Cytokine modulation of atopic dermatitis filaggrin skin expression

Abstract

Background: Atopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by a defective skin barrier function. Recent studies have reported mutations of the skin barrier gene encoding filaggrin in a subset of patients with AD.

Objective: We investigated whether reduced filaggrin expression was found in patients with AD who were not carriers of known filaggrin mutations and whether filaggrin expression was modulated by the atopic inflammatory response.

Methods: Filaggrin expression was measured in skin biopsies and cultured keratinocytes using real-time RT-PCR and immunohistochemistry. Filaggrin loss-of-function mutations were screened in a total of 69 subjects.

Results: Compared with normal skin, filaggrin expression was significantly reduced (P < .05) in acute AD skin, with further reduction seen in acute lesions from 3 European American subjects with AD who were heterozygous for the 2282del4 mutation. This was confirmed by using immunohistochemistry. AD skin is characterized by the overexpression of IL-4 and IL-13. Keratinocytes differentiated in the presence of IL-4 and IL-13 exhibited significantly reduced filaggrin gene expression (0.04 +/- 0.01 ng filaggrin/ng glyceraldehyde 3-phosphate dehydrogenase; P < .05) compared with media alone (0.16 +/- 0.03).

Conclusion: Patients with AD have an acquired defect in filaggrin expression that can be modulated by the atopic inflammatory response.

Clinical implications: The atopic immune response contributes to the skin barrier defect in AD; therefore, neutralization of IL-4 and IL-13 could improve skin barrier integrity.

Figure 1

Filaggrin deficiency in AD skin. RNA was isolated from the skin of normal subjects (n=15) and AD patients (n=16) with or without the 2282del4 mutation. Filaggrin gene expression was evaluated using real-time RT-PCR. * and ** indicate significant differences of p<0.05 and p<0.01, respectively.

Figure 2

Decreased filaggrin staining in AD skin. A. Representative paraffin embedded skin biopsies from normal subjects (n=11), AD patients (n=12), AD patients with the 2282del4 mutation (n=3), and patients with lichen planus (n=6) stained for filaggrin are shown. Images were collected at 40× magnification and the scale bar represents 50 μm. B. The intensity of the staining was graded visually on a scale from 0 (no staining) to 5 (the most intense staining). *, **, and *** indicate significant differences of p<0.05, p<0.01, and p<0.001, respectively.

Figure 2

Decreased filaggrin staining in AD skin. A. Representative paraffin embedded skin biopsies from normal subjects (n=11), AD patients (n=12), AD patients with the 2282del4 mutation (n=3), and patients with lichen planus (n=6) stained for filaggrin are shown. Images were collected at 40× magnification and the scale bar represents 50 μm. B. The intensity of the staining was graded visually on a scale from 0 (no staining) to 5 (the most intense staining). *, **, and *** indicate significant differences of p<0.05, p<0.01, and p<0.001, respectively.

Figure 3

Th2 cytokines down-regulate filaggrin gene expression. Primary human keratinocytes were cultured in 0.06 or 1.3 mM of CaCl₂ in the presence and absence of IL-4 and IL-13 or IFN-γ for five days. RNA was collected from the cells, and the levels of filaggrin were evaluated by real-time RT-PCR (A) or Immuno-dot Blot (B & C). *, **, and *** indicate significant differences of p<0.05, p<0.01, and p<0.001, respectively, between exposure groups.

Figure 3

Th2 cytokines down-regulate filaggrin gene expression. Primary human keratinocytes were cultured in 0.06 or 1.3 mM of CaCl₂ in the presence and absence of IL-4 and IL-13 or IFN-γ for five days. RNA was collected from the cells, and the levels of filaggrin were evaluated by real-time RT-PCR (A) or Immuno-dot Blot (B & C). *, **, and *** indicate significant differences of p<0.05, p<0.01, and p<0.001, respectively, between exposure groups.

Figure 3

Th2 cytokines down-regulate filaggrin gene expression. Primary human keratinocytes were cultured in 0.06 or 1.3 mM of CaCl₂ in the presence and absence of IL-4 and IL-13 or IFN-γ for five days. RNA was collected from the cells, and the levels of filaggrin were evaluated by real-time RT-PCR (A) or Immuno-dot Blot (B & C). *, **, and *** indicate significant differences of p<0.05, p<0.01, and p<0.001, respectively, between exposure groups.