Barrier function coordinately regulates epidermal IL-1 and IL-1 receptor antagonist mRNA levels

Abstract Disruption of the cutaneous permeability barrier increases mRNA levels for TNF, GM-CSF, FL-1 alpha, and IL-1 beta in the epidermis. We have hypothesized that the cylokines mediate the changes in lipid and DNA synthesis which occur following barrier disruption. To further characterize the cytokine response to barrier abrogation, we examined the levels of epidermal IL-Ira mRNA in two acute models and one chronic model in the hairless mouse. IL-Ira mRNA levels increased shortly after acute disruption of the barrier with acetone, reached a peak at 3–4 h after treatment, and returned to control levels by 8h. These changes in mRNA levels parallel those which occur for IL-1 alpha and beta. Furthermore, IL-Ira mRNA levels were elevated 5-fold and 4-fold, at 2.5 h and 4 h, respectively, following tape-stripping, a second acute model of barrier disruption. Finally, IL-Ira mRNA levels were elevated 2.5-fold in the epidermis of EFAD mice, which have a chronic barrier defect. Thus, the cutaneous response to barrier disruption includes mechanisms which increase IL-I and IL-Ira mRNA levels in a coordinate manner. The net result provides a regulatory mechanism for controlling the biological effects of increased IL-1 production.     

Alterations in cytokine regulation in aged epidermis: implications for permeability barrier homeostasis and inflammation. I. IL-1 gene family

Acute disruption of the cutaneous permeability barrier with either solvents or tape-stripping stimulates a homeostatic metabolic response in the subjacent nucleated layers of the epidermis that results in a rapid restoration of normal permeability barrier function. When the aged epidermal permeability barrier is stressed, it reveals a diminished capacity for recovery, in comparison to young epidermis, analogous to other organs in the aged when stressed. Although the signals that regulate this homeostatic response by the epidermis have not yet been resolved, acute permeability barrier disruption stimulates release of prestored IL-1alpha, and increased production of potentially regulatory cytokines, including IL-1alpha and TNFalpha in the epidermis. In these studies, we addressed the hypothesis that cytokine dysregulation explains the permeability barrier abnormality in aged epidermis, assessing the regulation of IL-1 and TNF signaling in aged vs young mice. To determine whether the IL-1 family of cytokines plays a key role in the permeability barrier abnormality of the aged, permeability barrier recovery rates were compared in transgenic mice lacking the functional IL-1 type 1 receptor vs wild-type mice at various ages. Knockout of the IL-1 type 1 receptor exacerbates the defect in permeability barrier homeostasis that is seen in age-matched, wild-type counterparts. Furthermore, the sluggish permeability barrier recovery in aged epidermis is associated with, and at least in part attributable to, altered expression of the IL-1 family of cytokines and receptors both under basal conditions and after acute barrier perturbations. Whereas modulations in cytokine expression with epidermal permeability barrier perturbation are qualitatively similar in aged epidermis, they greatly differ quantitatively. In contrast, examination of TNFalpha mRNA and protein basally, and following barrier perturbation revealed no alterations in aged epidermis. Together, these results show that selective alterations in the IL-1 family of cytokines occur with aging and that defects in IL-1 signaling may contribute to the epidermal permeability barrier abnormality of aged 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.     

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.     

Chemotaxis of freshly separated and cultured human keratinocytes

It is generally accepted that keratinocyte migration plays a critical role in the process of wound healing. A study was therefore made of the migratory response of freshly separated and cultured human keratinocytes to factors with chemotactic properties for a variety of cells. Interleukin-lα (IL-lα), interferon-γ (IFN-γ), interleukin-8 (IL-8), and tumour necrosis factor α (TNF-α) were tested for their chemotactic effectiveness in a modified Boydcn chamber assay. IFN-γ, IL-lα and IL-8 were demonstrated to serve as chemoattractants for freshly separated keratinocytes. For cultured cells, however, only IFN-γ was found to display chemotactic properties. The findings demonstrate that there is significant difference between the chemotactic behaviour of freshly separated and cultured cells.     

Serum levels of interleukin-8, tumor necrosis factor-α and γ-interferon in Egyptian psoriatic patients and correlation with disease severity

Psoriatic plaques have been shown to contain increased levels of pro-inflammatory cytokines. Also, serum levels of several cytokines have been reported elevated in psoriatic patients. It is postulated that changes in cytokine production both locally and systemically could be useful in monitoring disease activity. The aim of this study was to evaluate serum cytokine profile of interleukin (IL)-8, γ-interferon (IFN-γ) and tumor necrosis factor-α (TNF-α) in Egyptian psoriatic patients by enzyme-linked immunosorbent assay (ELISA) technique and to correlate these levels with disease severity. We analyzed serum samples from 60 Egyptian patients (31 females and 29 males) with a mean age of 40.2 ± 17.4 years with active psoriasis, and 21 healthy volunteers for major T-helper type 1 cytokines using the ELISA technique. The disease severity, including erythema, induration and scales, was assessed by Psoriasis Area and Severity Index (PASI) score. TNF-α and IFN-γ were markedly elevated in all sera from psoriatic patients. TNF-α was found a more efficient predictor for disease severity than IL-8 and IFN-γ using three receiver-operator curves with accuracy. IL-8 was also moderately elevated and correlated with the age of patients (r = 0.28). We have obtained evidence that TNF-α in our study was found to be more useful than the other two tested cytokines, IL-8 and IFN-γ as a follow-up marker for monitoring disease severity in Egyptian psoriatic patients. A positive correlation between lL-8 and the age of the patients was also noted.     

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.     

进行性斑状色素减少症发病机制初步探讨

目的探讨进行性斑状色素减少症(progressive macular hypomelanosis,PMH)患者皮损中分离出的痤疮丙酸杆菌对人角质形成细胞(human keratinocytes,HaCaT)细胞分泌干扰素(interferon,IFN)-γ、白细胞介素(interleukin,IL)-6、肿瘤坏死因子(tumor necrosis factor,TNF)-α的影响。方法体外培养HaCaT细胞株,分别加入分离自PMH的痤疮丙酸杆菌、痤疮丙酸杆菌标准菌株(NCTC737),酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)方法测定细胞上清液中IFN-γ、IL-6、TNF-α的分泌量。结果 PMH患者中分离的痤疮丙酸杆菌与角质形成细胞HaCaT细胞株共培养后,能促进角质形成细胞分泌IFN-γ和IL-6,与空白组比较,有明显升高,差异有统计学意义(P0.05);与痤疮丙酸杆菌标准菌株NCTC737相比也升高,差异有统计学意义(P0.05)。结论进行性斑状色素减少症皮损中分离出的痤疮丙酸杆菌可刺激HaCaT细胞分泌IFN-γ、IL-6、TNF-α,提示可能是皮损色素减退的原因之一。     

银屑病患者外周血单个核细胞Th_1/Th_2细胞因子表达研究

目的　探讨Th1/Th2 细胞因子在银屑病发病机理中的作用。方法　应用逆转录 聚合酶链反应法 (RT PCR)半定量分析 19例正常对照和 2 2例银屑病患者外周血单个核细胞中TNFα、IFN γ和IL 4、IL 10mRNA的表达。结果　银屑病患者TNFα和IFN γ表达显著上调 (P <0 .0 1) ,而IL 4显著降低 (P <0 .0 1) ,IL 10较正常降低但无显著差异(P >0 .0 5 )。结论　Th1型细胞因子在银屑病患者中过度表达 ,可能提示银屑病免疫病理机理中的T淋巴细胞活化模式。     

颅内接种热灭活隐球菌对小鼠隐球菌脑膜脑炎作用的研究

目的 研究脑内接种热灭活的隐球菌(H-CN)在中枢神经系统隐球菌感染中的作用及对小鼠脑和脾白介素-1β(IL-1β)、干扰素γ(IFN-γ)和肿瘤坏死因子α(TNF-α)基因表达的影响。方法 建立脑内接种隐球菌造成中枢神经系统感染的小鼠模型,分实验组(给予2次脑内接种H-CN)、对照组(给予2次脑内接种生理盐水),分别取2组小鼠的脑和脾用RT-PCR方法检测IL-1β、IFN-γ及TNF-α的mRNA表达,然后给予2组小鼠脑内接种致死量的隐球菌,观察2组小鼠的生存时间及脑组织中菌落形成单位的变化。结果 实验组与对照组相比小鼠生存时间显著延长,小鼠脑组织中隐球菌菌落形成单位明显减少。小鼠脑组织总RNA以IL-1β、IFN-γ、TNF-α引物进行的RT-PCR产物,实验组IL-1β表达阳性,IFN-γ、TNF-α均表达阴性;对照组IL-1β、IFN-γ、TNF-α均表达阴性。小鼠脾组织总RNA以IL-1β、IFN-γ、TNF-α引物进行的RT-PCR产物2组均表达阳性,差异无显著性。结论 给予小鼠脑内接种H-CN可提高中枢神经系统对隐球菌感染的抵抗力,中枢神经系统抗隐球菌机制很可能与H-CN引起局部细胞因子IL-1β的分泌有关。     

Cytokine and chemokine ligand expression in cutaneous lupus erythematosus

There is increasing evidence that cytokines as well as chemokines are important players in the pathogenesis of lupus erythematosus (LE). We aimed to compare cytokine and chemokine profiles in different types of cutaneous LE. We investigated lesional mRNA and protein expression of various cytokines and chemokines in patients with chronic discoid LE (CDLE, n=15), subacute cutaneous LE (SCLE, n=11), and lupus erythematosus tumidus (LET, n=21). TNF-α, INF-γ, TGF-β, IL-6, IL-10, IL-12p40, CXCL9, and CXCL10 mRNA expression were significantly increased in SCLE when compared to CDLE. Moreover, LET also showed significantly increased mRNA expression of TNF-α, TGF-β, IL-10, IL-12p40 and CXCL9, as compared to CDLE. In all LE subtypes, CXCL9 and CXCL10 mRNA expression significantly correlated with INF-γ mRNA expression, as indicated by r-values ranging from 0.71 - 0.87. Immunohistochemistry for TNF-α, INF-γ, and IL-10 gave support to our RT-PCR results. In conclusion, our results suggest that T helper 1, as well as T helper 2 cytokines are differentially expressed in CDLE, SCLE, and LET. Compared to CDLE, the highest cytokine and chemokine ligand profiles are found in SCLE followed by LET. Our correlation studies also support the importance of an IFN-driven inflammation in cutaneous LE.     

RANTES expression in psoriatic skin, and regulation of RANTES and IL-8 production in cultured epidermal keratinocytes by active vitamin D3 (tacalcitol)

The chemokine RANTES is a chemoattractant for eosinophils, T lymphocytes of memory phenotype and monocytes, suggesting that it plays an important part in chronic inflammatory and allergic diseases. In various types of cells, RANTES production is markedly induced by tumour necrosis factor (TNF)-α and interferon (IFN)-γ in combination. Psoriasis vulgaris is a chronic cutaneous inflammatory disease. Cytokines and chemokines produced by T cells and epidermal keratinocytes, such as interleukin (IL) 8, are involved in the pathogenesis of psoriasis. T-cell clones obtained from psoriatic skin have been shown to produce the Th1 cytokine IFN-γ. In addition, abnormal expression of proinflammatory cytokines including TNF-α has been observed in psoriatic lesions. These reports led us to hypothesis that psoriatic skin could provide epidermal keratinocytes with TNF-α and IFN-γ, so that keratinocytes could produce RANTES. In this study, we addressed the question as to whether RANTES was involved in psoriasis vulgaris. Immunohistochemistry of skin biopsies showed RANTES was present in the intercellular spaces between epidermal keratinocytes, in the fully developed lesions from the middle to the edge of psoriatic plaques, but not in the perilesional uninvolved and healthy control skin. Further, we confirmed the production of RANTES, together with IL-8, by cultured normal human epidermal keratinocytes, using an enzyme-linked immunosorbent assay. Stimulation with TNF-α and IFN-γ in combination synergistically increased the RANTES production in this system. These results clearly demonstrate the expression of RANTES in psoriatic lesions and suggest the involvement of this chemokine in the outcome of cutaneous inflammatory diseases. Tacalcitol (1α,24(R)-dihydroxyvitamin D₃), an active vitamin D₃ analogue, inhibited RANTES and IL-8 production in cultured normal epidermal keratinocytes. This result indicates that active vitamin D₃ is effective in the regulation of chemokine production by epidermal keratinocytes, which may partly account for its action as an antipsoriatic drug.     

天疱疮患者血清细胞因子的检测及其与病情的相关性

目的 研究天疱疮患者血清细胞因子的动态变化及其与病情严重程度的相关性.方法 用双抗体夹心ELISA方法检测31例急性发病期和治疗后缓解的天疱疮患者以及15例正常人对照血清白介素4(IL-4)、白介素10(IL-10)、γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)和可溶性IL-2受体(sIL-2R)水平,并以间接免疫荧光法检测患者血清抗体滴度,同时用烧伤面积九分法记录皮损面积.结果 天疱疮患者急性发病期IL-10水平低于正常人(P<0.05),且与皮损面积呈负相关(r=-0.25,P<0.05);sIL-2R和IFN-γ水平高于正常人(P<0.05),且与皮损面积呈正相关(分别为r=0.60,P<0.05;r=0.41,P<0.05);天疱疮抗体滴度与皮损面积无相关性(P>0.05).结论 天疱疮发病中存在细胞因子的异常,IL-10、sIL-2R和IFN-γ水平与病情严重程度有明显的相关性,其水平的检测可用于监控疾病的严重程度并指导临床治疗.