Peoniflorin suppresses tumor necrosis factor-α induced chemokine production in human dermal microvascular endothelial cells by blocking nuclear factor-κB and ERK pathway

Peoniflorin (PF) extracted from the root of Paeonia lactiflora pall displays anti-inflammation and antioxidant properties in several animal models. Chemokines are vital for directing the movement of circulating leukocytes to the sites of inflammation and are involved in the pathogenesis of various inflammatory skin diseases. Herein, we investigated the effects and potential mechanisms of PF on tumor necrosis factor-α (TNF-α) induced chemokine production in human dermal microvascular endothelial cells. Human dermal microvascular endothelial cell line (HMEC-1) was treated by TNF-α with or without PF. PF markedly attenuated TNF-α-induced chemokines (including CCL2, CCL5, CCL20, CXCL8, CXCL16 and CX3CL1) mRNA expression in HMEC-1. PF also reduced the secretion of these chemokines in culture supernatants. In addition, endothelial activation in the presence of PF markedly blocked the chemotactic activities of TNF-α-stimulated HMEC-1 supernatant on promyelocytic leukemia cell line (HL-60) or the acute mature monocytic leukemia cell line (THP-1) cell migration. Furthermore, Western blot data revealed TNF-α upregulated phosphorylation of inhibitor of κB-α (IκBα) and phosphorylation of extracellular signal-regulated kinase (ERK)1/2, which was almost completely reversed by PF. Finally, PF inhibited nuclear factor-κB (NF-κB) nuclear translocation to the nucleus. Taken together, our data provide the first evidence that PF has an anti-inflammatory ability against TNF-α-induced chemokine production and leukocyte migration, which may be at least partly related to the inhibition of NF-κB and ERK pathway. PF may be a candidate medicine for the treatment of inflammatory skin diseases.     

Co-culture of healthy human keratinocytes and T-cells promotes keratinocyte chemokine production and RORγt-positive IL-17 producing T-cell populations

BackgroundBoth keratinocytes and T-cells are crucial players in cutaneous immune responses. We hypothesized that direct interactions between keratinocytes and T-cell subsets could shape the nature or strength of the local immune response.ObjectiveWe investigated direct interactions between keratinocytes and T-cell subsets, focused on keratinocyte chemokine production and T-cell phenotype and cytokine production.MethodsA newly developed in vitro serum free co-culture model using primary keratinocytes and T-cells subsets from healthy human donors was used. Keratinocyte chemokine production was analyzed with luminex, T-cell phenotype and cytokine production were analyzed with flow cytometry.ResultsOur data show that upon co-culture with CD4^pos or CD8^pos T-cells primary human keratinocytes increased production of functionally active chemokines CCL2, CCL20 and CXCL10 and that regulatory T-cells did not regulate keratinocyte chemokine production. Next to that, we found that keratinocytes skewed CD4^pos and CD8^pos T-cell populations toward an IL-17^pos CCR6^pos RORγt^pos phenotype in a cell–cell contact independent manner, and that Treg were able to decrease the absolute number of IL-17 producing T-cells in keratinocyte/T-cell co-cultures. Correspondingly, freshly isolated skin-derived T-cell populations contained relatively high percentages of IL-17^pos cells.ConclusionWe provide evidence that keratinocyte/T-cell communication may regulate leukocyte influx in the skin, and that keratinocytes enrich T-cell populations for Th17/Tc17 cells. Accumulation of Th17/Tc17 cells, but not chemokine production, appears under the control of regulatory T-cells. Dysregulation of these processes may well contribute to the pathophysiology of inflammatory skin diseases.     

Comparative effects of polyphenols from green tea (EGCG) and soybean (genistein) on VEGF and IL-8 release from normal human keratinocytes stimulated with the proinflammatory cytokine TNFα

In skin inflammation, vascular endothelial growth factor (VEGF) and IL-8 play an important role and are produced by activated keratinocytes. Recently, some polyphenols have been reported to exhibit antiinflammatory and antiangiogenic properties. We therefore evaluated the effects of green tea, its major component epigallocatechin-3-gallate (EGCG) and an isoflavone derived from soybean (genistein) on the release of VEGF and IL-8 by activated normal human keratinocytes (NHK). NHK cultured in defined medium were stimulated for 48 h with the proinflammatory cytokine TNF with the addition or not of different concentrations of polyphenols. Levels of VEGF and IL-8 were measured in cell supernatants by enzyme-linked immunosorbent assays. The different constituents tested inhibited keratinocyte proliferation without inducing apoptosis. They reduced in a dose-dependent manner the basal release and the upregulation of VEGF in NHK. Green tea and EGCG were also potent inhibitors of IL-8 release by TNF-stimulated NHK, whereas genistein exerted only minor effects. These results underline the divergent pathways involved in the downregulation of VEGF and IL-8 by polyphenols in activated keratinocytes. They also suggest that polyphenols may contribute to moderate inflammatory processes in skin diseases associated with angiogenesis.     

Elevated levels of tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-10 in hidradenitis suppurativa skin: a rationale for targeting TNF-α and IL-1β

Background The pathogenesis of hidradenitis suppurativa (HS) is largely unknown and the disease is difficult to treat. Patients are in high need of an effective treatment. Although it is not known whether the levels of tumour necrosis factor (TNF)‐α are aberrant in HS skin, anti‐TNF‐α biologics are used, with variable clinical efficacy. Objectives To determine the cytokine profile in lesional and perilesional HS skin. Methods We cultured 20 lesional and 10 normal‐appearing perilesional HS skin samples, seven psoriasis and six healthy control skin samples in a transwell culture system. Two distinct cytokine bead arrays were used to measure the spectrum of inflammatory cytokines in the culture supernatant. Results from HS skin samples were compared with those of healthy and psoriasis skin. Results The proinflammatory cytokines interleukin (IL)‐1β and TNF‐α as well as the anti‐inflammatory cytokine IL‐10 were significantly elevated in HS skin. Elevated levels of these cytokines were also found in perilesional HS skin. Fold increases relative to control skin of IL‐1β, TNF‐α and IL‐10 in HS were 31, 5 and 34, compared with psoriasis: 4, 1 and 2, respectively. Levels of all three cytokines showed a trend towards a positive correlation with disease severity. IL‐2, IL‐4, IL‐5 and interferon‐γ were hardly detectable in HS or healthy control skin. Conclusions This study shows for the first time that IL‐1β, TNF‐α and IL‐10 levels are elevated in HS skin. These data provide a rationale for therapies with biologics targeting cytokines such as TNF‐α and IL‐1.     

Tumor necrosis factor alpha (TNFα) in human skin: a comparison of different antibodies for immunohistochemistry

Abstract Conflicting results have been reported regarding the localization and presence of TNFα in normal human skin. To study TNFα expression, we tested a panel of antibodies directed against human TNFα. First, antibodies were tested for immunoreactivity on cytospots of isolated LPS-stimulated peripheral blood mononuclear cells. Second, antibodies were tested to detect recombinant TNFα in Western blots. Some antibodies were found to be unable to detect recombinant TNFα in the blots. However, most antibodies were able to bind TNFα protein, but they did not bind to other irrelevant proteins that were also present in the blots. Finally, antibodies were tested on cryosections of normal human skin. Antibodies that did not react with TNFα in the blots were incubated with TNFα before the staining procedure to see whether these antibodies specifically bound TNFα. We found that, although all the antibodies bound TNFα, there were clear differences in staining patterns. This indicates that these antibodies may recognize distinct epitopes or different forms of TNFα. The differences found in this study and those reported previously could be the result of differences in the concentration of antibody used, the staining procedure or specificity of the antibody itself. So, for unambiguous interpretation of data, it is important to know the characteristics of the antibodies used. Received: 10 August 2000 / Revised: 25 October 2000 / Accepted: 1 February 2001     

Adalimumab (antitumour necrosis factor-α) treatment of hidradenitis suppurativa ameliorates skin inflammation: an in situ and ex vivo study

Background Hidradenitis suppurativa (HS) is a difficult‐to‐manage disease. Randomized controlled trials with antitumour necrosis factor (TNF)‐α biologics have been conducted and in most studies disease activity was reduced. However, the mechanism of action in HS skin is so far unknown. Objectives To assess whether anti‐TNF‐α treatment affects in situ cytokine production and frequency of inflammatory cell populations in HS lesional skin. Methods Nine patients with HS, participating in a larger placebo‐controlled, double‐blind phase IIb clinical trial on the efficacy and safety of adalimumab in patients with moderate to severe HS (M10‐467), were randomized and treated for 16 weeks. In a mechanism‐of‐action substudy, biopsies were obtained at fixed time points pre‐ and post‐treatment. One part of the biopsy was cultured for 24 h for cytokine release in the culture medium, while another part was used for in situ analysis. Results Secretion of cytokines, including interleukin (IL)‐1β, CXCL9 [monokine induced by interferon‐γ (MIG)], IL‐10, IL‐11, B‐lymphocyte chemoattractant (BLC) and IL‐17A, was significantly elevated in HS. Adalimumab treatment was associated with decreased production of cytokines in HS skin, especially IL‐1β, CXCL9 (MIG) and BLC. Treatment significantly reduced the number of CD11c+, CD14+ and CD68+ cells in HS lesional skin. The numbers of CD3+ and CD4+ T cells, and CD20+ and CD138+ B cells were also reduced by adalimumab treatment. Conclusions Adalimumab treatment inhibits important cytokines and inflammatory cell numbers in lesional HS skin, especially levels of IL‐1β and numbers of inflammatory CD11c+ dendritic cells.     

Influence of an anti-integrin α6 monoclonal antibody on the in vitro infection of human HaCaT keratinocytes with human papillomavirus (HPV) 6/11 virus particles

Objective To investigate the influence of an anti-integrin α6 monoclonal antibody (GoH3) on the in vitro infection of a human keratinocyte cell line HaCaT with HPV6/11 virus particles (VP). Methods HaCaT cells were infected in vitro with 4 different concentrations of HPV6/11 VP alone, HPV6/11 VP of 106 copies/ml after incubation with 6 different dilutions of GoH3, or 8 clinical isolates of HPV6/11 VP of 106 copies/ml before or after the incubation with 1∶ 100 dilution of GoH3. After additional culture, the infected HaCaT cells were collected and fluorescence quantitative (FQ)-PCR was performed to detect the HPV DNA load in cells. The inhibition rate of CoH3 on the infection was calculated. Results The viral load was different among the HaCaT cells infected with different concentrations of HPV6/11 VP (P < 0.01). The inhibition rate on the infection positively correlated with the concentration of CoH3 when the dilution was more than 1∶ 100; however, when the dilution was less than 1∶ 100, the increase in CoH3 concentration had no influence on the inhibition rate. The average viral load in HaCaT cells infected with clinical isolates of HPV6/11 VP was (5.81 ± 2.51) × 104 copies/ml in the absence of GoH3, (3.02 ± 1.21) × 104 copies/ml with the presence of CoH3, and the average inhibition rate of GoH3 was (46.9 ± 4.7)%. Conclusions GoH3 could partially suppress the adhesion of HPV6/11 VP to HaCaT cells, hinting that integrin a6 is an important HPV6/11 VP receptor on host cells.     

Influence of an anti-integrin α6 monoclonal antibody on the in vitro infection of human HaCaT keratinocytes with human papillomavirus (HPV) 6/11 virus particles

Objective To investigate the influence of an anti-integrin α6 monoclonal antibody (GoH3) on the in vitro infection of a human keratinocyte cell line HaCaT with HPV6/11 virus particles (VP). Methods HaCaT cells were infected in vitro with 4 different concentrations of HPV6/11 VP alone, HPV6/11 VP of 106 copies/ml after incubation with 6 different dilutions of GoH3, or 8 clinical isolates of HPV6/11 VP of 106 copies/ml before or after the incubation with 1∶ 100 dilution of GoH3. After additional culture, the infected HaCaT cells were collected and fluorescence quantitative (FQ)-PCR was performed to detect the HPV DNA load in cells. The inhibition rate of CoH3 on the infection was calculated. Results The viral load was different among the HaCaT cells infected with different concentrations of HPV6/11 VP (P < 0.01). The inhibition rate on the infection positively correlated with the concentration of CoH3 when the dilution was more than 1∶ 100; however, when the dilution was less than 1∶ 100, the increase in CoH3 concentration had no influence on the inhibition rate. The average viral load in HaCaT cells infected with clinical isolates of HPV6/11 VP was (5.81 ± 2.51) × 104 copies/ml in the absence of GoH3, (3.02 ± 1.21) × 104 copies/ml with the presence of CoH3, and the average inhibition rate of GoH3 was (46.9 ± 4.7)%. Conclusions GoH3 could partially suppress the adhesion of HPV6/11 VP to HaCaT cells, hinting that integrin a6 is an important HPV6/11 VP receptor on host cells.     

Influence of an anti-integrin α6 monoclonal antibody on the in vitro infection of human HaCaT keratinocytes with human papillomavirus (HPV) 6/11 virus particles

Objective To investigate the influence of an anti-integrin α6 monoclonal antibody (GoH3) on the in vitro infection of a human keratinocyte cell line HaCaT with HPV6/11 virus particles (VP). Methods HaCaT cells were infected in vitro with 4 different concentrations of HPV6/11 VP alone, HPV6/11 VP of 106 copies/ml after incubation with 6 different dilutions of GoH3, or 8 clinical isolates of HPV6/11 VP of 106 copies/ml before or after the incubation with 1∶ 100 dilution of GoH3. After additional culture, the infected HaCaT cells were collected and fluorescence quantitative (FQ)-PCR was performed to detect the HPV DNA load in cells. The inhibition rate of CoH3 on the infection was calculated. Results The viral load was different among the HaCaT cells infected with different concentrations of HPV6/11 VP (P < 0.01). The inhibition rate on the infection positively correlated with the concentration of CoH3 when the dilution was more than 1∶ 100; however, when the dilution was less than 1∶ 100, the increase in CoH3 concentration had no influence on the inhibition rate. The average viral load in HaCaT cells infected with clinical isolates of HPV6/11 VP was (5.81 ± 2.51) × 104 copies/ml in the absence of GoH3, (3.02 ± 1.21) × 104 copies/ml with the presence of CoH3, and the average inhibition rate of GoH3 was (46.9 ± 4.7)%. Conclusions GoH3 could partially suppress the adhesion of HPV6/11 VP to HaCaT cells, hinting that integrin a6 is an important HPV6/11 VP receptor on host cells.     

Subcutaneous administration of polymerized type I collagen downregulates interleukin (IL)-17A, IL-22 and transforming growth factor-β1 expression, and increases Foxp3-expressing cells in localized scleroderma

Background. Localized scleroderma (LS) is a disfiguring inflammatory autoimmune disease of the skin and underlying tissue. As in systemic sclerosis, a key feature is the presence of T cells in inflammatory lesions. Aim. To evaluate the effect of polymerized type I collagen vs. methylprednisolone (MP) in LS, and to determine the influence of this polymerized collagen (PC) on CD4+ peripheral T cells expressing interleukin (IL)‐4, IL‐17A, interferon‐γ and Forkhead box protein (Foxp)3, and on cells expressing transforming growth factor (TGF)‐β1, IL‐17A, IL‐22 and Foxp3 in the skin. Methods. In total, 16 patients with LS were treated for 3 months with monthly subcutaneous intralesional injections of 0.1 mL MP (giving a total dose of 20 mg/mL each month) and 15 patients were treated, with weekly subcutaneous intralesional injections of PC, ranging from 0.2 mL (equivalent to 1.66 mg collagen) for a lesion of 50 mm in size, up to a maximum of 1.0 mL (8.3 mg collagen) for a lesion > 100 mm in size, and followed up for a further 6 months. Skin biopsies were obtained from lesions at baseline (before treatment) and 9 months later (6 months after treatment end). Tissue sections were evaluated by histology and immunohistochemistry (IL‐17A, IL‐22, TGF‐β1 and Foxp3). CD4+ T‐cell subsets were determined in peripheral blood by flow cytometry. Results. Abnormal tissue architecture was seen in the biopsies taken from patients treated with MP, whereas the PC treatment restored normal skin architecture. PC downregulated pro‐inflammatory/profibrotic cytokine expression in peripheral cells, and upregulated the number of regulatory T cells (Tregs) in skin. PC was safe and well tolerated. Conclusions. PC is not only an antifibrotic/fibrolytic agent but also an immunomodulator biodrug that restores the balance between T helper (Th)1, Th2, Th17 and Tregs, downregulates production of pro‐inflammatory or profibrogenic cytokines (IL‐17A, IL‐22 and TGF‐β1), and renews skin architecture, without adverse effects.     

白细胞介素(IL)-12/23、IL-17和IL-23抑制剂治疗中度至重度斑块状银屑病的疗效和安全性的系统评价

目的:对白细胞介素(IL)抑制剂治疗中度至重度斑块状银屑病的疗效和安全性进行系统评价,为临床选用银屑病治疗药物提供参考依据.方法:通过计算机检索 PubMed、Cochrane Library、EMbase、Web of Science、CNKI、WanFang Data 和 CBM数据库2000年1月至2019年9月...     

Expression of vitamin D3 25-hydroxylase (CYP27) mRNA after induction by vitamin D3 or UVB radiation in keratinocytes of human skin equivalents – a preliminary study

Abstract The presence of both 1α-hydroxylase activity and 25-hydroxylase activity is a biochemical prerequisite for the enzymatic generation of 1α,25(OH)₂D₃ from vitamin D₃ in keratinocytes. In contrast to 1α-hydroxylase, however, activity of a 25-hydroxylase in keratinocytes has not previously been demonstrated. In this study, using RT-PCR we detected vitamin D₃ 25-hydroxylase mRNA in human keratinocytes. The expression of vitamin D₃ 25-hydroxylase mRNA in keratinocytes was identical to that of CYP27 expressed in mitochondria of human hepatoma HepG2 cells. CYP27 mRNA in keratinocytes is not constitutively expressed but is inducible both by vitamin D₃ (780 nM) and by UVB radiation (300 nm, 30 mJ/cm²). Received: 11 March 1999 / Received after revision: 28 May 1999 / Accepted: 11 June 1999