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.     

Production and Secretion of Platelet-Derived Growth Factor AB by Cultured Human Keratinocytes: Regulatory Effects of Phorbol 12-Myristate 13-Acetate,Etretinate, 1,25-Dihydroxyvitamin D3, and Several Cytokines

Platelet-derived growth factor (PDGF) is a potent mitogen for several mesenchymal cells and plays an important role in wound repair. Three PDGF isoforms, PDGF-AA, PDGF-BB, and PDGF-AB, have been found to be generated in various tissues. PDGF-AB production by normal human keratinocytes (NHKs), by human squamous cell carcinoma cell line (HSC-1) cells, and by human dermal fibroblasts (HDFs) was studied in the presence of agents which influence cell growth. Both NHKs and HSC-1 cells spontaneously produced and secreted PDGF-AB. NHKs grown in keratinocyte growth medium produced more PDGF-AB than did those grown in keratinocyte basic medium. Phorbol 12-myristate 13-acetate inhibited PDGF-AB production in NHKs but promoted its production in HSC-1 cells. 1,25-dihydroxyvitamin D₃ up-regulated PDGF-AB production, whereas etretinate did not. High levels of calcium in the culture medium induced little change in cellular PDGF-AB levels. Prostaglandin E₁ slightly inhibited PDGF-AB production, transforming growth factor β1 promoted PDGF-AB production and interferon-γ, interleukin-1α, and tumor necrosis factor-α failed to exert any influence at all. Cultured HDFs did not produce any detectable PDGF-AB. These results suggest that keratinocytes are a major source of cutaneous PDGF and that this factor may therefore play an important role in wound repair and in certain proliferative skin diseases.     

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.     

Prostaglandin I1 analogues,SM-10902 and SM-10906, affect human keratinocytes and fibroblasts in vitro in a manner similar to PGE1: therapeutic potential for wound healing

The newly synthesized prostaglandin (PG) I₁ analogues, SM-10902 and SM-10906, were compared with PGE₁ in terms of their biological effects on cultured normal human keratinocytes (NHKs) and human dermal fibroblasts (HDFs) in order to evaluate their therapeutic potential for cutaneous wound healing. The PGI₁ analogues had a direct effect on cell proliferation of HDFs as did PGE₁, but inibited cell growth of NHKs in contrast to the stimulatory effect observed with PGE₁. In contrast to NHKs stimulated with PGI₁ analogues, which exhibited low levels of adenosine 3,5-cyclic monophosphate (cAMP). HDFs stimulated with these analogues responded in a dose-dependent manner with extremely high levels of cAMP. Conditioned media (CM) derived from media in which HDFs had been incubated with both the PGI₁ analogues promoted NHK proliferation. HDF production of interleukin (IL)-6 increased in response to the PGI₁ analogues. Since IL-6 was shown to promote cell growth of NHKs, enhancement of NHK proliferation by CM was thought to be due to IL-6 derived from HDFs stimulated with the PGI₁ analogues.     

氮芥对体外培养HaCaT细胞白细胞介素分泌的影响

目的　观察氮芥对体外培养的HaCaT细胞细胞因子分泌的影响。方法　佛泊脂 /脂多糖 (PMA/LPS)刺激HaCaT细胞后培养 ,用ELISA法检测HaCaT细胞培养上清液中细胞因子的含量。结果　HaCaT细胞可自发性分泌IL 1α、IL 2 ,IL 6和IL 8。PMA/LPS刺激HaCaT细胞后 ,IL 1α分泌量下降 ,IL 6和IL 8的分泌量明显增加 ,与银屑病患者皮损中细胞因子的变化非常相似。加入氮芥后HaCaT细胞IL 6和IL 8的分泌量增加 ;在低浓度氮芥作用下IL 1α的分泌量无明显变化 ,高浓度时则可使IL 1α的分泌量培加。对于PMA/LPS刺激的HaCaT细胞 ,低浓度氮芥对其IL 6和IL 8的分泌量均有明显抑制作用 ,而IL 1α的分泌明显增加 ;高浓度氮芥则可使IL 1α、IL 6和IL 8的分泌量增加。结论　氮芥治疗银屑病的机制可能与IL 1α、IL 6和IL 8有关。     

Secretion of IL-2, IL-4, and IFN-γ for Dust Mite Antigens in a Patient with Atopic Dermatitis

In a 21-year-old female with severe atopic dermatitis, the secretion of interleukin-2 (IL-2), interleukin-4 (IL-4), and interferon-γ (IFN-γ) by her peripheral blood mononuclear cells (PBMC) was measured after incubation with/without antigens extracted from Dermatophagoides pteronyssinus (Dp), to which the patient had developed a positive patch-test reaction. Incubation with Dp antigen produced marked secretion of IL-2 and IFN-γ, but not IL-4. This may suggest that Dp-specific T lymphocytes present in the circulating blood cells are capable of producing IL-2 and IFN-γ, which may be relevant to the delayed-type allergic reaction occurring in the skin lesion.     

IL-10 secretion of allergen-specific skin-derived T cells correlates positively with that of the Th2 cytokines IL-4 and IL-5*

Abstract In atopic individuals, allergen-specific CD4+ T lymphocytes often belong to the T-helper 2 (Th2) subset as they secrete the marker cylokincs interleukin-4 (IL-4) and IL-5 but not intcrfcron-y (INF-y). IL-10 is a cytokine the production of which, in the mouse system has been described to be restricted to the Th2 subset, but in the human was found to be produced by both ThI and Th2 T cell clones (TCC). We have recently shown that house dust mite antigen (Dermatophagoides pteron-yssinus)-specific TCC isolated from skin of patients with atopic dermatitis have a more polarized Th2 cytokine production profile than TCC obtained from the peripheral blood of these patients. In this study, we report that skin-derived TCC secrete more IL-10, IL-4 and IL-5, than TCC isolated from the blood of the same individual (p < 0.05). The difference was more significant with specific TCC than with non-specific TCC. Furthermore, there was a positive correlation between the production of IL-10 and that of IL-4 and IL-5, respectively. In addition, the amount of IL-4 and IL-5 secreted by specific TCC from the skin correlated positively. These results were confirmed by the detection of mRNA by PCR. Finally, our data confirm that in human blood-derived TCC IL-10 secretion is not related to a particular cytokine production profile. We suggest that the skin of AD provides an unique environment for the development of aTh2-likc secretion pattern not only with respect to IL-4 and IL-5 but also regarding IL-10.     

Gingko biloba extract reduces VEGF and CXCL-8/IL-8 levels in keratinocytes with cumulative effect with epigallocatechin-3-gallate

In skin inflammation, vascular endothelial growth factor (VEGF) and CXCL-8/IL-8 play an important role and are produced by activated keratinocytes. Extracts from Ginkgo biloba leaves (GBE), widely used in phytotherapy, have been reported to exert antioxidant and anti-inflammatory properties in the skin. We therefore evaluated the effects of GBE on the release of VEGF and CXCL8/IL-8 by normal human keratinocytes (NHKs) activated by tumor necrosis factor α (TNFα). Moreover, as we previously showed that epigallocatechin-3-gallate (EGCG) reduces VEGF and CXCL8/IL-8 secretion in TNFα-activated NHKs, we also tested its effect in association with GBE. Our results showed that GBE exerted a potent inhibition on VEGF and CXCL8/IL-8 levels in activated cells. In association with EGCG, GBE down-regulated VEGF and CXCL8/IL-8 levels in a cumulative manner in TNFα-stimulated NHKs. These results suggest that GBE, alone or in association with EGCG may contribute to moderate inflammatory processes in skin diseases associated with angiogenesis.     

Diversity of immunobiological functions of T-cell lines established from patients with adult T-cell leukaemia

In order to understand the variety of HTLV-1-associated cutaneous diseases, we studied the cytological profile of HTLV-1-infected T-cell lines established from patients with adult T-cell leukaemia (ATL). Among four CD4⁺ cell lines, termed 16T(?), 35T(?), MH-1, and KS-2, the 16T(?) cells secreted elevated quantities of IL-4, IL-b and IFN-7, and expressed mRNA for each cytokine in the absence of exogenous stimulation. The 3ST(?) cells secreted IL-6 and a small amount of IFN-7, but not IL-4. The MH-1 and KS-2 cells secreted only 1L-6 in the absence of stimulation, hi response to stimulation with phorbol-12-myristate-13 acetate (PMA), the 16T(?) cells produced more IL-4 and IFN-γ, whereas the 35T(?) and MH-1 cells exhibited increased secretion of IFN-γ, but still no IL-4 or IL-4 mRNA production. Although neither IL-4 nor IFN-γ were found in the culture supernatant of KS-2 cells, the production of IL-4 mRNA was detected by RT PCR. Culture supernatants from the 16T(?) and 35T(?) cells induced the expression of intercellular adhesion molecule-1 (ICAM-1) and HLA-UR by cultured keratinocytes. This response was inhibited by pretreatment of the supernatant with anti-IFN-γ antibodies. These results indicate that some HTLV-1-infected T-cell lines constitutively secrete various cytokines, including biologically active IFN-γ. The diversity of immunobiological functions of the T-cell lines may be related to the variety of clinical features present in ATL patients.     

Pharmacological modulation of IL-6 and IL-8 secretion by the H1-antagonist decarboethoxy-loratadine and dexamethasone by human mast and basophilic cell lines

Abstract Mast cells and basophils are central effector cells of allergic reactions and are involved in inflammatory diseases. These cell types produce an array of mediators including a broad spectrum of cytokines. In order to examine whether antiallergic drugs modulate the release of these mediators, we have investigated the influence of dexamethasone and decarboethoxy-loratadine (DEL), the active metabolite of the H₁-blocking agent loratadine, on the release of IL-6 and IL-8 by the human mast cell line HMC-1 and the human basophilic cell line KU812 by ELISA. Dexamethasone (10^?6-10^?11 M) or Del (10^?5-10^?14 M) were added to the cells either 1 h prior to or simultaneously with PMA and Ca-ionophore A23187. When preincubated with the cells, DEL dose-dependently suppressed IL-6 release by up to 40% and IL-8 release by up to 50%. Dexamethasone potently suppressed secretion of both cytokines if simultaneously added to the cells with the stimuli by up to 60% and after preincubalion by up to 80%. Since both antihistamines and glucocorticoids are used for treatment of allergic diseases, the findings reported here indicate that these drugs may modulate allergic reactions via inhibition of cytokine release from mast cells and basophils.     

In vitro Comparative Study of the Antitumor Effects of Human Interferon-α, β and γ on the Growth and Invasive Potential of Human Melanoma Cells

We have studied the effects of interferon (IFN)-α, β, and γ in vitro on the growth and invasive potential of human melanoma SK-MEL-118 cells. The antiproliferative effects of IFNs were assessed by a quantitative regrowth assay in which cells were treated with IFNs at concentrations of 10², 10³ or 10⁴ IU/ml for 3 days (until day 4) and then further incubated without IFNs for 7 days (until day 11). The growth inhibitory effect of each IFN on melanoma cells was dose- and time-dependent. Among these three types of IFNs, however, IFN-β exerted the strongest inhibitory effect on cell growth. To assess the anti-invasive effect of each IFN on melanoma cells, we employed an in vitro assay system using matrigel-coated Transwell chambers. When cells were treated with 10², 10³, or 10⁴ IU/ml of the three types of IFNs for 24 hours, the amount of tritiated thymidine incorporated into cells generally increased, indicating that cell growth was not inhibited by the pretreatment When melanoma cells were treated for 24 hours with 10⁴ IU/ml of IFN-β or γ prior to the assay, the number of cells that invaded the filter decreased by 40%; this decrease was only 10% with the same amount of IFN-α. Simultaneous addition of IFNs during the invasion assay was not effective in any combination. Only when the cells were pretreated with IFNs, antiinvasive effects against melanoma cells were exerted. IFN-a was less inhibitory than IFN-β or γ on proliferation and not at all inhibitory on invasion. Considering both the antiproliferative and antiinvasive effects of IFNs, our results suggest that IFN-β has the strongest antitumoral effect on human melanoma cells. IFN-γ had a relatively strong inhibitory effect, especially on invasion.     

Cytokine Production of Peripheral Blood Mononuclear Cells in a Dermatophytosis Patient in Response to Stimulation with Trichophytin

Interferon-γ (IFN-γ), interleukin 2 (IL-2) and granulocyte/macrophage colony-stimulating factor (GM-CSF) were detected in the culture supernatant after 72 hours incubation with trichophytin in the peripheral blood mononuclear cells (PBMC) obtained from a patient who had a dermatophyte infection. These findings indicate that this patient has peripheral T-lymphocytes that produced IFN-γ, IL-2 and GM-CSF, which may play roles in the development of delayed-type hypersensitivity (DTH) reaction in the skin.     

Comparative analysis of CD80 and CD86 on human Langerhans cells: expression and function

Abstract Although both CD80 (B7–1) and CD86 (B7–2/B70) have been recently identified in cultured human Langerhans cells (LC), little is known of the role and regulatory properties of CD80 and CD86 on human LC. We present here the results of a study comparing the expression and function of CD80 and CD86 in human LC using the T-helper type-1 cytokines IL-2 and interferon γ (IFN)-γ, and the T-helper type-2 cytokines IL-10, IL-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Freshly isolated human LC expressed little CD80 and CD86 in vitro, but the expression of both molecules was rapidly induced during a 72-h incubation with cytokines and the expression of CD86 occurred much earlier and more strongly than that of CD80. The expression of both CD80 and CD86 was upregulated by GM-CSF and downregulated by IL-10, and the expression of CD86, but not that of CD80, was upregulated by both IL-4 and IFN-γ. Finally, pretreatment of LC with GM-CSF and IFN-γ, but not with IL-4, enhanced the alloreactive T-cell proliferation induced by the LC, and IL-10 pretreatment of LC decreased their capacity for alloreaction. These results indicate that the expression of both CD80 and CD86 on human LC may be regulated by these cytokines (IL-2, IL-4, GM-CSF, IFN-γ and IL-10) secreted from helper T cells infiltrating into the inflammatory microenvironment. Received: 4 December 1997     

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.     

Various peroxisome proliferator‐activated receptor (PPAR)‐γ agonists differently induce differentiation of cultured human keratinocytes

Peroxisome proliferator‐activated receptors (PPARs) are potentially useful for the treatment of skin diseases, because they stimulate keratinocyte differentiation, exert anti‐inflammatory effects and improve barrier function. We examined five PPAR‐γ agonists, including four thiazolidinediones (ciglitazone, troglitazone, rosiglitazone and pioglitazone) and an angiotensin‐II receptor blocker (telmisartan), for their ability to upregulate filaggrin and loricrin expression at both mRNA and protein levels in cultured normal human keratinocytes (NHKs). Troglitazone, rosiglitazone, pioglitazone and telmisartan significantly increased filaggrin expression at both mRNA and protein levels in calcium‐induced differentiated NHKs. Rosiglitazone and pioglitazone, but not troglitazone nor telmisartan, also significantly increased loricrin expression at both mRNA and protein levels in differentiated NHKs. These effects were not found in undifferentiated NHKs nor differentiated NHKs treated with ciglitazone. This study revealed differential effects of various PPAR‐γ agonists on epidermal differentiation, and the most potent of those are rosiglitazone and pioglitazone.     

SLE患者T淋巴细胞IL-13受体α1基因表达及其调节序列甲基化状态的研究

目的 研究SLE患者外周血T淋巴细胞IL-13受体α1（IL-13Rα1）基因mRNA的表达及IL-13Rα1基因调节序列甲基化状态。方法 免疫磁珠法（MACS）分离10例SLE患者和6例正常人外周血CD4+和CD8+ T细胞，采用实时荧光定量PCR检测T细胞中IL-13Rα1 mRNA的表达，并用甲基化特异性PCR（MSP）方法检测IL-13Rα1基因调节序列的甲基化水平。结果 活动期SLE患者CD4+ T细胞中IL-13Rα1 mRNA表达水平为2.224 ± 0.251，非活动期SLE患者为1.712 ± 0.132，正常人组为1.104 ± 0.044，三组间比较，差异均有统计学意义（P < 0.05）；CD8+ T细胞中IL-13Rα1 mRNA表达水平活动期、非活动期及正常人组分别为1.672 ± 0.142，1.410 ± 0.154，1.238 ± 0.106，活动期组与正常人组比较差异有统计学意义（P < 0.05），而非活动期组与正常人组、活动期组与非活动期组比较，差异均无统计学意义（P > 0.05）。CD4+ T细胞中IL-13Rα1基因甲基化指数活动期SLE患者为0.454 ± 0.023，非活动期为0.635 ± 0.065，正常人为0.844 ± 0.097，三组间比较，差异均有统计学意义（P < 0.05）；CD8+ T细胞中IL-13Rα1基因甲基化指数三组间比较，差异均无统计学意义（P > 0.05）。SLE患者外周血CD4+，CD8+ T细胞IL-13Rα1 mRNA的表达与疾病活动度（SLEDAI评分）呈正相关（r = 0.79，P < 0.01；r = 0.76，P < 0.05）；CD4+ T细胞的IL-13Rα1基因的甲基化水平与疾病活动度（SLEDAI评分）呈负相关（r = -0.89，P < 0.01）；CD4+ T细胞IL-13Rα1 mRNA表达与其调节序列的甲基化水平呈负相关（r = -0.84，P < 0.01）。结论 SLE的发生发展可能与DNA低甲基化导致SLE患者T细胞过度表达IL-13Rα1有关。     

Genetic association of IFN-γ +874T/A polymorphism in Mexican patients with drug-induced Stevens-Johnson syndrome/toxic epidermal necrolysis

Stevens–Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) are rare, but potentially life-threatening diseases, characterized by widespread epidermal necrosis and are predominantly drug induced. There is a paucity of data regarding the role of cytokine and cytokine receptors polymorphisms in the pathoimmunology of SJS/TEN. The aim of this study was to investigate the role of TNF-α-308, IFN-γ +874, IL-10-1082, IL-13 Arg130Gln, and IL-4R Gln551Arg gene polymorphisms in SJS/TEN in Mexican Mestizo patients. Twenty-nine unrelated SJS/TEN patients and 128 unrelated healthy individuals were studied. Genomic extraction was carried out from complete blood samples using the salting out method. The PCR–RFLP method was used to amplify the following polymorphisms: TNF-α-308, IFN-γ +874, IL-10-1082, IL-13 Arg130Gln, and IL-4R Gln551Arg. TNF-α-308, IL-10-1082, IL-13 Arg130Gln, and IL-4R Gln551Arg polymorphisms were not associated with the genetic susceptibility to SJS/TEN. The distribution of TT, TA, AA genotypes of IFN-γ +874 was significantly different in SJS/TEN patients compared with controls (pC = 0.012). TA and AA genotypes were grouped to highlight the differences between patients and controls given by the absence of the AA genotype in the group of patients (pC = 0.03, OR = 3.61 95 % CI 1.20–11.6). This preliminary study suggests that IFN-γ +874 T/A polymorphism is associated with SJS/TEN.     

Production of multiple cytokines by cultured human melanomas*

Abstract We screened a panel of 8 primary and 21 metastatic melanoma cell lines for constitutive secretion of cytokines. Melanomas expressed bioactivity for TGF-β (8/25 lines) and IFN (7/12), but not IL-2. Immu-noassays detected IL-1α (4/25), IL-1β (12/25), 1L-6 (13/29), IL-8 (29/ 29), TGF-β₂, (5/12) and GM-CSF (11 /29). but not IL-3, IL-4, TNF-α, or IFN-γ. There was no preferential association of cytokine production with cells cultured from primary versus metastatic disease, and only IL-8 was produced by all lines tested. These data demonstrate that cultured melanomas produce a variety of cytokines which are potentially capable of influencing tumor growth in vivo.