Ultraviolet Irradiation Downregulates the IL-1 Receptor in Pam 212 Keratinocytes

There is increasing evidence that keratinocytes, by virture of production of cytokines, may participate in inflammatory and immune responses in the skin. The expression of keratinocyte cytokines can be modulated by various exogenous and endogenous agents, including ultraviolet light (UV) and cytokines themselves. We have recently shown that augmentation of GM-CSF expression by UVB irradiation is mediated by UV-induced IL-1 in Pam 212 keratinocytes. This may suggest that an autocrine mechanism exists in this murine keratinocyte cell line. In order to further clarify the mechanism by which UV irradiation augments GM-CSF, this study was undertaken to assess the effect of UV on the expression of IL-1 receptor (IL-1R) in Pam 212 keratinocytes. UVB irradiation (35 or 70 J/m²) significantly downregulates the expression of IL-1R mRNA. IL-1R mRNA remains downregulated for 12h after UV, then slowly returns to the steady state level by 24h to 32h after UV. On the other hand, UV augments IL-1 mRNA in a biphasic pattern with an initial phase (by 6h after UV) and a late phase (24h and 48h after UV). The results of these studies indicate that modulation of IL-1R and IL-1 itself by UVB irradiation are important in mediating autocrine cytokine networks in keratinocytes.     

Augmentation of granulocyte/macrophage colony-stimulating factor expression by ultraviolet irradiation is mediated by interleukin 1 in Pam 212 keratinocytes

Keratinocytes are a potent source of a variety of cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF). In this study, we have shown that ultraviolet B (UVB) irradiation augments GM-CSF mRNA expression by murine keratinocytes. This is reflected in the increased production of GM-CSF protein by these cells. In the same cell population, exposure to UVB irradiation increases interleukin 1 alpha (IL-1 alpha) mRNA and IL-1 protein as detected by bioactivity. This increase in IL-1 alpha precedes the increase of GM-CSF mRNA. Addition of recombinant IL-1 alpha to the medium increases GM-CSF mRNA expression. Anti-IL-1 alpha antibodies can completely inhibit UV-augmented GM-CSF mRNA expression. These results demonstrate that UVB irradiation-induced augmentation of GM-CSF is mediated by UV-induced IL-1 alpha.     

IL-1对UVA辐射成纤维细胞基质金属蛋白酶表达的影响机制探讨

目的探讨IL1(IL1α,IL1β)对长波紫外线(UVA)辐射后成纤维细胞基质金属蛋白酶(matrixmetalloproteinases,MMPs)表达的影响机制。方法用ELISA法检测UVA辐射后成纤维细胞培养上清MMP1和MMP2的表达。接着用IL1α和IL1β分别处理UVA辐射后的成纤维细胞,用Western免疫印迹法检测其丝裂原活化蛋白激酶(MAPK)的活性;用RTPCR方法检测cfos和cjun的mRNA表达。结果不同剂量UVA(0,1,5,10J/cm2)辐射的成纤维细胞分泌MMP1逐渐上升,对MMP2分泌没有影响。IL1α和IL1β(0,1,10,100ng/ml)促进UVA(10J/cm2)辐射成纤维细胞的MAPK活性表达,并以剂量依赖方式促进cjun的mRNA表达。IL1α还显著增加cfosmRNA表达,但IL1β对cfosmRNA表达无明显影响。IL1α和IL1β促进UVA辐射成纤维细胞分泌MMP1,于100ng/ml时有显著性差异(P均<0.05),但对MMP2分泌无明显影响。结论UVA辐射成纤维细胞分泌MMP1增加,对MMP2分泌没有影响。IL1(IL1α和IL1β)通过促进MAPK活性和cjunmRNA表达,IL1α还促进cfosmRNA表达使UVA辐射成纤维细胞MMP1表达增加,表明IL1在皮肤光老化的真皮胶原过度降解中发挥着重要的作用。     

Regulation of epidermal cell interleukin-6 production by UV light and corticosteroids

Epidermal cells (EC) are well known as a source of cytokines, including interleukin (IL)-6. In the present study, we investigated whether ultraviolet (UV) light and corticosteroids (CS) affect IL-6 production by normal (HNK) or malignant (KB) human keratinocytes. Supernatants derived from UVB (100 J/m2)- but not from UVA (100-1500 kJ/m2)-exposed EC (HNK and KB) contained significantly increased levels of IL-6 activity. This was also confirmed by Western blot analysis, resulting in specific bands at 23 kD and 27 kD. Northern blot analysis revealed an enhanced IL-6 mRNA expression after UVB exposure. Addition of hydrocortisone, prednisolone, or dexamethasone immediately after UVB irradiation significantly blocked UVB or IL-1-induced IL-6 mRNA expression and production by EC. The suppressive effect was observed at doses in the physiologic (10(-7)-10(-9) M) as well as pharmacologic (10(-5)-10(-7) M) range. In contrast, the nonactive steroid prednisone did not affect EC IL-6 mRNA expression. These findings indicate that increased IL-6 production by EC after UVB irradiation may mediate local and systemic inflammatory reactions following extensive sun exposure. Thus, the therapeutic effect of corticosteroids observed in various inflammatory diseases may be partly due to their downregulating capacity of IL-6 production.     

Modulation of endothelin-1 in normal human keratinocytes by UVA1/B radiations, prostaglandin E2 and peptidase inhibitors

In the skin, keratinocytes synthesize and secrete endothelin-(ET-1), a potent vasoconstrictor peptide which acts also as a growth factor for most skin cells. The aim of the study was to test the effects of UVA1 and the associations UVA1/B on the expression of ET-1 in normal human keratinocytes and to determine whether exogenously added prostaglandin E2 (PGE2) regulated ET-1 expression. As ET-1 is susceptible to degradation, we also evaluated whether ET-1 secretion was modulated by peptidase inhibitors. Our results showed that UVA1 (365 nm) did not modify the levels of preproET-1 mRNA and protein. Moreover, the associations UVA1+UVB or UVB+UVA1 down-regulated the overexpression of secreted ET-1 induced by UVB alone. PGE2 at 10(-5) M reduced the expression of ET-1 at the mRNA and protein levels but did not exert any significant modification at lower concentrations from 10(-10) to 10(6) M. Phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, drastically decreased the amount of ET-1 accumulating in the culture medium in basal conditions or after UVB irradiation. Conversely, thiorphan, a specific inhibitor of neutral endopeptidase (NEP), rather increased the levels of ET-1 secretion mainly after UVB irradiation. Taken together, the results showed that normal human keratinocytes secrete and partly degrade ET-1 through ECE and NEP pathways and pointed out a differential regulation of ET-1 by UVB and UVA1 radiations without any noticeable role for PGE2.     

Opposing effects of ultraviolet B irradiation on interleukin-1 receptor antagonist and interleukin-1α messenger RNA expression in a human epidermoid carcinoma cell line

Interleukin-1 receptor antagonist (IL-1RA) is a cytokine that acts to antagonize IL-1 activity without agonist function. The expression of IL-1RA has been reported in many cell types, including the keratinocyte that covers the outer most part of the skin. However the modulation of IL-1RA by ultraviolet B (UVB), which is the most biologically active UV, has not been reported yet. We therefore selected a keratinocyte cell line with a cytokine-producing profile similar to that of keratinocytes and tested the effect of UVB on its ability to produce IL-1RA mRNA. IL-1RA mRNA was constitutively expressed in the cell line and began to be suppressed by 3 h after the UVB irradiation with 100 mJ/cm². The level of IL-1RA expression became lowest by 16 h after the irradiation with 100 mJ/cm². Simultaneously, IL-1α mRNA started to increase by 1 h and peaked by 3–16 h after the irradiation with 10–100 mJ/cm². The differential expression of IL-1α and IL-1RA mRNA following exposure to a high dose (100 mJ/cm²) of UVB may markedly potentiate the role of IL-1 in UV-induced inflammation.     

鳞状细胞癌细胞系12F细胞中白介素1αmRNA的表达及对紫外线照射的反应

目的 了解人角质形成细胞(KC)起源的鳞状细胞癌细胞系(SCC)12F细胞自发表达白介素1α(IL-1α)mRNA的能力以及紫外线照射对IL-1α分泌水平的影响。方法 采用RNA印迹法检测IL-1αmRNA的表达水平,采用ELISA方法检测IL-1α蛋白的分泌水平。结果 SCC12F细胞可在正常KC培养体系中自发表达IL-1αmRNA,其表达水平随细胞培养时间的延长而增强,在120h达到高峰值;经中波紫外线(UVB)照射后,IL-1α蛋白的分泌水平既随照射时间增加,亦随照射剂量增加。结论 SCC12F细胞在正常KC培养体系中可自发表达IL-1αmRNA,其IL-1α蛋白的分泌水平对UVB照射呈时效及量效反应。     

Modulation of IL-10, IL-12, and IFN-gamma in the epidermis of hairless mice by UVA (320-400 nm) and UVB (280-320 nm) radiation

We have observed recently that the suppression of contact hypersensitivity (CHS) induced in mice by UVB irradiation may be prevented by suberythemal exposure to UVA radiation. Because the UVB-immunosuppressed state is associated with an upregulation of the Th2-associated cytokines IL-10 and IL-4, and a deficiency in Th1-associated IL-2, IL-12, and IFN-gamma, and because UVA photoimmunoprotection appeared to be IFN-gamma- dependent, we tested the hypothesis that UVA immunoprotection results from an ability to prevent the UVB-induced cytokine disarray. This study describes changes in epidermal IL-10, IL-12 and IFN-gamma for 5 d following irradiation of hairless mice with the CHS-modulating doses of UVB, UVA, or UVA + UVB, using immuno-histochemical detection in paraffin embedded skin sections, followed by image analysis quantitation. We found that UVB, but not UVA exposure, caused an increase in epidermal IL-10 expression, peaking at 3 d. UVA irradiation, but not UVB, resulted in increased epidermal IL-12 expression, peaking at 3 d, and increased epidermal IFN-gamma expression peaking earlier at 1 d. Irradiation with UVA + UVB abrogated the UVB-enhanced expression of IL-10, and caused small but significant increases in IL-12 and IFN-gamma at 3 d and 1 d, respectively. These findings suggest that UVA photoimmunoprotection is mediated via prevention of IL-10 release, and thus the maintenance of the Th1/Th2 balance, probably by upregulation of IL-12 and IFN-gamma, which are known to antagonize IL-10 in numerous models. The time course suggests that IFN-gamma responds initially to UVA radiation, and may stimulate the increased expression of IL-12.     

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.     

紫外线对HaCaT细胞结合珠蛋白mRNA表达的影响

目的 观察紫外线照射对人角质形成细胞系HaCaT细胞表达结合珠蛋白的影响。方法 用逆转录-聚合酶链反应法检测长波紫外线UVA、中波紫外线UVB照射后HaCaT细胞各时相结合珠蛋白mRNA表达水平。结果 UVA12J/cm²照射后HaCaT细胞结合珠蛋白mRNA表达明显升高。照射后20min结合珠蛋白mRNA表达已开始升高,6h达第一高峰,48h出现第二高峰。照射后2h、4h、6h、24h、48h组与对照组比较,差异有统计学意义(P<0.05)UVB30mJ/cm²照射HaCaT细胞后结合珠蛋白mRNA表达也出现时相变化:4h达第一高峰,48h出现第二高峰。照射后20min、2h、4h、6h、12h、48h、72h组与对照组比较,差异有统计学意义(P<0.05)。结论 紫外线UVA、311nmUVB上调HaCaT细胞结合珠蛋白mRNA表达,随照射时间,结合珠蛋白mRNA表达呈时相变化。     

Gene expression profiles of TNF-alpha, TACE, furin, IL-1beta and matrilysin in UVA- and UVB-irradiated HaCat cells

BACKGROUND/PURPOSE: It is known that solar ultraviolet (UV) irradiation exerts multiple effects on mammalian skin tissues, one of which is the induction of local and systemic immunosuppression as well as inflammation. Tumor necrosis factor-alpha (TNF-alpha) and other cytokines are suggested to play a role in these responses. Quantitative real-time polymerase chain reaction (TaqMan RTPCR) was used to elucidate the effect of UVA and UVB irradiation on the expression of genes coding for TNF-alpha, IL-1beta, IL-10, FasL, matrilysin, TACE and furin in HaCaT cells over a 48 h period (IL-1beta, interleukin-1beta; FasL, Fas ligand). METHODS: Cultured HaCaT cells were either sham irradiated (control) or exposed to UVA (2000 and 8000 J/m2) or UVB (200 and 2000 J/m2) radiation. RNA was extracted from cells at 0, 4, 8, 12, 16, 24, 48 h post-irradiation and reverse transcribed to generate cDNA for subsequent real-time PCR amplification. RESULTS: Significant increases in the mRNA levels for all genes tested were detected in both UVA- and UVB-irradiated HaCaT cells compared with control (sham-irradiated) cells. TNF-alpha mRNA levels were immediately up-regulated (0 h) after irradiation, with maximal induction at 8 h post 2000 J/m2 UVA and 200 J/m2 UVB irradiation, at 4 h post 8000 J UVA irradiation and at 48 h post 2000 J/m2 UVB irradiation. No correlation was observed between TNF-alpha, TACE and furin mRNA induction in the different irradiated cohorts. CONCLUSION: Results suggest that time-distinct gene induction of TNF-alpha, furin, IL-1beta and matrilysin may be involved in UV-induced cellular responses, but not for TACE. In general, mRNA induction was dose dependent at some time points post-irradiation, but not throughout the whole time course tested. Our results show that quantitative real-time PCR is a useful tool in the analysis of quantitative changes of mRNA levels in cultured HaCaT cells after UV exposure.     

Increased expression of Fas on human epidermal cells after in vivo exposure to single-dose ultraviolet (UV) B or long-wave UVA radiation

BACKGROUND: Apoptosis has been proposed to act as an important mechanism for eliminating keratinocytes that have been irreversibly damaged by ultraviolet (UV) irradiation. One way to induce apoptosis in keratinocytes is through activation of the cell surface receptor Fas (CD95), either with the ligand (FasL) or directly with UV radiation. OBJECTIVES: To investigate the regulation of Fas and FasL expression in human skin and the formation of apoptotic cells after in vivo exposure to UVB or long-wave UVA radiation. METHODS: Volunteers were irradiated with either 3 minimal erythema doses (MED) of UVB (n = 6) or 3 MED of long-wave UVA (n = 6) on buttock skin 12, 24 and 72 h before skin punch biopsies were taken. Expression of Fas and FasL was demonstrated by immunohistochemistry on cryostat sections. Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labelling reaction. RESULTS: In five of six subjects, exposure to UVB radiation resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 24 and 72 h after irradiation. In all subjects, exposure to long-wave UVA resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 12 h after irradiation. In five of six subjects, exposure to UVB radiation resulted in temporarily decreased expression of FasL, but after 72 h the expression of FasL had returned to the preirradiation level. The expression of FasL on epidermal cells after exposure to long-wave UVA showed considerable variation. UVB irradiation was a stronger inducer of epidermal apoptosis than was UVA irradiation. The number of apoptotic epidermal cells did not correlate with expression of Fas or FasL. CONCLUSIONS: In human skin the expression of Fas on epidermal cells increases after in vivo exposure to UVB or long-wave UVA. Exposure to UVB causes a temporary decrease in the expression of FasL on epidermal cells.     

Changes of comedonal cytokines and sebum secretion after UV irradiation in acne patients

The aims of this study were to investigate the effect of UVA and UVB on comedones and sebum secretion in acne patients. Thirteen acne patients were irradiated by UVA (starting from 20J/cm2 and increasing 10% every day) and UVB (starting from 2/3 MED and increasing 10% every day). IL-1alpha, IL-6, IL-1 receptor antagonist (IL-1ra), IL-10 and GM-CSF were measured by ELISA. Measurement of sebum level was also performed. Sebum level was increased in the first three days by UVA (18.4 --> 37.6 microg/cm2) and UVB (19.1 --> 40.0 microg/cm2), but subsequently returned to normal values. Production of IL-1alpha, IL-1ra, IL-6, and IL-10 was generally higher on day 5 than on day 10. GM-CSF was not detected from all comedones. After UV irradiation, clinically stationary acne patients showed a higher increase in cytokine production compared with improved acne patients. It is suggested that IL-10 & IL-1ra have key roles in this cytokine network as the anti-inflammatory comedonal cytokines. They may play important roles in the immuno-regulation, which may be disturbed in stationary acne patients.     

Ultraviolet B radiation-induced production of interleukin 1α and interleukin 6 in a human squamous carcinoma cell line is wavelength-dependent and can be inhibited by pharmacological agents

Ultraviolet (UV) B irradiation induces keratinocytes to produce among others the proinflammatory cytokines interleukin (IL) 1 and IL-6. The wavelength dependence of this UVB effect has not yet been assessed. We evaluated the potential of different UVB wavelength regions to release cytokines from the squamous carcinoma cell line SCL II and also assessed the effect of various putative inhibitors. Confluent monolayers of the cells were irradiated with 0.5–2.0 mJ/cm² UVB at 280, 290, 300, 310 or 320 (each ± 5) nm. In additional experiments dexamethasone (10^?9–10^?5 mol/L), ascorbic acid, d-α-tocopherol or indomethacin (each 10^?7–10^?4 mol/L) were added to the culture medium 24 h before, immediately after or combined before and after irradiation with 1 mJ/cm² UVB at 280 ± 5 nm. Supernatants of the cell cultures were recovered at 24 h after irradiation, and IL-1α or IL-6 were determined by an enzyme-linked immunosorbent assay (ELISA). IL-1α and IL-6 production were induced by UVB at 280, 290 and 300 nm, the production depended on the UV dose and decreased with increasing wavelengths. Irradiation at 310 or 320 nm did not induce cytokine production up to the maximum dose used. The production of IL-1 α/IL-6 was inhibited up to 80/89% (10^?7–10^?6 mol/L before and after irradiation) by dexamethasone in a concentration-dependent manner and with all conditions of incubation. Release of cytokines was also suppressed by indomethacin, d-α-tocopherol or ascorbic acid, but concentration dependence was not always evident. These results show that particularly shorter UVB radiation, which is expected to increase due to stratospheric ozone depletion, induces prominent production of proinflammatory cytokines, indicating major biological effects. Different pharmacological compounds can interfere with this effect and seem worth further evaluation with regard to their clinical effects.     

Contrasting effects of an ultraviolet B and an ultraviolet A tanning lamp on interleukin-6, tumour necrosis factor-α and intercellular adhesion molecule-1 expression

BACKGROUND: Recent studies have demonstrated that a tanning lamp emitting predominantly ultraviolet (UV) A induces significant yields of the type of potentially mutagenic DNA damage that are associated with the onset of skin cancer (i.e. cyclobutane pyrimidine dimers). UV-induced immunosuppression is also an important event leading to skin cancer. OBJECTIVES: To the modulation of key immunological molecules following exposure to a broad-spectrum UVB lamp and a predominantly UVA-emitting tanning lamp using model in vitro systems. METHODS: We compared secretion and mRNA expression of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in normal human epidermal keratinocytes, and interferon (IFN)-gamma-induced intracellular adhesion molecule (ICAM)-1 in normal human fibroblasts irradiated in vitro with a broad-spectrum UVB lamp or with a Philips 'Performance' tanning lamp. RESULTS: With broad-spectrum UVB irradiation, upregulation of IL-6 and TNF-alpha mRNA was detected 6 h after irradiation, and a dose-dependent increase of cytokines in the supernatants of irradiated cells was found 24 h after irradiation. In contrast, there was no cytokine secretion and little evidence for mRNA upregulation following exposure to a tanning lamp. When cells were exposed first to broad-spectrum UVB, then the tanning lamp, UVB-induced cytokine secretion was inhibited, although mRNA levels were upregulated to a level close to that observed with UVB alone. By using a Schott WG 320 nm filter to attenuate the level of UVB relative to UVA emitted by the tanning lamp, the inhibition of cytokine secretion was shown to be associated with UVA exposure. Both UV sources inhibited IFN-gamma-induced ICAM-1 mRNA expression in a dose-dependent fashion. By using a Schott WG 335 nm filter, inhibition of ICAM-1 mRNA expression by the tanning lamp was shown to be associated with UVB exposure. CONCLUSIONS: These results suggest that UV sources emitting different levels of UVA and UVB have differential effects on the modulation of different immunoregulatory molecules, and indicate that there are potential interactions between these wavelengths.     

Inhibition of the elicitation phase of contact hypersensitivity by thymidine dinucleotides is in part mediated by increased expression of interleukin-10 in human keratinocytes

The production of immunomodulatory cytokines such as interleukin-10 (IL-10) from keratinocytes and other target cells in the skin plays a crucial role in UV-induced immunosuppression. Substantial evidence supports an association between DNA damage and immunomodulation. It is also known that small DNA fragments such as thymidine dinucleotides (pTpT) can mimic several UV-induced effects, including inhibition of the induction phase of the contact hypersensitivity response and up-regulation of tumor necrosis factor-alpha (TNF-alpha). To determine whether pTpT also induces IL-10 secretion by keratinocytes, and by inference whether IL-10 production after UV irradiation is a response to DNA damage, we compared the effects of pTpT with those of UV irradiation on primary human keratinocyte cultures. Subconfluent cultures of primary human keratinocytes were treated either with 10 micro M or 100 micro M pTpT or diluent alone, or exposed to solar-simulated light (100 J/m2 of UVB) or sham irradiated. An increase in IL-10 mRNA expression was observed 6-24 h after irradiation and at 24-48 h after treatment with pTpT. Detection of secreted IL-10 protein coincided with up-regulation of IL-10 gene expression at 48 and 72 h as determined by ELISA. Conditioned media from human keratinocytes treated with pTpT, like that from irradiated cells, significantly inhibited lymphocyte proliferation in the allogeneic-mixed lymphocyte reaction (MLR) assay. To determine whether pTpT mimics the suppressive influence of UVB on the elicitation phase of contact hypersensitivity, believed to result largely from IL-10 release, we compared the effects of topical application of pTpT with those of UVB irradiation on C57Bl/6 mice sensitized with dinitrofluorobenzene. Sensitized mice treated with pTpT or UVB irradiation showed markedly suppressed elicitation of ear-swelling responses. These results demonstrate that increased keratinocyte IL-10 mRNA level and IL-10 protein release are among the effects of pTpT and support the hypothesis that pTpT treatment triggers many of the biologic effects of UV irradiation by mimicking UV-induced DNA damage. Finally, regardless of mechanism, the data suggest that topical treatment with pTpT may provide a novel means of suppressing contact hypersensitivity or other lymphocyte-mediated reactions in skin.