Paeoniflorin suppresses vascular damage and the expression of E‐selectin and ICAM‐1 in a mouse model of cutaneous Arthus reaction

Paeoniflorin (PF) extracted from the root of Paeonia lactiflora pall, displays anti‐inflammation properties in several animal models. Adhesion molecules are important for the recruitment of leucocyte to the vessel wall and involved in the pathogenesis of various autoimmune and inflammatory diseases. Herein, we investigate the effects of PF on adhesion molecule expression in a mouse model of cutaneous Arthus reaction and cultured human dermal microvascular endothelial cells (HDMECs). We showed that PF significantly ameliorated the immune complex (IC) induced vascular damage, leucocyte infiltrates and adhesion molecules expression. Furthermore, PF markedly blocked tumor necrosis factor‐α (TNF‐α)‐induced E‐selectin and intercellular adhesion molecule‐1 (ICAM‐1) expression in HDMECs at both mRNA and protein levels. PF also suppressed TNF‐α‐induced adhesion of polymorphonuclear leucocytes (PMNs) to HDMECs. Finally, western blot data revealed that PF can inhibit the phosphorylation of p38, JNK in TNF‐α‐treated HDMECs. These data suggest that PF, as an anti‐inflammatory agent, can downregulate adhesion molecules expression. PF may be a candidate medicine for the treatment of IC‐induced inflammatory response.     

Immature mast cells exhibit rolling and adhesion to endothelial cells and subsequent diapedesis triggered by E‐ and P‐selectin,VCAM‐1 and PECAM‐1

Please cite this paper as: Immature mast cells exhibit rolling and adhesion to endothelial cells and subsequent diapedesis triggered by E‐ and P‐selectin, VCAM‐1 and PECAM‐1. Experimental Dermatology 2010; 19: 424–434. Abstract: Mast cell numbers are markedly increased at sites of chronic inflammation. However, the underlying mechanisms of mast cell accumulation including mast cell progenitor trafficking remain to be identified in detail. Thus, the aim of this study was to identify the adhesion molecules involved in rolling, firm adhesion and transendothelial diapedesis of murine bone marrow‐derived cultured mast cells (BMCMC) as a model for immature mast cells. We could show that BMCMCs exhibit in vivo rolling on skin vessel walls and strong adhesion to skin endothelial cells (ECs) in vitro under static and flow conditions. Interestingly, interaction of BMCMC with the EC adhesion molecules E‐ and P‐selectin, vascular cell adhesion molecule‐1 (VCAM‐1) and platelet endothelial cell adhesion molecule‐1 (PECAM‐1) is required to mediate rolling and firm adhesion to ECs. The adhesion of BMCMCs to skin ECs is further enhanced by TNF, IL‐4, IL‐15 and vascular endothelial cell growth factor. Furthermore, BMCMCs exhibit directed and dose‐dependent transmigration across an endothelial barrier, mediated by a PECAM‐1‐dependent mechanism. Our results demonstrate that BMCMCs can undergo a tightly regulated extravasation cascade consisting of rolling on and adhesion to endothelium and followed by directed diapedesis and reveal selectins, VCAM‐1 and PECAM‐1 as required adhesion molecules. These processes may contribute to mast cell accumulation in chronic inflammatory skin diseases and reveal opportunities to modulate peripheral tissue numbers of mast cells.     

Cytotoxic T‐lymphocyte‐associated protein 4 expressed by melanoma cells does not affect melanoma‐specific cytotoxic T lymphocytes in the effector phase

Cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) is one of the important molecules that regulate the anti‐melanoma T‐cell response. Currently, there are some reports showing that CTLA‐4 is expressed not only by T cells but also by various kinds of tumor cells, including melanoma cells. However, there is no report that shows the role of CTLA‐4 expressed by melanoma cells in melanoma‐specific cytotoxic T‐lymphocyte (CTL) response. In this report, we confirmed substantial CTLA‐4 expression and the localization of CTLA‐4 in melanoma cell lines and tissues. Also, we examined its impact on melanoma‐specific CTL in vitro, and found that CTLA‐4 expressed by melanoma cells does not affect melanoma‐specific CTL in the effector phase. Our findings suggest the importance of elucidating the role of CTLA‐4 expressed by melanoma cells, particularly in anti‐CTLA‐4 antibody therapy.     

内皮素1对人A375黑素瘤细胞株细胞黏附及细胞黏附分子1表达的影响

目的 探讨内皮素（ET）－1对人A375黑素瘤细胞增殖、黏附、迁移及细胞黏附分子（ICAM）－1表达的影响。方法 MTT法观察ET－1对人A375黑素瘤细胞生长的影响,黏附实验检测细胞黏附,Transwell迁移系统检测细胞迁移,流式细胞仪检测对人A375黑素瘤细胞ICAM－1表达的影响。结果 ET-1在0.002 ～ 0.2 μg/mL浓度范围可促进黑素瘤细胞的增殖、在纤连蛋白上的黏附、通过微孔滤膜及抑制ICAM－1的表达（P < 0.01）,在0.2 μg/mL时作用最强,细胞代谢活性（24 h）、细胞黏附率、ICAM-1含量（24 h）分别为0.327 ± 0.009、163.31 ± 4.05、4.667 ± 0.551;在0.2 ～ 2 μg/mL浓度范围作用相反。结论 ET-1可能通过抑制ICAM-1的表达、促进细胞增殖、黏附及迁移而增强细胞代谢、增加色素形成。     

Comparison of changes in endothelial adhesion molecule expression following UVB irradiation of skin and a human dermal microvascular cell line (HMEC-1)

We have assessed the pattern of dermal endothelial adhesion molecule expression following broadband UVB irradiation in vivo and in vitro. Skin biopsies were taken from 4 human volunteers at baseline and at 4, 8 and 24 h post-irradiation with 2.5 minimal erythema doses of UVB. Sections were stained immunohistochemically for E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD31 and neutrophil elastase. The effect of direct UVB irradiation on E-selectin, ICAM-1 and VCAM-1 was examined in a human dermal microvascular endothelial cell line, HMEC-1. Cultured HMEC-1 were irradiated with 2.5–40 mJ/cm² of UVB, and assessed for adhesion molecule expression by immunofluorescence microscopy and fluorescence-activated cell sorter analysis. In vivo, E-selectin was minimally expressed on EC at baseline and was induced by 4 h following irradiation, P<0.01. ICAM-1 was moderately expressed at baseline and appeared mildly induced at 24 h, although this did not reach statistical significance. VCAM-1 was weakly expressed in unirradiated skin while CD31 was moderately expressed, but neither was induced by UVB irradiation. A significant neutrophilic infiltrate appeared by 8 h and was maximal at 24 h, P<0.05. Neutrophil infiltration correlated with E-selectin expression, r=0.96. In HMEC-1, ICAM-1 was upregulated at 24 h post-irradiation, with an increase in mean channel fluorescence from 100% at baseline to 145 (SD12)%> at 24 h, P<0.05. No change was seen in expression of E-selectin, VCAM-1 or CD31. These studies support the involvement of endothelial adhesion molecules E-selectin and ICAM-1 in UVB-induced inflammation. Whereas ICAM-1 is upregulated by direct irradiation of endothelial cells, E-selectin stimulation appears to be an indirect effect.     

Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes

Please cite this paper as: Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes. Experimental Dermatology 2010; 19 : e44–e49. Abstract: Toll‐like receptors (TLRs) on epidermal keratinocytes are the first line of defense against microbe invasion, and matrix metalloproteases (MMPs) regulate inflammation, cell migration and wound healing. In this study, we demonstrate that the mRNA and protein expressions of MMP‐1 and MMP‐9 in human epidermal keratinocytes are induced by ligands for TLR2, TLR3 and TLR5 [Pam3CSK4, Poly(I:C) and flagellin, respectively] in a dose‐dependent manner. We also found that the ligands for TLR2, TLR3 and TLR5 activate the MAP kinases, JNK and p38 MAPK, but not ERK1/2. Furthermore, treatment with the ligands for TLR2, TLR3 and TLR5 also induced the degradation of IκB‐α and activated the nuclear translocation of NF‐κB. MMP‐1 induction by the ligands for TLR2, TLR3 and TLR5 was inhibited by pretreatment with BAY11‐7082 (NF‐κB inhibitor) or SP600125 (JNK inhibitor), whereas MMP‐9 expression was inhibited by pretreatment with BAY11‐7082, SP600125 or SB203580. These findings demonstrate that the activation of TLR2, TLR3 or TLR5 induces the expression of MMP‐1 and MMP‐9 in human epidermal keratinocytes. In addition, NF‐κB or JNK mediated the MMP‐1 expression induced by TLR2, TLR3 and TLR5, whereas NF‐κB, JNK or p38 MAPK mediated the MMP‐9 expression induced by TLR2, TLR3 and TLR5.     

Expression of CD80 (B7/BB-1) and CD28 in human white blood cells treated with urocanic acid

Urocanic acid (UCA) is formed in the epidermis where it accumulates to be converted fromtrans-tocis-UCA by ultraviolet (UV) radiation. The two isomers modulate immune functions in several experimental systems. In particular,cis-UCA has been shown to induce antigen-specific immune tolerance, but the molecular mechanism of this effect is unknown. The present investigation was instituted to disclose any effect of UCA isomers on the cellular expression of the costimulatory antigens CD80 (B7/BB-1) and CD28. CD80 expression was efficiently induced in monocytic (CD14⁺) cells by human interferon-γ, while CD28 levels on lymphocytes remained unchanged, as detected by flow cytometry. Neither UCA isomer showed any effect on the expression patterns of these costimulatory molecules. The results obtained suggest that the mode of action for epidermal UCA-induced tolerogenesis may not involve modulation of CD80 (B7/BB-1) or CD28 expression.     

Efficacy of anti‐programmed cell death‐1 immunotherapy for skin carcinomas and melanoma metastases in a patient with xeroderma pigmentosum

Xeroderma pigmentosum (XP) is an orphan disease of poor prognosis. We report one case of parallel efficacy with anti‐programmed cell death‐1 (PD‐1) antibody on both melanoma and skin carcinoma in a patient with XP. A 17‐year‐old patient presented with metastatic melanoma and multiple nonmelanoma skin cancers. He was treated with pembrolizumab, a monoclonal anti‐PD‐1 antibody, at a dose of 2 mg kg^?1, every 3 weeks. Parallel therapeutic efficacy of anti‐PD‐1 was observed in metastatic melanoma and skin carcinomas, and maintained at week 24. This observation suggests anti‐PD‐1 may be considered in patients with XP and metastatic melanoma in addition to advanced nonmelanoma skin cancer.     

Increased serum levels of soluble vascular cell adhesion molecule-1 and soluble E-selectin in patients with polymyositis/dermatomyositis

BACKGROUND: Elevated levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble E-selectin (sE-selectin) have previously been reported in patients with various inflammatory diseases, but not in patients with polymyositis/dermatomyositis (PM/DM). OBJECTIVES: To determine serum levels and significance of sVCAM-1 and sE-selectin in patients with PM/DM. PATIENTS AND METHODS: Serum samples from 36 PM/DM patients, 30 patients with systemic sclerosis and 25 healthy control subjects were examined using specific enzyme-linked immunosorbent assay systems. RESULTS: The serum levels of sVCAM-1 in the PM/DM patients were significantly higher than those in the healthy controls. The elevated serum sVCAM-1 levels were correlated with the values of elevated erythrocyte sedimentation rate and elevated serum hyaluronate levels in the PM/DM patients. The serum sE-selectin levels in the PM/DM patients were also significantly higher than those in the healthy controls. The elevated serum sE-selectin levels were correlated with the incidence of elevated creatine kinase activities. The concentrations of serum sE-selectin were correlated with the serum tissue inhibitor of metalloproteinase-1 concentrations in the PM/DM patients (r = 0.53). CONCLUSIONS: These results suggest that serum sVCAM-1 and sE-selectin levels might be useful for detecting disease activity in patients with PM/DM.     

NF1 gene silencing induces upregulation of vascular endothelial growth factor expression in both Schwann and non‐Schwann cells

Neurofibromatosis type I (NF1) is associated with typical hypervascular tumors, including neurofibroma, glioma, malignant peripheral nerve sheath tumors (MPNST) and glomus tumors. Previously, we and other groups reported that neurofibromas showed high‐level expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor involved in neovascularization. However, the molecular mechanism underlying the upregulation of VEGF in neurofibromas remains unclear. In this study, we examined the effects of Nf1 gene silencing on VEGF expression in Schwann cell and non‐Schwann cell line and the upstream mTOR‐HIF‐1α – VEGF pathway in Schwann cell line. The results indicated that Nf1 gene silencing by lentiviral‐mediated RNA interference resulted in elevated expression of VEGF, HIF‐1α and phosphorylated mTOR at the protein level. The results obtained from Nf1 gene silencing in murine Schwann cell line analogously suggest that NF1 gene haploinsufficiency in human tumor Schwann cells may directly elicit upregulation of VEGF expression without the tumor microenvironment by activation of the mTOR‐HIF‐1α – VEGF pathway. We also showed that interleukin‐6 is upregulated in Nf1 gene knock‐down Schwann cells at the protein level.     

Ultraviolet B‐induced apoptosis of human skin fibroblasts involves activation of caspase‐8 and ‐3 with increased expression of vimentin

Background: After irradiation with a high dose of ultraviolet B (UVB), cells undergo apoptosis. Caspase‐8 and ‐3 are key mediators of apoptosis in many cells. Vimentin, an important cytoskeleton component, can be cleaved by caspase‐3, ‐6, ‐7 and ‐8. Cell apoptosis is promoted via caspase‐triggered proteolysis of vimentin. In this study, we explored the roles of caspase‐8 and ‐3 and the changes in vimentin expression in UVB‐induced apoptosis of human dermal fibroblasts. Methods: Skin fibroblasts were irradiated with 150 mJ/cm² UVB and cell death was monitored by the 3‐(4,5)‐dimethylthiahiazo(‐z‐y1)‐3,5‐diphenytetrazoliumromide assay and Hoechst staining. Caspase‐8 and ‐3 activities were detected by the caspase activity assay. Vimentin expression was assessed by immunofluorescence and Western blot. Results: Caspase‐8 and ‐3 were activated by 150 mJ/cm² UVB irradiation. Caspase‐8 and ‐3 activities changed in a time‐dependent way after UVB irradiation to induce apoptosis of fibroblasts, and caspase‐8 and ‐3 interacted with each other in this process. However, their substrate, vimentin, showed an enhanced expression over time after UVB irradiation. Conclusions: UVB‐triggered apoptosis of fibroblasts was dependent on the activation of caspase‐8 and ‐3 with an increased expression of vimentin.     

Human papillomavirus 16 E6/E7‐immortalized human gingival keratinocytes with epithelial mesenchymal transition acquire increased expression of cIAP‐1, Bclx and p27Kip1

Abstract: Epithelial mesenchymal transition (EMT) has been implicated in neoplastic invasion and metastasis. We have previously generated six cell lines from human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus type 16. Ldk and NuB1 lines represented EMT phenotype and other four lines represented cobblestone non‐EMT phenotype. These cell lines were utilized as model cells to determine whether inhibitors of apoptosis proteins and cell‐cycle regulators were molecular players during EMT. EMT cells exhibited increased expression of vimentin, vascular endothelial growth factor (VEGF) receptor1 and the ability to form tubules on Matrigel as well as to grow anchorage independently. We found that EMT cells expressed significantly elevated levels of cIAP‐1, Bclx and p27^kip higher than non‐EMT cells. These proteins could therefore be used as intrinsic indicators of EMT of human gingival keratinocytes.     

Orf virus infection of human keratinocytes and dermal fibroblasts: Limited virus detection and interference with intercellular adhesion molecule‐1 up‐regulation

Orf virus (Parapoxvirus ovis, ORFV) is a dermatotropic virus causing pustular dermatitis in small ruminants and humans. We analysed isolated human primary keratinocytes (KC) and dermal fibroblasts (FB) for cell death and virus replication by infection with a patient‐derived ORFV isolate. ORFV infection was associated with rapid induction of cell death in KC allowing for considerable virus removal. Upon infection with ORFV, KC and FB harboured intracytoplasmic ORFV and showed viral protein presence; however, missing virus spread indicated an abortive infection. Upon ORFV exposure, KC but not FB secreted the pro‐inflammatory cytokine interleukin (IL)‐6. ORFV infection enhanced the frequency of KC expressing intercellular adhesion molecule (ICAM)‐1 which was independent of IL‐6. Interestingly, ORFV inhibited ICAM‐1 up‐regulation on infected but not on non‐infected KC. Even interferon‐γ, a potent inducer of ICAM‐1, up‐regulated ICAM‐1 only on non‐infected KC. Transfer of ORFV‐free supernatant from infected to non‐infected KC induced ICAM‐1 on non‐infected KC pointing to the involvement of soluble mediator(s). Similarly as in KC, in FB interference with ICAM‐1 up‐regulation by ORFV infection was also observed. In conclusion, we shed light on epidermal and dermal defense mechanisms to ORFV infection and point to a novel ICAM‐1‐related immune evasion mechanism of ORFV in human skin.     

Small‐animal PET imaging analysis with [18F]FHBG in a mouse model of HSV1‐tk gene expression in melanoma

The purpose of this study was to establish a small‐animal model for molecular imaging and to acquire basic data on assessing the efficacy of candidate melanoma drugs using small‐animal PET imaging analysis with [¹⁸F]FHBG for herpes simplex virus 1‐thymidine kinase (HSV1‐tk) gene expression in a melanoma mouse model. The B16 melanoma cell line was transduced with a recombinant lentiviral vector containing the HSV1‐tk gene and inoculated into the back skin of C57BL/6J mice. [¹⁸F]FHBG PET imaging showed better contrast for HSV1‐tk(+) melanomas compared to brain, heart, gall bladder, intestine and kidney than did [¹⁸F]FDG PET imaging.