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.     

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.     

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.     

Modulation of intercellular adhesion molecule-1 expression on human melanocytes and melanoma cells: evidence for a regulatory role of IL-6, IL-7, TNF beta, and UVB light.

Intercellular adhesion molecule-1 (ICAM-1) is involved in cell-cell interactions of leukocytes and parenchymal cells and thus plays an important role in immunologic and inflammatory reactions. The expression of ICAM-1 that is found on many different cells such as melanocytes and melanoma cells is induced by various cytokines, including interferon-gamma (IFN gamma), interleukin (IL)-1 and tumor necrosis factor alpha (TNF alpha). Because expression of ICAM-1 in melanoma was found to correlate with increased risk of metastasis, the regulation of ICAM-1 expression on human melanocytes and melanoma cells was investigated. Foreskin-derived melanocytes and melanoma cell lines (A375, G361) were incubated with different cytokines and ICAM-1 expression was evaluated by fluorescence-activated cell sorter. IFN gamma, IL-1, IL-7, TNF alpha, and TNF beta significantly upregulated ICAM-1 expression in a dose-dependent manner. Most interestingly, the cytokine IL-6, which does not influence adhesion-molecule expression on other cells, significantly upregulated melanocyte and melanoma cell ICAM-1 expression. This effect was dose dependent and could be blocked by an IL-6 antibody. Irradiation with ultraviolet (UVB) light did not influence constitutive ICAM-1 expression on melanoma cells and melanocytes, but suppressed cytokine-induced ICAM-1 expression when cells were harvested 16 h after irradiation. These findings were further confirmed by Northern blot analysis, showing a marked accumulation of ICAM-1 mRNA after cytokine treatment, which was reduced by irradiation with UVB light. However, when UVB-exposed melanoma cells were cultured for at least 48 h induction of ICAM-1 expression was observed. These data indicate that, similar to other cells, ICAM-1 expression on melanoma cells and melanocytes is regulated by cytokines and that UVB light affects ICAM-1 expression on melanocytic cells in a biphasic manner.     

Tumor necrosis factor beta and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression

Intercellular adhesion molecule-1 (ICAM-1) functions as a ligand of leukocyte function-associated antigen-1 (LFA-1), as well as a receptor for human picorna virus, and its regulation thus affects various immunologic and inflammatory reactions. The weak, constitutive ICAM-1 expression on human keratinocytes (KC) can be up-regulated by cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha). In order to further examine the regulation of KC ICAM-1 expression, normal human KC or epidermoid carcinoma cells (KB) were incubated with different cytokines and/or exposed to ultraviolet (UV) radiation. Subsequently, ICAM-1 expression was monitored cytofluorometrically using a monoclonal anti-ICAM-1 antibody. Stimulation of cells with recombinant human (rh) interleukin (IL) 1 alpha, rhIL-4, rhIL-5, rhIL-6, rh granulocyte/macrophage colony-stimulating factor (GM-CSF), rh interferon alpha (rhIFN alpha), and rh transforming growth factor beta (TGF beta) did not increase ICAM-1 surface expression. In contrast, rhTNF beta significantly up-regulated ICAM-1 expression in a time- and dose-dependent manner. Moreover, the combination of rhTNF beta with rhIFN gamma increased the percentage of ICAM-1-positive KC synergistically. This stimulatory effect of rhTNF beta was further confirmed by the demonstration that rhTNF beta was capable of markedly enhancing ICAM-1 mRNA expression in KC. Finally, exposure of KC in vitro to sublethal doses of UV radiation (0-100 J/m2) prior to cytokine (rhIFN tau, rhTNF alpha, rhTNF beta) stimulation inhibited ICAM-1 up-regulation in a dose-dependent fashion. These studies identify TNF beta and UV light as potent regulators of KC ICAM-1 expression, which may influence both attachment and detachment of leukocytes and possibly viruses to KC.     

55-kd tumor necrosis factor receptor is expressed by human keratinocytes and plays a pivotal role in regulation of human keratinocyte ICAM-1 expression.

Tumor necrosis factor alpha (TNF alpha) is a potent modulator of human keratinocyte intercellular adhesion molecule-1 (ICAM-1) expression. TNF alpha is known to exert its biologic effects by binding to specific cell-surface receptors. Two distinct TNF binding molecules, the 55-kd and the 75-kd TNF receptor (TNFR) recently have been found to be expressed by human cells. These two receptor types are independently regulated and differ markedly in their intracellular regions, indicating functional dichotomy. In order to gain further insight into the mechanisms underlying ICAM-1 regulation in human keratinocytes, in the present study, the receptor molecules mediating TNF alpha induced ICAM-1 upregulation in human keratinocytes was defined. Human keratinocyte TNFR expression was assessed using monoclonal antibodies that specifically recognize the 55-kd or the 75-kd TNFR. Using FACS analysis, normal (HNK) as well as transformed (KB) human keratinocytes were found to react with anti-55-kd TNFR, but not anti-75-kd TNFR antibodies. These immunofluorescence data were confirmed by Northern blot analysis revealing clearly detectable amounts of mRNA specific for the 55-kd TNFR in KB cells. Incubation of human keratinocytes with anti-55-kd TNFR antibodies at 37 degrees C for 24 h increased ICAM-1 expression in a TNF alpha-like fashion. Moreover, the well known synergistic effect of IFN gamma plus TNF alpha on keratinocyte ICAM-1 induction could be mimicked by stimulation of cells with IFN gamma plus anti-55-kd TNFR antibodies. Synergistic ICAM-1 induction was not associated with increased expression of the 55-kd TNFR in IFN gamma-stimulated human keratinocytes. These studies indicate that human keratinocytes express the 55-kd TNF receptor and that this surface molecule may play an important role in regulation of human keratinocyte ICAM-1 expression.     

Differential induction of intercellular adhesion molecule-1 in human skin by recombinant cytokines

We examine the effects of the recombinant epidermal cytokines interleukin 1α (IL-1α), interleukin 1β (IL-1β), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), tumor necrosis factor alpha (TNF), and the effect of the lymphokine gamma interferon (γ-IFN) on the expression of ICAM-1 in short term organ cultures of newborn human foreskins. In normal skin, ICAM-1 was detected immunohistochemically exclusively on endothelial cells. In addition to enhanced ICAM-1 expression by endothelial cells (at 1 h) and keratinocytes (by 6 h) exposed to γ-IFN, increased endothelial cell expression also resulted at 24 h after exposure to IL-6 and GM-CSF and at 48 h to M-CSF. Dermal dendritic cell reactivity for ICAM-1 was observed at 24 h after supplementation with IL-1α, IL-1β and IL-6, and at 48 h with GM-CSF and M-CSF. Incubation with culture medium alone or with IL-3 resulted in no change in baseline ICAM-1 expression on any cell type, and incubation with TNF resulted in enhanced ICAM-1 expression only by endothelial cells. Thus, in skin explains (as opposed to isolated cell in culture), ICAM-1 is induced on various cell types by a wide range of epidermal cytokines, including IL-6, GM-CSF, M-CSF, IL-1α and IL-1β Endogenous production and release of these cytokines which may stimulate adhesion molecule expression directly or indirectly may govern lymphoid adhesion to specific dermal as well as epidermal types of skin cells under physiologic and pathologic conditions.     

Characterization of intercellular adhesion molecule-1 and HLA-DR expression in normal and inflamed skin: modulation by recombinant gamma interferon and tumor necrosis factor

Lymphocytes bind to cultured keratinocytes that are treated with interferon gamma (IFN-gamma) and tumor necrosis factor (TNF). When the lymphocytes are preincubated with antibody to lymphocyte function associated antigen-1 (LFA-1), this adherence is inhibited. Because intercellular adhesion molecule-1 (ICAM-1) is a ligand for LFA-1, we studied the cellular expression of ICAM-1, as well as two other IFN-gamma-inducible antigens, (HLA) human lymphocyte antigens DR and DQ, in both normal and diseased skin. The modulation of these cell surface antigens by IFN-gamma and TNF with the use of short-term organ cultures of skin was compared with isolated keratinocytes grown in a conventional tissue culture system. While in normal skin, keratinocytes did not express HLA-DR, DQ, or ICAM-1, when organ cultures were supplemented with IFN-gamma, rapid induction of keratinocyte ICAM-1 expression occurred after 24 hours; HLA-DR but not DQ expression occurred after 48 hours. TNF also induced keratinocyte ICAM-1 expression (although to a lesser degree than IFN-gamma) but did not induce either keratinocyte HLA-DR or DQ expression. There was good correlation of keratinocyte expression of ICAM-1 and HLA-DR by IFN-gamma and TNF when the epidermis of the organ culture system was compared with the isolated keratinocytes grown in tissue culture. The presence of intraepidermal lymphocytes correlated extremely well with keratinocyte ICAM-1 expression but not with keratinocyte HLA-DR expression in psoriasis, atopic dermatitis, lichen planus, and mycosis fungoides. The intensity of endothelial cell expression of ICAM-1 correlated with the degree of dermal inflammation. We conclude that IFN-gamma, once produced by activated T lymphocytes in the dermis, may be of importance in lymphocyte trafficking in the epidermis by the induction of keratinocyte ICAM-1 expression. The use of the short-term organ culture system, in which there is inducible ICAM-1 expression, provides an experimental bridge between purely in vitro and in vivo investigations to further our understanding of the molecular basis for lymphocyte apposition to keratinocytes in the skin.     

Effects of dexamethasone and sex hormones on cytokine-induced cellular adhesion molecule expression in human endothelial cells

The cause or mechanism of the female predisposition in systemic lupus erythematosus and progressive systemic sclerosis is largely unknown. Accumulating evidence shows that dysfunction or activation of endothelial cells plays an important role in these conditions. In this study, we investigated the influence of various steroid hormones on the IL-1beta (50 U/mL)/TNF-alpha (50 U/mL) stimulated human dermal microvascular endothelial cell line (HMEC-1) and human umbilical vein endothelial cells (HUVEC). Dexamethasone showed significant inhibition of cytokine-induced ICAM-1 expression in HMEC-1, and E-selectin, VCAM-1 and ICAM-1 expressions in HUVEC. Androgens, especially dihydrotestosterone had a small, but statistically significant suppressive effect in HMEC-1 only. Estrogen exhibited no regulatory function in either cell line. No obvious expression of estrogen and androgen receptors could be demonstrated in either cell by immunostaining. Our study provided some pharmacological evidence that the superior anti-inflammatory effect of glucocorticoids on vasculitis was partly due to their inhibition of the CAM expression in endothelial cells. More studies are needed to determine if androgens could have a protective effect in vasculitis or vasculopathy associated with connective tissue diseases.     

Immunophenotypic analysis of human endothelial cells

Very Little is known about the immunological attributes of human endothelial cells. In this study, we performed immunologic phenotypic analysis of cultured human dermal microvascular endothelial cells in comparison with human umbilical vein endothelial cells and examined the ability of various biologic response modifiers to alter the phenotypes. Using FACS analysis, both types of the cells appear to lack many of the cell surface markers of immunologically proficient cells, E.G. OKT4, OKT8, Leu7, FcIgG receptor, complement receptors, IL-2 receptor and HLA-Dr, but they possess beta 2-microglobulin and DAF. HLA-Dr antigens can be induced on both types of endothelial cells by gamma-IFN in a dose and time dependent manner. Both types of endothelial cells possess several kinds of Cell Adhesion Molecules (CAMs), such as ICAM-1, CD44, LFA-3, but not LFA-1 or CD2. ICAM-1 but not LFA-3 or CD44 can be upregulated by exposure of both types of endothelial cells to gamma-IFN, IL-1 and TNF. These data suggest that endothelial cells of the dermal microvasculature may play central roles in a variety of different cutaneous inflammation.     

SLE患者皮损部ICAM-1 LFA-1 VCAM-1 ELAM-1的表达

以37例SLE患者为实验对象，应用LSAB试剂盒，用ABC法研究了SLE患者皮损部I－CAM－1、LFA－1、VCAM－1、ELAM－1的表达。结果表皮细胞表面ICAM－1、血管内皮细胞表面ELAM-1、VCAM－1、ICAM－1及浸润细胞表面ICAM－1、LFA－1的表达均增强。说明细胞表面粘附分子与SLE皮损发生有关。     

Comparative analysis of surface antigens in cultured human outer root sheath cells and epidermal keratinocytes: persistence of low expression of class I MHC antigens in outer root sheath cells in vitro

In the anagen human hair follicle, the epithelial cells from the infrainfundibular portion and the hair matrix cells express markedly lower numbers of major histocompatibility complex class I molecules than interfollicular epidermal keratinocytes. During the catagen phase of the hair cycle, class I expression on these cells increases, and activated macrophages aggregate around the follicle, which has led to the hypothesis that the cells to be resorbed are recognized by virtue of their low class I antigen expression. In the present study, we showed that, in vitro, outer root sheath cells also maintain a lower constitutive expression of MHC class I molecules compared with epidermal keratinocytes. In contrast, other surface antigens such as HLA-DR, -DP and -DQ, ICAM-1, LFA-3 and CD29, which are all known to participate in leucocyte-keratinocyte interactions, were similarly expressed in both cell types. Furthermore, interferon gamma strongly upregulated MHC class I and II and ICAM-1 expression in both cell types, whereas CD29 and LFA-3 remained unaffected. Tumour necrosis factor alpha, to a lesser extent, also upregulated MHC class I and ICAM-1 expression, but not class II expression. The differences in constitutive surface antigen expression of infrainfundibular outer root sheath cells compared with interfollicular epidermal keratinocytes emphasizes a distinct role of this cell type in the hair cycle, and possibly also in alopecia areata.     

Increased ICAM-1 expression causes endothelial cell leakiness, cytoskeletal reorganization and junctional alterations

Tumor necrosis factor (TNF)-induced ICAM-1 in endothelial cells (EC) promotes leukocyte adhesion. Here we report that ICAM-1 also effects EC barrier function. Control- or E-selectin-transduced human dermal microvascular EC (HDMEC) form a barrier to flux of proteins and to passage of current (measured as transendothelial electrical resistance or TEER). HDMEC transduced with ICAM-1 at levels comparable to that induced by TNF show reduced TEER, but do so without overtly changing their cell junctions, cell shape, or cytoskeleton organization. Higher levels of ICAM-1 further reduce TEER, increase F/G-actin ratios, rearrange the actin cytoskeleton to cause cell elongation, and alter junctional zona occludens 1 and vascular endothelial-cadherin staining. Transducing with ICAM-1 lacking an intracellular region also reduces TEER. TNF-induced changes in TEER and shape follow a similar time course as ICAM-1 induction; however, the fall in TEER occurs at lower TNF concentrations. Inhibiting NF-kappaB activation blocks ICAM-1 induction; TEER reduction, and shape change. Specific small-interfering RNA knockdown of ICAM-1 partially inhibits TNF-induced shape change. We conclude that moderately elevated ICAM-1 expression reduces EC barrier function and that expressing higher levels of ICAM-1 affects cell junctions and the cytoskeleton. Induction of ICAM-1 may contribute to but does not fully account for TNF-induced vascular leak and EC shape change.     

Modulation of melanocyte intercellular adhesion molecule-1 by immune cytokines

Human melanocyte expression of intercellular adhesion molecule-1 (ICAM-1) with or without stimulation by interferon gamma (IFN-G), tumor necrosis factor alpha (TNF-alpha), or interleukin-1-alpha (IL-1 alpha), was measured utilizing direct immunofluorescence and fluorescence-activated cell sorting (FACS). Melanocytes grown in vitro expressed low levels of ICAM-1, which could be increased by exposing the cells to IFN-G, TNF-alpha, or IL-1 alpha. Each cytokine caused an enhancement of melanocyte ICAM-1 expression in a dose-dependent fashion. The lowest dose necessary to cause a significant increase in melanocyte ICAM-1 expression was 1 U/ml IFN-G, 0.3 ng/ml TNF-alpha, or 3 U/ml IL-1 alpha. Melanocytes were most sensitive to TNF-alpha stimulation, with the greatest levels of ICAM-1 expression following 30 ng/ml or more TNF-alpha. When IFN-G was added to melanocyte cultures in combination with TNF-alpha or IL-1 alpha, there was an additive increase in ICAM-1 expression but no synergy was noted with the combined cytokines. To our knowledge, this is the first report of melanocyte ICAM-1 induction by TNF-alpha and IL-1 alpha and by physiologically relevant doses of IFN-G. Because of the importance of ICAM-1 in the regulation of immune cell-target interactions, the study of ICAM-1 expression by melanocytes may help us to better understand immune mechanisms of melanocyte injury.