Cytokine regulation of proliferation and ICAM-1 expression of human dermal microvascular endothelial cells in vitro.

The effects of recombinant human interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF) on the cell proliferation and the expression of intercellular adhesion molecule-1 (ICAM-1) were assessed in cultured human dermal microvascular endothelial cells (HDMEC). IL-1 alpha and IL-1 beta stimulated the proliferation of HDMEC in a dose-dependent manner, whereas in control experiments using human umbilical vein endothelial cells (HUVEC), IL-1 alpha and IL-1 beta did not stimulate HUVEC growth. Also GM-CSF stimulated the proliferation of HDMEC, whereas IL-6 did not affect endothelial cell growth in vitro. Treatment with IL-1 alpha, IL-1 beta, and TNF markedly increased the expression of ICAM-1 on HDMEC in a time- and dose-dependent manner, in contrast to IL-6 and GM-CSF. By pre-embedding immunoelectron microscopy, membrane-bound expression of ICAM-1 was visualized with pronounced labeling in areas of microvillous cell protrusions. The TNF-induced expression of ICAM-1 on HDMEC was blocked by co-incubation with a neutralizing antibody against TNF, but not with neutralizing antibodies against IL-1 alpha, IL-1 beta, or IL-6. In addition, co-incubation of HDMEC with TNF and the retinoid compound acitretin, dexamethasone, or indomethacin did not abrogate the TNF-induced ICAM-1 expression. These results disclose IL-1 as a major, multifunctional endothelial cell-targeted cytokine and further confirm the concept that pro-inflammatory cytokines exert differential regulatory effects on dermal microvascular endothelial cell proliferation and expression of cell-adhesion molecules.     

The effect of herpesvirus infection on the expression of cell adhesion molecules on cultured human dermal microvascular endothelial cells

Cell-mediated immune response to herpes simplex virus (HSV) may be important in the pathogenesis of herpes keratitis, erythema multiforme or Behcet's disease. We examined whether herpesvirus infection regulates the expression of cell adhesion molecules on cultured human dermal microvascular endothelial cells (HDMEC) and the regulation of T-lymphocytes binding to HDMEC. The expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), or E-selectin on HDMEC increased significantly after treatment with HSV-1, HSV-2, or measles virus on HDMEC. Anti-IL-1 alpha antibody or anti-TNF alpha antibody partially inhibited the expression of ICAM-1, VCAM-1, or E-selectin on HDMEC. The binding of T-lymphocytes to HDMEC increased significantly after the treatment of HSV-1 or measles virus on HDMEC. The binding of T-lymphocytes to HDMEC was significantly inhibited after 16 h of incubation following treatment with anti-ICAM-1 antibody, anti-IL-1 alpha antibody or anti-TNF alpha antibody to HDMEC. These study results suggest that HSV induces the increased expression of ICAM-1, or induction of VCAM-1 and E-selectin on HDMEC and that among these adhesion molecules, the expression of ICAM-1 on HDMEC mainly regulates the binding of T-lymphocytes to HDMEC. The data also suggest that IL-1 alpha or TNF alpha which was produced by HSV infected HDMEC may be related to these events.     

VCAM-1-, ELAM-1-, and ICAM-1-independent adhesion of melanoma cells to cultured human dermal microvascular endothelial cells.

We have examined the mechanisms by which tumor cells bind to endothelial cells utilizing cultured melanoma cells and microvascular endothelial cells derived from human dermis (HDMEC). The ability of biologic response modifiers (BRM) to modulate the adhesion of melanoma cells to HDMEC was defined and those results were compared with results from human umbilical vein endothelial cells (HUVEC). SK-MEL-2, WM266-4, and Hs 294T melanoma cells all bound to HDMEC and HUVEC monolayers and adherence of melanoma cells was enhanced in a dose- and time-dependent manner by the treatment of HDMEC with interleukin 1 (IL-1) alpha or tumor necrosis factor (TNF) alpha. Similar increases in binding to HDMEC or HUVEC were induced after BRM stimulation, although baseline melanoma cell binding to HUVEC tended to be slightly higher than to HDMEC. In contrast, whereas phorbol 12-myristate 13-acetate (PMA) augmented melanoma cell adherence to HDMEC, PMA failed to increase adherence to HUVEC. The alterations in melanoma cell binding were induced only after pretreatment of endothelial and not melanoma cells with PMA. Studies of the expression of cell adhesion molecules (CAM) on HDMEC and HUVEC using enzyme-linked immunosorbent assay showed that vascular cell adhesion molecule 1 (VCAM-1) is not induced by PMA on HDMEC and intercellular adhesion molecule 1 (ICAM-1) is downregulated on HDMEC by PMA treatment. Endothelial leukocyte adhesion molecule 1 (ELAM-1) is induced by PMA, IL-1 alpha, or TNFalpha, but its expression does not correlate with increased melanoma cell binding MoAb recognizing VCAM-1-inhibited TNFalpha-induced increases in melanoma cell binding to HUVEC. However, anti-VCAM-1 antibody failed to clock melanoma cell binding to PMA or IL-1 alpha-stimulated HDMEC and only partially inhibited melanoma cell binding to TNF alpha-stimulated HDMEC. This study demonstrates that PMA and IL-1 alpha-induced increases in melanoma cell adherence to HDMEC are not mediated via known CAM, including ICAM-1, VCAM-1, or ELAM-1, and may be affected through microvessel-specific novel proteins not previously described on endothelial cells.     

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.     

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.     

Corticosteroids induce proliferation but do not influence TNF- or IL-1Β-induced ICAM-1 expression of human dermal microvascular endothelial cells in vitro

The effects of hydrocortisone, dexamethasone, betamethasone 17-valerate and clobetasol propionate at concentrations of 10^–5–10^–12 M on the proliferation of human dermal microvascular endothelial cells (HDMEC) were studied in vitro. In addition, confluent HDMEC were treated with TNF (1000 U/ml) or IL-1 (1000 U/ml) alone or in combination with the corticosteroids (10^–5 M, 10^–6 M) for 24 h, and cytokine-induced expression of intercellular adhesion molecule-1 (ICAM-1) was assessed by immunocytochemistry. Controls were treated either with growth medium or with the corticosteroids alone. All tested corticosteroids stimulated HDMEC growth after 4 and 6 days of treatment in a dose-dependent manner, as assessed by ³H-thymidine incorporation and the 4-methyl-umbelliferyl heptanoate (MUH) assay. The minimal effective concentration was 10^–9 M for hydrocortisone, 10^–10 M for dexamethasone and betamethasone, and 10^–12 M for clobetasol. In untreated and in corticosteroid-treated cultures, less than 5% of the cells expressed ICAM-1. TNF and IL-1 were strong inducers of ICAM-1 expression on 74% and 82% of the cells, respectively. None of the tested corticosteroids significantly influenced cytokine-induced ICAM-1 expression, suggesting that the anti-inflammatory effects of corticosteroids are not related to ICAM-1 modulation on HDMEC.     

Presence of circulating antibodies to a disease-specific antigen on cultured human dermal microvascular endothelial cells in patients with Behçet’s disease

Abstract Beh?et’s disease is a chronic, multisystem disorder characterized by a recurrent inflammatory reaction. Antiendothelial cell antibodies have been detected in the serum from patients with autoimmune diseases with presenting vasculitis and it is assumed that they can induce damage to the endothelial cells. In this study, we detected antiendothelial cell antibodies in the serum from patients with Beh?et’s disease using an enzyme-linked immunosorbent assay, electrophoresis and immunoblotting. The cytolysis of human dermal microvascular endothelial cells (HDMEC) was measured using a cytotoxicity assay. The serum from 37.4% of Beh?et’s disease patients showed IgM antibodies against unstimulated HDMEC while the serum from 18.4% of patients showed an increase in IgM antibody titer after IFN-γ pretreatment. The frequency of vasculitis was higher in the IgM-positive Beh?et’s disease patients than in the IgM-negative patients. In Western blotting, IgM-positive Beh?et’s disease serum reacted with the 44 kDa HDMEC surface antigen, whereas IgM-positive systemic lupus erythematosus serum reacted with the 81 kDa HDMEC surface antigen. The reactivity to the 44 kDa protein band was also observed in cultured human umbilical vein endothelial cells but not in fibroblasts, A431 cells or SK-MEL-2 cells. Serum from Beh?et’s disease patients incubated with human complement or mononuclear cells produced no significant lysis of HDMEC, and cultured HDMEC were resistant to antibody-dependent cell-mediated cytotoxicity. The results suggest that antibodies against antigens on the surface of endothelial cells may play a role in inducing vasculitis in Beh?et’s disease, not through a direct toxic effect of an antiendothelial cell antibody but by an indirect effect involving the activation of endothelial cells to produce cytokines. Received: 23 October 1998 / Received after revision: 2 March 1999 / Accepted: 12 March 1999     

Effects of rIFN alpha, beta, and gamma on the morphology, proliferation, and cell surface antigen expression of human dermal microvascular endothelial cells in vitro

The influence of recombinant human interferon alpha 2a (rIFN alpha), recombinant human interferon beta 1 (rIFN beta), and recombinant human interferon gamma (rIFN gamma) on human dermal microvascular endothelial cells (HDMEC) cultured in vitro was studied in various rIFN concentrations (0.1 IU/ml-10(4) IU/ml) over 2, 3, 4, 6, 8, and 10 d. Cell morphology and ultrastructure, cell proliferation, expression of class II alloantigens (HLA-DR and HLA-DQ), and intercellular adhesion molecule-1 (ICAM-1) were investigated using an in vitro technique established in our laboratory. All rIFN tested induced alterations of typical HDMEC morphology; the cells became spindle-shaped and fibroblastoid, although they maintained their endothelial cell marker expression. Also, all IFN dose- and time-dependently inhibited the proliferation of HDMEC in vitro (rIFN alpha greater than beta greater than gamma), whereby rIFN alpha exerted the strongest growth-inhibitory effect. Alkaline phosphatase anti-alkaline phosphatase (APAAP) immunocytochemistry of the cultured cells showed dose- and time-dependent stimulation of ICAM-1 and class II antigen expression only by rIFN gamma (HLA-DR greater than HLA-DQ), rIFN alpha and beta did not exert any immunomodulatory activity on HDMEC in vitro. These results indicate that HDMEC are an important target for the action of IFN. Besides growth inhibition, it seems that rIFN gamma in particular may be involved in the modulation of leucocyte adhesion and trafficking by altering the immunophenotype of the endothelial cell population.     

Cultivation of human dermal microvascular endothelial cells in vitro: Immunocytochemical and ultrastructural characterization and effect of treatment with three synthetic retinoids

Summary A new reliable and reproducible technique to culture endothelial cells from the small vessels and capillaries of human skin is introduced, and proliferation and differentiation of the growing cells are characterized. The endothelial origin of the culture cells was confirmed by light- and electron microscopy and by labelling with Ulex europaeus Agglutinin I and an antibody against Factor VIII-related antigen. Further immunocytochemical characterization showed that 92–100% of the cells were positive for ₂-microglobulin and the entire cell population expressed vimentin, whereas cytokeratins, desmin, HLA-DR antigen, Leu 6 and S 100 protein, could not be detected. As vascular endothelium is a common site of inflammation and retinoids have been shown to be of good clinical efficacy in some chronic inflammatory skin diseases, we investigated the influence of etretinate, etretin and isotretinoin on proliferation and antigen expression of our culture cells. All retinoids applied inhibited proliferation of endothelial cells in a dose- and time-dependent manner whereas they induced neither HLA-DR nor intercellular adhesion molecule-1 (ICAM-1). Furthermore, none of the retinoids applied influenced the -interferon-induced expression of these surface antigens on endothelial cells. Our results suggest that the action of retinoids in skin inflammation is not mediated by modulation of HLA-DR or ICAM-1. The cell culture technique described here is an interesting and reliable model for studying the influence of drugs on endothelial cell growth and differentiation in vitro.This work was partly presented at the ESDR-JSID-SID Tri-continental Meeting, Washington D. C., April 26–30, 1989     

OX40 ligand and OX40 are increased in atopic dermatitis lesions but do not correlate with clinical severity

Background The interaction between the OX40 ligand (OX40L) and OX40 has been suggested to have pathogenetic significance in atopic dermatitis (AD). Objective The purpose of this study was to investigate the expression and relevance of OX40L and OX40 in AD skin. Methods OX40L and OX40 were stained immunohistochemically on the cryosections of the lesional and non‐lesional skin of 17 subjects with moderate‐to‐severe AD and of 10 patients with psoriasis vulgaris. Phorbol myristate acetate (PMA) stimulated keratinocytes and cell membrane preparations from PMA‐stimulated keratinocytes or LAD‐2 mast cells were incubated with peripheral blood mononuclear cells (PBMC) in the presence or absence of blocking monoclonal antibodies to OX40L, CD30L or ICAM‐1. Results We show for the first time that the staining intensity of OX40L and the number of OX40⁺ cells are significantly greater in the lesional dermis than in the healthy‐looking dermis in AD (P < 0.001 in both comparisons) and also in psoriasis (P = 0.01 and P < 0.001 respectively), but neither molecule correlate significantly with the clinical severity of AD. Living keratinocytes and cell membranes from LAD‐2 mast cells and keratinocytes increased the PBMC proliferation response. Anti‐OX40L antibody inhibited, in a similar fashion as anti‐ICAM‐1 and anti‐CD30L, PBMC proliferation induced by LAD‐2 membranes, but stimulated that induced by keratinocytes. Conclusion Our findings provide evidence for the involvement of OX40 and OX40L in the pathogenesis of AD though they are not specific to AD and in vitro results suggest complex interaction.     

K252a, a high-affinity nerve growth factor receptor blocker, improves psoriasis: an in vivo study using the severe combined immunodeficient mouse-human skin model

The peripheral nervous system, in addition to its sensory and motor functions, can induce a local inflammatory response known as neurogenic inflammation. This phenomenon plays a critical role in several inflammatory diseases, e.g., asthma, atopy, rheumatoid arthritis, psoriasis, and ulcerative colitis. Neurogenic inflammation and the role of nerve growth factor (NGF) have been extensively studied in psoriasis. There are increased levels of NGF in the keratinocytes and upregulation of NGF receptor (NGF-R) in the cutaneous nerves of psoriatic plaques. NGF can influence all the salient pathologic events noticed in psoriasis such as proliferation of keratinocytes, angiogenesis, T cell activation, expression of adhesion molecules, proliferation of cutaneous nerves, and upregulation of neuropeptides. In this double-blinded, placebo-controlled study, we addressed the role of NGF/NGF-R in psoriasis in an in vivo system using the severe combined immunodeficient (SCID) mouse-human skin model of psoriasis. The transplanted psoriatic plaques on the SCID mice (n=12) were treated with K252a, a high-affinity NGF receptor blocker. Psoriasis significantly improved following 2 wk of therapy. The length of the rete pegs changed from 308.57+/-98.72 to 164.64+/-46.78 microm (p<0.01, Student's t test). A similar improvement of psoriasis was observed by directly inhibiting NGF with NGF-neutralizing antibody. NGF-neutralizing antibody in normal saline at 10 ng (n=4) and 20 ng (n=4) per kilogram of body weight doses were used. Both doses of NGF-neutralizing antibody reduced rete peg lengths significantly, e.g., from 298.5+/-42.69 to 150.52+/-32.93 microm (p<0.05, Student's t test). This study provides evidence for the role of NGF and its high-affinity receptor in the pathogenesis of psoriasis and insights to develop novel therapeutic modalities.     

Mast cells of psoriatic and atopic dermatitis skin are positive for TNF-α and their degranulation is associated with expression of ICAM-1 in the epidermis

Abstract The release of cytokines from cutaneous cells may be of major importance in the initiation and development of many inflammatory skin disorders. For example, tumor necrosis factor-alpha (TNF-α), which in healthy skin is found preformed only in mast cells, is able to induce the expression of several adhesion molecules including intercellular adhesion molecule-1 (ICAM-1). Increased expression of ICAM-1 occurs in keratinocytes in lesional skin of psoriasis and atopic dermatitis (AD) and it is considered to be an important initiator of leucocyte/keratinocyte interactions in skin inflammation. We counted the mast cells showing TNF-α immunoreactivity using a double-staining method in nonlesional and lesional skin sections from 12 patients with AD and 12 patients with psoriasis. The percentage of TNF-α⁺ mast cells in lesional and nonlesional AD skin was 36 ± 22% and 21 ± 15% (P < 0.018, paired t-test), respectively, and in psoriatic skin was 16 ± 25% and 15 ± 15%, respectively (P < 0.89, paired t-test). We also cultured whole skin biopsies taken from the healthy-looking skin of psoriatic and AD patients in the presence of mast cell degranulator compound 48/80, which resulted in focal expression of ICAM-1 in the epidermis. In cultured keratinocytes, both histamine and an extract of a human mast-cell line (HMC-1) induced ICAM-1 immunostaining only in occasional cells, but the combination of histamine and the HMC-1 extract resulted in intense ICAM-1 staining in numerous cells. This enhancement of ICAM-1 staining was abolished by preincubation of the HMC-1 extract with anti-TNF-α antibody. These results suggest that the degranulation of mast cells induces the expression of ICAM-1 in keratinocytes probably via TNF-α and histamine. Received: 8 August 1997     

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.     

Pentoxifylline inhibits human T-cell adhesion to dermal endothelial cells

Pentoxifylline (PTX), a methyl xanthine derivative with phosphodiesterase inhibitory activity, has been shown to have antiinflammatory effects. Previous studies have demonstrated that PTX can suppress TNFα production and function, and can inhibit the adhesion of neutrophils and monocytes to endothelial cells. In the present study, we sought to determine whether PTX also interferes with the adhesion of human peripheral blood T lymphocytes to cells of the human dermal endothelial cell line HMEC-1. Using a cell adhesion immunoassay, the effect of different doses of PTX (10 ^–5 –10 ^–2 M ) on the binding of unactivated or PMA-activated T cells to unstimulated or TNFα-stimulated endothelial cells was investigated. In addition, blocking experiments with monoclonal antibodies against pairs of adhesion molecules known to be involved in endothelial cell/T-cell adhesion were performed. Unactivated T cells showed minimal adhesion to unstimulated endothelial cells. PMA-activated T cells showed an eightfold increased binding to TNFα-stimulated endothelial cells, which was found to be mediated largely by LFA-1/ICAM-1. PTX inhibited the binding of PMA-activated T cells to TNFα-stimulated endothelial cells in a dose-dependent manner. This inhibition was only found when PTX was present during the adhesion assay. A similar inhibition was found when PTX was replaced by isobutylmethylxanthine, another methyl xanthine derivative, or by a combination of two cAMP analogues. The results suggest that interference with T-cell/endothelial cell adhesion, which forms an essential step in the migration of T cells from the peripheral blood into sites of inflammation, may be another explanation for the beneficial effect of PTX in several inflammatory dermatoses. Received: 22 May 1996     

IL-10 inhibits ICAM-1 expression on human Langerhans cells but not on keratinocytes, dermal endothelial cells or fibroblasts

Abstract Intercellular adhesion molecule-1 (ICAM-1) is regularly expressed or inducible on all major cutaneous cell populations including Langerhans cells, keratinocytes, endothelial cells and dermal fibroblasts. ICAM-1 is induced in the skin under inflammatory conditions and plays an important role in the activation of T cells. Interleukin-10 (IL-10) is a pluripotent immunosuppressive cytokine that inhibits proliferation of T cells via inhibition of antigen-presenting cells including Langerhans cells. We demonstrates that IL-10 inhibits baseline and also cytokine-stimulated ICAM-1 expression on human Langerhans cells, which has previously been shown in the murine system. No effect of IL-10 was seen on human dermal vascular endothelial cells, which like Langerhans cells are also able to present antigen. Additionally, no inhibitory effect of IL-10 was observed on the ICAM-1 expression of keratinocytes and dermal fibroblasts. As IL-10 only weakly suppresses MCH II on human Langerhans cells, inhibition of ICAM-1 and other accessory molecules by IL-10 seems to be an important mechanism inhibiting the antigen-presenting function of human Langerhans cells. Received: 9 December 1997     

Calcitonin gene-related peptide and adrenomedullin modulate cytokine-induced microvascular endothelial cell cellular adhesion molecule expression and NF-κB activation

AM and the sensory neuropeptide CGRP are potent vasoactive mediators that activate high-affinity G-protein-coupled receptors consisting of receptor-activity modifying proteins (RAMPs) and a seven-transmembrane domain calcitonin receptor-like receptor (CRLR) with RAMP-1/CRLR as CGRP and RAMP2 or -3/CRLR as AM receptors. In this study, we have examined the possibility that AM or CGRP modulate dermal microvascular EC adhesion molecule (ICAM-1 and VCAM-1) expression. Primary HDMEC or cells of the EC line HMEC-1 were transfected with cDNA expression vectors for an EGFP control, RAMP-1, RAMP-2 and CRLR by electroporation, or left untransfected. Stimulation of EC-overexpressing R1/CRLR or R2/CRLR with CGRP or AM (0.01–1000 n m ) resulted in a dose-dependent upregulation of intracellular cAMP. Importantly, when HDMEC transfected with R1/CRLR or R2/CRLR were treated with TNFα in combination with CGRP or AM, these peptides interfered with the TNF-induced expression of ICAM-1 and VCAM-1 as well as the adhesion of lymphoblastoid cell lines to HDMEC monolayer in a biphasic manner. Likewise, AM and CGRP modulated the activation of nuclear factor κB (NF-κB) partly by inhibiting the TNFα-induced degradation of cytosolic IκBα. Neither transfection with the orphan CRLR nor RAMPs alone was capable of mediating a full reduction of TNFα-induced ICAM-1 or VCAM-1 expression. In conclusion, CGRP and more pronounced AM are capable of modulating TNFα-induced EC CAM expression, which may be of importance for the regulation of leucocyte–endothelial cell interaction during cutaneous neurogenic inflammation.
This study was supported by the "Medizinische Forschungsgesellschaft Salzburg" and a grant of the Austrian Science Foundation (P14906).     

复方昆明山海棠对TNF-α诱导的人血管内皮细胞ICAM-1表达影响的试验研究

目的通过复方昆明山海棠对TNF-α诱导的人血管内皮细胞ICAM-1表达影响的研究,探讨复方昆明山海棠的抗炎机制。方法采用体外细胞培养、免疫组化技术观察复方昆明山海棠对TNF-α诱导的内皮细胞黏附分子ICAM-1表达的影响。结果复方昆明山海棠（0.05～2）mg/ml预处理30min,可不同程度地抑制TNF-α诱导的内皮细胞黏附分子ICAM-1的表达,与空白对照组相比较有统计学意义（P〈0.05）;到2mg/ml时ICAM-1的表达减到最弱（P〈0.01）。结论复方昆明山海棠能明显抑制TNF-α诱导的人血管内皮细胞表面黏附分子ICAM-1表达,（0.05-2）mg/ml范围内其抑制作用的强弱与浓度有关。提示复方昆明山海棠有可能是通过抑制人血管内皮细胞表面黏附分子ICAM-1的表达从而减少白细胞与血管内皮的黏附来发挥其抗炎作用的。