Interleukin-18 is elevated in the horny layer in patients with atopic dermatitis and is associated with Staphylococcus aureus colonization

Background An increase in interleukin (IL)‐18 production from epidermal cells has been reported in an atopic dermatitis (AD) mouse model, and subsequent topical application of Staphylococcus aureus results in severe dermatitis. Objectives To reveal the relationship between S. aureus colonization of skin lesions and keratinocyte IL‐18 production, particularly in AD with relatively low serum IgE levels. We also aimed to establish a simple and noninvasive method of assaying IL‐18 produced by epidermal keratinocytes to evaluate local skin inflammation and therapeutic effects in patients with AD. Methods IL‐18 in the horny layer of the skin was collected via a tape‐stripping method and measured in 95 patients with AD and 40 healthy controls by enzyme‐linked immunosorbent assay (ELISA). Clinical severity, blood data and S. aureus skin colonization were evaluated before and after treatment. Results IL‐18 levels in the horny layer were significantly higher in the skin lesions of patients with AD than in healthy controls and correlated with SCORAD, levels of serum IL‐18, IgE, lactate dehydrogenase, thymus and activation‐regulated chemokine, blood eosinophils and transepidermal water loss. In the AD group with serum IgE < 1500 IU mL^?1, significantly higher IL‐18 levels were observed in the horny layer of patients colonized with S. aureus compared with those who were not. Conclusions Epidermal IL‐18 production was associated with the severity of AD. Staphylococcus aureus colonization seems to contribute to this IL‐18 production, especially in the AD group with relatively low IgE production. Tape stripping provides an easy and noninvasive method to assess epidermal IL‐18 production by ELISA.     

Effects of a ceramide‐containing water‐in‐oil ointment on skin barrier function and allergen penetration in an IL‐31 treated 3D model of the disrupted skin barrier

Atopic dermatitis (AD) is a chronically relapsing, pruritic inflammation of the skin with dryness and disturbed skin barrier function. Recently, we established that IL‐31 treatment of human 3D skin models resulted in a disrupted skin barrier phenotype resembling AD. In this model, we found that IL‐31 interferes with the differentiation of keratinocytes and inhibits the expression of terminal differentiation markers. In the present study, we investigated the effects of a ceramide‐containing water‐in‐oil skin care ointment on the physical skin barrier structure and function in disrupted skin barrier models, generated either by using primary normal human epidermal keratinocytes (NHEK) or HaCaT cells. We observed that the physical skin barrier of the models recovered after daily topical treatment with the ceramide‐containing ointment. Topical application of the ointment prevented downregulation of filaggrin and disorganization of other differentiation markers, such as keratin 10 and β4‐integrin, as demonstrated by immunohistological analysis. The expression of Ki67 was also upregulated in response to the ointment. Furthermore, functional studies revealed that local application of the ointment diminished the increased uptake of fluorescently labelled recombinant allergens of timothy grass (phl p1) in our model. In conclusion, our data revealed that topical application of a ceramide‐containing skin care ointment reduced IL‐31 induced impairments of the physical skin barrier and skin barrier function in an in vitro model of the disrupted skin barrier. This standardized model can be utilized in the future to monitor ex vivo effects of various topical therapies on skin morphology, physiology, and gene expression.     

Role of epidermal γδ T‐cell‐derived interleukin 13 in the skin‐whitening effect of Ginsenoside F1

Ginsenoside F1 (GF1) is a metabolite of ginsenoside Rg1. Although GF1 has several benefits for skin physiology, the effect of GF1 on skin pigmentation has not been reported. We found that a cream containing 0.1% GF1 showed a significant whitening effect on artificially tanned human skin after 8 weeks of application. However, GF1 did not inhibit mRNA expression of tyrosinase or dopachrome tautomerase (DCT) in normal human epidermal melanocytes (NHEMs) or cocultured NHEMs/normal human epidermal keratinocytes. Interestingly, GF1 enhanced production of interleukin 13 (IL‐13) from human epidermal γδ T cells. IL‐13 significantly reduced the mRNA expression and protein amount of both tyrosinase and DCT and reduced melanin synthesis activities in NHEMs, resulting in visible brightening of NHEM pellet. These results suggest that enhancement of IL‐13 production by GF1 from epidermal γδ T cells might play a role in the skin‐whitening effect of GF1 via the suppression of tyrosinase and DCT.     

Role of interleukin-17 in the pathogenesis of vitiligo

Background. Skewing of the immune response towards T helper (Th)1 or Th17 and away from regulatory T cells (Tregs) and Th2 cells may be responsible for the development and progression of autoimmune disease. An autoimmune theory has been proposed in the pathogenesis of vitiligo. No previous reports have investigated alterations in IL‐17 produced by Th17 cells in lesional skin in vitiligo. Aim. To investigate the role of IL‐17 in the pathogenesis of vitiligo by assessing its levels in lesional skin and serum of patients with vitiligo compared with controls. Methods. In total, 30 patients with vitiligo and 20 controls matched for age and gender were enrolled in the study. Serum and tissue IL‐17 levels were measured by ELISA and compared between both groups for correlations with age, gender, family history, disease duration, activity of vitiligo and percentage of involved body surface area. Results. A significant difference between patients and healthy controls was found for both serum and tissue IL‐17 levels (P < 0.001 for both). Significant positive correlations were found between disease duration and IL‐17 level in both serum (r = 0.42, P = 0.02) and lesional skin (r = 0.45, P < 0.015); between extent of vitiligo and IL‐17 levels in both serum (r = 0.65, P < 0.001) and skin (r = 0.48, P < 0.05); and between the serum and the tissue IL‐17 levels in patients with vitiligo (r = 0.54, P = 0.002). Conclusions. Multiple factors have been implicated in the pathogenesis of vitiligo. The increased levels of IL‐17 we found in serum and lesional skin suggest an important role for this cytokine in the pathogenesis of vitiligo.     

Increased levels of serum IL‐31 in chronic spontaneous urticaria*

Please cite this paper as: Increased levels of serum IL‐31 in chronic spontaneous urticaria. Experimental Dermatology 2010; 19: 464–466. Abstract: IL‐31 represents a novel cytokine involved in pruritic skin diseases including atopic dermatitis (AD). We, therefore, aimed at investigating IL‐31 levels in chronic spontaneous urticaria (CU). We included 46 patients with CU, 26 non‐atopic skin healthy subjects as negative and 28 patients with AD as positive controls. IL‐31 serum levels were analysed using commercial ELISA kit. IL‐31 serum levels were higher in patients with CU compared to healthy controls (P < 0.001), but lower compared to patients with AD (P < 0.001). There was no difference in IL‐31 serum levels in autologous serum skin test positive or negative CU patients and patients with infectious trigger factors including helicobacter pylori infection. IL‐31 serum levels may play a role in the pathophysiology of CU. This is supported by the finding that not all patients with CU respond to antihistamine treatment but to the treatment with immunosuppressive drugs.     

Tacrolimus decreases the expression of eotaxin, CCR3, RANTES and interleukin-5 in atopic dermatitis

Background There is a lack of studies on the effect of tacrolimus on eosinophils and related molecules including eotaxin, CCR3, RANTES and interleukin (IL)‐5. Objectives To investigate the effects of tacrolimus on in vivo eosinophil counts and on the related molecules eotaxin, CCR3, RANTES and IL‐5 in patients with atopic dermatitis (AD). Methods Lesional skin specimens and sera were obtained from 15 patients with AD and from 15 normal controls. For 8 weeks, the patients with AD applied 0·03% tacrolimus ointment to all affected areas twice daily. Blood sampling and skin biopsies were then repeated. We evaluated serum eotaxin and IL‐5 levels, and tissue eotaxin, CCR3, RANTES and IL‐5 levels. Additionally, tissue levels of eotaxin and CCR3 mRNA were measured. Results After treatment with topical tacrolimus twice daily for 8 weeks, significant decreases were found in serum IL‐5 levels, immunoreactive cell counts of eotaxin, IL‐5, CCR3 and RANTES in AD skin, and tissue eosinophil counts. However, the change in the serum eosinophil count was not statistically significant, and mRNA levels of eotaxin and CCR3 were not decreased significantly after treatment. Conclusions Topical tacrolimus reduces the number of eosinophils in tissue and suppresses the expression of eotaxin, CCR3, RANTES and IL‐5 related to proliferation, recruitment, activation and survival of eosinophils.     

Time‐dependent progression from the acute to chronic phases in atopic dermatitis induced by epicutaneous allergen stimulation in NC/Nga mice

Atopic dermatitis (AD) is a complicated skin condition influenced by genetic background and environmental factors. In this study, we applied Dermatophagoides farinae body extract (DfE) to the barrier‐disrupted skin of NC/Nga mice twice a week for 8 weeks to identify the clinical and immunological factors in AD progression. Repeated application of the DfE to the skin of NC/Nga mice showed the similar consequences for the natural course of progression in human AD, histologically and immunologically. We confirmed that the AD‐like skin lesions in NC/Nga mice did not last for the whole period of our experiment in spite of repeated topical applications of DfE twice a week. Topical DfE stimulation increased the skin mRNA expressions of Th1‐, Th2‐ and Th17‐related cytokines in the acute phase. The expression patterns of IL‐4 and IL‐13 in splenic T cells and skin lesions were consistent with the time course alterations of clinical features of AD‐like skin symptoms. We also showed that there was a remission phase either just before or right after the chronic phase in this experimental model. Interestingly, splenic T‐cell‐derived IL‐5 expression began to increase in the chronic phase, while skin‐derived IL‐5 mRNA expression increased in the acute phase. In conclusion, our results suggest that we should pay attention to the characteristics of each stage of AD progression and choose a suitable corresponding stage of animal model not only to elucidate the pathogenesis of AD but also to develop and evaluate therapeutic drugs for AD.     

Stimulated human melanocytes express and release interleukin‐8, which is inhibited by luteolin: relevance to early vitiligo

Vitiligo is a disorder of depigmentation, for which the pathogenesis is as yet unclear. Interleukin (IL)‐8 (CXCL8) is a key inflammatory chemokine. We investigated the regulation of IL‐8 production in human melanocytes, and the IL‐8 serum levels and skin gene expression in patients with vitiligo and in controls. Cultured melanocytes were stimulated for 24 h with tumour necrosis factor (TNF) 100 ng/mL and IL‐1β 10 ng/mL, with or without pretreatment with luteolin 50 μmol/L for 30 min, and IL‐8 release was measured by ELISA. Serum cytokines were measured by a microbead array. Skin biopsies were taken from healthy subjects (n = 14) as well as from marginal lesional and nonlesional skin from patients with vitiligo (n = 15). IL‐8 gene expression was evaluated by quantitative real time PCR. Both TNF and IL‐1β stimulated significant IL‐8 release (P < 0.01) from melanocytes, whereas pretreatment with luteolin significantly inhibited this effect (P < 0.01). IL‐8 gene expression was significantly increased in vitiligo compared with control skin (P < 0.05). IL‐8 may be involved in vitiligo inflammation. Inhibition by luteolin of IL‐8 release could be useful for vitiligo therapy.     

Gene expression study of IL10 family genes in vitiligo skin biopsies, peripheral blood mononuclear cells and sera

Background Vitiligo is a pigmentation disorder, the cause of which is complex and not yet fully understood. There is a significant change of epidermal cytokines in involved skin of patients with vitiligo compared with uninvolved skin and skin of healthy controls, thus suggesting a possible involvement of cytokines in the pathogenesis of vitiligo. Objectives To evaluate potential roles of IL10 family cytokines (IL10, IL19, IL20, IL22 and IL24) in vitiligo. Along with the selected cytokines, we investigated subunits of the receptors (IL10RA, IL10RB, IL20RA and IL22RA1) which are involved in the signalling pathway of the cytokines. Methods Quantitative real‐time polymerase chain reaction was used to detect mRNA expression levels in samples extracted from skin biopsies and peripheral blood mononuclear cells and an enzyme‐linked immunosorbent assay was used to measure protein concentrations in serum from patients with vitiligo and healthy controls. Results IL22 is significantly associated with vitiligo, especially with the active stage of vitiligo, as shown by results of mRNA expression and supported by results of protein level in sera. IL22 may provoke inflammation which leads to destruction of melanocytes. Conclusions The actual role of IL22 during pathogenesis of vitiligo remains to be better characterized. Signal transductions of other investigated cytokines seem to be regulated on the expression level of their receptor complex subunits.     

Increased interleukin‐36γ expression in skin and sera of patients with atopic dermatitis and mycosis fungoides/Sézary syndrome

Interleukin (IL)‐36γ is expressed by keratinocytes and functions as a key initiator of inflammation in the skin. IL‐36γ expression is enhanced by tumor necrosis factor‐α and IL‐17A, having a strong association with psoriasis. In this study, we examined the role of IL‐36γ in atopic dermatitis (AD) and mycosis fungoides (MF)/Sézary syndrome (SS). Serum levels of IL‐36γ in AD patients and MF/SS patients were elevated compared with those of healthy controls. Importantly, serum IL‐36γ levels in AD patients positively correlated with Eczema Area and Severity Index and those of MF/SS patients positively correlated with serum soluble IL‐2 receptor levels. IL‐36γ mRNA levels in AD skin and MF/SS skin were significantly higher than those of normal skin. IL‐36γ mRNA levels in MF/SS skin positively correlated with IL‐17A mRNA levels. Immunohistochemical staining revealed that IL‐36γ was highly expressed in keratinocytes in lesional skin of AD and MF/SS. Taken together, our study demonstrated that IL‐36γ expression was increased in sera and skin of patients with AD and MF/SS as was reported in psoriatic patients.     

Effect of topical application and intraperitoneal injection of oregonin on atopic dermatitis in NC/Nga mice

Please cite this paper as: Effect of topical application and intraperitoneal injection of oregonin on atopic dermatitis in NC/Nga mice. Experimental Dermatology 2010; 19 : e37–e43. Abstract: The diarylheptanoid, oregonin (ORE), which was isolated from the bark of Alnus japonica Steudel that grows natively in Korea, has been known to exert antioxidative, anti‐inflammatory, anti‐cancer and immune response inhibitory effects. The antioxidative effect of ORE was observed on the superoxide and 1,1‐diphenyl‐2‐picrylhydrazyl radical, as well as on the expression of inducible nitric oxide synthase and cyclooxygenase‐2 in lipopolysaccharide‐treated RAW264.7 macrophages. The statistically significant inhibitory action of ORE against production of cytokines induced by bacterial products or by interleukin (IL)‐1β, free radicals and nitrogen species, and a corresponding increase in cellular calcium concentration because of ORE were confirmed in bone marrow and spleen dendritic cells that are known to play important functions in the development and advancement of atopic dermatitis (AD). It was thus expected that ORE would exert a beneficial effect in the treatment of AD. A study on the pharmaceutical benefits of ORE against AD has not yet been conducted in vivo. We therefore used an in vivo AD animal model, namely the NC/Nga mice, and by applying ORE onto the animals through skin application as well as intraperitoneal injection, we attempted to evaluate the benefits of ORE in this system. Evaluation of ORE was conducted by following the SCORE method to score the effect, as well as by measuring the Th2 cytokines IL‐4, IL‐5 and IL‐13 levels from serum and lymphocytes, and IgE and eosinophil levels from serum. Additionally, the expression of mRNA and protein levels was estimated using real‐time polymerase chain reaction and Western blotting analysis. The following categories of clinical evaluation, Th2 cytokines IL‐4, IL‐5 and IL‐13 values, serum IgE levels, serum eosinophil levels, and mRNA and protein expression levels of iNOS and COX‐2, were evaluated from topical application and intraperitoneal injection groups of ORE. The effects of ORE on AD in NC/Nga mice were confirmed as being similar to the positive control group, while a significant difference with the negative control group was observed. The results presented in this report suggest that ORE might be beneficial in the treatment of AD.     

Mechanism of pathogenesis of imiquimod‐induced skin inflammation in the mouse: A role for interferon‐alpha in dendritic cell activation by imiquimod

Topical application of imiquimod (IMQ), a Toll‐like receptor (TLR)7 ligand, can induce and exacerbate psoriasis, a chronic inflammatory skin disorder. In a mouse model of IMQ‐induced psoriasis‐like skin inflammation, T‐helper (Th)17 cells and interleukin (IL)‐17/IL‐22‐producing γδ‐T cells have been shown to play a pivotal role. However, the mechanisms of induction of the Th17 pathway and development of psoriasis‐like skin inflammation by IMQ treatment remain unclear. In this study, we investigated pathogenic mechanisms of IMQ‐induced psoriasis‐like skin inflammation in mice. We first confirmed that, together with an increase in IL‐17 and IL‐22 production, application of IMQ to mouse skin induced the expression of cytokines required for activation of the Th17 pathway, and pro‐inflammatory mediators involved in the pathology of psoriasis. Analysis of Tlr7^?/? mice demonstrated that most of the in vivo effects of IMQ were mediated via TLR7. In an in vitro study using plasmacytoid dendritic cells (DCs), IMQ induced production of interferon (IFN)‐α, IL‐23, IL‐6 and tumor necrosis factor (TNF)‐α. Furthermore, when we analyzed in vitro‐generated bone marrow‐derived DCs with features similar to TNF‐α and inducible nitric oxide synthase (iNOS)‐producing DCs, IL‐23, IL‐6, IL‐1β, TNF‐α and iNOS/NO production was weakly induced by IMQ alone and further enhanced after co‐stimulation with IMQ and IFN‐α. These in vitro effects of IMQ were also mediated via TLR7 and the synergistic effect of IMQ, and IFN‐α was suggested to be caused by upregulation of TLR7 expression by IFN‐α. These results demonstrate part of the mechanism by which the Th17 pathway and psoriasis‐like skin inflammation are induced by IMQ and IFN‐α in a mouse model.     

Platinum and palladium nanoparticle‐containing mixture,PAPLAL, does not induce palladium allergy

Palladium (Pd) is a common metal found in jewellery and dental appliances, but it has been shown to be likely to cause metal allergy. We previously reported that platinum (nPt) and palladium (nPd) nanoparticle‐containing mixture (PAPLAL) has both superoxide dismutase and catalase activities and that the topical application of PAPLAL improved skin atrophy induced by chronic oxidative damage in an ageing mouse model. However, the safety of PAPLAL for preventing Pd allergy remains unclear. In the present study, we investigated whether or not PAPLAL induces Pd allergy. We found that PAPLAL treatment caused no skin inflammation, while nPd administration caused only slight skin inflammation compared to the palladium chloride‐induced severe reaction in an experimental metal allergy model. A gene expression analysis revealed that PAPLAL treatment significantly suppressed the expression of Inf‐γ, Il‐1β and Tnfα genes. Even in human clinical trials using patches containing metal nanoparticles, nPd and PAPLAL failed to induce significant skin inflammation. These results suggest that mixing with nPt in PAPLAL suppresses the inflammation response of nPd. PAPLAL can be expected to be applied to various skin treatments as a safe topical substance.     

Tandem repeated irritation in aged skin induces distinct barrier perturbation and cytokine profile in vivo

Background The barrier perturbation pattern and molecular markers of inflammation upon tandem repeated irritation in chronologically aged skin have not been previously studied. Objectives We aimed to investigate the barrier impairment kinetic and in vivo cytokine profile following sequential irritation with sodium lauryl sulfate (SLS) and undiluted toluene (Tol) in aged compared with young skin. Methods Four fields on the volar forearm of healthy aged and young volunteers (median age, respectively, 63·9 and 32·6 years) were sequentially exposed to 0·5% SLS and undiluted toluene in a controlled tandem repeated irritation test; an adjacent nontreated field served as control. The permeability barrier function was monitored by repeated measurements of transepidermal water loss (TEWL), capacitance and erythema every 24 h up to 96 h. The stratum corneum cytokines were harvested by sequential tape stripping and quantified by multiplex bead array and enzyme‐linked immunosorbent assay. Results Compared with young skin, aged skin was characterized by delayed and/or less pronounced alterations in the visual irritation score, TEWL, chromametry a*‐value and capacitance, assessed by the respective Δ‐values for each parameter and monitoring time point. In both groups, exposure to SLS/SLS, SLS/Tol and Tol/SLS resulted in decreased interleukin (IL)‐1α levels, whereas the application of Tol/Tol induced an increase in IL‐1α. Furthermore, decreased IL‐1 receptor antagonist (IL‐1RA) levels and a lower IL‐1RA/IL‐1α ratio were found following repeated exposure to the irritants. Conclusions Our results provide evidence for selective alterations in the cytokine profile and distinct barrier impairment kinetic following tandem repeated irritation with SLS and Tol in aged compared with young skin in vivo.     

Lipocalin‐2 exacerbates psoriasiform skin inflammation by augmenting T‐helper 17 response

Lipocalin‐2 (LCN2) is an antimicrobial protein and adipokine associated with insulin resistance, obesity and atherosclerotic disease. Psoriasis is a T‐helper (Th)1/Th17‐mediated, chronic inflammatory dermatosis related to metabolic syndromes and serum LCN2 levels are elevated in psoriatic patients. We examined the in vivo effects of LCN2 on topical imiquimod (IMQ)‐induced psoriasiform skin in BALB/c mice and in vitro on human keratinocytes (KC). Clinically, i.p. injected LCN2 exacerbated erythema and scaling in IMQ‐treated murine skin compared with phosphate‐buffered saline injection alone, and it augmented interleukin (IL)‐17A, IL‐17F, IL‐22, IL‐23p19, IL‐12p40, CCL20, tumor necrosis factor‐α, chemokine (C‐X‐C motif) ligand (CXCL)1, CXCL2, DEFB4, DEFB14, LCN2 and S100A7 mRNA levels of IMQ‐treated murine skin while it did not increase the mRNA levels of interferon‐γ, IL‐12p35 or CXCL10. LCN2 in synergy with IL‐17 increased mRNA levels of CCL20, LCN2 and DEFB4A but not of CXCL10 in human KC in vitro. These results suggest that LCN2 enhances the expression of Th17 cytokines/chemokines and antimicrobial peptides in murine IMQ‐treated psoriatic skin and KC. LCN2 may potentiate the development of psoriasis via the enhancement of Th17‐ and antimicrobial peptide‐mediated inflammation.     

Role of CPI‐17 in restoring skin homoeostasis in cutaneous field of cancerization: effects of topical application of a film‐forming medical device containing photolyase and UV filters

Cutaneous field of cancerization (CFC) is caused in part by the carcinogenic effect of the cyclobutane pyrimidine dimers CPD and 6‐4 photoproducts (6‐4PPs). Photoreactivation is carried out by photolyases which specifically recognize and repair both photoproducts. The study evaluates the molecular effects of topical application of a film‐forming medical device containing photolyase and UV filters on the precancerous field in AK from seven patients. Skin improvement after treatment was confirmed in all patients by histopathological and molecular assessment. A gene set analysis showed that skin recovery was associated with biological processes involved in tissue homoeostasis and cell maintenance. The CFC response was associated with over‐expression of the CPI‐17 gene, and a dependence on the initial expression level was observed (P = 0.001). Low CPI‐17 levels were directly associated with pro‐inflammatory genes such as TNF (P = 0.012) and IL‐1B (P = 0.07). Our results suggest a role for CPI‐17 in restoring skin homoeostasis in CFC lesions.     

Long‐term maintenance of skin immune system in a NOD‐Scid IL2rγnull mouse model transplanted with human skin

We developed a NOD‐Scid IL2rγ^null mouse model transplanted with human skin that brings fundamental insight on in vivo cellular mechanisms of intradermal immunization and antigen presentation by dermal dendritic and epidermal Langerhans cells for skin T‐cell immunity. Indeed, T‐cell immunity is a crucial checkpoint for the induction of in vivo rapid control of skin infection. With the long‐term preservation of a complete human skin immune system, this model offers the unique opportunity not only to better understand mechanisms of skin immune response but also to test new compounds and devices for cutaneous routes of vaccination, as well as new therapeutics approach for skin diseases, allergies or infections.