Barrier disruption increases gene expression of cytokines and the 55 kD TNF receptor in murine skin

Abstract The signalling mechanisms that regulate epidermal permeability barrier homeostasis arc not known. Previous Northern blot analysis showed that both acute and chronic barrier disruption increase mRNA levels of several cytokines in murine epidermis. To further characterize the epidermal response to barrier abrogation, we used more sensitive, multi-probe RNasc protection assays to measure the mRNA levels of additional cytokines. as well as cytokine receptors in acute and chronic models of barrier disruption. Normal mouse epidermis expressed interleukin (IL)-lα, interferon-γ (IFN-γ). tumor necrosis factor-α (TNF-α) and IL-6 mRNAs. Following tape-stripping, only the mRNA levels for TNF-α, IL-lα, IL-lβ and IL-6 increased at 2.5 and 7 h, and returned toward normal levels by IX h. No mRNAs encoding TNF-β. IL-2, IL-3. IL-4 or IL-5, were detected in the epidermis either under basal conditions or after tape-stripping. Similarly, in a chronic model, essential fatty acid deficiency, epidermal levels of TNF-α, IL-lα, IL-lβ and IL-6 mRNAs, but not IFN-γ mRNA. were elevated over controls; and again, mRNAs for the remaining probed cytokines were not detected. In contrast, in the dermis. only IL-Iβ mRNA levels increased 2.5 h after tape-stripping, and remained elevated at I8 h. mRNAs encoding the IL-1 (p60), IFN-γ ami IL-6 receptors were present in epidermis, but their levels remained unchanged following either acute or chronic barrier disruption. In contrast, epidermal TNF (p55) receptor mRNA levels were increased by 87% (P<0.01) at 2.5 h, returned to control levels at 7 h and were increased by 68% (p<0.03) at 18 h after tape-stripping. The increase at 2 h was confirmed by Northern blot analysis and was not prevented by latex occlusion performed immediately after tape-stripping. mRNAs for the IL-I (p80) receptor and TNF (p75) receptor were not detected in epidermis. Low levels of TNF (p55) receptor mRNA were present in the dermis. and they remained unchanged after tape-stripping. The presence of specific receptor mRNAs in the epidermis and dermis suggests that these tissues are capable of responding in an autocrine and/or paracrine fashion to the cognate cytokines. These results suggest that epidermal cytokines produced after barrier disruption may initiate a cytokine cascade which could regulate cytokine and cytokine receptor production and/or inflammatory responses.     

TNF-α和IL-1β对HaCaT细胞诱生型一氧化氮合酶表达的影响

目的　探讨炎症性细胞因子肿瘤坏死因子 α(tumornecrosisfactor α ,TNF α)协同白细胞介素 1β(inter leukin 1β ,IL 1β)对体外培养角质形成细胞 (keratinocye ,KC)株HaCaT细胞诱生型一氧化氮合酶 (induciblenitricoxidesyn thase ,iNOS)mRNA和蛋白表达的调节作用 ,以及地塞米松对TNF α和IL 1β作用的影响。 方法　用RT PCR、Westernblotting和免疫组织化学 (SP)方法检测HaCaT细胞iNOSmRNA和蛋白表达情况。结果　正常培养HaCaT细胞iNOS微弱表达或不表达 ,TNF α协同IL 1β显著上调HaCaT细胞iNOSmRNA和蛋白表达 ,地塞米松可显著抑制TNF α和IL 1β的作用。结论　推测TNF α和IL 1β可能通过上调角质形成细胞表达iNOS合成释放的一氧化氮 (niricoxide ,NO)参与皮肤免疫和炎症反应 ;地塞米松的治疗效应可能部分与其能抑制iNOS表达有关。     

苯并芘与肽聚糖体外协同对人皮脂腺细胞炎症因子表达的影响

目的：检测苯并芘（Bap）和革兰氏阳性菌细菌壁肽聚糖（PGN)体外对SZ95人皮脂腺细胞炎症因子表达的影响。方法：体外培养SZ95永生化人皮脂腺细胞,分为空白对照组、Bap组、PGN组和（Bap+PGN）组, PGN浓度为20μg/mL,Bap浓度 为10-5mol/L。 RT-PCR和ELISA检测SZ95永生化人皮脂腺细胞分泌白介素(IL-1α、IL-1β）、TNF-α mRNA和蛋白表达水平。结果：（Bap+PGN）组IL-1α,IL-1β和TNF-αmRNA及蛋白表达水平高于空白对照组、Bap组和PGN组,差异具有统计学意义（P<0.01）。结论：苯并芘协同增强PGN诱导的皮脂腺细胞炎症因子IL-1α,IL-1β,TNF-α的表达,这可解释为什么环境污染可加重或诱导痤疮 的发生。     

Epidermal stratification reduces the effects of UVB (but not UVA) on keratinocyte cytokine production and cytotoxicity

Ultraviolet (UV) radiation induces cytokine release from cultured keratinocytes as well as from epidermis in vivo. The purpose of this study was to determine whether differentiation of cultured keratinocytes into stratified epithelium decreases the effects of UVA and UVB radiation on cytokine release. Interleukin-1 (IL-1)α, IL-1β and tumor necrosis factor (TNF)-α release from human keratinocytes and reconstituted human epidermis was measured after exposure to UVA or UVB radiation. Release of IL-1α, IL-1β, and TNF-α was induced by both UVA and UVB radiation from both keratinocytes and reconstituted epidermis. Release of these cytokines was correlated with cytotoxicity. Keratinocyte cultures were far more sensitive to UVB radiation than reconstituted epidermis, in terms of both cytotoxicity and cytokine release. In contrast, epidermal stratification/differentiation had much less effect on the sensitivity to UVA radiation. We conclude that epidermal stratification and the formation of a stratum corneum provide protection against UVB radiation but have limited barrier effect against UVA radiation.     

Investigating the release of inflammatory cytokines in a human model of incontinence-associated dermatitis

Incontinence-associated dermatitis (IAD) is a painful complication in elderly patients, leading to reduced quality of life. Despite recent attention, its underlying inflammatory mechanisms remain poorly understood. This study was designed to quantify the release of inflammatory cytokines in a human model of IAD. The left volar forearm of ten healthy volunteers was exposed to synthetic urine and synthetic faeces for 2 h, simulating the effects of urinary and faecal incontinence, respectively, and the subsequent cytokine response compared to that of an untreated control site. Inflammatory cytokines were collected using both the Sebutape® absorption method and dermal microdialysis and quantified using immunoassays. Results from the former demonstrated an upregulation in IL-1α, IL-1RA and TNF-α. Synthetic urine caused a higher median increase in IL-1α from baseline compared to synthetic faeces, whereas synthetic faeces were associated with significantly higher median TNF-α levels compared to synthetic urine (p = 0.01). An increase in IL-1α/IL-1RA ratio was also observed with significant differences evident following exposure to synthetic urine (p = 0.047). Additionally, microdialysis revealed a time-dependent increase in IL-1β and IL-8 following exposure of up to 120 min to synthetic urine and synthetic faeces, respectively. This study demonstrated the suitability of both sampling approaches to recover quantifiable cytokine levels in biofluids for the assessment of skin status following exposure to synthetic fluids associated with incontinence. Findings suggest some differences in the inflammatory mechanisms of IAD, depending on moisture source, and the potential of the cytokines, IL-1α and TNF-α, as responsive markers of early skin damage caused by incontinence.     

Cytokines in leprosy, I. Serum cytokine profile in leprosy

Background Leprosy is a chronic infectious disease characterized by a broad spectrum of clinical forms depending on the patient's immune response, in particular cell-mediated immune response. Methods Cytokines can play a role in the cell-mediated immune response. Serum levels of interferon-gamma (IFN-γ), interleukin-2 (IL-2), interleukin-2 receptor (IL-2R), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA) in 55 untreated leprosy patients and 35 reactional leprosy patients, in addition to 20 age- and sex-matched healthy controls. Results Leprosy patients showed significantly higher serum levels of the studied cytokines (except IL-2) compared with healthy controls. When the two poles were compared, tuberculoid leprosy (TT) patients showed significantly higher levels of IFN-γ and TNF-α with significant negative correlations with the bacterial index (BI), whereas lepromatous leprosy (LL) patients showed significantly higher serum levels of IL-2R, IL-10, and IL-1β with significant positive correlations with the BI. Both type I and type II reactional patients showed significantly higher serum IFN-γ, IL-2R, and IL-1β, in addition to IL-10 in type II reactional patients, compared with nonreactional leprosy patients. When compared with each other, type I reactional patients showed increased levels of IFN-γ, whereas type II reactional patients showed increased levels of IL-10. Conclusions In leprosy patients, both IFN-γ and TNF-α are immunoprotective, whereas IL-2R, IL-10, and IL-1β are immunosuppressive. Our results indicate that type I reaction, with increased levels of IFN-γ, is a cell-mediated immune response, whereas type II reaction, with increased levels of IL-10, is essentially an immune complex disease.     

Differential regulation of IL-1 and IL-1 receptor antagonist in HaCaT keratinocytes by tumor necrosis factor-α and transforming growth factor-β1

Abstract Cytokines such as TNFα and TGFβ1 have potent effects on keratinocyte differentiation and have been implicated in cutaneous injury, immunologic reactions, and wound healing. To determine whether such conditions might alter the balance of epidermal keratinocyte IL-1 and the IL-1 receptor antagonist (IL-1ra), TNFα and TGFβ1 were added to HaCaT cells, a human adult keratinocyte cell line. mRNA levels of IL-1α, IL-1β, and IL-IRa were detected by polymerase chain reaction (PCR) on reverse transcribed RNA extracts, followed by Southern blot of the PCR products, ³⁵S-labeled probe hybridization, and quantification against standard curves. TNFα (100 ng/ml) at the 3-h time point significantly induced increases in mRNA expression of Iliα (9.2±2.9 fold increase) and IL-1β (2.5±0.7 fold increase) (n=7) which were concordant with increases in IL-1α protein (7.1±1.3 fold increase) and II-β protein (4.4±1.0 fold increase) measured by ELISA 24 h after stimulation. By contrast, icIL-IRa mRNA and protein levels were not affected by TNFα. TGFβl induced a mild increase in IL-lα mRNA (3.8±1.8 fold) and protein (3.5±1.2 fold). TGFβl did not affect IL-1α mRNA levels but caused variable increases in IL-1β protein levels. TGFβ1 did not alter icIL-1Ra mRNA or protein levels. Inhibition of RNA synthesis with actinomycin D demonstrated that the rate of degradation of IL-1β mRNA was reduced by treatment with TNFα. This stabilization of IL-1β mRNA was specific, because TGFP I did not stabilize IL-1β mRNA, and TGFβ1 and TNFα did not increase the stability of II-1α mRNA. icIL-l Ra mRNA was fairly stable over a 20 hour period and its slow degradation was not affected by treatment with either TNFα or TGFβ1, indicating a higher steady state stability of icIL-1ra mRNA relative to IL-1 mRNA's. Given the high rate of degradation of IL-1α and IL-1β mRNA, levels of these mRNAs may rapidly decrease while the icIL-1ra mRNA levels remain constant, thus allowing for rapid dampening of IL-1 activity soon after the stimuli provoking an inflammatory or reparative response have abated. In conclusion, TNFα and TGFβl, cytokines with potent effects on inflammation and differentiation, both induce keratinocyte IL-1α mRNA and protein levels, but differentially regulate IL-1β mRNA. They both exert little effect on IL-1 Ra levels, which were constitutively highly stable. Such differential regulation provides mechanisms for separately controlling the relative activity of these cytokines under normal and disordered conditions.     

Inter-regulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis

Accumulating evidence indicates that IL-1 family members and Th17 cytokines have a pathogenic role in psoriasis. We investigated the regulatory interactions of the IL-1-like IL-36 cytokine family and the Th17 cytokines in the context of skin inflammation. We observed increased gene expression of all three IL-36 cytokines in a Th17-dominant psoriasis-like animal model. The induction was downregulated by neutralizing IL-22. Expression of the IL-36s was also induced in cultured primary human keratinocytes (KC) by IL-17A and tumor necrosis factor (TNF)-α, and IL-22 synergized with IL-17A and TNF-α. Furthermore, the IL-36s directly induced their own expression and the production of proinflammatory mediators (TNF-α, IL-6, IL-8) in KC. These functions were markedly enhanced with the addition of IL-17A or TNF-α to the cultures. Similarly, IL-36α and IL-36β augmented IL-17A-mediated induction of antibacterial peptides. Finally, we show that the increased gene expression of IL-36 correlated with Th17 cytokines in the lesions of psoriatic patients. Our results indicate that the IL-36 cytokines are not only regulated by Th17 cytokines, but that they themselves can regulate the expression and enhance the function of Th17 cytokines. We propose that a feedback loop between the IL-36 and Th17 cytokines is involved in driving cytokine expression in psoriatic tissues.     

Effects of low concentrations of cis- and trans-urocanic acid on cytokine elaboration by keratinocytes

The urocanic acid cis isomer (cis-UCA) is a possible cutaneous photoreceptor for the immunomodulatory phenomena that follow ultraviolet B irradiation. Several experiments in animals show an inhibitory action of cis-UCA on cellular immunity. However, the action of cis-UCA on the synthesis of cytokines in keratinocytes remains unknown. Long-term cultures of normal human keratinocytes were prepared in a serum-free medium, and stimulated with 1 μg/ml of phorbol 12-myristate 13-acetate (TPA) and UCA or UVB-UCA (10–100 μg/ml). Synthesis of the following cytokines was measured using ELISA and Northern blot techniques: TNF-α, IL-1α, IL-1β, IL-6, IL-8 and TGF-β1. TPA increased TNF-α protein levels in culture supernatants. No changes in IL-1α and IL-1β protein levels were detected in basal culture supernatant after TPA stimulus. TPA augmented RNA expression for TNF-α, IL-1α, IL-1β and TGF-β1. UCA isomers did not induce cytokine changes in protein synthesis. Expression of IL-1α and IL-1β genes was increased after exposure to 100 μg/ml UVB-UCA (70 μg/ml cis-UCA). A slight increase in TNF-α RNA expression was detected when the dose of UVB-UCA reached 100 μg/ml. No effects on cytokine synthesis were found after UCA stimulus. These results suggest that low doses of cis-UCA do not effect cytokine synthesis by keratinocytes.     

孕酮在淋球菌引起的中性粒细胞炎症反应中的调节作用

目的 研究孕酮在淋球菌引起的中性粒细胞炎症反应中的作用。方法 提取正常人外周血中性粒细胞,根据是否加入孕酮,将其分为孕酮组、淋球菌组、干预组（孕酮 + 淋球菌）及对照组。荧光定量RT-PCR分别测定在0、3、8、12 h各组中性粒细胞中诱导型一氧化氮合酶（iNOS）、TNF-α、IL-1β mRNA含量,并用Western印迹测定iNOS蛋白水平。结果 淋球菌组和干预组中iNOS、TNF-α、IL-1β mRNA表达水平均升高;淋球菌组在8 h达到高峰,以后逐渐下降;干预组三者水平明显低于淋球菌组（P < 0.05）。而孕酮组、对照组各因子含量无明显变化。Western印迹结果显示,淋球菌组和干预组iNOS蛋白表达水平亦升高,前者明显高于后者（P < 0.05）。结论 孕酮下调中性粒细胞iNOS、TNF-α及IL-1β的表达,抑制淋球菌引起的中性粒细胞炎症反应,这一机制可能在女性无症状感染中起作用。     

The role of mitogen- and stress-activated protein kinase 1 and 2 in chronic skin inflammation in mice

Mitogen- and stress-activated protein kinase 1 and 2 (MSK1/2) are two kinases phosphorylated by both ERK1/2 and p38 MAPK. Recently, MSK1 and 2 have been reported to act as negative regulators of acute inflammation. In this study, we investigated the role of MSK1/2 in chronic skin inflammation using an oxazolone-induced allergic contact dermatitis model in MSK1/2 knockout mice and wild-type mice. MSK1/2 knockout mice were demonstrated to have significantly increased inflammation compared with wild-type mice. This was measured by an increased ear thickness, elevated infiltration of neutrophils in the skin and increased inflammatory histological changes. Furthermore, we found significantly elevated levels of the proinflammatory cytokines Tumor necrosis factor-α (TNF-α), IL-1β and IL-6 at both mRNA and protein levels in MSK1/2 knockout mice compared with wild-type mice after oxazolone treatment. In addition, the mRNA expression of the chemokine Thymus and activation regulated chemokine (TARC) was demonstrated to be significantly elevated in oxazolone-treated MSK1/2 knockout mice compared with wild-type mice. The increased expression of TARC was paralleled by increased infiltration of cells positive for the TARC receptor, CCR4, in the dermis of MSK1/2 knockout mice. Our results indicate that MSK1/2 are involved in the activation of feedback mechanisms that dampen oxazolone-induced skin inflammation.     

Interleukin-1 family members are enhanced in psoriasis and suppressed by vitamin D and retinoic acid

Interleukin (IL)-1 family comprise 11 members that play an important role in immune regulation and inflammatory process. Retinoids exert complex effects on the immune system, having anti-inflammatory effects in chronic dermatological diseases. Vitamin D (vitD) and analogs have been shown to suppress TNF-α-induced IL-1α in human keratinocytes (KCs). In the present study, we investigated IL-1 family members in psoriasis and the effects of vitD and retinoic acid (RA) on these members. We analyzed IL-1 family members gene expression in psoriatic skin and in ex vivo skin organ culture exposed to TNF-α, IL-17 or broadband UVB; afterwards, treatment with vitD or RA was performed and IL-1 family members mRNA was evaluated. Similarly, KCs were stimulated with IL-17 and subsequently treated with vitD. IL-1 family members were enhanced in psoriatic skin and in ex vivo skin organ cultures after pro-inflammatory stimuli (TNF-α, IL-17 and UVB). RA and vitD were able to suppress this enhancement.     

microRNA-125a在寻常型银屑病皮损中的表达及其对HaCaT细胞增殖的影响

【摘要】 目的 检测寻常型银屑病皮损中microRNA-125a（miR-125a）的表达与皮损处炎症因子水平的相关性及其对人永生化角质形成细胞（HaCaT）增殖的影响。方法 收集2017—2018年沈阳市第七人民医院40例寻常型银屑病患者皮损及相邻非皮损组织,采用反转录实时荧光定量PCR检测组织中miR-125a的表达及皮损组织中肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、IL-6、IL-17 mRNA的表达。将miR-125a过表达质粒、过表达对照质粒、miR-125a干扰质粒、干扰对照质粒转染HaCaT细胞,在转染后0、24、48、72 h采用CCK8法检测各组HaCaT细胞增殖能力,采用双抗体夹心酶联免疫吸附法（ELISA）检测质粒转染后HaCaT细胞上清液中TNF-α、IL-1β、IL-6、IL-17水平。相关性分析采用Spearman等级相关检验分析,两组间均数比较采用t检验。结果 寻常型银屑病皮损区miR-125a相对表达水平（2-ΔΔCt,0.389 ± 0.354）低于非皮损区（1.106 ± 0.396,t = 7.717,P < 0.001）。银屑病皮损组织中miR-125a表达与TNF-α、IL-1β、IL-17 mRNA的表达呈负相关（r = -0.447、-0.424、 -0.436,均P < 0.01）。转染相应质粒后0、24 h时,细胞增殖能力在过表达miR-125a组与过表达对照组（t = 0.282、1.445,均P > 0.05）、干扰miR-125a组与干扰对照组（t = 0.120、1.543,均P > 0.05）间差异无统计学意义;转染后48、72 h时,过表达miR-125a组细胞的增殖能力低于过表达对照组（t = 3.222、4.563,均P < 0.05）,干扰miR-125a组高于干扰对照组（t = 3.036、3.269,均P < 0.05）。MiR-125a过表达组TNF-α、IL-1β的表达水平低于过表达对照组,差异有统计学意义（t = 4.318、3.813,均P < 0.05）。结论 寻常型银屑病患者皮损中miR-125a低表达,miR-125a抑制角质形成细胞增殖,可能在银屑病发生发展中发挥保护作用。