Human interferon-γ induces expression of HLA-DR on keratinocytes and melanocytes

Summary Primary human epidermal cell cultures composed of keratinocytes and melanocytes were exposed to supernatants of phytohaemagglutinin (PHA)-stimulated T cells, various lymphokines and interferon-, and checked for the emergence of HLA-DR antigen using immunofluorescence and immunoelectron microscopy. HLA-DR expression was induced by the supernatants and human recombinant interferon- (rIFN-), whereas recombinant ₂, interleukin-2 and non-recombinant human interferon- had no such effect. The threshold concentration of rIFN- required to induce this phenomenon was 10 IU/ml; no further increase of reaction intensity was observed using doses of more than 100 IU/ml. Maximum reaction intensity was achieved after 72 h of incubation; a minimum of 3 h of incubation with rIFN- followed by 72 h incubation in rIFN--free medium proved sufficient to induce HLA-DR expression. The inductive effect of the supernatants and rIFN- could be completely abrogated by pretreatment with excess doses of the monoclonal antibody GZ4 specific for human IFN-. Keratinocytes and melanocytes reacted in an identical fashion both qualitatively and quantitatively in all experiments. These data indicate that IFN- possesses specific signal functions in the induction of HLA-DR expression on epidermal cells.Abbreviations IFN- interferon- - rIFN- recombinant interferon- - r IFN-₂ recombinant interferon-₂ - nrIFN- nonrecombinant interferon- - IL-2 interleukin-2 - EC epidermal cells - K keratinocytes - M melanocytes     

The expression of RXRα in epidermis of psoriasis vulgaris

目的:研究维A酸类X受体α(RXRα)在寻常性银屑病患者表皮角质形成细胞中的表达和意义.方法:采用免疫组化方法(SP法)检测RXRα在5例正常人表皮、17例寻常性银屑病患者皮损、非皮损表皮中的表达,并用图像分析方法进行半定量分析.结果:RXRα在正常人表皮角质形成细胞中呈细胞核表达;在银屑病非皮损及皮损表皮中的表达面积均较正常人明显增加(P ＜ 0.01＝,并伴随着由胞核向胞膜的移位;而RXRα在银屑病皮损中的表达强度较非皮损明显下降(P ＜ 0.001＝.结论:RXRα在表皮的正常角化以及稳态维持中可能发挥一定作用.在银屑病表皮中,RXRα由细胞核移位至细胞膜,其表达强度的下降,尤其是细胞核中表达的缺失,可能是使银屑病表皮失去正常生长分化调控的重要原因.     

Obligatory roles of filamin A in E-cadherin-mediated cell–cell adhesion in epidermal keratinocytes

BackgroundExtracellular Ca²⁺ (Ca_o²⁺)-induced E-cadherin-mediated cell–cell adhesion plays a critical role in promoting differentiation in epidermal keratinocytes. Our previous studies show that the calcium-sensing receptor (CaR) regulates keratinocyte cell–cell adhesion and differentiation via Rho A-mediated signaling. CaR forms a protein complex with Rho A, guanine nucleotide exchange factor Trio, and a cytoskeletal actin-binding protein, filamin A, at the cell–cell junctions in response to elevated Ca_o²⁺ levels. Filamin A has the ability to interact directly with CaR, Trio, and Rho and mediate CaR-dependent signaling events.ObjectiveThis study was conducted to investigate the roles of filamin A and Trio in regulating Ca_o²⁺-induced Rho activation and intercellular adhesion.MethodsExpression of filamin A and Trio in keratinocytes was inhibited by siRNA. Its effects on Ca_o²⁺-dependent junction formation and adhesion complex formation were evaluated by fluorescence immunostaining and immunoprecipitation. Endogenous Rho activity and expression of keratinocyte differentiation markers were also examined. The significance of the physical interactions of filamin A with Trio and Rho was assessed in dominant-negative inhibition studies.ResultsInhibiting filamin A expression blocked the formation of CaR-Rho A-Trio-E-cadherin protein complex. Knockdown of filamin A or Trio inhibited Ca_o²⁺-induced membrane localization and activation of Rho A, formation of the E-cadherin–catenin adhesion complex, and keratinocyte terminal differentiation. Expressing dominant-negative peptides disruptive to the endogenous filamin–Trio, filamin–Rho, and CaR–filamin interactions suppressed the formation of adherens junctions.ConclusionThrough physical interactions with CaR, Trio and Rho, filamin A generates a scaffold for organizing a signaling complex that promotes E-cadherin-mediated cell–cell adhesion and keratinocyte differentiation.     

Protective effects of (−)-epicatechin-3-gallate on UVA-induced damage in HaCaT keratinocytes

(–)-Epigallocatechin-3-gallate (EGCG), a constituent of green tea, has been extensively studied and shown to be a powerful antioxidant protecting skin cells against photodamage. In this study, however, we demonstrated that another gallated catechin, (–)-epicatechin-3-gallate (ECG), was also able to protect human keratinocytes against damage induced by ultraviolet A (UVA) light. We found that ECG dose-dependently inhibited UVA-induced keratinocyte death as determined by cell viability assay. Moreover, ECG had similar potency to EGCG in inhibiting UVA-induced cell death. Therefore, the mechanism of action of ECG was further investigated. As assayed by flow cytometry, UVA-induced hydrogen peroxide (H₂O₂) production in keratinocytes was inhibited by ECG in a concentration-dependent manner, suggesting that ECG can act as a free radical scavenger while keratinocytes were photodamaged. The scavenging effect of ECG was confirmed by the fact that ECG treatment attenuated cell damage induced by H₂O₂ and hypoxanthine-xanthine oxidase. In a parallel experiment, UVA-induced activation of extracellular signal-regulated kinase in keratinocytes was blocked by ECG. We provided here the first evidence that ECG is a potent protectant that protects keratinocytes from photodamage. Because ECG is abundant in green tea, we believe that this compound is beneficial for skin care.     

Organ culture of psoriatic skin: effect of TGF-α and TGF-Β on epidermal structure in vitro

Summary Normal skin and uninvolved and involved psoriatic skin specimens were maintained in vitro in organ culture. The 3–4 mm punch-biopsied skin specimens were put freely into the culture medium with or without fetal calf serum, under an atmosphere of 95% O₂ plus 5% CO₂, and rotated at 60 rpm at 37C. In the serum-free culture medium (vitamin A-free) granular layers appeared in the involved psoriatic epidermis in culture. Addition of TGF- caused normal skin and uninvolved and involved psoriatic skin specimens to become acanthotic and to degenerate easily almost to the full thickness of the epidermal layer in proportion to increasing concentrations of TGF- as well as with the duration of the culture, but without disappearance of their granular layers. TGF- caused the normal skin and uninvolved psoriatic skin specimens to become thinned without disappearance of granular layers, but caused the involved psoriatic skin specimens to be thinned without appearance of granular layers in serumcontaining medium or with their disappearance in the serum-free medium. TGF- also antagonized the acanthotic and degenerative effect of TGF-. The results suggest that TGF- and TGF- may partially be related to the induction of psoriatic epidermal lesions.     

Proliferation–differentiation relationships in the expression of heparin-binding epidermal growth factor-related factors and erbB receptors by normal and psoriatic human keratinocytes

The topological relationships between erbB receptors and ligands of the epidermal growth factor family were characterized by immunocytochemistry in normal and psoriatic epidermis and in proliferating and differentiating human keratinocytes in culture. Spatial colocalization of receptors and ligands was assessed by dual immunostaining. Expression of epidermal growth factor receptor (EGFr), erbB2, and erbB3, but not erbB4, was detected throughout the epidermis, although labeling for erbB2 and erbB3 was accentuated in the upper spinous layers, and EGFr was more strongly labeled in basal cells. Of the tested growth factors, heparin-binding epidermal growth factor (HB-EGF) was diffusely expressed throughout normal and psoriatic epidermis and sparsely colocalized with EGFr in all viable epidermal layers, with increased colocalization in psoriatic epidermis. In contrast, betacellulin and heregulin/neu differentiation factor (NDF) alpha were largely restricted in their distribution to the upper spinous and granular layers. Betacellulin was downregulated in psoriatic keratinocytes. Although heregulin/NDF-beta was undetectable in normal epidermis, it was upregulated in psoriasis. Betacellulin and heregulin/NDF-alpha strikingly colocalized with EGFr and erbB3 receptors in the granular layer and in a declining gradient from the granular zone to the basal layer, respectively. Similar patterns were observed in cultured keratinocytes under proliferative conditions and upon differentiation in high-calcium medium. These morphological data collectively suggest divergent functions for members of the growth factor family, and in particular, we propose that betacellulin and heregulin/NDF-alpha are involved in epidermal morphogenesis and/or in maintenance of the differentiated phenotype.     

The effects of prostaglandins E1 and F2α on epidermal growth

Summary The effects of prostaglandins E₁ and F₂ (PGE₁ and PGF_2a) on the growth of guinea pig epidermis have been studied in vivo. The prostaglandins were injected intradermally into guinea pigs, and tritiated thymidine was injected intraperitoneally prior to sacrifice. The autoradiographic labelling indices (L.I.) were assessed and a significant increase was found 4 b after injection of PGE₁-an effect which lasted for up to 72 h. PGF_2a injections had no significant effect on the L.I.

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Zusammenfassung Der Effekt der Prostaglandine E₁ und F₂ auf das Wachstum der Epidermis von Meerschweinchen wurde in vivo untersucht. Die Prostaglandine wurden intradermal injiziert; mit Tritium markiertes Thymidin wurde intraperidonial vor dem Töten der Tiere injiziert. Die autoradiographischen Daten (labelling indices) wurden bestimmt. Ein signifikanter Anstieg wurde 4 h nach der Injektion von PGE₁ ermittelt, ein Effekt der mindestens 72 h anhielt. PGF_2a-Injektionen hatten keinen Einfluß auf den L.I.

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Based on a paper read at meeting of the Investigative Group of the British Association of Dermatologists, January 1975     

Roxithromycin downregulates production of CTACK/CCL27 and MIP-3α/CCL20 from epidermal keratinocytes

Cutaneous T cell-attracting chemokine (CTACK)/CCL27 and macrophage inflammatory protein (MIP)-3α/CCL20 are the major inflammatory chemokines involved in skin inflammation. The present study showed that roxithromycin (RXM) suppressed the TNFα-induced production of CCL27 and CCL20 in HaCaT keratinocytes and normal human keratinocytes (NHKs) in a dose-dependent manner. The production of CCL20 induced by TNFα was suppressed by the addition of inhibitors of nuclear factor kappa B (NFκB). RXM suppressed NFκB activity induced by TNFα. RXM, by regulating CCL27 and CCL20, may contribute to the modulation of inflammation.     

Oxytocin is expressed in epidermal keratinocytes and released upon stimulation with adenosine 5'-[γ-thio]triphosphate in vitro

Oxytocin is a neuropeptide produced primarily in the hypothalamus and is best known for its roles in parturition and lactation. It also influences behaviour, memory and mental state. Recent studies have suggested a variety of roles for oxytocin in peripheral tissues, including skin. Here we show that oxytocin is expressed in human skin. Immunohistochemical studies showed that oxytocin and its carrier protein, neurophysin I, are predominantly localized in epidermis. RT-PCR confirmed the expression of oxytocin in both skin and cultured epidermal keratinocytes. We also show that oxytocin is released from keratinocytes after application of adenosine 5'-[γ-thio]triphosphate (ATPγS, a stable analogue of ATP) in a dose-dependent manner. The ATPγS-induced oxytocin release was inhibited by removal of extracellular calcium, or by the P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). These results suggest that oxytocin is produced in human epidermal keratinocytes and is released in response to calcium influx via P2X receptors.     

Involvement of RAGE, MAPK and NF-κB pathways in AGEs-induced MMP-9 activation in HaCaT keratinocytes

Advanced glycation end products (AGEs) exert divergent effects on the pathogenesis of diabetes complications. Excessive expression of matrix metalloproteinases-9 (MMP-9) is deleterious to the cutaneous wound-healing process in the context of diabetes. However, the effect of AGEs on MMP-9 induction in skin cells and the exact molecular mechanisms involved are still poorly understood. In this study, we investigated the effect of AGEs on the production of MMP-9 in HaCaT keratinocytes and characterized the signal transduction pathways activated by AGEs that are involved in MMP-9 regulation. We showed that AGE-BSA increased MMP-9 expression in HaCaT cells at both the protein and mRNA levels. The stimulatory effect of AGE-BSA on MMP-9 was attenuated by inhibitors of extracellular-signal-regulated kinase (ERK1/2, U0126), p38 mitogen-activated protein kinase (MAPK, SB203580) and NF-κB, but not c-Jun N-terminal kinase. Furthermore, receptor for advanced glycation end products (RAGE) was expressed in keratinocytes, and incubation with AGE-BSA resulted in a significant upregulation of RAGE expression in a dose-dependent manner. Silencing of the RAGE gene prevented AGE-BSA-induced MMP-9 activation and the phosphorylation of ERK1/2 and p38 MAPK. We also observed the involvement of NF-κB in AGE-BSA-induced MMP-9 activation, which was not blocked by U0126 and SB203580. These results suggest that AGEs may play an important role in the impairment of diabetic wound healing by upregulating MMP-9 expression in keratinocytes via the RAGE, ERK1/2 and p38 MAPK pathways; activation of NF-κB is also involved in this process. These pathways may represent potential targets for drug interventions to improve diabetic wound healing, a process in which MMP-9 plays a critical role.