Normal response to tumor necrosis factor-alpha and transforming growth factor-beta by keratinocytes in psoriasis

Abstract Normal and chronic plaque psoriatic keralinocyte cultures were tested for their in vitro response to 2–200 ng/nil TNF-α and 0.1–10 ng/ml TGF-β in a serum-free culture system. All normal and lesional psoriatic epidermal cell cultures showed a dose- and lime-dependent inhibition of growth in response to TNF-α and TGF-β. Inhibition in individual cultures was first seen at a concentration of 2 ng/ml for TNF-α and 0.1 ng/ml for TGF-β at day 2, but became significant at 20 ng/ml and 1 ng/ ml for TNF-α and TGF-β respectively at days 2-6. This effect was statistically significant at days 3–4 for the group of normal (TNF-α and TGF-β, n = 10, p<0.01 and psoriatic cultures (TNF-α. n = 9, p<0.0l; TGF-β, n = 7, p<0.05). Epidermal cells from normal and psoriatic skin were inhibited to the same extent at the same optimal concentrations by each cytokine. Inhibition was abolished by the addition of specific antibody to each cytokine, whilst antibody to a different cytokine had no effect. Nuclear and/or nuclear membrane staining was observed with antibody to the p55 TNF receptor both in cultured keralinocyles and in the tipper epidermal layers of both normal and psoriatic skin. In contrast, plasma membrane and cytoplasmic expression of the p55 TNF receptor was observed on macrophages and lymphocytes infiltrating psoriatic der-mis. This study has shown that the growth of normal and psoriatic keratinocytes was equally inhibited by TNF-α and TGF-βin vitro. The expression of TNF receptor by psoriatic keratinocytes in vivo may permit regulation of epidermal growth by administration of TNF-α in this disease.     

Contrasting effects of ultraviolet A1 and ultraviolet B exposure on the induction of tumour necrosis factor-α in human skin

Ultraviolet B (UVB) irradiation of the skin causes immunosuppression which is relevant to the induction of skin cancer. The mechanism of this immunomodulation is unclear but various regulatory molecules have been implicated, including cis-urocanic acid (cis-UCA) and the cytokines tumour necrosis factor-α (TNF-α) and interleukin 10 (IL-10). Whether ultraviolet A (UVA) induces similar changes has not been investigated fully. We studied the effect of in vivo UVB and long-wave UVA (UVA1) exposure on the induction of TNF-α, IL-10 and cis-UCA in human skin. Volunteers were irradiated with three minimal erythema doses (MED) of UVB or UVA1. At different times after irradiation, suction blisters were raised from irradiated and from non-irradiated (control) skin. The TNF-α and IL-10 protein concentration, and the percentage of cis-UCA in the blister fluid, were then determined. UVB irradiation of human skin led to a rapid and significant increase in TNF-α concentration in suction-blister fluid, with maximal values 6 h after irradiation (n = 6, P < 0.05). In contrast, UVA1 irradiation led to a decrease in TNF-α concentration in the suction-blister fluid compared with non-irradiated skin, with the lowest values 6 h after irradiation (n = 6, P < 0.05). Both UVB and UVA1 exposure of the skin induced a slight increase in IL-10 concentration. However, the increase in IL-10 was only significant after UVB irradiation (UVB, n = 6, P < 0.05; UVA, n = 7, P < 0.1). As previously shown, both UVB and UVA1 result in the photo-isomerization of trans-UCA and an increased percentage of cis-UCA was found in the suction-blister fluid. Thus the results show differential effects of UVB and UVA1 irradiation on the induction of immunoregulatory molecules, which may help to explain the variation in immune responses after UVB and UVA1 exposure of human skin.     

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.     

Production of multiple cytokines by cultured human melanomas*

Abstract We screened a panel of 8 primary and 21 metastatic melanoma cell lines for constitutive secretion of cytokines. Melanomas expressed bioactivity for TGF-β (8/25 lines) and IFN (7/12), but not IL-2. Immu-noassays detected IL-1α (4/25), IL-1β (12/25), 1L-6 (13/29), IL-8 (29/ 29), TGF-β₂, (5/12) and GM-CSF (11 /29). but not IL-3, IL-4, TNF-α, or IFN-γ. There was no preferential association of cytokine production with cells cultured from primary versus metastatic disease, and only IL-8 was produced by all lines tested. These data demonstrate that cultured melanomas produce a variety of cytokines which are potentially capable of influencing tumor growth in vivo.     

TGF-β1对人工皮肤光损伤炎症因子IL-1α、IL-6、TNF-α影响的研究

目的探讨TGF-β1对人工皮肤光损伤中炎症因子IL-1α、IL-6、TNF—α的影响。方法将角质形成细胞和成纤维细胞接种到胶原支架中体外构建人工皮肤,将人工皮肤分为四个实验组,即阴性对照组,紫外线照射,TGF-β1干预组,以及TGF—β1干预后再进行UV照射,采用ELISA法检测各实验组上清液中炎症因子IL-1α、IL-6、TNF—α．的浓度变化。结果体外培养的人工皮肤分为表皮和真皮层,与正常皮肤结构相似。与阴性对照组相比,UV照射组炎症因子IL-1仪、IL-6、TNF—α明显增加,有显著性差异（p〈0．01）;TGF-β1对IL-1α、IL-6、TNF-α的浓度没有明显影响（P〉0．05）;但是TGF—β1干预的uV组跟UV照射组比较则是IL—1α、IL-6、TNF-α浓度增加减少（p〈0．01）。结论TGF-β1对紫外线所致的人工皮肤光损伤的炎症因子IL-1α、IL-6、TNF-α产生有一定抑制作用,即对光损伤模型有保护作用。     

Trans-urocanic acid, a natural epidermal constituent, inhibits human natural killer cell activity in vitro

Abstract UV irradiation has been reported to influence NK cell function both in vitro and in vivo. Since urocanic acid may mediate UV-induced immune modulation we tested the effect of trans- and cis-urocanic acid (UCA) on the cylotoxic activity of human peripheral blood lymphocytes against the erythroleukemic target cell line K562 in vitro. Trans-UCA was found to be a strong inhibitor of NK cell activity whereas cis-UCA had no effect. Trans-UCA also partially inhibited cytotoxic function of IL-2-activated NK cells and reduced IL-2-induced activation of NK cells. This is the first report describing trans-UCA to be active, and cis-UCA inactive, in regulating an immune function. In the skin, a decrease in epidermal trans-urocanic acid concentration by UV radiation could produce a favorable milieu for NK cell activity, and thus counteract the impairment of antigen-specific immune surveillance, induced by increased cis-urocanic acid concentrations.     

Differences in responses of interleukin-1 and tumor necrosis factor α production and secretion to cyclosporin-A and ultraviolet B-irradiation by normal and transformed keratinocyte cultures

Abstract Among epidermal cytokines, IL-1 and TNFα are involved in inflammatory skin reactions and suspected of modulation by immuno-suppressive treatment (e.g., cyclosporin A, CsA) or UVB-irradiation, 2 mediators probably being involved in epithelial carcinogenesis. We evaluated the effects of 8 μg/ml CsA and 100 J/m² UVB-irradiation on the production and secretion of IL-1 and TNFα on normal human epidermal keratinocytes (NHK) and epidermal keratinocyte cell lines either spontaneously transformed (HaCaT) or transformed by human papillomavirus (HPV) type 16 or 18 (EK I6 and EK 18), by using FLISA test. Normal and immortalized keratinocytes constitutively produced and released IL-lα IL-lβ and IL-1 receptor antagonist (IL-IRA) but IL-I synthesis by NHK was significantly higher than by cell lines. All the cells spontaneously excreted low amounts of TNFα. Different responses to treatments were evidenced between NHK and cell lines. CsA modified significantly the production and secretion of ILI in most cells whereas slight changes were observed with TNFα secretion. UVB irradiation had no effect on the intracellular ILI pool of any cells but increased the release of IL1 and TNFα. The association CsA-UVB did not result in additive effects on synthesis and secretion of IL1; the release of TNFα by the cells remained poor except for EK18 cells. Taken together, these results show that, in immortalized keratinocytes, the IL-1 and TNFα expression was differently affected by treatments wilh CsA and/or UVB-irradiation as compared to NHK. In addition, spontaneously transformed keratinocytcs. HaCaT, reacted differently from HPV-transformed keratinocyles, EK I6 and EK I8.     

Cyclosporin A inhibits keratinocyte cytokine gene expression

Summary The immunosuppressive peptide cyclosporin A (CyA) is an extremely effective therapy for severe recalcitrant psoriasis, although its mechanism of action is unknown. In this study, we examined the effect of CyA on keratinocyte growth and cytokine expression, and showed that CyA inhibits the growth of murine and human keratinocytes (KC) and KC cell lines. In addition, CyA inhibits the expression of cytokine genes in a dose-dependent fashion. After 2 days' incubation with 20 μM CyA, interleukin-lα (IL- α), interleukin-1β (IL-l β), and interleukin 8 (IL-8) mRNA were decreased by 4-fold, 3·3-fold and 3·3-fold, respectively, in COLO-16, a keratinocyte cell line. IL-1 biological activity recovered from COLO-16 culture supernatants decreased to one-fifth of that of controls. In the murine KC cell line PAM 212, 10 μ CyA treatment for 2 days downregulated IL-1α, tumour necrosis factor-α (TNF-α) and IL-1 receptor by 60%, but had no effect on the message for interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), ornithine decarboxylase and β-actin. Cells cultured for 5 days in the presence of CyA required much lower concentrations (2 μM) to achieve the same degree of inhibition of IL-1α. Similar tissue concentrations of CyA have been reported in psoriatics undergoing CyA therapy. The inhibition of KC growth caused by CyA treatment could be partially overcome by the addition of 0·l–1·0 ng/ml of recombinant IL-1α simultaneously with CyA, suggesting that the growth inhibitory effect of CyA on KC cultures is due in part to loss of autocrine cytokine stimulation. Collectively, these data suggest that CyA may exert some of its therapeutic effects in psoriasis by inhibiting the release of KC cytokines, so terminating epidermal hyperproliferation. As some of these cytokines (IL-1, IL-8 and TNF-α) are chemotactic for leucocytes, or induce endothelial adhesion molecules, infiltration and inflammation may also be reduced by their inhibition.     

Treatment of contact hypersensitivity with urocanic acid

In order to investigate the effect of cis-urocanic acid (UCA) on a delayed-type hypersensitivity response in humans, a contact hypersensitivity reaction was induced on four test sites on the back of 33 volunteer subjects. The first test site was pretreated with cis-UCA immediately before application of the allergen. The second and third test sites were post-treated on the second and third days of the hypersensitivity response with cis-UCA and a class III corticosteroid, respectively. The fourth test site was used as a positive control. The cutaneous blood flow of the test sites was measured using laser Doppler flowmetry. Pretreatment with cis-UCA reduced the hypersensitivity response significantly. It is possible that cis-UCA could be used in the preventive treatment of contact hypersensitivity responses.     

Expression of CD80 (B7/BB-1) and CD28 in human white blood cells treated with urocanic acid

Urocanic acid (UCA) is formed in the epidermis where it accumulates to be converted fromtrans-tocis-UCA by ultraviolet (UV) radiation. The two isomers modulate immune functions in several experimental systems. In particular,cis-UCA has been shown to induce antigen-specific immune tolerance, but the molecular mechanism of this effect is unknown. The present investigation was instituted to disclose any effect of UCA isomers on the cellular expression of the costimulatory antigens CD80 (B7/BB-1) and CD28. CD80 expression was efficiently induced in monocytic (CD14⁺) cells by human interferon-γ, while CD28 levels on lymphocytes remained unchanged, as detected by flow cytometry. Neither UCA isomer showed any effect on the expression patterns of these costimulatory molecules. The results obtained suggest that the mode of action for epidermal UCA-induced tolerogenesis may not involve modulation of CD80 (B7/BB-1) or CD28 expression.     

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.     

烟曲霉刺激下TLR4对大鼠肺泡巨噬细胞释放TNF-α和IL-1β作用

目的观察在烟曲霉孢子刺激下Toll样受体4(TLR4)介导大鼠肺泡巨噬细胞(PAM)释放肿瘤坏死因子α(TNF-α)和白介素1β(IL-1β)的水平并分析其意义。方法应用体外培养的Wistar大鼠PAM,设置抗TLR4抗体组(阴性对照组)、单纯烟曲霉孢子组(阳性对照组)、烟曲霉和抗TLR4抗体混合组(实验组),于刺激0.5h,1h和2h后分别用酶联免疫吸附试验(ELISA)检测各组上清中TNF-α和IL-1β的水平。结果0.5h,1h和2h后,实验组抗TLR4单抗浓度为20μg/mL的A3组的TNF-α和IL-1β分别为(120±12.4)PG/ML,(160±13.2)PG/ML,(240±16.6)PG/ML和(18±2.3)PG/ML,(58±4.2)PG/ML,(92±9.4)PG/ML,显著低于阳性对照组(P<0.05),高于阴性对照组(P<0.05),且呈剂量依赖性。结论TLR4在烟曲霉孢子诱导大鼠PAM释放TNF-α和IL-1β中发挥着重要作用,监测TNF-α、IL-1β水平的动态变化有利于曲霉病的早期发现和防治。     

米非司酮对胚胎生长及TNF-α、TGF-β表达及性激素浓度的影响分析

目的:分析孕妇服用米非司酮对体内肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)以及腹内胚胎生长情况、性激素浓度的影响。方法:临床纳入人工流产的妊娠女性120例,按随机数字表法将患者分为两组各60例。其中60例患者给予米非司酮口服治疗作为研究组,另60例患者未给米非司酮口服治疗作为对照组。结果:两组妊娠女性绒毛中TGF-β1以及受体TGF-β1R基因表达差异无统计学意义(P0.05);研究组蜕膜中TGF-β1以及受体TGF-β1R基因表达分别为(1.64±0.59)、(1.52±0.48),对照组蜕膜中TGF-β1以及受体TGF-β1R基因表达分别为(0.98±0.44)、(0.87±0.28),有统计学差异(P0.05);研究组服药前血清TNF-α水平为(0.53±0.39)μg/L,服药后血清TNF-α水平为(0.85±0.12)μg/L,有统计学差异(P0.05);研究组服用后的FSH、E2、PRL、LH、P等激素指标与对照组比较,差异有统计学意义(P0.05)。结论:孕妇服用米非司酮会下调蜕膜中的TGF-β1、TGF-β1R受体,提高血清TNF-α水平,使免疫系统失调,抑制胚胎的生长、发育。     

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。