Long TSLP transcript expression and release of TSLP induced by TLR ligands and cytokines in human keratinocytes

Background: Thymic stromal lymphopoietin (TSLP), highly expressed in keratinocytes in atopic dermatitis patients and bronchial epithelial cells in asthma patients, plays a key role in allergic diseases. Two forms of TSLP mRNA (long and short) have been reported. Objective: We compared the expression of the long-form and total TSLP transcripts in primary human keratinocytes. Methods: Primary human keratinocytes were stimulated with Toll-like receptor (TLR) ligands, cytokines, and vitamin D receptor agonists. Gene expression was analyzed by quantitative real-time PCR. The amount of TSLP released was measured by ELISA. Results: Polyl:C (TLR3 ligand), FSL-1 (TLR2-TLR6 ligand) and flagellin (TLR5 ligand) upregulated long-form TSLP expression, which predominantly contributed to upregulation of total TSLP expression. A glucocorticoid or an endosomal acidification inhibitor inhibited the polyl:C-dependent upregulation of total TSLP and the decrease of the total TSLP was due to the decrease of the long-form. An atopic cytokine milieu (TNF-alpha + IL-4 + IL-13) or TNF-alpha alone also upregulated the long-form. These stimuli also induced the release of TSLP. In contrast, a high concentration of calcitriol (1,25-dihydroxyvitamin D-3, the active form of vitamin D-3) or its analog MC903 upregulated total TSLP significantly but not the long-form, and did not induce the release of TSLP. Conclusion: TLR ligands or cytokines predominantly upregulate the gene expression of the long TSLP form, which contributes to the TSLP protein production, in primary human keratinocytes. Specific measurement of the long-form rather than total TSLP should be useful for accurate detection of functional human TSLP gene expression. (C) 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.