Tetracycline suppresses ATP gamma S-induced CXCL8 and CXCL1 production by the human dermal microvascular endothelial cell-1 (HMEC-1) cell line and primary human dermal microvascular endothelial cells

Abstract:  Tetracyclines (TCN) have powerful anti-inflammatory properties in addition to their anti-microbial effects. These anti-inflammatory effects are thought to play a role in inhibiting cutaneous inflammation in patients with rosacea and acne; however, the mechanism(s) of this action remains poorly understood. We have previously shown that adenosine-5'-triphosphate (ATP)γS, a hydrolysis-resistant ATP analogue, augments secretion of pro-inflammatory messengers by a human dermal microvascular endothelial cell line (HMEC-1). ATP released by the sympathetic nerves during stress may stimulate release of pro-inflammatory chemokines by dermal vessel endothelial cells, resulting in recruitment of inflammatory cells and exacerbation of inflammatory skin disease. Here we demonstrate that TCN inhibits ATPγS-induced release of pro-inflammatory mediators by HMEC-1 cells and primary human dermal microvascular endothelial cells. TCN dose-dependently inhibited ATPγS-induced augmentation of CXCL8 (interleukin-8) and CXCL1 (growth-regulated oncogene-α) production by HMEC-1 cells and primary human dermal endothelial cells in vitro . TCN and ATPγS did not affect HMEC-1 cell viability as determined by trypan-blue exclusion and cell counts. Inhibition of production of inflammatory mediators by endothelial cells may be one mechanism by which TCN improves inflammatory skin diseases. The ability to inhibit release of inflammatory mediators induced in HMEC-1 cells by purinergic agonists may be a useful way to screen for potential therapeutic agents for cutaneous inflammation.     

Effect of UVB 311 nm irradiation on normal human skin

Ultraviolet radiation B (UVB) on the skin induces erythema, inflammation and modifications of the immune system. These changes have been reported after excessive short-term or long-term exposure to broad spectrum UVB. In this study, we examined the effects of local repetitive UVB irradiation of 311 nm wavelength on the skin of seven young volunteers. Skin biopsies were taken before and after UVB irradiation, and we immunohistochemically analyzed the expression of CD1a and HLA-DR antigens of Langerhans cells (LC), the possible infiltration of dermis/epidermis by CD11b macrophages, the modifications or the induction of intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1) involved in the binding of leukocytes to the endothelial surface and the development of perivascular infiltrates of LFA-1⁺ mononuclear cells. We also determined the expression of substance P receptors (SPR) using biotinylated substance P (SPB). Exposure of UVB 311 nm induced a drastic reduction of CD1a⁺ cells and a moderate increase of HLA-DR⁺ dendritic cells in the epidermis without infiltration by CD11b macrophages. An increase of the binding of SPB to upper layer epidermal cells was noted in five of seven biopsies. In the dermis, vessel-associated ICAM-1 expression increased and an induction of E-selectin occurred on nearly 20 to 40% of endothelial cells, but VCAM-1 expression remained undetectable. The percentage of LFA-1⁺ cells did not change significantly after irradiation. These observations may be compatible with a selective role of UVB 311 nm on the skin immune response.     

蘘荷氯仿提取物对人真皮微血管内皮细胞表面黏附分子的表达及淋巴细胞黏附作用的影响

【摘要】 目的 探讨蘘荷对人真皮微血管内皮细胞（HDMEC）表面黏附分子的表达及淋巴细胞黏附作用的影响。方法 用蘘荷氯仿提取物（CFMG）与肿瘤坏死因子（TNF）-α/白介素（IL）-1α分别诱导或按不同顺序联合诱导HDMEC不同时间,用酶联免疫吸附试验（ELISA）检测细胞间黏附分子-1（ICAM-1）、血管细胞间黏附分子-1（VCAM-1）和E选择素表达水平。使用γ计数器测定HDMEC对T淋巴细胞和Ramos细胞的黏附能力。用Sigma Plot 12软件包对数据进行配对t检验。 结果 与0.2%二甲基亚砜相比,CFMG轻度下调HDMEC表面ICAM-1、VCAM-1和E选择素表达水平（均P > 0.05）;与细胞培养液相比,TNF-α显著上调ICAM-1、VCAM-1、E选择素的表达水平（均P < 0.01）,IL-1α上调ICAM-1和E选择素的表达水平（均P < 0.01）,对VCAM-1轻度上调（P > 0.05）。CFMG预处理可显著抑制TNF-α和IL-1α对黏附分子表达水平的上调作用（均P < 0.01）,但是CFMG后处理对经TNF-α和IL-1α刺激后升高的黏附分子表达水平影响不大（均P > 0.05）。与0.2% DMSO相比,CFMG轻度降低HDMEC对T淋巴细胞和Ramos细胞黏附率（均P > 0.05）,而与细胞培养液相比,TNF-α和IL-1α显著提高其黏附率（均P < 0.01）。 结论 蘘荷脂溶性粗提物可能通过阻遏前炎症细胞因子TNF-α和IL-1α对HDMEC表面黏附分子的上调作用,抑制组织中炎细胞浸润从而起到抗炎作用。 【关键词】 蘘荷; 内皮细胞; 细胞黏附分子; 细胞因子     

Studies of the modulation of MHC antigen and cell adhesion molecule expression on human dermal microvascular endothelial cells

Interactions between leukocytes and endothelial cells, particularly in the microvasculature, are important for the initiation and regulation of tissue inflammation. These interactions are regulated by the recognition of specific cell adhesion molecules (CAM) on both leukocytes and endothelial cells. In this study, we examined the modulation of cell surface expression of MHC antigens and the CAM intercellular adhesion molecule 1 (ICAM-1), lymphocyte function antigen 3 (LFA-3), and CD44 on human dermal microvascular endothelial cells (HDMEC) both grown in monolayers and differentiated into capillary-like structures on the basement membrane-like substrate matrigel. HDMEC grown in monolayers or differentiated on matrigel express comparable cell surface MHC class I, LFA-3, CD44, and ICAM-1. ICAM-1, but not LFA-3 or CD44, was increased in expression in a dose- and time-dependent manner by interleukin 1 (IL-1) alpha, tumor necrosis factor (TNF) alpha, lipopolysaccharide (LPS), or interferon (IFN) gamma. Comparable upregulation was observed both in cells grown in monolayers and cells differentiated on matrigel. IL-1 alpha, TNF alpha, and LPS increased ICAM-1 expression on average 100-200% whereas IFN gamma was somewhat less potent. Comparative studies with human umbilical vein endothelial cells (HUVEC) demonstrated consistently lower levels of ICAM-1 expression on HUVEC, but greater increases after cytokine stimulation. Pretreatment with dexamethasone or transforming growth factor (TGF) beta did not affect baseline expression of ICAM-1 or inhibit upregulation of ICAM-1 on HDMEC by IL-1 alpha, TNF alpha, LPS, or IFN gamma. Both IFN gamma and TNF alpha, but not IL-1 alpha increased MHC class I expression, whereas only IFN gamma induced the expression of HLA-DR on HDMEC. The effect of IL-1 alpha, TNF alpha, or IFN gamma was inhibited by antibody to the specific cytokine, but was unaffected by antibody to other cytokines. Additionally, IFN alpha or beta inhibited upregulation of HLA-DR by IFN gamma, but had no effect on the increased MHC class I or ICAM-1 expression mediated by this cytokine. These data demonstrate that the expression of CAM and MHC antigens on small vessel-derived endothelial cells is different from that observed on large-vessel HUVEC, is regulated by the presence of multiple cytokines operating via distinct pathways, and the expression and regulation of these proteins appear to be similar on cells that have been grown in monolayers to those morphologically differentiated into blood vessel-like structures.     

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。     

UVB irradiation decreases the magnitude of the Th1 response to hapten but does not increase the Th2 response

Exposure of murine skin to low doses of ultraviolet-B (UVB) radiation before sensitization with hapten reduces the ability of antigen presenting cells (APC) in the draining lymph nodes to initiate contact hypersensitivity responses in vivo and results in the induction of hapten-specific suppressor T cells. In the present study, we tested the hypothesis that exposure of skin to UVB radiation suppresses T cell responses to hapten in vivo by altering the functions of APC, resulting in decreased stimulation of Th1 lymphocytes, which mediate contact hypersensitivity responses, and preferential activation of Th2 cells. C3H/HeN mice were exposed to either a single 2 kJ/m² dose of UVB or to 400 J/m² of UVB daily from FS40 sunlamps for four consecutive days and sensitized with fluorescein isothiocyanate on UV-irradiated skin. Draining lymph node cells were collected 18 h after sensitization and co-cultured with nylon wool-purified T cells from naive or fluorescein-immunized mice. Unseparated lymph node cells or sorter-purified fluorescein-bearing APC from UV-irradiated mice induced less T cell proliferation than APC from non-UV-exposed mice. Lymph node cells produced less Th1 and Th2-associated cytokines, interferon-gamma and interleukin-4, respectively, in response to APC from UV-irradiated animals compared with APC from unirradiated, fluorescein-sensitized mice. Thus, low doses of UV radiation do not result in preferential stimulation of Th2 response in lymph nodes, and results from cloned cell lines may incompletely reflect T cell responses in vivo.     

Knockdown of paraoxonase 1 expression influences the ageing of human dermal microvascular endothelial cells

Skin is one of the most commonly studied tissues for microcirculation research owing to its close correlation of cutaneous vascular function, ageing and age‐related cardiovascular events. To elucidate proteins that determine this correlation between endothelial cell function and ageing in the vascular environment of the skin, we performed a proteomic analysis of plasma samples from six donors in their 20s (young) and six donors in their 60s (old). Among identified proteins, paraoxonase 1 (PON1) was selected in this study. To elucidate the role of PON1 on skin ageing and determine how it controls cellular senescence, the characteristics of PON1 in human dermal microvascular endothelial cells (HDMECs) were determined. When the expression of endogenous PON1 was knocked‐down by small interfering RNA (siRNA) targeting PON1, HDMECs showed characteristic features of cellular senescence such as increases in senescence‐associated β‐galactosidase stained cells and enlarged and flattened cell morphology. At 48 h post‐transfection, the protein expression of p16 in PON1 siRNA‐treated HDMECs was higher than that in scrambled siRNA‐treated HDMECs. In addition, the expressions of moesin and rho GTP dissociation inhibitor, additional age‐related candidate biomarkers, were decreased by PON1 knock‐down in HDMECs. In conclusion, these results suggest that PON1 functions as an ageing‐related protein and plays an important role in the cellular senescence of HDMECs.     

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.     

Malassezia furfur invasiveness in a keratinocyte cell line (HaCat): effects on cytoskeleton and on adhesion molecule and cytokine expression

Abstract The lipophilic yeast Malassezia furfur is a member of the cutaneous microbiota, also associated with several chronic diseases such as pityriasis versicolor, folliculitis, seborrhoeic dermatitis, and some forms of atopic dermatitis, psoriasis and confluent and reticulate papillomatosis. In this study we determined the immunomodulatory and invasive capacity of M. furfur in a human keratinocyte cell culture, HaCat. At a yeast cell to HaCat ratio of 30 : 1, M. furfur penetration was only 30% with poor phagolysosome fusion and with cytoskeleton modification. Transglutaminase I gene expression was also inhibited, supporting the hypothesis that M. furfur causes an initial break in the barrier function of the epidermis. Moreover, we demonstrated that M. furfur modulates proinflammatory and immunomodulatory cytokine synthesis by downregulating IL-1α and by inhibiting IL-6 and TNF-α and by upregulating IL-10 and TGF-β1. The suppressed inflammatory response induced by M. furfur may play a role in chronic disease. Received: 18 September 2000 / Revised: 24 April 2001 / Accepted: 16 June 2001     

皮肤白细胞碎裂性与淋巴细胞性血管炎某些粘附分子表达的比较研究

目的：比较细胞粘附分子ＩＣＡＭ－１、ＶＣＡＭ－１和ＥＬＡＭ－１在皮肤白细胞碎裂性和淋巴细胞性血管炎的表达及其意义。方法：采用免疫组化方法研究两型血管料的粘附分子表达情况。结果：角质形成细胞ＩＣＡＭ－１在淋巴细胞性血管炎（８５．７１％）表达明显高于白细胞碎裂性血管炎（１１．１１％（Ｐ〈０．０００１）,ＩＣＡＭ－１、ＶＣＡＭ－１、ＥＬＡＭ－１在两型血管炎血管内皮中表达无区别（Ｐ值分别为１,０．０６８,０．４６）。结论：无论在白细胞碎裂性还是淋巴细胞性血管炎、粘附分子表达均上调。两种血管炎粘附分子表达率的不同反应了局部细胞因子释放的不同,这对揭示血管炎发病机理将具有重要意义。     

Expression of psoriasis-associated fatty acid-binding protein in senescent human dermal microvascular endothelial cells

Aging is associated with the progressive pathophysiologic modification of endothelial cells. In vitro endothelial cell senescence is accompanied by proliferative activity failure and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in aging organisms. In order to observe the changing patterns of protein expression in senescent human dermal microvascular endothelial cells (HDMECs), proteins obtained from both early- and late-passaged HDMECs were separated by two-dimensional electrophoresis, visualized by silver staining, and quantified by image processing. Proteins of interest were extracted by in-gel digestion with trypsin and quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), by searching the National Center for Biotechnology Information protein-sequence database. More than 2000 spots were detected by 2D electrophoresis within a linear pH range of 3-10. Twenty-two major differentially expressed spots were observed in serially passaged HDMECs and identified with high confidence by MALDI-TOF-MS. One of these spots was found to be a 14-15 kDa psoriasis-associated fatty acid-binding protein (PA-FABP) with high affinity for long-chain fatty acids. The expression of PA-FABP was confirmed to be elevated in senescent HDMECs (passage 20) by fluorescence-activated cell sorting (FACS), confocal laser microscopy, and by immunohistochemistry in aged human skin tissue. Our results suggest that the overexpression of FABP in cultured senescent HDMECs is closely related to skin aging.     

Different mechanisms of adhesion molecule expression in human dermal microvascular endothelial cells by xanthoma tissue-mediated and copper-mediated oxidized low density lipoproteins

BACKGROUND: Oxidation of low density lipoprotein (LDL) has been implicated in infiltration of foam cells derived from circulating monocytes. Monocyte adhesion to endothelial cells and migration into dermis are essential steps for infiltration of foam cells. OBJECTIVE: We investigated the role of adhesion molecules contributing to the process of monocyte adhesion to human dermal microvascular endothelial cells (HDMEC). Special attention was paid to the signal transduction for adhesion molecule expression induced by two distinct types of oxidized LDL. METHODS: HDMEC were incubated with xanthoma tissue-modified LDL (x-LDL), a model of extravasated LDL oxidized in xanthoma lesions, or Cu(2+)-treated LDL (Cu-LDL), a model of oxidized LDL. Adhesion of U937 cells, a human monocytic leukemia cell line, to HDMEC and expression of endothelial cell adhesion molecules on HDMEC were examined. Signal transduction pathways for the adhesion molecule expression were evaluated by employing specific inhibitors. RESULTS: x-LDL induced adhesion of U937 cells to HDMEC through vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by activating tyrosine kinase pathway. Cu-LDL up-regulated the adhesion through not only VCAM-1 and E-selectin but also intercellular cell adhesion molecule-1 (ICAM-1) by activating G(i) protein pathway. CONCLUSION: Extravasated and oxidized LDL in xanthoma lesions contributes to foam cell recruitment by activating tyrosine kinase pathway and inducing adhesion of monocytes to HDMEC through VCAM-1 and E-selectin. Cu-LDL, on the other hand, activates G(i) protein pathway and induces the adhesion through ICAM-1, VCAM-1 and E-selectin.     

The effect of methotrexate on the expression of cell adhesion molecules and activation molecule CD69 in psoriasis

BACKGROUND: The mechanism of the action of methotrexate (MTX) in the treatment of psoriasis has not been completely elucidated. OBJECTIVE: To assess the effect of MTX on the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, activation molecule CD69 and T-cell phenotype in skin specimens from patients with psoriasis. METHODS: We performed an immunohistochemical analysis of the expression of T-cell phenotype and cell adhesion/activation molecules in skin biopsies from patients with psoriasis treated with a fixed dose of MTX (12.5 mg/week). To determine data on the epidermal/dermal T-cell infiltration we carried out a manual quantification. RESULTS: Skin samples prior to therapy showed a moderate to severe inflammatory infiltrate, mainly due to T lymphocytes with a helper/inducer (CD4) phenotype. Most of these cells also expressed ICAM-1 and VCAM-1. Blood vessels showed expression of E-selectin and VCAM-1, and keratinocytes were positive for ICAM-1 staining. The cell infiltrate was reduced after therapy, as well as the expression of cell adhesion molecules. However, we also noted the persistence of the T lymphocyte phenotype CD8(+), expressing the CD69 activation molecule, after the MTX treatment. CONCLUSIONS: MTX downregulates the expression of some adhesion molecules, a phenomenon that may contribute to its anti-inflammatory therapeutic effect in psoriasis. The infiltrating T cells post-treatment have an activated cytotoxic phenotype, which may suggest a pathogenic role in the continuation and/or recurrence of psoriasis.     

Effect of nerve growth factor on endothelial cell biology: proliferation and adherence molecule expression on human dermal microvascular endothelial cells

Abstract In addition to its effect on the central nervous system, nerve growth factor (NGF) appears to play a key role in the initiation and maintenance of inflammation in many organs. NGF degranulates mast cells, recruits inflammatory cellular infiltrates and activates T cells. Extravascular migration of leukocytes is initially controlled by the interaction of cell surface adhesion molecules of leukocytes and endothelial cells. A marked upregulation of NGF in keratinocytes is also observed in conditions characterized by angiogenesis such as psoriasis and wound healing. In this study we investigated the role of NGF in inflammation by studying its effects on endothelial cell proliferation and intracellular adhesion molecule expression by endothelial cells. The effect of NGF on human dermal microvascular endothelial cell (HDMEC) proliferation was measured using the hexosaminidase assay. ICAM-1 expression on HDMEC was measured by ELISA. The function of ICAM-1 was assessed by adherence of peripheral blood mononuclear cells (PBMC) to HDMEC using ⁵¹Cr-labeled PBMC. There was a significant increase in proliferation of HDMEC stimulated with NGF as compared to unstimulated HDMEC (P < 0.001). NGF-neutralizing antibody decreased the mitogenic effect of NGF significantly (P < 0.05). NGF also increased ICAM expression on HDMEC as compared to unstimulated HDMEC (P < 0.05). NGF-neutralizing antibody decreased ICAM expression on NGF-stimulated HDMEC (P < 0.05). The percentage of PBMC adherence was higher in NGF-stimulated HDMEC (P < 0.001). Anti-ICAM antibody decreased PBMC adherence. In the study reported here, the role of NGF in two important aspects of inflammation, i.e. angiogenesis and inflammatory cell recruitment at the site of inflammation, was investigated . Received: 7 December 2000 / Revised: 10 December 2000 / Accepted: 24 February 2001