Whole-body UVB (TL-01) or UVA-1 irradiation does not alter the levels of immunomodulatory cytokines in the serum of human volunteers

BACKGROUND/PURPOSE: Ultraviolet (UV) exposure of mammalian skin induces local and systemic immunosuppression. In mice it has been proposed that systemic immunosuppression is mediated by an UV-induced cytokine cascade involving systemic interleukin (IL)-4 and IL-10 and a reduction in IL-12 activity. To investigate whether there was a parallel mechanism in humans we examined the effect of whole-body narrowband ultraviolet B (UVB) (311-313 nm; TL-01) and ultraviolet A (UVA)-1 (340-400 nm) on serum cytokine levels. METHODS/RESULTS: In a first study, five male psoriatic subjects were whole-body irradiated with three sessions of a standard UVB (TL-01) phototherapy regimen previously shown to cause downregulation of natural killer cell activity and T helper 1 (Th1) and Th2 cytokine production by peripheral blood mononuclear cells. Enzyme-linked immunoabsorbent assay (ELISA) of sera taken before and after the third session showed no effect of phototherapy on IL-10 and tumour necrosis factor-alpha (TNF-alpha). In a second study, five healthy subjects received three whole-body exposures of UVB (TL-01) and five other healthy subjects received three exposures of UVA-1 on alternate days (total 22 J/cm(2)). Blood samples were taken before the first irradiation and at 0, 4, 8, 12, 14, 24 and 48 h after the third irradiation. The sera were subsequently analysed for IL-10, IL-12, IL-8, IL-1beta and TNF-alpha, by ELISA. The levels of IL-1beta and TNF-alpha were below detection limits (<5 pg/ml), while no significant change in the levels of IL-10, IL-12 or IL-8 was detected as a result of either TL-01 or UVA-1. CONCLUSIONS: It seems unlikely that a modulation in these circulating cytokines assessed in this study accounts for systemic UV-induced immunosuppression in human subjects.     

Eicosapentaenoic acid, a n-3 polyunsaturated fatty acid differentially modulates TNF-alpha, IL-1alpha, IL-6 and PGE2 expression in UVB-irradiated human keratinocytes

In response to UVB-irradiation keratinocytes release a variety of cytokines and prostaglandins, including tumor necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha), interleukin 6 (IL-6), and prostaglandin E2 (PGE2). n-3 Polyunsaturated fatty acids (PUFA), mainly present in fish oil, can modulate cytokine synthesis, as predominantly studied in macrophages. In order to investigate the immune modulating actions of n-3 PUFA on the UVB response in human skin, we investigated the effect of eicosapentaenoic acid (EPA), a n-3 PUFA and a precursor of eicosanoid biosynthesis, on UVB-modulated TNF-alpha, IL-1alpha, IL-6, and PGE2 expression in normal human keratinocytes (NHK). We show that cultured NHK can efficiently take up EPA. Basal TNF-alpha expression is very low in NHK. IL-1alpha on the contrary is significantly present in untreated cultured NHK. Upon UVB-irradiation (32 mJ per cm2) TNF-alpha mRNA expression and secretion is induced and IL-1alpha mRNA expression is reduced, although IL-1alpha secretion is induced. EPA treatment results in higher TNF-alpha and IL-1alpha expression, both in nonirradiated and UVB-irradiated keratinocytes. Moreover EPA and UVB appear to act synergistically to superinduce TNF-alpha expression. EPA treatment results also in lipid peroxidation and in decreased PGE2 and IL-6 secretion after UVB-irradiation. In contrast to EPA, oleic acid (monounsaturated fatty acid) and linoleic acid (n-6 PUFA) treatment did not result in higher TNF-alpha or IL-1alpha levels in nonirradiated or UVB-irradiated NHK, indicating that the observed effects are specific for EPA. In conclusion, these results show that EPA can differentially modulate UVB-induced cytokine and prostaglandin synthesis in NHK.     

Effect of ultraviolet radiation on the expression of pp38MAPK and furin in human keratinocyte-derived cell lines

Background: Ultraviolet radiation (UVR) is known to induce the activation of stress‐inflammation signal transduction pathways, and to induce the activity of many proteases in skin cells. It is unknown whether the activation of proteases such as furin is related to changes in the phosphorylation status of p38MAPK. Methods: The effect of UVR on immortalized keratinocyte (HaCaT) and squamous cell carcinoma (Colo16) cells was investigated with respect to cell survival, phosphorylation of p38MAPK, and the proprotein convertase, furin. The cells were exposed to either a low or a high dose of UVA and/or UVB and the viability was monitored over 48 h, along with changes in the intracellular expression of p38MAPK and furin. Results: Low‐dose UVA (2 kJ/m²) and/or UVB (0.2 kJ/m²) radiation had no effect on cell viability, except in UVA‐irradiated Colo16 cells. High UVA (20 kJ/m²) caused a loss of cell viability in HaCaT cells, but not in Colo16 cells. The opposite effect was seen in cells exposed to a high UVB dose (2 kJ/m²). The viability of both cell cultures decreased when exposed to high‐dose UVA+B radiation. UV irradiation downregulated the expression of phosphorylated p38 (pp38) in HaCaT cells irrespective of the UV dose and type. In Colo16 cells, UV radiation induced pp38 expression in the cells following exposure, with the highest increase in cells exposed to high‐dose UVA. The expression of furin in UV‐irradiated HaCaT cells was similar to that seen for pp38 expression. In Colo16 cells, UV radiation induced furin expression, with the highest increase seen in cells 24 h after exposure to both high‐dose UVB and UVA+B radiation. Conclusion: The results show that there are differences between the effect of UV types and doses on cell function in the keratinocyte‐derived cell lines examined in this study. The level of furin expression in Colo16 cells correlated to changes in pp38 levels in the cells following exposure to UV radiation, but not in HaCaT cells. From an improved understanding of the signalling pathways and their downstream events and how these may differ as a result of tumorigenesis, it may enable the development of inhibitors, which may have therapeutic applications.     

Subcutaneous administration of collagen-polyvinylpyrrolidone down regulates IL-1beta, TNF-alpha, TGF-beta1, ELAM-1 and VCAM-1 expression in scleroderma skin lesions

In this study the effect of collagen-polyvinylpyrrolidone (collagen-PVP) vs. triamcinolone acetonide (Triam) in scleroderma (SSc) skin lesions was evaluated. Ten SSc patients were treated weekly with subcutaneous injections of 0.2 mL Triam (8 mg/mL) or 0.2 mL collagen-PVP (1.66 mg collagen). Skin biopsies were obtained from lesions before and after treatment. Tissue sections were evaluated by histology and immunohistochemistry (ELAM-1, VCAM-1, IL-1beta, TNF-alpha, TGF-beta1 and PDGF). The corticoid-treated group showed abnormal tissue architecture while the biodrug-treatment restored cutaneous appendages and type I/III collagen proportion. Cytokine and adhesion molecule expression was almost inhibited with Triam, while collagen-PVP down-regulated it. Collagen-PVP improved the tissue architecture of SSc lesions and down-regulated some proinflammatory parameters, without the side effects induced by corticoids.     

Effect of gamma-interferon on IL-1 alpha, beta and receptor antagonist production by normal human keratinocytes

Abstract In inflammatory dermatoses. activated T cells produce inter-feron-gamma (IFN-γ), which interacts with keratinocytes and contributes to the direct activation of these cells by inducing, among other factors, the expression of HLA-DR antigens and intercellular adhesion molecule-1. However, the action of IFN-γ on epidermal cell cytokine production is not known. Our aim was to assess the effect of IFN-γ on the production of IL-1 by normal human keratinocytes cultured in low calcium medium (MCDB153). In comparison with controls, the addition of nontoxic IFN-yγ concentrations (50-500 U/ml) to cell cultures induced a significant increase of IL-1α and IL-1β production predominantly after 100 U/ml treatment in the cell extracts as well as in the supernatants at 24h and 48h. The production of the antagonist. IL-1RA, was also enhanced and the effect of the critical concentration (100 U/ml) was more evident. However, the absence of a characteristic dose response could not be explained by an antiproliferative effect of high IFN-γ concentrations (250 and 500 U/ml) on cultured keratinocytes or by the induction of the nuclear stress protein, Hsp72. two phenomena known to down-regulate IL-1 biosynthesis. In conclusion, the modifications in keratinocyte IL-1 production under IFN-γ stimulation can contribute to activate the epidermal cells and thus involve them in the local immune response.     

Differential effects of UV irradiation on nuclear retinoid receptor levels in cultured keratinocytes and melanocytes

A major risk factor for skin cancer is UV irradiation, which not only damages DNA and other photosensitive compounds like vitamin A, but may also perturb cellular signaling, e.g. via the retinoid receptor system believed to be important for cancer protection. We used cultured normal human keratinocytes and melanocytes to examine the effects of UV irradiation on the expression of the predominant retinoid receptors in the human skin (RARalpha, RARgamma and RXRalpha) and the AP-1 protein c-Jun; mRNA levels were studied by real-time PCR and protein levels by Western blot. In keratinocytes, a single dose of UVB (50 mJ/cm2) caused a rapid drop in the expression of all three receptors (mRNA levels minus 35-50% after 4 h; protein levels minus 20-45% after 8 h), which was followed over the next 40 h by a variable response, leading to full normalization for RARalpha only. In contrast, the levels of c-Jun did not change significantly after UV exposure. In melanocytes, UVB caused a similar drop of the retinoid receptor levels as in keratinocytes but this was soon followed by an increased expression leading to a complete normalization of all receptor levels within 1-3 days. The c-Jun levels in melanocytes increased 1 day after UV exposure and remained high (plus 50%) thereafter. In both cell types, a approximately 3-fold increase in apoptosis (measured by DNA fragmentation) was observed 8-48 h after UVB irradiation. In conclusion, a depletion of vitamin A and retinoid receptors by UV irradiation, together with unchanged or even increased c-Jun levels, might seriously interfere with retinoid signaling and thus promote future tumor development, especially in keratinocytes.     

Increased expression of TRPV1 channel in intrinsically aged and photoaged human skin in vivo

Abstract:  Transient receptor potential vanilloid type 1 (TRPV1) is activated by various stimuli including capsaicin, heat and acid. While TRPV1 has been localized in the epidermis, little is known about the physiological role of TRPV1 in the skin, especially in skin ageing. In this study, we investigated the effect of acute UV irradiation on TRPV1 expression in human skin and the changes in TRPV1 mRNA and protein in intrinsic ageing and photoageing using human sun-protected (upper inner arm) and sun-exposed (forearm) skin of young and elderly subjects.
Western blot analysis of UV-irradiated young buttock skin revealed that the expression of TRPV1 protein was increased at 24 h (2.3-fold) and 48 h (2.4-fold) after UV irradiation. Real-time PCR analysis also showed that the mRNA level of TRPV1 was augmented by 2.4-fold at 4 h after UV irradiation. TRPV1 protein was expressed at higher levels by 2.6-fold in the sun-protected skin of the elderly subjects than in that of young people according to western blotting, real-time PCR analysis and immunohistochemical staining. In addition, the photoaged skin of elderly showed increased expression of TRPV1 mRNA and protein compared with that of the sun-protected skin of the same individuals. Also, we found increased expression of TRPV1 in nerve fibres of elderly persons using double staining of TRPV1 and nerve fibres.
Based on the above results, our data suggest that the expression of TRPV1 is affected by both the intrinsic ageing and photoageing processes.     

产妇宫内感染对早产儿IL-1β、IL-6、IL-8、IL-10和TNF-α影响情况研究

目的:探讨产妇宫内感染对早产儿IL-1β、IL-6、IL-8、IL-10和TNF-α的影响。方法:回顾性分析2015年8月至2016年10月在我院接受分娩的宫内感染产妇的早产儿的临床资料,同时选取同期在我院分娩的非宫内感染产妇的早产儿作为对照。观察两组早产儿血清炎症因子水平、NIHSS评分、肾功能、心肌酶指标和出生时一般情况的差异。结果:宫内感染组早产儿的IL-1β、IL-6、IL-8、IL-10和TNF-α水平均高于对照组(t=7.044、12.208、7.289、20.185、5.421,P0.001);宫内感染组早产儿的Sp O2、Pa O2低于对照组(t=-6.353、-35.142,P0.001),Pa CO2水平高于对照组(t=35.720,P0.001);宫内感染组早产儿的NIHSS评分高于对照组(t=50.424,P0.001),出生1min和5min的Apgar评分低于对照组(t=-3.475、-4.398,P0.001);宫内感染组早产儿的尿素氮、肌酐和CK-MB水平均高于对照组(t=49.014、11.611、16.458,P0.001);Pearson相关分析法结果显示,宫内感染早产儿血清IL-1β、IL-6、IL-8、IL-10和TNF-α水平与NIHSS评分、肾功能、心肌酶指标水平正相关,与Apgar评分负相关。结论:宫内感染产妇分娩的早产儿的血IL-1β、IL-6、IL-8、IL-10和TNF-α水平较高,且与患儿的NIHSS评分、肾功能、心肌酶指标密切相关。     

Suppressed alloantigen presentation, increased TNF-alpha, IL-1, IL-1Ra, IL-10, and modulation of TNF-R in UV-irradiated human skin

Cytokines induced in skin by ultraviolet radiation cause local and systemic immunosuppression. Tumor necrosis factor alpha, interleukin-1, and interleukin-10 are key mediators in the mouse, but less is known about cytokine synthesis and function in ultraviolet-irradiated human skin. We exposed human skin to 3 minimal erythema doses of solar-simulated radiation and raised suction blisters at intervals to 72 h. Alloantigen presentation was suppressed in a mixed epidermal cell-lymphocyte reaction by 69% from 4 to 15 h post-solar-simulated radiation, but recovered to control values by 24 h. Tumor necrosis factor alpha was raised at 4 h after solar-simulated radiation, reached a maximum 8-fold increase at 15 h, then rapidly declined to control values. Interleukin-1alpha and interleukin-1beta were first increased at 15 h, and remained raised to 72 h, although interleukin-1beta declined from its 15 h maximum. Interleukin-10 increased a maximum 2-fold between 15 and 24 h, coincident with recovery of mixed epidermal cell-lymphocyte reaction responses and downregulation of tumor necrosis factor alpha and interleukin-1beta. Solar-simulated radiation differentially affected soluble tumor necrosis factor alpha receptors; soluble tumor necrosis factor-RI was suppressed 33% at 8-15 h whereas soluble tumor necrosis factor-RII increased 2-fold from 15 to 48 h. Interleukin-1 receptor antagonist was raised at all times post-irradiation. Interleukin-12 was not detectable in control or irradiated skin. These kinetics suggest the tumor necrosis factor alpha network has primary importance in ultraviolet-damaged human skin. The small increase in interleukin-10 implies that 3 minimal erythema doses of solar-simulated radiation is the threshold dose for its induction and local, rather than systemic, functions for interleukin-10 in immunosuppression and regulation of other cytokines.     

Human keratinocytes constitutively express IL-4 receptor molecules and respond to IL-4 with an increase in B7/BB1 expression

Abstract In certain pathological conditions, such as atopic dermatitis, interleukin-4 (IL-4) can be detected in the skin. As the rôle of this cytokine in inflammatory skin lesions is not completely clear, we investigated its biological effects on skin keratinocytes. It was found that freshly isolated as well as cultured keratinocytes obtained from normal individuals express mRNA for the IL-4 receptor (IL-4R) and produce IL-4R protein, as determined by How cytometry. Moreover, IL-4 induced a proliferative response in keratinocyles after 1 day of culture and enhanced B7/BB1 expression in these cells. B7-2 (CD86) mRNA and protein were neither detected on untreated nor IL-4 treated keratinocytes. In contrast to interferon-γ (IFN-γ), IL-4 did not induce ICAM-1 (CD54) or HLA-DR-expression. Keratinocytes which had been treated with IL-4 showed an enhanced ability to stimulate allogeneic T-cell proliferation in the presence of staphylococcus enterotoxin B (SEB),(p<0.01). Neutralizing anti- B7/BBI monoclonal antibodies did not block this effect. These results indicate that other molecules than B7/BB-I, HLA-DR or ICAM-1 on IL-4-activated keratinocytes may be involved in T-cell stimulation. In conclusion our results suggest, that locally produced IL-4, besides modulating keratinocyte membrane molecules, may enable keratinoeytes to interact with skin infiltrating lymphocytes.     

Ultraviolet-B irradiation decreases IFN-gamma and increases IL-4 expression in psoriatic lesional skin in situ and in cultured dermal T cells derived from these lesions

Type 1 cytokine producing T cells play an important role in the pathogenesis of psoriasis. Ultraviolet-B (UVB) irradiation is effective in the treatment of this disease. In normal skin, UVB causes a change in dermal microenvironment, leading to a decrease of IFN-gamma expressing type 1 T cells and a concurrent increase of IL-4 expressing type 2 T cells. The aim of this study was to show whether UVB irradiation causes a like-wise shift of type 1 and type 2 responses in psoriatic skin. For this purpose, biopsies were obtained from the lesional skin of psoriatic patients before, 2 days and 14 days after a single exposure to 4 MED UVB. Sections from these biopsies were immunostained (CD3, IFN-gamma and IL-4) or RNA was extracted and analyzed for the expressions of IFN-gamma and IL-4 by PCR. In addition, primary cultures of T cells from dermal cell suspensions were stained intracellularly for IFN-gamma and IL-4 expression and CD4+ and CD8+ T subsets were analyzed by flow cytometry. IFN-gamma was abundantly expressed in situ before irradiation and decreased in all patients after UVB irradiation, whereas IL-4 expression was variably expressed before irradiation and increased in different degrees after irradiation. Cytokine mRNA expressions determined by PCR showed a clear decrease of IFN-gamma and increase of IL-4 following UVB irradiation. Both CD4+ and CD8+ dermal T cells were found to produce less IFN-gamma and more IL-4 following UVB irradiation as determined by flow cytometry. Decrease in IFN-gamma expression and increase in IL-4 expression of dermal T cells in psoriatic lesions after UVB irradiation may lead to decrease in local immunoreactivity. These changes could be part of the therapeutic effects of UVB on psoriasis.     

UV irradiation induces downregulation of bcl-2 expression in vitro and in vivo

Abstract Recently, the proto-oncogenes bcl-2 and bax have emerged as important regulators of the apoptotic form of cell death. We examined UV irradiation-elicited apoptosis and regulation of bcl-2 and bax expression both in vivo in human skin and in vitro in HeLa cells. Using flow cytometric analysis, HeLa cells were found to undergo apoptosis at the 12-h time-point after exposure to UVB irradiation (100 mJ/cm²). The expression of bcl-2 mRNA was found to decrease after a single dose of UVB radiation (doses 10–200 mJ/ cm²). In contrast, the expression of bax mRNA was not significantly changed. When human skin was irradiated with a single dose of solar-simulated radiation (40 mJ/cm²), Bcl-2-positive cells were significantly reduced in the epidermis at the 3- and 6-h time-points. Our results suggest that UV irradiation downregulates bcl-2 expression both in vitro at the mRNA level and in vivo at the protein level, and that downregulation of bcl-2 constitutes a mechanism of potential importance in UV-induced apoptosis in human epidermis. Received: 1 July 1998 / Received after revision: 12 October1998 / Accepted: 2 November 1998     

Co-culture of human melanocytes and keratinocytes in a skin equivalent model: effect of ultraviolet radiation

Melanocytes grown in pure monolayer culture lack the three-dimensional organization and many of the cellular interactions that exist in vivo. This can be partially overcome by growing melanocytes together with other epidermal cells in skin equivalent models. In this study skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in various ratios onto de-epidermized dermis. They were cultured in DMEM/Ham's F12 (31) for 3 days and then lifted to the air-liquid interface and maintained for 11 days. Histological examination revealed a structure that closely resembled human interfollicular epidermis. Melanocytes, identified by their dendritic appearance, positive dopa reaction and positive staining with a melanocyte-specific antibody (MEL5), were located in the basal layer. Melanin was seen both in melanocytes and in neighbouring keratinocytes. Whilst the skin equivalent became more pigmented following UV irradiation (total UVB 4760 J/m² over 3 days), the quantity and distribution of melanin at the light microscopic level appeared to be unchanged. However, the number and dendricity of melanocytes increased, as did their staining with dopa and MEL5. These results indicate that melanocytes are functional and capable of responding to UV irradiation.     

The maturation-dependent production of interleukin-16 is impaired in monocyte-derived dendritic cells from atopic dermatitis patients but is restored by inflammatory cytokines TNF-alpha and IL-1beta

BACKGROUND: Maturation of dendritic cells (DCs) influences important DC functions such as production of cytokines. Recently, DCs were identified as a source of interleukin-16 (IL-16), a chemotactic factor for DCs themselves, CD4+ T cells, and eosinophils. There is evidence that DC-derived IL-16 may contribute to the pathogenesis of atopic dermatitis (AD). OBJECTIVE: To investigate the production of IL-16 during differentiation of monocytes into DCs in healthy individuals and patients with AD. METHODS: IL-16 production was investigated by quantitative real-time RT-PCR, intracellular cytokine staining, immunoblotting, and ELISA. RESULTS: DCs generated from peripheral monocytes by 5-day culture in the presence of IL-4 and granulocyte/macrophage colony-stimulating factor acquired the capability to synthesize, store, and secrete IL-16. Storage and release of IL-16 was further enhanced during final DC maturation induced by additional 3-day culture with tumor necrosis factor-alpha (TNF-alpha) and monocyte-conditioned medium. Maturation, as determined by up-regulation of CD83 and CD86 surface expression, and production of IL-16, but not production of IL-10 and IL-12p40 was impaired in day 8 DCs derived from AD patients compared to those from healthy donors. Stimulation of day 8 DCs from AD patients with TNF-alpha and IL-1beta enhanced the expression of CD83 and CD86 and restored the production of IL-16. CONCLUSIONS: Signals involved in the activation and maturation of DCs enhance their capacity to produce IL-16. Functional abnormalities present in patients with AD at the monocyte level may account for impaired maturation and IL-16 production of monocyte-derived DCs.     

Modulation of intercellular adhesion molecule-1 expression on human melanocytes and melanoma cells: evidence for a regulatory role of IL-6, IL-7, TNF beta, and UVB light.

Intercellular adhesion molecule-1 (ICAM-1) is involved in cell-cell interactions of leukocytes and parenchymal cells and thus plays an important role in immunologic and inflammatory reactions. The expression of ICAM-1 that is found on many different cells such as melanocytes and melanoma cells is induced by various cytokines, including interferon-gamma (IFN gamma), interleukin (IL)-1 and tumor necrosis factor alpha (TNF alpha). Because expression of ICAM-1 in melanoma was found to correlate with increased risk of metastasis, the regulation of ICAM-1 expression on human melanocytes and melanoma cells was investigated. Foreskin-derived melanocytes and melanoma cell lines (A375, G361) were incubated with different cytokines and ICAM-1 expression was evaluated by fluorescence-activated cell sorter. IFN gamma, IL-1, IL-7, TNF alpha, and TNF beta significantly upregulated ICAM-1 expression in a dose-dependent manner. Most interestingly, the cytokine IL-6, which does not influence adhesion-molecule expression on other cells, significantly upregulated melanocyte and melanoma cell ICAM-1 expression. This effect was dose dependent and could be blocked by an IL-6 antibody. Irradiation with ultraviolet (UVB) light did not influence constitutive ICAM-1 expression on melanoma cells and melanocytes, but suppressed cytokine-induced ICAM-1 expression when cells were harvested 16 h after irradiation. These findings were further confirmed by Northern blot analysis, showing a marked accumulation of ICAM-1 mRNA after cytokine treatment, which was reduced by irradiation with UVB light. However, when UVB-exposed melanoma cells were cultured for at least 48 h induction of ICAM-1 expression was observed. These data indicate that, similar to other cells, ICAM-1 expression on melanoma cells and melanocytes is regulated by cytokines and that UVB light affects ICAM-1 expression on melanocytic cells in a biphasic manner.