Type I IL-1 receptor mediates IL-1 and intracellular IL-1 receptor antagonist effects in skin inflammation

The IL-1 system plays a key role in skin physiology and pathology. In this study, we used mutant mice lacking the type I IL-1 receptor (IL-1RI), lacking IL-1 receptor antagonist (IL-1Ra), or overexpressing the human intracellular (ic) IL-1Ra1 isoform, as well as combinations thereof, to dissect the role of the IL-1 system in phorbol 13-myristate 12-acetate (PMA)-induced skin inflammation. In wild-type (WT) mice, PMA application induced epidermal thickening and dermal inflammation. Skin IL-1alpha production and circulating levels of the acute-phase protein serum amyloid A (SAA) were elevated. In mice lacking IL-1RI or overexpressing icIL-1Ra1, PMA induced similar epidermal thickening as in WT mice, but dermal inflammation was partially prevented. Skin IL-1alpha mRNA expression was similar in PMA-treated IL-1RI-/- and WT mice, whereas the increase in serum SAA was suppressed in IL-1RI-/- mice. Interestingly, PMA-induced IL-1alpha mRNA expression was further enhanced by icIL-1Ra1 overexpression in an IL-1RI-dependent manner. Finally, IL-1Ra-/- mice spontaneously displayed skin lesions characterized by high IL-1beta, but not IL-1alpha, expression. In conclusion, PMA-induced epidermal thickening and skin IL-1alpha expression were independent of IL-1 signaling, in contrast to dermal inflammation and systemic inflammatory response.     

Novel functions of intracellular IL-1ra in human dermal fibroblasts: implications in the pathogenesis of fibrosis

Intracellular IL-1 receptor antagonist (icIL-1ra) is reportedly involved in functions independent of blocking IL-1 receptor signaling. Fibroblasts derived from the involved skin of patients with systemic sclerosis (SSc) are predominantly of the myofibroblast phenotype, with higher levels of icIL-1ra compared to normal skin fibroblasts. We examined the effect of overexpression of icIL-1ra on the phenotype and function of normal fibroblasts with respect to the expression of alpha smooth muscle actin (alpha-SMA), a specific marker for myofibroblasts, and plasminogen activator inhibitor (PAI), a protein involved in fibrogenesis and expressed at higher levels in myofibroblasts, and the production of collagenase (matrix metalloproteinase-1 (MMP-1)), the major enzyme involved in the degradation of native collagen in the skin. Normal human foreskin fibroblasts overexpressing icIL-1ra showed higher levels of alpha-SMA and PAI and had lower levels of collagenase and MMP-1 mRNA induced by inflammatory cytokines. By contrast, levels of mRNA for tissue inhibitor of metalloproteinase-1 in the transfected cells were not different from the control cells. Pretreatment of the ic-IL-1ra-transfected cells with antisense oligonucleotide directed against the mRNA of icIL-1ra restored MMP-1 expression induced by stimulation with IL-1beta. Our data indicate novel functions for icIL-1ra, which might be relevant to the genesis of fibrotic diseases such as SSc.     

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。     

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。     

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。     

尖锐湿疣组织中TGFβ1,TGFβRⅡ及Cyclin E的表达

目的研究转化生长因子β1(TGFβ1)、转化生长因子βⅡ型受体(TGFβRⅡ)和细胞周期素E(Cyclin E)蛋白在尖锐湿疣(CA)组织中的表达。方法采用免疫组织化学SP法和图像分析系统检测30例CA患者皮损、15例正常人皮肤(包皮)组织中TGFβ1,TGFβRⅡ及Cyclin E表达及分布。结果(1)TGFβ1,CyclinE在CA组织中的表达(分别为0.26380±0.01965,0.29554±0.01650)均显著高于正常皮肤组织(分别为0.20088±0.03815,0.24330±0.06417,P<0.05);TGFβRⅡ在CA组织中的表达(0.09730±0.01760)显著低于正常皮肤组织(0.23407±0.04883,P<0.05);(2)TGFβ1与Cyclin E在CA组织中的表达呈正相关(P<0.05);而TGFβRⅡ与Cyclin E在CA组织中的表达呈负相关(P<0.05)。结论CA组织中TGFβ1表达上调,而TGFβRⅡ表达下调,使TGFβ信号传导通路受阻,致TGFβ1缺乏对细胞增殖负性调节作用,导致Cyclin E过表达使细胞周期调控异常,细胞增殖。     

In vitro mechanism(s) of ultraviolet-induced tumor necrosis factor-α release in a human keratinocyte cell line

It has been demonstrated that ultraviolet (UV) irradiation is able to induce both in vivo and in vitro, tumor necrosis factor-α (TNF) release. The purpose of the present study was to evaluate, using a human keratinocyte cell line NCTC 2544, the mechanism(s) of UV-induced TNF release and the ability of commonly used sunscreens to modulate UV-induced TNF release. TNF release can be partially prevented both by adding an anti-human IL-1α antibody after irradiation, suggesting an autocrine effect of IL-1α in inducing TNF release, and by adding antioxidants indicating also a role of oxidant species. TPCK, a Iκ-Bα protease inhibitor, was able to virtually abolish UV-induced TNF release, indicating that UV-induced TNF release requires NF-κB activation. Anti-human IL-1β antibody was ineffective as expected, considering that keratinocytes are unable to process pre-IL-1β to its active form. To evaluate the sunscreen's modulation on UV-induced TNF release, confluent cells were irradiated, in the presence or absence of the tested sunscreens (Uvinul MS40, Uvinul P25 and Uvinul DS49). Different IC₅₀ values could be calculated, which may be related to different UV absorption spectrums: Uvinul MS40 offers great protection by virtue of its broader absorption spectrum, closely followed by Uvinul P25 and finally by Uvinul DS49.     

Interleukin-1 receptor antagonist production by human keratinocytes.

Human keratinocytes produce biologically active pro-IL-1 alpha and inactive pro-IL-1 beta with most protein remaining intracellular. IL-1 receptor antagonist (IL-1ra) is a newly described member of the IL-1 family that is secreted by stimulated monocytes and binds competitively to IL-1 receptors without stimulating target cells. We examined the characteristics of IL-1ra production by cultured human keratinocytes. By ELISA, keratinocyte lysates contained 390 ng IL-1ra/mg total protein with little IL-1ra detected in supernatants. In contrast, monocytes produced 297 ng IL-1ra/mg total protein during 24 h of culture on adherent IgG with about half of the IL-1ra detected in supernatants. By Western blot analysis, keratinocyte IL-1ra was approximately 20 kD in size and was slightly larger than recombinant monocyte IL-1ra. In contrast to monocytes, human keratinocyte IL-1ra was not secreted in 22-25-kD molecular weight glycosylated forms. Affinity-purified keratinocyte IL-1ra exhibited identical biologic activity to recombinant monocyte IL-1ra, each inhibiting IL-1-dependent augmentation of murine thymocyte proliferation to the same degree per amount of protein. An IL-1ra mRNA of 1.8 kb was detected by Northern blot analysis in RNA extracted from keratinocytes. In order to determine the effect of differentiation on IL-1 and IL-1ra production, human keratinocytes were cultured for 72 h in low (0.03 mM), medium (0.15 mM), or high (1.0 mM)-calcium concentrations. The absolute amounts of IL-1ra increased twofold and the ratio of IL-1ra to IL-1 alpha in keratinocyte lysates increased from approximately 12:1 to 25:1 during differentiation. These results indicate that keratinocytes constitutively produce large amounts of a biologically active intracellular variant of IL-1ra that increase with differentiation. IL-1ra released during keratinocyte damage may be important in modifying the inflammatory effects of IL-1 alpha in human skin.     

miR-31靶向抑制IL1RN促进HaCaT细胞中IL-1β的分泌

目的研究miR-31/IL1RN途径在银屑病发生发展中的功能作用。方法分析miR-31在银屑病皮损组织及其对照正常组织中的表达差异,然后在HaCaT细胞中研究miR-31表达变化对IL-1Ra蛋白表达的影响,双荧光素酶实验验证miR-31与IL1RN mRNA 3′UTR的靶向结合关系,最后检测miR-31和IL1RN基因对炎症因子IL-1β分泌的影响。结果 miR-31在银屑病皮损组织中的表达高于非皮损组织,而体外细胞实验显示,miR-31过表达可抑制细胞中IL-1Ra蛋白表达,干扰miR-31则可上调细胞中IL-1Ra蛋白表达。双荧光素酶实验结果表明,miR-31可靶向结合IL1RN的3′UTR区。功能实验结果显示,miR-31过表达可促进IL-1β的分泌,而IL1RN过表达则可下调IL-1β的分泌,并可削弱miR-31对IL-1β分泌的促进作用。结论 miR-31通过靶向IL1RN的3′UTR区域抑制IL-1Ra的表达,促进IL-1β分泌,从而参与银屑病的进展。     

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.     

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.     

银屑病皮损中IL—1受体拮抗物的蛋白和mRNA表达

采用抗人ＩＬ－１ｒａ单抗和地高辛标记的ＲＮＡ探针,在银屑病破损和正常人皮肤切片上进行ＡＰＡＡＰ染色和原位杂交,检测并比较ＩＬ－Ｉｒａ的蛋白表达和ｍＲＮＡ转录水平。结果发现,正常人皮肤中ＩＬ－１ｒａ蛋白染色阴性,ＩＬ－１ｒａｍＲＮＡｗ信号微弱;相比之下,银屑病皮损中ＩＬ－１ｒａ蛋白和ｍＲＮＡ的表达均异常增高,统计学比较相差显著。上述结果显示：ＩＬ－１ｒａ在银屑病皮损中增强表达,提示银屑病病理过程中Ｉ     

Effects of Etretinate on Keratinocyte Proliferation and Secretion of Interleukin-1α (IL-1α) and IL-8

Etretinate has proven to be effective in the treatment of psoriasis. Since abnormal proliferation and cytokine secretion are well-known features of psoriatic epidermis, we studied the in vitro effects of etretinate on these two processes using human keratinocytes. Etretinate promoted proliferation of normal human keratinocytes (NHKs) grown in keratinocyte growth medium (KGM) but not in growth factor-deficient keratinocyte basic medium (KBM). Moreover, etretinate partly overcame growth inhibition by PMA. Etretinate was shown to have an effect on either IL-1α or IL-8 secretion in unstimulated NHKs. In HSC-1, a human squamous cell carcinoma cell line cultured in 20% FCS/DMEM, inhibited IL-1α secretion and enhanced IL-8 secretion. These results indicate that the effects of etretinate on keratinocyte proliferation and cytokine secretion may depend on cell type and culture conditions. Stimulation of NHKs with PMA significantly enhanced IL-1α and IL-8 secretion, and these effects were inhibited by etretinate. However, etretinate failed to inhibit rTNFα-induced IL-8 secretion, suggesting that etretinate regulation of NHK cytokine secretion may also depend on the stimulus. As treatment of keratinocytes or epidermis with PMA can induce psoriasis-like changes, so might the experimental “anti-PMA” activity of etretinate be related to its therapeutic benefit in the treatment of psoriasis.