The stratum corneum and epidermis serve as a protective physical barrier against invading allergens and pathogens. This physical barrier is significantly compromised in the skin of AD patients; resulting in increased allergen sensitization, as well as viral and bacterial infections [24
]. Previous studies have shown that increased penetration of allergens and human pathogens augment the atopic inflammatory response which can exacerbate the disease [6
]. It is not fully understood how this additionally affects the epidermal barrier. The epidermal differentiation complex (EDC) is a cluster of genes located on chromosome 1q21 that encode a number of proteins important for barrier function [27
]. LOR and IVL are two proteins encoded within the EDC, therefore these studies evaluated the relationship between their expression and the atopic inflammatory response.
In our current study, we demonstrate that both LOR and IVL expression are down-regulated in lesional and non-lesional AD skin as compared to skin from normal subjects. This is contradictory to previous studies by Sugiara et al [28
] and Jensen et al. [29
]. Using gene microarrays, Sugiara et al. [28
] observed increased IVL and decreased LOR gene expression in lesional AD skin. Jensen et al. [29
] observed increased LOR and decreased IVL expression using immunohistochemistry and western blotting. Important differences in experimental design may explain the discrepancies between our observations and those previously published: 1) while gene microarrays provide a significant amount of information, they are known to have limitations and can be falsely positive or negative [30
]. Our study used real-time RT-PCR, which has a higher degree of sensitivity, to examine the genetic expression of LOR and IVL and immunohistochemistry to confirm the observation on the protein level. 2) The lesional skin used in our current study is collected solely from acute atopic lesions. This factor is not clearly defined in either of the previous studies [28
]. AD lesions are bi-phasic with acute lesions having more of a Th2 cytokine milieu and chronic lesions having more of a Th1 cytokine milieu [19
]. The relationship between cytokine milieu and barrier integrity is not completely understood. In fact, our current study is the first to investigate the effect of the cytokine milieu on LOR and IVL expression.
Keratinocytes are the primary cell of the epidermis and migrate through the basal, spinous, and granular regions to differentiate terminally into cornified cells and form the CE [15
]. For that reason, we used primary human keratinocytes to investigate why LOR and IVL expression is decreased in AD skin. Acute AD skin is characterized by the over-expression of IL-4 and IL-13 [19
]. Therefore, we differentiated normal primary keratinocytes in the presence and absence of IL-4 and/or IL-13 for 5 days and evaluated the gene expression of LOR and IVL. The induction and expression of LOR and IVL was significantly inhibited by IL-4 and IL-13, suggesting that expression of LOR and IVL is down-regulated in AD skin due to the over-expression of Th2 cytokines in AD skin.
IL-4 and IL-13 signal through IL-4 receptor α1 and α2 to activate the STAT-6 signaling pathway [22
]. This pathway has previously been shown to down-regulate important components of the innate immune response [33
], therefore we investigated the role of STAT-6 in the Th2 mediated down-regulation of LOR and IVL. Skin biopsies from STAT-6 transgenic mice exhibited significantly lower levels of both LOR and IVL as compare to their wild type controls. This suggests that IL-4 and IL-13 modulation of LOR and IVL is mediated by STAT-6 activation.
The current study demonstrates that gene and protein expression of LOR and IVL are decreased in the skin from AD patients. In addition, we demonstrate that gene expression of LOR and IVL is down-regulated by IL-4 and IL-13 through STAT-6. Taken together, our data suggest that decreased expression of LOR and IVL is caused, in part, by the over-expression of Th2 cytokines that down-regulate LOR and IVL expression during the differentiation into CE. Therefore, our current study suggests that cutaneous sensitization by various allergens and pathogens in AD is due, in part, to defect in epidermal skin barrier and may result in frequent skin inflammation and infection in AD skin.