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.     

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.     

Effects of low concentrations of cis- and trans-urocanic acid on cytokine elaboration by keratinocytes

The urocanic acid cis isomer (cis-UCA) is a possible cutaneous photoreceptor for the immunomodulatory phenomena that follow ultraviolet B irradiation. Several experiments in animals show an inhibitory action of cis-UCA on cellular immunity. However, the action of cis-UCA on the synthesis of cytokines in keratinocytes remains unknown. Long-term cultures of normal human keratinocytes were prepared in a serum-free medium, and stimulated with 1 μg/ml of phorbol 12-myristate 13-acetate (TPA) and UCA or UVB-UCA (10–100 μg/ml). Synthesis of the following cytokines was measured using ELISA and Northern blot techniques: TNF-α, IL-1α, IL-1β, IL-6, IL-8 and TGF-β1. TPA increased TNF-α protein levels in culture supernatants. No changes in IL-1α and IL-1β protein levels were detected in basal culture supernatant after TPA stimulus. TPA augmented RNA expression for TNF-α, IL-1α, IL-1β and TGF-β1. UCA isomers did not induce cytokine changes in protein synthesis. Expression of IL-1α and IL-1β genes was increased after exposure to 100 μg/ml UVB-UCA (70 μg/ml cis-UCA). A slight increase in TNF-α RNA expression was detected when the dose of UVB-UCA reached 100 μg/ml. No effects on cytokine synthesis were found after UCA stimulus. These results suggest that low doses of cis-UCA do not effect cytokine synthesis by keratinocytes.     

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.     

Effects of interleukins, colony-stimulating factor and tumour necrosis factor on human hair follicle growth in vitro: a possible role for interleukin-1 and tumour necrosis factor-α in alopecia areata

The immune system may be involved in the regulation of normal hair follicle growth as well as in the pathogenesis of some hair diseases. Immunomodulatory cytokines not only act as mediators of immunity and inflammation but also regulate cell proliferation and differentiation and. as such, may play an important part in regulating hair growth. We have investigated the effects of a number of interleukins (IL). colony stimulating factors and tumour necrosis factors (TNF) on hair follicle growth in vitro. Dose-response studies showed that IL-1α. IL-1ß and TNF-o were potent inhibitors of hair follicle growth. The histology of hair follicles maintained with inhibitory doses of IL-1α. IL-1ß and TNF-α showed similar changes in hair follicle morphology, resulting in the formation of dystrophic anagen hair follicles. These changes in histology were characterized by the condensation and distortion of the dermal papilla, marked vacuolation of the hair follicle matrix, abnormal keratinization of the follicle bulb and inner root sheath, disruption of follicular melanocytes and the presence of melanin granules witbin the dermal papilla. Moreover, these changes in hair follicle morphology are similar to those reported in alopecia areata and suggest that IL-1α, IL-1ß and TNF-α may play an important part in the pathophysiology of inflammatory hair disease.     

An increased ratio of interleukin-1 receptor antagonist to interleukin-1α in inflammatory skin diseases

Abstract: IL-1 receptor antagonist (IL-1ra) is a cytokine that competitively binds the IL-1 receptor to antagonize IL-1 activity without any agonist function. Previous experiments indicated that the ratio of IL-1ra to IL-1α in the normal stratum corneum (SC) was much higher in the sunexposed face than in the sun-protected area, upper arms. It was also reported by another laboratory that IL-1ra is increased in the lesional skin of psoriatic patients. This study was designed to measure the contents of IL-1α and IL-1ra in non-lesional and pathological SC obtained from inflammatory skin diseases including psoriasis and non-psoriatic dermatoses such as atopic dermatitis. The SC materials were obtained with a noninvasive tape-stripping method. Their soluble fractions were prepared and assayed for IL-1α and IL-1ra by enzyme-linked immunosorbent assays. As a result we confirmed the previous findings that the ratio of IL-1ra to IL-1α in the normal SC was much higher in the face than in the sun-protected sites, the trunk as well as extremities. Next, we found that IL-1α contents were significantly reduced in the SC samples obtained from inflammatory skin regardless of whether their IL-1ra contents increased or unchanged. Moreover, we noted that an increased ratio of IL-1ra to IL-1α in the SC was not specific to psoriasis, but was also found in other inflammatory skin diseases including atopic dermatitis. This ratio was found to become lower after successful treatment of these skin lesions with topical glucocorticoids. We conclude from these observations that the increased ratio of IL-1ra to IL-1α in the SC is a non-specific phenomenon that can occur in any inflammatory skin diseases regardless of the inflammatory pattern, probably reflecting a skin regulation process against various kinds of inflammation.     

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.     

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。     

Tumour necrosis factor alpha is pro-inflammatory in normal human skin and modulates cutaneous adhesion molecule expression

Tumour necrosis factor a (TNF-α) is a potent immunoregulatory cytokine produced by many cutaneous cells, including kcratinocytes, mast cells and Langerhans cells. To explore its potential role in inflammatory skin disease, we have studied immunohistochemically the effects of intradermal recombinant human TNF-α (rHuTNF-α) on cutaneous inflammatory cells, adhesion molecules and Langerhans cells in normal human skin. Volunteers received rHuTNF-α 100U (group A), 5000 U (group B), or 100 U daily for 5 days (group C), and biopsies were taken at 6 h (groups A and B), or 6 h after the final injection (group C). An inflammatory cell infiltrate developed in all cases: following single injections of either 100 or 5000 U rHuTNF-α this was predominantly neutrophilic, whereas following multiple injections of 100 U few neutrophils were seen, although many lymphocytes (CD3⁺, CD4⁴) were present. In all groups there was an increase in cells of monocyte/macrophage lineage (CD36⁾⁺. TNF-α induced a dose- and time-dependent decrease in CDla⁺ epidermal Langerhans cell numbers and an increase in dermal CDla⁴ cells, suggesting migration of Langerhans cells away from the epidermis. TNF-α induced endothelial E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in all groups, and adhesion molecule expression by interstitial dermal dendritic cells (ICAM-1 and VCAM-1) and keratinocytes (ICAM-1) was observed. These findings indicate that TNF-α is a potent modulator of cutaneous immune function in vivo, and this central role in the cutaneous immune response suggests that TNF-α may be an attractive target for therapeutic inhibition.     

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。     

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.     

Investigating the release of inflammatory cytokines in a human model of incontinence-associated dermatitis

Incontinence-associated dermatitis (IAD) is a painful complication in elderly patients, leading to reduced quality of life. Despite recent attention, its underlying inflammatory mechanisms remain poorly understood. This study was designed to quantify the release of inflammatory cytokines in a human model of IAD. The left volar forearm of ten healthy volunteers was exposed to synthetic urine and synthetic faeces for 2 h, simulating the effects of urinary and faecal incontinence, respectively, and the subsequent cytokine response compared to that of an untreated control site. Inflammatory cytokines were collected using both the Sebutape® absorption method and dermal microdialysis and quantified using immunoassays. Results from the former demonstrated an upregulation in IL-1α, IL-1RA and TNF-α. Synthetic urine caused a higher median increase in IL-1α from baseline compared to synthetic faeces, whereas synthetic faeces were associated with significantly higher median TNF-α levels compared to synthetic urine (p = 0.01). An increase in IL-1α/IL-1RA ratio was also observed with significant differences evident following exposure to synthetic urine (p = 0.047). Additionally, microdialysis revealed a time-dependent increase in IL-1β and IL-8 following exposure of up to 120 min to synthetic urine and synthetic faeces, respectively. This study demonstrated the suitability of both sampling approaches to recover quantifiable cytokine levels in biofluids for the assessment of skin status following exposure to synthetic fluids associated with incontinence. Findings suggest some differences in the inflammatory mechanisms of IAD, depending on moisture source, and the potential of the cytokines, IL-1α and TNF-α, as responsive markers of early skin damage caused by incontinence.     

Effects of prostaglandin E1 on human keratinocytes and dermal fibroblasts: A possible mechanism for the healing of skin ulcers

Abstract The effects of prostaglandin E, (PGE) on cell growth, cytokine production and interaction of cultured normal human keratinocytes (NHKs) and human dermal fibroblasts (HDFs) were investigated. When NHKs were treated with PGE, directly, only a slight increase in cell growth and a transient decrease in interleukin 1 alpha (IL-lα) secretion were observed. No IL-6 was detected either before or after PGE, treatment. In addition, IL-8 and transforming growth factor alpha (TGFα) production were uninfluenced by PGE. The response of HDFs to PGE, differed from that of NHKs. Following PGE₁, treatment, IL-lα and TGFα. from HDFs remained undetectable while IL-6 production was enhanced markedly. IL-8 production was also slightly enhanced. Exposure of HDFs to PGE, for 96 hours significantly promoted cell proliferation. Two kinds of conditioned media (CM) were prepared by a brief feeding of HDFs with keratinocyte basic medium or Dulbecco's modified Eagle's medium supplemented with 5% PCS with or without PGE. NHKs proliferated more rapidly in CM than in corresponding basic medium. Moreover, CM prepared with PGE, treatment showed a stronger effect in promoting NHK proliferation than CM without PGE, treatment. This promoting effect was inhibited by anti-human IL-6 monoclonal antibody dose-dependently. These results indicate that fibroblasts are more sensitive than keratinocytes in response to PGE, and that, upon PGE, stimulation. HDF-derived IL-6 may play an essential role in NHK cell proliferation which may at least partly account for the beneficial effects of PGE, in the treatment of cutaneous liberations.     

Distribution of interleukin 1 receptor antagonist protein (IRAP), interleukin 1 receptor, and interleukin 1α in normal and psoriatic skin. Decreased expression of IRAP in psoriatic lesional epidermis

The distribution of interleukin 1 alpha (IL-1 alpha), type 1 interleukin 1 receptor (IL-1R), and the interleukin 1 receptor antagonist protein (IRAP), was investigated in biopsies of normal skin, and in uninvolved and lesional skin of patients with psoriasis, using specific monoclonal antibodies. We report the novel finding that IRAP is distributed throughout the living layers of the epidermis in normal skin, and is also associated with sebaceous glands and eccrine sweat glands. Our finding that the inhibitor protein IRAP is present in areas where the pro-inflammatory cytokine IL-1 alpha is distributed provides strong evidence in favour of a cytokine regulatory system in normal skin. We further document for the first time that IL-1R in normal skin is localized to the living layers of the epidermis, sebaceous and eccrine glands, as well as to a prominent network of dermal dendritic cells, and upper dermal blood vessels. There was a consistent reduction in the level of IRAP expression in lesional compared with uninvolved skin in biopsies from six out of seven psoriasis patients. Decreased IRAP expression in lesions of psoriasis indicates that alterations in the level of this inhibitor protein may be important in the pathogenesis of inflammatory skin conditions.     

尿道炎后慢性前列腺炎患者精浆中TNF-α及IL-18的检测及临床意义

目的检测尿道炎后慢性前列腺炎患者精浆中肿瘤坏死因子(TNF-α)、白细胞介素18(IL-18)的浓度并探讨其临床意义。方法采用ELISA法测定精浆中IL-18浓度;放射免疫法测定精浆中TNF-α浓度。结果尿道炎后慢性前列腺炎(Ⅱ型、Ⅲ型)患者精浆中TNF-α浓度与对照组比较,差异均有显著性(P<0.01);而在慢性细菌性前列腺炎(Ⅱ型)和慢性非细菌性前列腺炎(Ⅲ型)之间比较,差异无显著性(P>0.05)。尿道炎后慢性前列腺炎(Ⅱ型、Ⅲ型)患者精浆中IL-18浓度与对照组、Ⅱ型和Ⅲ型比较,差异均有显著性(P<0.01)。结论尿道炎后慢性前列腺炎患者精浆中TNF-α、IL-18明显升高,提示TNF-α,IL-18在尿道炎后慢性前列腺炎的发病过程中起了重要的作用。