Figure 1. Decreased HBD-3 gene expression in lesional EAD and IAD skin. RNA was isolated from the skin of normal subjects (n = 6), EAD (n = 9), IAD (n = 7), and psoriasis (n = 8) patients and the levels of HBD-3 evaluated by real-time RT-PCR.

Figure 2. Anti-CD3 stimulation of PBMC induces pro-inflammatory cytokines which induce HBD-3 in human keratinocytes. PBMC were separated from the whole blood of normal subjects (n = 5) and stimulated with 100 ng/ml of anti-CD3 for 24 h. (A) Levels of IL-1β, IL-6, TNF-α, and IFN-γ were evaluated in the PBMC supernatants using the BioRad Multiplex assay. (B) HBD-3 expression was evaluated by real-time RT-PCR in HaCaT cells that were stimulated with purified IL-1β (20 ng/ml), IL-6 (20 ng/ml), TNF-α (20 ng/ml), and IFN-γ (20 ng/ml). (C) HBD-3 expression was evaluated by real-time RT-PCR in HaCaT cells that were stimulated with supernatants from anti-CD3 stimulated PBMC.

Figure 3. Pro-inflammatory cytokines induce HBD-3 in normal skin, but not AD skin. Supernatant from anti-CD3 stimulated PBMC was added to skin explants from normal subjects (n = 4) or EAD patients (n = 4) for 24 h. HBD-3 expression in the skin explants was evaluated by real-time RT-PCR.

Figure 4. Reduced HBD-3 expression is an acquired defect in primary keratinocytes from AD patients. The levels of HBD-3 were evaluated by real-time RT-PCR in differentiated keratinocytes obtained from normal subjects (n = 6), EAD patients (n = 3), IAD patients (n = 3), and psoriatic patients (n = 6). HBD-3 was induced by 24-h stimulation with TNF-α plus IFN-γ. Samples treated with media alone are indicated by NT.

Figure 5. HBD-3 expression in HaCaT keratinocytes is down-regulated by Th2 cytokines. (A) RNA was isolated from keratinocytes stimulated with the supernatants from normal PBMC (n = 6) cultured with media or anti-CD3 (100 ng/ml) in the presence or absence of Th2 cytokines. (B) RNA was isolated from keratinocytes stimulated with the supernatants from AD PBMC (n = 3) cultured with media or anti-CD3 (100 ng/ml) in the presence or absence of Th2 cytokines. Levels of HBD-3 evaluated by real-time RT-PCR.

Figure 6. Neutralization of Th2 cytokines in AD skin augments HBD-3 expression. Lesional skin from EAD patients (n = 5) was cultured in the presence and absence of anti-IL-4 (1 μg/ml), IL-10 (1 μg/ml), and IL-13 (1 μg/ml) for 24 h and the level of HBD-3 evaluated by real-time RT-PCR.