Abstract
Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Although the pathogenesis of AD is not fully understood, we and others have shown that IL-4 plays a key role. In this study we aimed to identify keratinocyte genes regulated by IL-4 that may play important roles in the pathophysiology of AD. HaCat cells were treated with IL-4 at various concentrations for 24h, and PCR gene array on inflammation/autoimmunity was performed three times for analysis of differential gene expression. Of all the 370 genes examined, 32 and 53 genes are up- and down-regulated, respectively. Specifically related to AD, chemokines CCL3L1, CCL8, CCL24, CCL25, CCL26, CXCL6 and CXCL16 are up-regulated by IL-4. Pro-inflammatory factors, such as IL-19, IL-20, IL-1α, IL-12Rβ2, IL-25, IL-31RA, OSMR and nitric oxide synthase 2, are also up-regulated. In addition, IL-4 up-regulates VEGFA, a pro-angiogenic factor. In contrast, antimicrobial peptides (AMPs) or factors involved in APM production, such as IFN-κ, S100s, Toll-like receptors, and several chemokines are down-regulated. Similarly IL-4 also down-regulates TNF-α, lymphotoxin-β, an IgE suppressor, TNFSF18, a T-cells function regulator, and the glucocorticoid receptor. On the in vivo level, real-time RT-PCR on the selected genes confirmed that IL-4 up-regulates chemokines, proinflammatory cytokines while it suppresses AMP production related genes in the skin obtained from IL-4 Tg mice. Detailed examination of these genes will delineate their specific roles in chemotaxis, inflammation, angiogenesis and AMP production, all of which may contribute to the development and progression of AD.
Highlights
Atopic dermatitis (AD), a chronic inflammatory skin disease, affects 10-20% of children and 1-3% of adults in developed countries [1]
Since keratinocytes are a dominant epidermal cell type that is known to participate in immune responses [8], and they are located adjacent to the IL-4 expression milieu in both human AD [8] and the IL-4 Tg mice [9], we aim to identify the keratinocyte genes that are directly regulated by IL-4
Materials IL-4, cell culture medium DMEM, fetal bovine serum (FBS), charcoal stripped FBS, Penicillin/Streptomycin, nonessential amino acids and sodium pyruvate were obtained from Invitrogen (Carlsbad, CA); RNA isolation kit was from Qiagen (Valencia, CA); First strand kit, qPCR master mix, and Inflammatory Response and Autoimmunity 384HT PCR Arrays were purchased from SABiosciences
Summary
Atopic dermatitis (AD), a chronic inflammatory skin disease, affects 10-20% of children and 1-3% of adults in developed countries [1]. Through over-expressing IL-4 in the basal epidermis using a basal keratinocyte-specific keratin-14 promoter/enhancer, we have generated a mouse AD animal model, IL-4 transgenic (Tg) mice [9] These Tg mice spontaneously develop skin lesions, which satisfy the clinical and histological diagnostic criteria for human AD, while the non-Tg littermates are lesion-free [9]. Using these Tg mice, we have shown in vivo that IL-4 over-expression in the epidermis leads to up-regulation of several proinflammatory cytokines [7], B cell activation molecules [10], angiogenic factors [5], and critical adhesion molecules [11]. Selected real-time RT-PCR on IL-4 Tg mice confirmed our in vitro findings
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