Cytokines In Human Keratinocytes Research Articles

Anti-Inflammatory Artificial Extracellular Vesicles with Notable Inhibition of Particulate Matter-Induced Skin Inflammation and Barrier Function Impairment.

Particulate matter (PM) exposure disrupts the skin barrier, causing cutaneous inflammation that may eventually contribute to the development of various skin diseases. Herein, we introduce anti-inflammatory artificial extracellular vesicles (AEVs) fabricated through cell extrusion using the biosurfactant PEGylated mannosylerythritol lipid (P-MEL), hereafter named AEVP-MEL. The P-MEL has anti-inflammatory abilities with demonstrated efficacy in inhibiting the secretion of pro-inflammatory mediators. Mechanistically, AEVP-MEL enhanced anti-inflammatory response by inhibiting the mitogen-activated protein kinase (MAPK) pathway and decreasing the release of inflammatory mediators such as reactive oxygen species (ROS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines in human keratinocytes. Moreover, AEVP-MEL promoted increased expression levels of skin barrier proteins (e.g., involucrin, IVL) and water-proteins (e.g., aquaporin 3, AQP3). In vivo studies revealed that repeated PM exposure to intact skin resulted in cutaneous inflammatory responses, including increased skin thickness (hyperkeratosis) and mast cell infiltration. Importantly, our data showed that the AEVP-MEL treatment significantly restored immune homeostasis in the skin affected by PM-induced inflammation and enhanced the intrinsic skin barrier function. This study highlights the potential of the AEVP-MEL in promoting skin health against PM exposure and its promising implications for the prevention and treatment of PM-related skin disorders.

Effects of Ultra-Weak Fractal Electromagnetic Signals on Malassezia furfur

Malassezia spp. are dimorphic, lipophilic fungi that are part of the normal human cutaneous commensal microbiome. However, under adverse conditions, these fungi can be involved in various cutaneous diseases. In this study, we analysed the effect of ultra-weak fractal electromagnetic (uwf-EMF) field exposure (12.6 nT covering 0.5 to 20 kHz) on the growth dynamics and invasiveness of M. furfur. The ability to modulate inflammation and innate immunity in normal human keratinocytes was also investigated. Using a microbiological assay, it was possible to demonstrate that, under the influence of uwf-EMF, the invasiveness of M. furfur was drastically reduced (d = 2.456, p < 0.001), while at the same time, its growth dynamic after 72 h having been in contact with HaCaT cells both without (d = 0.211, p = 0.390) and with (d = 0.118, p = 0.438) uwf-EM exposure, were hardly affected. Real-time PCR analysis demonstrated that a uwf-EMF exposure is able to modulate human-β-defensin-2 (hBD-2) in treated keratinocytes and at the same time reduce the expression of proinflammatory cytokines in human keratinocytes. The findings suggest that the underlying principle of action is hormetic in nature and that this method might be an adjunctive therapeutic tool to modulate the inflammatory properties of Malassezia in related cutaneous diseases. The underlying principle of action becomes understandable by means of quantum electrodynamics (QED). Given that living systems consist mainly of water and within the framework of QED, this water, as a biphasic system, provides the basis for electromagnetic coupling. The oscillatory properties of water dipoles modulated by weak electromagnetic stimuli not only affect biochemical processes, but also pave the way for a more general understanding of the observed nonthermal effects in biota.

Treponema pallidum FlaA2 inducing the release of pro-inflammatory cytokines is mediated via TLR2 in keratinocytes

BackgroundSyphilis, caused by Treponema pallidum (T. pallidum), is a multi-organ, multiple systems, multi-stage sexually transmitted diseases with various clinical manifestations, among of which pathological lesions of skin and mucosa are the typical clinical manifestations of syphilis. However, the immunopathogenesis of this process is poorly understood. T. pallidum flagellin FlaA2, as a part of the important organelle responsible for the causative agent's motility, may contributes to the host skin inflammatory response. ObjectivesTo determine the mechanisms of T. pallidum FlaA2 stimulating the expression of pro-inflammatory cytokines in human keratinocytes. MethodsRecombinant FlaA2 protein was performed to stimulate human keratinocytes. The mRNA transcription levels and protein expression levels of IL-6 and IL-8 were detected by qRT-PCR and ELISA, respectively. Western blot was used to detect the total protein and phosphorylation levels of ERK, p38, JNK and NF-κB, respectively. The intracellular location of NF-κB p65 was detected by immunofluorescence staining. ResultsRecombinant FlaA2 could considerably induced the expression of pro-inflammation cytokines IL-6 and IL-8 in HaCaT cells, and FlaA2-induced IL-6 and IL-8 secretion could be decreased by inhibiting TLR2 using pZERO-hTLR2. Further investigation showed that FlaA2 could activate the phosphorylation of ERK, p38 and IκBα and FlaA2-stimulated secretion of IL-6, IL-8 were attenuated by ERK, p38 and NF-κB inhibitors in HaCaT cells. Moreover, FlaA2 activates the ERK, p38 and NF-κB pathways through TLR2 signaling pathway in HaCaT cells. ConclusionsFrom the findings above, these results confirm that T. pallidum FlaA2 activates ERK, p38 and NF-κB signaling pathway through TLR2 pathway to induce the production of IL-6 and IL-8, which could contribute to enhance the understanding of the skin inflammatory response induced by the pathogen in syphilis patients.

Sphingosine 1-Phosphate Signaling at the Skin Barrier Interface.

Simple SummarySphingosine 1-phosphate is a bioactive phospholipid derived from cell membranes. It is biosynthesized and metabolized within the cell and released extracellularly to trigger various cellular responses via sphingosine 1-phosphate receptors. Keratinocyte and mast cell functions are responsible for skin barrier acquired and innate immunity, and express sphingosine 1-phosphate receptors. These receptors regulate not only cell differentiation and proliferation, but also immune responses such as cell migration and cytokine secretion, through G-protein-mediated signaling pathways. Sphingosine 1-phosphate and its receptor signaling pathways are also involved in several skin diseases along with various receptor agonists and are being investigated as potential therapeutic agents. Here, we focus on the association of sphingosine 1-phosphate and its receptors within the skin barrier interface.Sphingosine 1-phosphate (S1P) is a product of membrane sphingolipid metabolism. S1P is secreted and acts via G-protein-coupled receptors, S1PR1-5, and is involved in diverse cellular functions, including cell proliferation, immune suppression, and cardiovascular functions. Recent studies have shown that the effects of S1P signaling are extended further by coupling the different S1P receptors and their respective downstream signaling pathways. Our group has recently reported that S1P inhibits cell proliferation and induces differentiation in human keratinocytes. There is a growing understanding of the connection between S1P signaling, skin barrier function, and skin diseases. For example, the activation of S1PR1 and S1PR2 during bacterial invasion regulates the synthesis of inflammatory cytokines in human keratinocytes. Moreover, S1P-S1PR2 signaling is involved in the production of inflammatory cytokines and can be triggered by epidermal mechanical stress and bacterial invasion. This review highlights how S1P affects human keratinocyte proliferation, differentiation, immunoreaction, and mast cell immune response, in addition to its effects on the skin barrier interface. Finally, studies targeting S1P-S1PR signaling involved in inflammatory skin diseases are also presented.

城市大气细颗粒物PM2.5对人角质形成细胞屏障相关蛋白表达的影响

Objective To evaluate the effect of atmospheric fine particulate matter (PM2.5) on the expression of skin barrier-associated proteins and proinflammatory cytokines in human keratinocytes. Methods Atmospheric PM2.5 samples were given by Professor Yufeng Zhou in Children′s Hospital of Fudan University. Human primary keratinocytes were isolated from circumcised foreskins of 5 males, and subjected to culture. These human primary keratinocytes were divided into several groups to be stimulated with PM2.5 at different concentrations of 0 (control group) , 10, 50, 100, 200 mg/L for 24 hours, and cell counting kit (CCK) -8 assay was performed to determine the survival rates of keratinocytes. Fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of filaggrin, keratin-14 and claudin-1 in these keratinocytes respectively, and enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of interleukin (IL) -1α, thymic stromal lymphopoietin (TSLP) and IL-33 in the culture supernatant of these keratinocytes. Statistical analysis was carried out with SPSS13.0 software by using one-way analysis of variance, least significant difference (LSD) -t test and Pearson correlation analysis. Results After 24-hour treatment with different concentrations of PM2.5, there was no significant difference in the survival rate of keratinocytes between the 10-mg/L PM2.5 group and control group (P > 0.05) , while the survival rates of keratinocytes were significantly lower in the 50-, 100-, 200-mg/L PM2.5 groups than in the control group (all P 0.05) . The 10-, 50-, 100- and 200-mg/L PM2.5 groups showed significantly increased keratin-14 expression compared with the control group (all P < 0.05) . The claudin-1 expression did not differ between the 10-mg/L PM2.5 group and control group (P = 0.87) , but significantly higher in the 50-, 100- and 200-mg/L PM2.5 groups than in the control group (all P < 0.05) . The stimulation with PM2.5 at 10, 50, 100 and 200 mg/L could induce an increase in the supernatant levels of TSLP, IL-1α and IL-33 (all P < 0.01) . Pearson correlation analysis showed that the supernatant levels of the proinflammatory cytokines TSLP, IL-1α and IL-33 were positively correlated with the concentration of PM2.5 (r = 0.57, 0.67, 0.91 respectively, all P < 0.05) . Conclusion The exposure to PM2.5 can induce decreased survival rate of keratinocytes, aberrant expression of filaggrin, keratin-14 and claudin-1, and elevated secretion of the proinflammatory cytokines TSLP, IL-1α and IL-33, which may lead to impaired skin barrier function. Key words: Keratinocytes; Particulate matter; Cell survival; Keratin-14; Claudins; Interleukin-1alpha; PM2.5; Barrier-associated proteins; Filaggrin; Interleukin-33; Thymic stromal lymphopoietin

Effect of 2nd and 3rd generation PAMAM dendrimers on proliferation, differentiation, and pro-inflammatory cytokines in human keratinocytes and fibroblasts.

BackgroundPoly(amidoamine) (PAMAM) dendrimers are of considerable interest when used as a carrier for topical drugs for the skin, although little is known about their possible side effects. Therefore, our study was about the impact of 2nd and 3rd generation PAMAM dendrimers on human keratinocytes and fibroblasts cells.MethodsThe effect of the tested compounds on collagen biosynthesis was determined using 5[3H]-proline incorporation bioassay. Morphological changes accompanying cell growth inhibition were observed using a confocal microscope. To evaluate the percentage of apoptotic/necrotic cells and the cell growth dynamic of apoptotic features, we performed Annexin V/PI double staining assay, assessed caspase activity, and performed cell cycle analysis by flow cytometry. The flow cytometry method was also used to determine the effect of dendrimers on pro-inflammatory cytokines (IL-6, IL-8 IL-1β).ResultsThe obtained results showed that as the concentration and the generation of dendrimers increased, collagen biosynthesis decreased. We also observed abnormalities in cell differentiation, which may have caused disturbed secretion of pro-inflammatory cytokines. We found that dendrimers cause chronic inflammation which may cause adverse changes in the skin, ultimately– leading to apoptosis in the case of dendrimers in lower concentrations or necrosis at higher concentrations (especially 3rd generation dendrimers). In addition, the inflammatory path induced by the tested compounds was caused by damage in the mitochondria, which we observed as a significant decrease in the mitochondrial membrane potential.ConclusionThe results of our study showed that PAMAM dendrimers can cause disorders of cell proliferation and differentiation and may be the cause of cell cycle deregulation and chronic adverse inflammation.

957 TNFα- and IL-17A-mediated induction of IL-36 cytokines in human keratinocytes

957 TNFα- and IL-17A-mediated induction of IL-36 cytokines in human keratinocytes

TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes.

As the outermost physical barrier of an organism, the skin is diurnally exposed to UV radiation (UVR). Recent studies have revealed that the skin exhibits a circadian rhythm in various functions, and this oscillation is disturbed and reset via a strong environmental cue, the UVR. However, a molecular link between circadian perturbation by UVR and UVR-induced cellular responses has not been investigated. We identified tissue inhibitor of metalloproteinase (TIMP)-3 as a novel circadian locomotor output cycles kaput (CLOCK)–dependent diurnal gene by using a CLOCK-knockdown strategy in human keratinocytes. Among dozens of identified transcripts down-regulated by CLOCK knockdown, TIMP3 displayed a rhythmic expression in a CLOCK-dependent manner, in which the expression of matrix metalloproteinase (MMP)-1 and inflammatory cytokines, such as TNF-α, chemokine (C-X-C motif) ligand (CXCL)-1, and IL-8, were inversely regulated. Upon UVB exposure, the expression of CLOCK and TIMP3 was down-regulated, which led to an up-regulation of secretion of MMP1 and TNF-α proteins and in the transcription of CXCL1 and IL-8 via CCAAT-enhancer binding protein (C/EBP)-α. UVB-induced TNF-α secretion increased further or decreased by knockdown or overexpression of TIMP3, respectively, as well as by CLOCK. As a novel CLOCK-dependent diurnal gene, TIMP3 inhibits the expression of inflammatory cytokines that are up-regulated by UV irradiation in human keratinocytes. Thus, our work suggests a molecular link between circadian perturbation by UVR and UVR-induced inflammation.—Park, S., Kim, K., Bae, I.-H., Lee, S. H., Jung, J., Lee, T. R., Cho, E.-G. TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes.

Elevation of serum squamous cell carcinoma antigen 2 in patients with psoriasis: associations with disease severity and response to the treatment.

Squamous cell carcinoma antigen (SCCA) belongs to the ovalbumin-serpin family and is a known tumour marker. Expression of SCCA is upregulated in the serum and skin of patients with psoriasis. The aim of this study was to determine SCCA2 levels in association with disease severity and treatment efficacy in patients with psoriasis. Patients with psoriasis (n=123) and healthy controls (n=25) were enrolled in this prospective cross-sectional study. Enzyme-linked immunosorbent assay (ELISA) analysis was performed to determine serum SCCA2 levels. SCCA2 expression in skin was evaluated using immunohistochemical analysis. Serum SCCA2 levels were compared with Psoriasis Area Severity Index (PASI) scores. The effect of treatment on serum SCCA2 levels was assessed using serial examinations. Induction of SCCA2 by several psoriatic cytokines in human keratinocytes was evaluated. The serum levels of SCCA2 were significantly higher in patients with psoriasis than healthy controls and correlated well with disease severity. Increased SCCA2 staining was observed in lesional skin but not in nonlesional skin of patients with psoriasis. In addition, SCCA2 expression levels in skin correlated with serum concentrations of SCCA2. SCCA2 significantly decreased according to improvement of PASI scores. Interleukin (IL)-17 and IL-22 synergistically increased the production of SCCA2 at both mRNA and protein levels in human keratinocytes. Significant elevation of SCCA2 is associated with disease severity and reflects treatment efficacy. SCCA2 may be a useful biomarker in psoriasis, reflecting T-helper 17-type inflammation - the main determinant of the severity of psoriasis.

버섯류 추출물의 피부각질세포(HaCaTcell) 내 염증성 사이토카인 억제효과

Mushroom is known for anti-inflammatory and anti-oxidative potential. This study provides evidence that the inhibitory effect of mushroom on the expression of pro-inflammatory cytokines in human keratinocytes, HaCaT cells. To define the underlying mechanisms of action, tumor necrosis factorα/IFNγ-activated human keratinocytes model was used. Mushroom significantly inhibited the expression of cytokines in HaCaT cells. Taken together, the results demonstrate that mushroom inhibited inflammtion, suggesting that mushroom (DW extract: Grifola frondosa Cordyceps militaris), (Ethanol extract: Ganoderma lucidum, Lentinus edodes, Cordyceps militaris, Flammulina velutipes) might be a candidate for the treatment of skin inflammation.

Dual oxidase 2 is essential for house dust mite-induced pro-inflammatory cytokine production in human keratinocytes.

House dust mites (HDMs) are known to trigger chronic inflammation through Toll-like receptors (TLRs) and their signalling cascades. In this study, we found that TLR2 ligation by HDMs induced the activation of dual oxidase 2 (Duox2) and nuclear factor-κB (NF-κB), leading to the production of pro-inflammatory cytokines in human keratinocytes. Stimulation of human keratinocytes with HDMs resulted in increases in interleukin-8 (IL-8) and chemokine (C-C motif) ligand 20 (CCL20) levels. However, pro-inflammatory cytokine production was abolished in keratinocytes transfected with TLR2 siRNA, indicating that HDM-induced cytokine production was mediated via TLR2 signalling. We also examined the function of Duox1/2 isozymes, which are primarily expressed in keratinocytes, in HDM-mediated pro-inflammatory cytokine production. Human keratinocytes transfected with control siRNA or Duox1 siRNA showed no inhibition of IL-8 or CCL20 production in response to HDMs, whereas the silencing of Duox2 expression resulted in a failure to induce cytokine production. Moreover, the phosphorylation and nuclear localization of RelA/p65, a component of NF-κB, were induced by HDMs in human keratinocytes. Transfection of human keratinocytes with TLR2 siRNA or Duox2 siRNA resulted in the complete abolishment of RelA/p65 nuclear localization in response to HDMs. Taken together, these results indicate that the HDM-dependent TLR2-Duox2 signalling axis indeed promotes NF-κB activation, which induces IL-8 and CCL20 production and mediates epidermal keratinocyte inflammation.

Suppression of skin inflammation in keratinocytes and acute/chronic disease models by caffeic acid phenethyl ester.

Skin inflammation plays a central role in the pathophysiology and symptoms of diverse chronic skin diseases including atopic dermatitis (AD). In this study, we examined if caffeic acid phenethyl ester (CAPE), a skin-permeable bioactive compound from propolis, was protective against skin inflammation using in vitro cell system and in vivo animal disease models. CAPE suppressed TNF-α-induced NF-κB activation and expression of inflammatory cytokines in human keratinocytes (HaCaT). The potency and efficacy of CAPE were superior to those of a non-phenethyl derivative, caffeic acid. Consistently, topical treatment of CAPE (0.5 %) attenuated 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced skin inflammation on mouse ear as CAPE reduced ear swelling and histologic inflammation scores. CAPE suppressed increased expression of pro-inflammatory molecules such as TNF-α, cyclooxygenase-2 and inducible NO synthase in TPA-stimulated skin. TPA-induced phosphorylation of IκB and ERK was blocked by CAPE suggesting that protective effects of CAPE on skin inflammation is attributed to inhibition of NF-κB activation. Most importantly, in an oxazolone-induced chronic dermatitis model, topical application of CAPE (0.5 and 1 %) was effective in alleviating AD-like symptoms such as increases of trans-epidermal water loss, skin thickening and serum IgE as well as histologic inflammation assessment. Collectively, our results propose CAPE as a promising candidate for a novel topical drug for skin inflammatory diseases.

Signaling via the IL-20 receptor inhibits cutaneous production of IL-1β and IL-17A to promote infection with methicillin-resistant Staphylococcus aureus

Staphylococcus aureus causes the majority of human skin and soft tissue infections, and is a major infectious cause of mortality. Host defense mechanisms against S. aureus are incompletely understood. Interleukin (IL)-19, -20 and -24 signal through type I and type II IL-20 receptors and are associated with inflammatory skin diseases such as psoriasis and atopic dermatitis. We show here that these cytokines promote cutaneous S. aureus infection in mice by downregulating IL-1β- and IL-17A-dependent pathways. Similar effects of these cytokines were seen in human keratinocytes after S. aureus exposure, and antibody blockade of IL-20 receptor improved outcomes in infected mice. Our findings identify an immunosuppressive role for these cytokines during infection that could be therapeutically targeted to alter susceptibility to infection.

Illicium verum extract inhibits TNF-α- and IFN-γ-induced expression of chemokines and cytokines in human keratinocytes

Ethnopharmacological relevanceIllicium verum Hook. f. (star anise) has been used in traditional medicine for treatment of skin inflammation, rheumatism, asthma, and bronchitis.This study investigated the anti-inflammatory effects of Illicium verum extract (IVE) in the human keratinocyte HaCaT cell line. Materials and methodsWe investigated the effectiveness of IVE in tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-induced human keratinocytes. To measure the effects of IVE on chemokine and pro-inflammatory cytokine expression in HaCaT cells, we used the following methods: cell viability assay, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, and luciferase reporter assay. ResultsIVE inhibited the expression of TNF-α/IFN-γ-induced mRNA and protein expression of thymus and activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22), interleukin (IL)-6, and IL-1β. Furthermore, IVE decreased TNF-α/IFN-γ-induced mRNA expression of intercellular adhesion molecule-1 (ICAM-1). IVE inhibited nuclear factor (NF)-κB translocation into the nucleus, as well as phosphorylation and degradation of IκBα. IVE inhibited TNF-α/IFN-γ-induced NF-κB and signal transducer and activator of transcription (STAT)1 activation in a dose-dependent manner. In addition, IVE significantly inhibited activation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and Akt. Furthermore, IVE contained 2.14% trans-anethole and possessed significant anti-inflammatory activities. ConclusionsIVE exerts anti-inflammatory effects by suppressing the expression of TNF-α/IFN-γ-induced chemokines, pro-inflammatory cytokines, and adhesion molecules via blockade of NF-κB, STAT1, MAPK, and Akt activation, suggesting that IVE may be a useful therapeutic candidate for inflammatory skin diseases, such as atopic dermatitis.

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

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. We compared the expression of the long-form and total TSLP transcripts in primary human keratinocytes. 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. PolyI: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 polyI: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-α+IL-4+IL-13) or TNF-α 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. 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.

Effects of AV119, a natural sugar from avocado, on Malassezia furfur invasiveness and on the expression of HBD‐2 and cytokines in human keratinocytes

AV119 is a patented blend of two sugars from avocado that can induce human beta-defensin-2 production by normal human keratinocytes. In this study, we analysed the effect of AV119 on growth and invasiveness of Malassezia furfur, a dimorphic, lipid-dependent yeast that is part of the normal human cutaneous commensal flora. The ability to modulate the expression of the proinflammatory and immunomodulatory cytokines in normal human keratinocytes was also investigated. Microbiological assay demonstrated that this sugar induced the aggregation of yeast cells and inhibited the invasiveness of M. furfur, without affecting its growth. Real-time PCR analysis demonstrated that AV119 was able to modulate the HBD-2 response in treated keratinocytes, reaching a maximum after 48-h treatment, and to induce the recovery of a satisfactory proinflammatory response in human keratinocytes. As AV119 can induce aggregation of yeast cells, thus inhibiting their penetration into the keratinocytes, the sugar could be used in the preparation of cosmetics or pharmacological drugs to inhibit colonization of the skin by pathogenic strains of M. furfur.

Broad-spectrum sunscreens prevent the secretion of proinflammatory cytokines in human keratinocytes exposed to ultraviolet A and phototoxic lomefloxacin

The combination of phototoxic drugs and ultraviolet (UV) radiation can trigger the release of proinflammatory cytokines. The present study measured the ability of sunscreens to prevent cytokine secretion in human keratinocytes following cotreatment of these cells with a known photoreactive drug and UVA. Keratinocytes were treated for 1 h with increasing concentrations of lomefloxacin (LOM) or norfloxacin (NOR), exposed to 15 J/cm2 UVA, and incubated for 24 h. NOR, owing to the absence of a fluorine atom in position 8, was non-phototoxic and used as a negative control. Cell viability and the release of 3 cytokines were assessed, namely interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-alpha). The measurement of these cytokines may be a useful tool for detecting photoreactive compounds. To measure their ability to prevent cytokine secretion, various sunscreens were inserted between the UVA source and the cells. Treatment with NOR, NOR plus UVA, or LOM had no effect on the cells. LOM plus UVA, however, had an effect on cell viability and on cytokine secretion. IL-1alpha levels increased with LOM concentration. The release of TNF-alpha and IL-6 followed the same pattern at lower concentrations of LOM but peaked at 15 micromol/L and decreased at higher concentrations. Sunscreens protected the cells from the effects of LOM plus UVA, as cell viability and levels of cytokines remained the same as in the control cells. In conclusion, the application of broad-spectrum sunscreen by individuals exposed to UVA radiation may prevent phototoxic reactions initiated by drugs such as LOM.

Cutaneous sarcoidosis treated with medium-dose UVA1

An 82-year-old woman presented with a 2-year history of progressive, itching skin lesions, affecting more than 70% of her body surface. In addition, she had multiple internal diseases such as breast cancer, lung fibrosis after radiation, heart failure, hypertension, and diabetes mellitus. Physical examination revealed multiple, painless, red-brown, elevated, partially confluent papules and plaques disseminated over the whole body (Fig 1). Diascopy of the lesions revealed a grey-yellow infiltrate. All blood tests were within reference limits. Angiotensin-converting enzyme was increased at 43 U/L (normal: 6-32 U/L). Chest radiography; electrocardiography; abdominal sonography; and internal, neurologic, and ophthalmologic examinations revealed no systemic signs of sarcoidosis. Histology of a papule showed noncaseating epitheloid cell granulomas with multinucleated giant cells confirming the diagnosis of cutaneous sarcoidosis. Topical glucocorticosteroids and tacrolimus ointment were used without effect. Oral therapeutic approaches were not induced as a result of absence of systemic involvement of sarcoidosis and the risk of side effects in the patient who is multimorbid. Thus, we treated our patient 4 times a week with medium-dose UVA1 irradiation (Sellamed 2400A, Dr Sellmayer, Sellas, Gevelsberg, Germany), starting with a dose of 20 J/cm2 for the first 3 treatment sessions and 40 J/cm2 for the subsequent 12 sessions. The final dose of 60 J/cm2 was given 35 times. After 50 sessions of medium-dose UVA1 and a total dose of 2640 J/cm2, nearly all lesions disappeared (Fig 2). Adverse effects were not observed during and after UVA1 irradiation. A biopsy specimen 5 months after therapy showed normal skin and a serum angiotensin-converting enzyme level within reference limit. Sarcoidosis is a multisystem disorder characterized histologically by epitheloid cell granulomas without caseating necrosis. Pathophysiologically it has been proposed that formation of sarcoid granulomas occurs as a series of immune events consisting of the triggering of CD4+ T-helper cells by local antigen-presenting cells and the accumulation of immunocompetent cells at sites of disease. It is speculated that accumulated T-helper cells, cooperating with macrophages, contribute to the development of granulomas in sarcoidosis. Diverse therapeutic approaches have been used in the therapy of sarcoidosis. UVA1 phototherapy represents an increasingly used therapeutic modality. Disparate diseases such as atopic dermatitis, urticaria pigmentosa, and lichenoid graft-versus-host disease have been treated with success. Various immunomodulating and immunosuppressive effects of UVA1 are known, which may be responsible for the therapeutic effort in cutaneous sarcoidosis. Long-wave UVA induces apoptosis in T cells.1Morita A Werfel T Stege H Ahrens C Karmann K Grewe M et al.Evidence that singlet oxygen-induced human T helper cell apoptosis is the basic mechanism of UVA radiation phototherapy.J Exp Med. 1997; 186: 1763-1768Crossref PubMed Scopus (237) Google Scholar UVA1 irradiation, similar to UVB, has been shown to induce the expression of immunsuppressive cytokines in human keratinocytes including tumor necrosis factor-α and IL-10.2Krutmann J, Stege H, Morita A. UVA1 phototherapy: indications and mode of action. In: Dermatological phototherapy and photodiagnostic methods. New York: Springer; 2001. p. 261-78Google Scholar Graefe et al3Graefe T Konrad H Barta U Elsner P Successful UVA1 treatment of cutaneous sarcoidosis.Br J Dermatol. 2001; 145: 354-355Crossref PubMed Scopus (32) Google Scholar described an improvement in cutaneous sarcoidosis by a female patient with plaques on her forehead treated with high-dose UVA1. Our patient had generalized disease involving more than 70% of her skin. Medium-dose (ie, up to 60 J/cm2) UVA1 phototherapy was applied, on the basis of our experience and publications on positive effects of medium-dose UVA1 in other cutaneous diseases.4Pinton P.C Capezzera R Zane C De Panfilis G Medium-dose UVA1 therapy for pityriasis lichenoides et varioliformis acuta and pityriasis lichenoides chronica.J Am Acad Dermatol. 2002; 47: 410-414Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Full remission was achieved without any side effects. However, the long-term effects such as carcinogen risk of UVA1 therapy have to be followed up.5Sterenbroigh H.C.J.M van der Leun J.C Tumorgenesis by long wave-length UVA source.Photochem Photobiol. 1990; 51: 325-330Crossref PubMed Scopus (126) Google Scholar

Induction of Transcription Factor AP-2 by Inflammatory Cytokines in Human Keratinocytes

Activator protein-2 is an important transcription factor for the activation of a number of genes. Here we report the induction of activator protein-2 in response to inflammatory cytokines such as interleukin-6 in keratinocytes. Immunoblotting and semiquantitative reverse transcriptase-polymerase chain reaction assays using normal human keratinocytes revealed that interleukin-6 caused a time- and concentration-dependent induction of activator protein-2 mRNA and protein. The increase of activator protein-2 mRNA was detected at 30 min after stimulation and that of activator protein-2 protein was at 2 h. Their levels were lower than the control levels at 24 h. The interleukin-6-dependent induction of activator protein-2 mRNA was completely blocked by adding actinomycin D, whereas it was approximately 50% affected by cycloheximide. Co-incubation with neutralizing antibodies against various inflammatory cytokines resulted in inhibition of the interleukin-6-dependent activator protein-2 induction at varying degrees, indicating an involvement of various cytokines in the activator protein-2 induction. The activator protein-2 induction was observed in keratinocytes derived from lesional skins with psoriasis or squamous cell carcinoma, and the high levels of activator protein-2 were histochemically detected in these lesions. Furthermore, a gel mobility shift assay using the nuclear extracts from interleukin-6-treated cells showed that interleukin-6 induced the functional activator protein-2 protein for the gene activation. These findings suggest a possible regulation mechanism of activator protein-2 through a complex cytokine system, which is conceivably the initial reaction leading to skin inflammation, and resultant keratinocyte growth and carcinogenesis.

Regulation of Inducible Nitric Oxide Synthase mRNA Levels by Differentiation and Cytokines in Human Keratinocytes

Inducible nitric oxide synthase (iNOS) transcription is regulated by cytokines and immune stimuli in various cell types. However, there are few data available on iNOS expression and regulation in keratinocytes, even though it can play a role in keratinocyte proliferation during inflammation following wound healing. Our studies demonstrated a differentiation-dependent regulation of iNOS transcription in human keratinocytes from epidermis but not in buccal mucosal cells, which are less differentiated. Different interferon (IFN) types (α and γ) regulated skin keratinocyte iNOS mRNA levels in different ways, predominantly in a differentiation-dependent manner. TGFβ1 and IFNα can antagonize IFNγ related iNOS induction. However, the antagonistic effects of IFNα on IFNγ differ from those of TGFβ1. Our studies demonstrate: (a) the presence of iNOS mRNA in human keratinocytes; (b) inducibility of iNOS mRNA by different cytokines; and (c) antagonism between certain cytokines in induction of iNOS mRNA. Further studies are needed to determine biological and therapeutical relevance of our findings.