epicutaneous sensitization because OVA-specific IgG1 and IgE antibody levels and OVA-driven splenocyte secretion of cytokines in these mice were comparable with those seen in wild-type control animals.

Conclusions: CRTH2 promotes allergic skin inflammation in response to cutaneous exposure to antigen in previously sensitized mice.

Figure 1. Mouse models of allergic skin inflammation

(A) Skin site subjected to repeated EC sensitization. (B) Acute skin challenge by application of OVA to tape stripped previously unsensitized skin of EC sensitized mice.

Figure 2. Levels of PGD2 levels in tape stripped skin and in chronically EC sensitized skin

(A) PGD2 levels in tape stripped skin of WT BALB/c mice (n=4 mice per group). (B) PGD2 levels in unsensitized skin of BALB/c mice taken immediately after shaving, and in skin taken at the end of 7 week sensitization with saline or OVA (n=4–5 mice per group). Experiments in A and B were performed at different times. (C) PGD2 levels in tape stripped skin of WBB6F1/J-Kit^W/Kit^W-v mice (n=3 mice per group). *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Allergic skin inflammation in skin sites repeatedly sensitized with OVA is intact in CRTH2^−/− mice

(A) Dermal infiltration with eosinophils and CD4⁺ cells. (B) Q-PCR analysis of mRNA levels of IL-4, IL-5 and IL-13 and IFN-γ Results are expressed as fold induction relative to saline sensitized skin of WT mice (n=5–6 mice per group). **p<0.01.

Figure 4. Impaired allergic skin inflammation in response to acute skin challenge with OVA in EC sensitized CRTH2^−/− mice

(A) PGD2 levels in challenged previously unsensitized skin from BALB/c mice previously EC sensitized with OVA. Measurement was performed 24 hrs after tape stripping and challenge with OVA or saline. (B) Representative skin histology. Magnification 200X. (C) Skin infiltrating CD4⁺ T cells and eosinophils. (D) Q-PCR analysis of mRNA levels of IL-4 and IL-13. Results are expressed as fold induction relative to saline challenged skin of EC sensitized WT mice (n=4–5 mice per group). *p<0.05, **p<0.01.

Figure 5. Intact systemic immune response to OVA in EC sensitized CRTH2^−/− mice

(A) Serum levels of OVA-specific immunoglobulin isotypes. (B) Cytokine secretion by splenocytes in vitro in response to OVA stimulation (n=4–5 mice per group). N.D.= not detectable

Figure 6. Expression of CCL17/TARC and CCL11/eotaxin in chronically sensitized mouse skin

CCL17/TARC (A) and CCL11/eotaxin (B) mRNA levels in skin from CRTH2^−/− mice and WT BALB/c controls, chronically sensitized with saline or OVA sensitized. Results are expressed as fold induction relative to saline sensitized skin of WT mice (n=4–5 mice per group).