The initial step in Langerhans cell (LC) migration from the epidermis to the lymph node involves migration of maturing LC into the dermis. Here, we investigated the migration of LC out of the epidermis after exposure of the skin to contact allergens. Ex vivo intact human skin, epidermal sheets, and LC derived from the MUTZ-3 cell line (MUTZ-LC) were used to determine whether dermal fibroblasts play a role in mediating LC migration towards the dermis. Exposure of epidermal sheets or MUTZ-LC to allergens (nickel sulphate, 2,4-dinitrochlorobenzene, and cinnamaldehyde) or a cytokine maturation cocktail resulted in LC migration towards dermal fibroblasts. This was due to upregulation of CXCR4 on maturing LC and secretion of CXCL12/stromal derived factor-1 chemokine by fibroblasts. Neutralizing antibodies to either CXCL12 or CXCR4 completely blocked migration. Injection of CXCL12 neutralizing antibodies into intact human skin totally inhibited LC migration into the dermis. In contrast, neutralizing antibodies to CCL19/ CCL21 did not inhibit migration into the dermis. We describe a novel and essential role of dermis-derived CXCL12 in initiating migration of maturing human LC to the dermis thus permitting their further journey to the draining lymph nodes.Key words: Allergy . Chemotaxis . Cytokines . DC . Skin See accompanying commentary by Villablanca and Rodrigo IntroductionHuman skin is the external barrier to harmful environmental factors. Upon environmental assault (e.g. exposure to pathogens or allergens), Langerhans cells (LC) residing in the epidermis start to mature and migrate through the dermis [1,2]. Pathogenor allergen-exposed LC then migrate further into the afferent lymphatics towards the local lymph node where they are able to initiate a T-cell-mediated response [3]. LC migration is regulated by the sequential and differential expression of chemokines and their receptors [4,5]. Although migration to the lymph node is well documented, the chemokine ligands/receptors involved in the initial step of human LC migration out of the epidermis and into the dermis are as yet unknown.Several studies describe an essential role of CCR7 in mediating migration of mature LC towards the lymphatics [6][7][8][9][10]. CCR7 is highly expressed on fully mature, but not on immature LC, and is the chemokine receptor for the ligands CCL19 (MIP-3b) and CCL21 (6Ckine) [11][12][13]. Both of these chemokines are upregulated by lymphatic endothelial cells in response to allergen exposure [14]. Simultaneously, the chemokine receptor CCR6 on LC is downregulated upon maturation [15]. CCR6 is the receptor for CCL20 , which is mainly produced by keratinocytes (KC) in clinically normal human skin. CCR6/CCL20 is thought to 3050be of importance for epidermal homeostasis and homing of immature LC [16][17][18][19]. Taken together these reports show that loss of CCR6 and gain of CCR7 play important roles in the migration of mature LC out of the epidermis and into the lymphatic vessels. However, it is probable that other as yet unidentified sig...

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In this report, the construction of a functional, immunocompetent, full-thickness skin equivalent (SE) is described, consisting of an epidermal compartment containing keratinocytes, melanocytes, and human LCs derived from the MUTZ-3 cell line (MUTZ-LC) and a fibroblast-populated dermal compartment. The CD1a(+)Langerin(+)HLA-DR(+) MUTZ-LCs populate the entire epidermis at a similar density to that found in native skin. Exposure of the SE to subtoxic concentrations of the allergens NiSO(4) and resorcinol resulted in LC migration out of the epidermis toward the fibroblast-populated dermal compartment. A significant dose-dependent up-regulation of the DC maturation-related CCR7 and IL-1β transcripts and of CD83 at the protein level upon epidermal exposure to both allergens was observed, indicative of maturation and migration of the epidermally incorporated LC. We have thus successfully developed a reproducible and functional full-thickness SE model containing epidermal MUTZ-LC. This model offers an alternative to animal testing for identifying potential chemical sensitizers and for skin-based vaccination strategies and provides a unique research tool to study human LC biology in situ under controlled in vitro conditions.

Skin irritation is generally not considered to be an immunological event; however, alterations in the density of Langerhans cells (LC) in the epidermis do occur, which is indicative of LC migration. In this study, we investigated the migration of LC out of the epidermis after skin exposure to contact irritants and identified the chemokines involved. With the aid of ex vivo-intact human skin and epidermal sheets we show that dermal fibroblasts play a role in mediating LC migration towards the dermis. Exposure of ex vivo-intact human skin to a panel of seven irritants (SDS, salicylic acid, phenol, isopropanol, DMSO, TritonX, or benzalkonium chloride) resulted in decreased numbers of CD1a 1 cells in the epidermis and the accumulation of CD1a 1 cells in the dermis. In contrast to allergen exposure, neutralizing antibodies to either CXCL12 or CCL19/CCL21 did not inhibit LC migration out of the epidermis. Exposure of epidermal sheets to the prototypical irritant SDS resulted in a TNF-a-dependent LC migration towards dermal fibroblasts. This was a result of CCL2/MCP-1 and CCL5/RANTES chemokine secretion by fibroblasts: injection of CCL2-and CCL5-neutralizing antibodies into intact human skin totally inhibited LC migration into the dermis. We have thus identified a novel role for TNF-a-inducible dermis-derived CCL2 and CCL5 in initiating migration of irritant-exposed human LC out of the epidermis.Key words: Chemokine . DC . Irritant-contact dermatitis IntroductionEpidermal Langerhans cells (LC) are known to be capable of taking up and presenting antigenic chemicals to T lymphocytes and to be directly involved in the induction of allergic-contact dermatitis (ACD). In contrast, irritant-contact dermatitis (ICD) is considered to be an immunologically non-specific, local inflammatory reaction caused by substances found in, e.g. the workplace or consumer products that come into direct contact with the skin. Irritant substances penetrate the outer layer of the skin, whereupon they cause a localized acute inflammation. Morphologic studies have demonstrated that, despite ICD's supposed independence of adaptive immunity, alterations in the density and morphology of LC do occur concomitant with the clinical manifestation of inflammation. Several groups showed that upon irritant exposure, LC residing in the epidermis migrate into the dermis [1][2][3][4]. Whereas relatively much is known about allergeninduced migration of maturing LC out of the epidermis, very little is known about the underlying mechanisms of migration of LC exposed to non-maturing stimuli, such as irritants, out of the epidermis. Irritancy may result from perturbation or disruption of the lipid bilayers of the epidermis together with increased hydration [5,6]. Disruption of the skin barrier leads to the release of 2026cytokines such as IL-1a, . TNF-a is described to be one of the key cytokines in irritant dermatitis. Upon its release by keratinocytes, it induces strong gelatinolytic activity and down-regulates E-cadherin, which in turn decreases the attachment o...

Langerhans cells (LCs) migrate after topical exposure of the skin to irritants, despite the supposed independence of irritant contact dermatitis from adaptive immunity. Whereas allergen-activated LCs are known to migrate to the draining lymph nodes (LNs), the fate of migrated LCs upon topical irritant exposure is unknown. Here, we identified a phenotypic switch of LCs after their migration into the dermis upon irritant exposure. With the aid of ex vivo intact human skin and epidermal sheets, we show that dermal fibroblasts are necessary for an IL-10-dependent postmigrational phenotypic switch of LCs into macrophage-like cells. Exposure of ex vivo skin to a panel of seven irritants resulted in a decrease in the number of CD1a(+) cells and an increase in CD14(+)/CD68(+) cells in the dermis. Neutralizing antibodies against IL-10 totally inhibited the phenotypic LC-to-macrophage transition, but did not influence the migration of CD1a(+) cells. Exposure of epidermal sheets to irritants resulted in a fibroblast-dependent LC-to-CD14(+)/CD68(+) switch coinciding with migration, which could be totally inhibited by neutralizing antibodies against either IL-10 or CCL2/CCL5 (two chemokines responsible for epidermal-to-dermal migration). We have thus identified an IL-10-dependent phenotypic switch of LCs into macrophage-like cells upon irritant exposure and emigration from the epidermis.

Usher syndrome type III (USH3) characterized by progressive deafness, variable balance disorder, and blindness is caused by destabilizing mutations in the gene encoding the clarin-1 protein (CLRN1). Here we report a novel strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1N48K that involved a cell-based high-throughput screening of small molecules capable of stabilizing CLRN1N48K, a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure activity relationships. This resulted in the identification of BF844. To test the efficacy of BF844, a mouse model was developed that mimicked the progressive hearing loss of USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the human CLRN1N48K mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.

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Histone deacetylase inhibitors (HDACi) have been discovered as potential drugs for cancer treatment. The effect of BL1521, a novel HDACi, on the cell cycle distribution and the induction of apoptosis was investigated in a panel of MYCN single copy and MYCN amplified neuroblastoma cell lines. BL1521 arrested neuroblastoma cells in the G1 phase and induced up to 30% apoptosis. Downregulation of CDK4, upregulation of p21 WAF1/CIP1 and an increase of hypophosphorylated retinoblastoma protein were observed, indicating a possible mechanism for the cell-cycle arrest. BL1521 also induced downregulation of p27, which may underlie the observed induction of apoptosis.