Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is overexpressed in inflammatory skin diseases and affects epidermal morphology in constitutive knockout mice and murine 3D skin models.

Inter-α-trypsin inhibitors are protease inhibitors that are thought to be important regulators in various acute-phase processes. They are composed of one light chain (bikunin) and different heavy chains (ITIHs). The only function known so far of ITIHs is the covalent linkage to hyaluronan (HA). As there is virtually no knowledge on the distribution and function of ITIH proteins in skin tissue, we performed a systematic characterization of ITIH expression in healthy and diseased skin. Using GeneChip(®) Human Exon 1.0 ST expression profiling, we found that ITIH5 represents the major ITIH family member expressed in human skin. Moreover, the use of quantitative reverse transcription PCR and a customized ITIH5-specific antibody indicated that ITIH5 is predominantly produced by dermal fibroblasts. Immunohistochemical analysis revealed a clearly detectable ITIH5 protein expression in normal skin. Interestingly, ITIH5 expression was significantly up-regulated in inflammatory skin diseases. Furthermore, 3D skin models employing murine Itih5(-/-) epidermal keratinocytes and dermal fibroblasts as well as skin specimens of Itih5(-/-) mice revealed a significantly altered epidermal structure compared to wild-type controls. Hence, we can strengthen the presumption that ITIH5 may constitute a novel regulatory molecule of the human skin that could play an important role in inflammation via its interaction with HA.

Knockdown of lecithin retinol acyltransferase increases all-trans retinoic acid levels and restores retinoid sensitivity in malignant melanoma cells.

Retinoids such as all-trans retinoic acid (ATRA) influence cell growth, differentiation and apoptosis and may play decisive roles in tumor development and progression. An essential retinoid-metabolizing enzyme known as lecithin retinol acyltransferase (LRAT) is expressed in melanoma cells but not in melanocytes catalysing the esterification of all-trans retinol (ATRol). In this study, we show that a stable LRAT knockdown (KD) in the human melanoma cell line SkMel23 leads to significantly increased levels of the substrate ATRol and biologically active ATRA. LRAT KD restored cellular sensitivity to retinoids analysed in cell culture assays and melanoma 3D skin models. Furthermore, ATRA-induced gene regulatory mechanisms drive depletion of added ATRol in LRAT KD cells. PCR analysis revealed a significant upregulation of retinoid-regulated genes such as CYP26A1 and STRA6 in LRAT KD cells, suggesting their possible involvement in mediating retinoid resistance in melanoma cells. In conclusion, LRAT seems to be important for melanoma progression. We propose that reduction in ATRol levels in melanoma cells by LRAT leads to a disturbance in cellular retinoid level. Balanced LRAT expression and activity may provide protection against melanoma development and progression. Pharmacological inhibition of LRAT activity could be a promising strategy for overcoming retinoid insensitivity in human melanoma cells.

P-glycoprotein (ABCB1) expression in human skin is mainly restricted to dermal components.

Several transport proteins are constitutively expressed in skin cells, but the putative role of the ABC transporter P-glycoprotein (P-gp) in human skin is yet unknown. Therefore, we analysed mRNA and protein expression and localization of P-gp in human skin. Using qRT-PCR, we demonstrated a strong MDR1 mRNA expression in whole skin specimens and dermis, whereas the expression of MDR1 in epidermis, epidermal keratinocytes or dermal fibroblasts was only weak. Immunohistochemistry confirmed mRNA data and revealed a marked expression of P-gp within sweat ducts, vessels, nerve sheaths and muscles of human skin and a moderate expression in basal epidermis. Our findings closely correlate with previous studies in murine skin supporting the role of P-gp in the uptake of compounds from the epidermal compartment and their secretion into the bloodstream and sweat ducts. It may also prevent the uptake of xenobiotics into the skin by functioning as a barrier located in the dermal vasculature.

Active transport of contact allergens in human monocyte-derived dendritic cells is mediated by multidrug resistance related proteins.

The multidrug resistance related proteins (MRPs) function as efflux transporters of a variety of large organic anions or their conjugates. In recent studies we demonstrated that antigen-presenting cells express a specific pattern of MRPs. MRP-mediated efflux activity of human monocyte-derived dendritic cells (moDCs) was analyzed using an in vitro transport assay. The efflux transport of radiolabeled contact allergens was inhibited using the specific MRP inhibitor indomethacin. Treatment with indomethacin increased intracellular concentration of [³H] eugenol and [³H] isoeugenol in moDCs. In addition by using MRP1 expressing inside-out membrane vesicles we revealed that the transport of eugenol is mediated by MRP1. Human DCs were employed to assess the sensitizing potential of contact allergens and alters their cytokine gene expression profile. Hence, to survey the functionality of indomethacin after stimulation with contact allergens IL-8 and TRIM16 regulation was measured by a DC-based in vitro assay. Incubation with isoeugenol after pre-treatment with indomethacin leads to increased IL-8 and TRIM16 gene expression. These results strongly support the functional role of MRPs in the active efflux of contact allergens also in antigen-presenting cells like moDCs, a novel mechanism which could possibly play a role in the pathogenesis of contact allergy.

Research in practice: the second barrier of the human skin.

A major function of human skin is to form an effective barrier between the environment and the inside of the organism. Especially important for this function is the activity of the physical barrier of the skin, which is mainly located in the stratum corneum. To improve this barrier function of the skin, skin protection agents are used. Recent studies have revealed that application of skin protection agents before exposition to xenobiotics does not generally reduce the percutaneous uptake of these compounds. These findings indicate that besides new study designs and improved test systems, there seems to be a need for new therapeutic approaches for more effective skin protection. In this light, new findings regarding a second barrier function of the human skin, the biochemical/toxicological barrier, could be of interest. A crucial part of this barrier function are members of the cytochrome P450 (CYP) family and efflux-transport proteins of the multidrug resistance-related protein family (MRPs), which are mainly expressed by basal layer keratinocytes. Recent studies have revealed that besides the physiological and protective function of these transport proteins and CYP enzymes in skin cells, the same proteins also play a role in the transport of contact allergens and activation of prohaptens to haptens causing contact dermatitis. Inhibition of this metabolism mediated activation of prohaptens and stimulation of the active elimination of contact allergens from skin cells could represent novel mechanisms improving the established tools for skin protection.

Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts.

Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 microg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 microg/ml of pantothenate compared with untreated cells using the GeneChip Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)-6, IL-8, Id1, HMOX-1, HspB7, CYP1B1 and MARCH-II. Regulation of these genes was subsequently verified by quantitative real-time polymerase chain reaction analysis. Induction of HMOX-1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts.

Cutaneous metabolic activation of carvoxime, a self-activating, skin-sensitizing prohapten.

Bioactivation of low molecular weight compounds in the skin can cause contact sensitization. We have previously shown that the alpha, beta-R-unsaturated oxime R-carvoxime [1, (R)-2-methyl-5-isopropenylcyclohex-2-enone oxime] is bioactivated to two diastereomeric highly reactive and strongly sensitizing alpha, beta-epoxy oxime metabolites. To investigate if this metabolic activation is catalyzed by the major cytochrome P450 (P450) enzymes found in human skin, incubations of 1 with a skinlike P450 cocktail in the presence of glutathione were carried out. We identified three glutathione conjugates in the incubation mixture arising from two diasteomeric alpha, beta-epoxy oxime metabolites of 1, thus showing that the metabolic activation of 1 is P450-mediated. A P450 identification study using the individual P450 enzymes present in the skinlike P450 cocktail showed the involvement of P450 1A1 and 1B1 and also to some extent 2B6. P450 1B1 metabolism of 1 was found to be stereoselective as glutathione conjugates from only one of the alpha, beta-epoxyoxime metabolites were identified (metabolite 2). Additionally, 1 was found to be an inducer of P450 1B1 (but not 1A1) in human monocyte-derived dendritic cells (moDCs) and to some extent in normal human epidermal keratinocytes. A further transcriptional gene expression change observed in moDCs was a 44-fold upregulation of IL-8, a marker often used for assessment of sensitizing potential of contact allergens. The autoinduction of P450 1B1 by 1 may be a key event in the development of contact allergy to 1 and may also explain why only metabolite 2, and not 3, was found to elicit an allergic response in mice sensitized to 1. Our data show that the alpha, beta-unsaturated oxime 1 is bioactivated by human cutaneous P450, thus forming highly allergenic metabolites, and has the potential to induce its own bioactivation pathway, particularly in antigen-presenting cells.

Topographical control of human macrophages by a regularly microstructured polyvinylidene fluoride surface.

In this study we investigated the influence of surface topography on the inflammatory response of human macrophages. We generated different polyvinylidene fluoride (PVDF) surfaces including (i) a smooth surface of PVDF spherulites as a control, (ii) a randomly nanotextured surface with alumina particles, and (iii) a microstructure using laser ablation. The identical chemistry of all PVDF surfaces was demonstrated by X-ray photoelectron spectroscopy. The topography was evaluated by white light interferometry and X-profile analysis. Macrophages were cultured on the different surfaces including lipopolysaccharide (LPS) treatment as an inflammatory activator. Our results demonstrate that the microstructured surface but not the nanotexured significantly affects the activation of primary human macrophages by inducing a specific cytokine and gene expression pattern. This activation resulted in a subtype of macrophages with pro- but also anti-inflammatory properties. Interestingly, the response on the topography differed from that triggered by LPS, pointing to a different activation state of the cells. Our data clearly show that a particular topography induces an inflammatory response. This suggests that the modification of topography could influence the inflammatory potency of a biomaterial and hence could affect the biocompatibility of implants.

Differential expression of influx and efflux transport proteins in human antigen presenting cells.

Human macrophages (M Phi) express cytochrome P450 enzymes verifying their capacity to metabolize a variety of endogenous and exogenous substances. Here we analysed the mRNA and protein expression of transport proteins involved in the uptake or export of drugs, hormones and arachidonic acid metabolites in dendritic cells (DC) and M Phi compared to their precursors - blood monocytes - using cDNA microarray, RT-PCR, Western-blot and immunostaining techniques. The transport proteins studied included members of the solute carrier organic anion transporter family (SLCO) and the multidrug resistance associated proteins (MRP) 1-6 belonging to the ATP-binding cassette subfamily C (ABCC). We found that only mRNA for SLCO-2B1, -3A1, and -4A1 were present in monocytes, M Phi and DC. Most interestingly the expression of SLCO-2B1 was markedly enhanced in M Phi as compared to monocytes and DC. The presence of mRNA for ABCC1, 3, 4, 5 and 6 in all three cell types was demonstrated. On protein level ABCC1/MRP1 which has been identified as leukotriene C(4) transporter was found to be the most abundant transporter in M Phi and DC. Blocking the ABCC1/MRP1 activity with the specific inhibitor MK571 resulted in a phenotypic change in DC but not in M Phi. Our data show that human blood monocytes and monocyte derived M Phi as well as DC express a specific profile of transporters involved in uptake and export of exogenous molecules like allergens or drugs, but also of endogenous substances in particular of inflammatory lipid mediators like leukotrienes and prostaglandins.

Reactivity of in vitro activated human T lymphocytes to p-phenylenediamine and related substances.

BACKGROUND: Patch tests to p-phenylenediamine (PPD) and related substances often show concurrent reactions that can be attributed to separate sensitization or cross-reactivity. OBJECTIVES: In order to understand the health risks associated with cross-reactivity, we studied cross-reactivity of eight chemicals in vitro by measurement of T-cell proliferation of peripheral blood mononuclear cells (PBMC), T-cell lines (TCL), and T-cell clones (TCC) of subjects with a positive patch test result to PPD. PATIENTS/METHODS: We studied PBMC from 13 patients and were able to generate TCL from seven and TCC from four patients. Their proliferative responses to the chemicals were estimated. RESULTS: Concurrent reactions to these compounds on the polyclonal and monoclonal level were found. A restricted T-cell receptor (TCR) Vbeta16-usage was observed (5/8 clones). A detailed analysis of 34 TCL showed broad cross-reactivity (64.7%) between PPD, p-toluenediamine, Bandrowski's Base, and p-aminoazobenzene. More restricted patterns were found in 8.8%, which responded only to compounds with two or three benzene rings, whereas 26.5% of the clones reacted specifically only to one compound. CONCLUSION: More than 60% of the clones showed a broad cross-reactivity pattern. Hence, clinically observed cross-reactivity between different para-amino compounds can be based on a TCR recognizing similar epitopes of these compounds with low specificity.

The RGD coupling strategy determines the inflammatory response of human primary macrophages in vitro and angiogenesis in vivo.

Surface modifications of implants are frequently done using bioactive peptides. However, immune cells such as macrophages might evoke a rejection of an implant due to an undesired activation by the materials. Here, the influence of different strategies for peptide immobilization onto (poly)-vinylidene fluoride (PVDF) on inflammation and angiogenesis is studied. The inflammatory response of human primary macrophages is investigated by analyzing inflammatory cytokine expression. Surface roughness and adsorptive coupling have only minor effects on macrophage activation. Acrylic acid (AAc)-based covalent RGD-coupling leads to the most favorable cellular reaction, indicated by increased VEGF release. Chemical vapor deposition treated surfaces are inert, but additional covalent coupling of RGD induces a pronounced proinflammatory reaction. An in vivo angiogenesis study reveals that covalent coupling of RGD results in delayed but increased angiogenesis. It is concluded that for implant decoration with peptides, the substrate material has to be selected carefully to prevent inflammatory immune responses.

Downregulation of STRA6 expression in epidermal keratinocytes leads to hyperproliferation-associated differentiation in both in vitro and in vivo skin models.

Retinoids are known to affect skin cell proliferation and differentiation and are key molecules that target retinoid and retinoic acid receptors (RXRs and RARs), leading to physiological and pharmacologic effects. Our aim was to elucidate the role of the retinol-binding protein receptor STRA6, mediating cellular uptake of retinol, on skin structure and function. Our results indicate that STRA6 is constitutively expressed in human epidermal keratinocytes and dermal fibroblasts and is regulated via RAR/RXR-mediated pathways. HaCaT (Human adult low Calcium high Temperature) cells with stable STRA6 knockdown (STRA6KD) showed increased proliferation. Consistently, human organotypic 3D skin models using stable STRA6KD HaCaT cells showed a significantly thicker epidermis and enhanced expression of activation, differentiation, and proliferation markers. The effects were reversible after treatment with free retinol. Human skin reconstitution employing STRA6KD HaCaT cells leads to massive epithelial thickening under in vivo conditions in SCID mice. We propose that STRA6KD could lead to cellular vitamin A deficiency in keratinocytes. Consequently, STRA6 has a role for regulating retinoid homeostasis and in helping to program signaling that drives proliferation and differentiation of human skin cells. By its influence on hyperproliferation-associated differentiation, STRA6 could also have a role in skin regeneration and could be a target for pharmacological approaches to improve wound healing.

Expression and function of macrophage migration inhibitory factor in the pathogenesis of UV-induced cutaneous nonmelanoma skin cancer.

Chronic skin exposure to ultraviolet light stimulates the production of cytokines known to be involved in the initiation of skin cancer. Recent studies in mouse models suggested a role for macrophage migration inhibitory factor (MIF) in the UVB-induced pathogenesis of nonmelanoma skin cancer (NMSC). Our studies aimed at defining the pathophysiological function of MIF in cutaneous inflammatory reactions and in the development and progression of NMSC. Immunohistochemical analysis revealed a moderate expression of MIF in normal human skin samples but an enhanced expression of this cytokine in lesional skin of patients with actinic keratosis or cutaneous SCC. Enzyme-linked immunosorbent assay studies showed a time-dependent increase in MIF secretion after a moderate single-dose UVB irradiation in NHEKs and SCC tumor cells. MIF is known to interact with CXCR2, CXCR4 and CD74. These receptors are not constitutively expressed in keratinocytes and HaCaT cells and their expression is not induced by UVB irradiation either. However, stimulation with IFNγ upregulated CD74 surface expression in these cells. Affymetrix(®) Gene Chip analysis revealed that only keratinocytes prestimulated with IFNγ are responsive to MIF. These findings indicate that MIF may be an important factor in the pathogenesis of NMSC tumorigenesis and progression in an inflammatory environment.