Intercellular Adhesion Molecule-1 In Response Research Articles

548-P: Baicalin Attenuates Fibrosis Process in Human Renal Proximal Tubular Cells in Hyperglycemic Condition

Background: Baicalin, isolated from Scutellaria baicalensis, has been reported to have anti-inflammatory effect by inhibiting expression of cell adhesion molecules and migration of leukocytes in vascular cells. However, there is a limited evidence on its anti-inflammatory effect in hyperglycemia-associated milieu in other cells. Hyperglycemia and its associated factors are known to induce diabetic nephropathy by exacerbating inflammation, especially, in proximal convoluted tubule. Herein, we investigated whether baicalin exerts anti-inflammatory and anti-fibrosis effects on human renal proximal tubular cells in hyperglycemia induced conditions. Method: HK-2 cell was maintained in normal glycemic (5.78mM glucose) or in hyperglycemic (30mM glucose) condition with or without advanced glycation end-products (AGE) and tumor necrosis factor alpha (TNFα). In each condition, baicalin was treated at 10uM. Cell viability was determined by MTT assay. Gene and protein expression of multiple inflammatory and fibrosis markers including toll-like receptor 2 (TLR2), TLR4, fibronectin, collagen IV α1 (ColIVα1), collagen IV α2 (ColIVα2), transforming growth factor β (TGFβ), intercellular adhesion molecule 1 (ICAM1), and vascular adhesion molecule 1 (VCAM1) were evaluated by PCR and WB, respectively. Results: Baicalin had no effect on cell viability. Baicalin significantly decreased gene expression of ColIVα2 in response to TNFα stimulation in normal glycemic condition (p=0.038). In hyperglycemic condition, baicalin decreased gene expressions of ICAM1 (p=0.049) and VCAM1 (p=0.049) significantly in AGE treated cells; and it significantly decreased gene expressions of TGFβ (p=0.038) and ICAM1 (p=0.017) in TNFα treated cells. Baicalin also decreased protein expression of ICAM1 in response to AGE (p=0.048) and TNFα (p=0.040) stimulation in hyperglycemic condition. Conclusion: Baicalin attenuated fibrosis process by hyperglycemia associated factors in HK-2 cell. Disclosure J. Nam: None. J. Kim: None. K. Park: None. S. Lee: None. J. Nam: None. J. Park: None. S. Park: None. Y. Kim: None. C. Ahn: None.

Bilirubin Inhibits the Transmigration of Monocytes Across Endothelial Cell Monolayers: Implications for Atherosclerosis Prevention

Monocyte infiltration into the vascular endothelium, a critical early step in the formation of atherosclerotic plaques, is mediated primarily by the binding of α and β integrins to endothelial cell adhesion molecules Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intercellular Adhesion Molecule-1 (ICAM-1). Our lab previously has shown that bilirubin, a potent, chain-breaking antioxidant, disrupts VCAM-1-dependent processes by scavenging reactive oxygen species (ROS) signaling intermediates. Therefore, we examined the effect of bilirubin on the transmigration of THP-1 human monocytic cells across TNF-α-activated human umbilical vein endothelial cell (HUVEC) monolayers. Bilirubin, at physiological concentrations (0 – 20 µM) was found to inhibit the movement of THP-1 cells across HUVEC monolayers in a dose-dependent manner. Bilirubin did not alter endothelial cell expression of VCAM-1 or ICAM-1 in response to TNF-α, nor did it inhibit the binding of THP-1 cells to HUVEC monolayers, supporting that bilirubin exerts its effect at the level of cellular transmigration. Studies employing blocking antibodies support that VCAM-1 is the principal adhesion molecule mediating monocyte transmigration. In light of epidemiological studies demonstrating an inverse association between serum bilirubin levels and the risk of cardiovascular disease, we speculate that bilirubin prevents plaque formation by inhibiting monocyte migration across the vascular endothelium through disruption of VCAM-1 signaling.

Benzoate X receptor zinc-finger gene switches for drug-inducible regulation of transcription

Targeted zinc-finger (ZF) DNA-binding domains in conjunction with nuclear receptor ligand-binding domains (LBDs) produce chemically inducible gene switches that have applications in gene therapy and proteomic and genomic research. The benzoate X receptor-β (BXRβ) LBD was used to construct homodimer and single-chain ZF transcription factors (ZF(TF)s). These ZF(TF)s specifically regulated the transcription of target genes in response to two ligands, ethyl-4-hydroxybenzoate and propyl-4-hydroxybenzoate, in a dose-dependent manner. The ZF(TF)s also regulated the expression of endogenous intercellular adhesion molecule-1 in response to either ligand. The advantage of BXRβ-based ZF(TF)s is that the ligands are inexpensive and easily synthetically modified, making the system a base for creation of orthogonal ligand-receptor pairs and expanding the gene-switch toolbox.

Generation and characterisation of human saphenous vein endothelial cell lines

Primary human endothelial cells have a finite life span in vitro. After 3–4 passages, they tend to de-differentiate and eventually reach senescence. This limits their use in studies of endothelial cell function. To overcome this, we have developed human saphenous vein endothelial cell lines (HSVEC lines). Two cell lines were produced by infection with pZipSVtsA58-U19 which encodes the simian virus 40 large T-antigen, and one cell line was obtained by transfection with pLXSN16E6E7, which encodes the human papillomavirus type 16 E6 and E7 genes. Two of the three HSVEC lines exhibited an extended life span in vitro and retained characteristic endothelial ‘cobblestone’ morphology. These cell lines expressed the known endothelial markers CD31 and vascular endothelial cadherin, and were able to bind Ulex europaeus lectin I, but they did not retain the expression of von Willebrand factor. Furthermore, one cell line was able to functionally up-regulate the expression of intercellular adhesion molecule-1 in response to stimulation with tumor necrosis factor α and was also able to incorporate acetylated low-density lipoprotein. Our results suggest that this latter HSVEC line will provide a useful resource to investigate selected responses of the vascular endothelium to physiological and pathological situations.

Santonin-related compound 2 inhibits the expression of ICAM-1 in response to IL-1 stimulation by blocking the signaling pathway upstream of IκB degradation

Santonin-related compounds (SRCs) were synthesized from the starting material l-α-santonin and tested for the biological activity on the expression of intercellular adhesion molecule-1 (ICAM-1) in response to IL-1 stimulation on human adenocarcinoma cells. One of the bromoketone derivatives termed SRC2 [(11S)-2α-bromo-3-oxoeudesmanno-13,6α-lactone] strongly inhibited the ICAM-1 expression at an IC50 value of 5.9 μM, whereas l-α-santonin itself was totally inactive up to 100 μM. The blockage of ICAM-1 expression by SRC2 was not due to the direct inhibition of de novo RNA and protein synthesis. The nuclear translocation of NF-κB subunit p65 was markedly prevented by SRC2. Moreover, IκBα degradation upon IL-1 stimulation was strongly inhibited by SRC2. These observations suggest that SRC2 blocks the IL-1 signaling pathway upstream of IκB degradation.

Effects of TNF-alpha on expression of ICAM-1 in human airway epithelial cells in vitro. Signaling pathways controlling surface and gene expression.

Signaling pathways associated with tumor necrosis factor (TNF)-alpha-induced intercellular adhesion molecule 1 (ICAM-1) surface and gene expression were investigated in well differentiated normal human bronchial epithelial (NHBE) cells in air-liquid interface primary culture. Cells were exposed to human recombinant TNF-alpha (hrTNF-alpha; 0.015 to 150 ng/ml [specific activity, 2.86 x 10(7) U/mg]). TNF-alpha enhanced ICAM-1 surface expression (measured by flow cytometry) and steady-state messenger RNA (mRNA) levels (assessed by Northern hybridization) in concentration- and time-dependent manners. TNF-alpha-induced ICAM-1 surface and gene expression were both blocked by the RNA polymerase II inhibitor actinomycin D (0.1 microg/ml), and surface expression was attenuated by a neutralizing monoclonal antibody directed against the TNF-alpha receptor p55 (TNF-RI). The intracellular signaling pathway leading to enhanced expression appeared to involve activation of a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC) because D609, a specific PC-PLC inhibitor, attenuated TNF-alpha-induced increases in production of diacyl-glycerol (DAG), a hydrolysis product of PC-PLC, and also attenuated TNF-alpha enhancement of ICAM-1 surface and gene expression. Because DAG formed by action of PC-PLC can activate protein kinase C (PKC), involvement of PKC was investigated. The specific PKC inhibitor calphostin C blocked both surface and gene expression of ICAM-1 in response to TNF-alpha in a concentration-dependent manner. Finally, TNF-alpha stimulated binding of p65 and/or c-rel complexes to the nuclear factor (NF)-kappaB consensus binding site found on the ICAM-1 promoter, and binding of these complexes was inhibited by D609. The results support the following pathway, whereby TNF-alpha enhances expression of ICAM-1 in NHBE cells: TNF-alpha --> TNF-RI --> PC-PLC --> DAG --> PKC --> (NF-kappaB?) --> ICAM-1 mRNA --> ICAM-1 surface expression.

Invasion of human mucosal epithelial cells by Neisseria gonorrhoeae upregulates expression of intercellular adhesion molecule 1 (ICAM-1).

Infection of the mucosa by Neisseria gonorrhoeae involves adherence to and invasion of epithelial cells. Little is known, however, about the expression by mucosal epithelial cells of molecules that mediate cellular interactions between epithelial cells and neutrophils at the site of gonococcal infection. The aim of this study was to determine the expression of intercellular adhesion molecule 1 (ICAM-1) by epithelial cells during the process of gonococcal invasion. The highly invasive strain FA1090 and the poorly invasive strain MS11 were incubated with human endometrial adenocarcinoma (HEC-1-B) or human cervical carcinoma (ME-180) epithelial cells, after which ICAM-1 expression was measured by flow cytometry. After 15 h of infection with FA1090, expression of ICAM-1 increased 4.7- and 2.1-fold for HEC-1-B and ME-180 cells, respectively, whereas 15 h of infection of HEC-1-B cells with MS11 increased ICAM-1 expression only 1.6-fold. ICAM-1 expression was restricted to the cell surface, since no soluble ICAM-1 was detected. The distribution of staining was heterogeneous and mimicked that seen after treatment of HEC-1-B cells with the ICAM-1 agonist tumor necrosis factor alpha (TNF-alpha) in the absence of bacteria. PCR and dot blot analyses of ICAM-1 mRNA showed no change in levels over time in response to infection. Although TNF-alpha was produced by HEC-1-B cells after infection, the extent of ICAM-1 upregulation was not affected by neutralizing anti-TNF-alpha antiserum. Dual-fluorescence flow cytometry showed that the cells with the highest levels of ICAM-1 expression were cells with associated gonococci. We conclude that epithelial cells upregulate the expression of ICAM-1 in response to infection with invasive gonococci. On the mucosa, upregulation of ICAM-1 by infected epithelial cells may function to maintain neutrophils at the site of infection, thereby reducing further invasion of the mucosa by gonococci.

Regulation of RANTES and ICAM-1 expression in murine mesangial cells.

Chemokines and adhesion molecules play a pivotal role in leukocyte infiltration during tissue injury. RANTES (regulated upon activation, normal T cell expressed and secreted) is a monocyte chemoattractant that induces the expression of CD11/CD18 integrins on leukocytes for which intercellular adhesion molecule-1 (ICAM-1) is the ligand. Both RANTES and ICAM-1 can be expressed by mesangial cells (MC) in culture and in glomeruli during immune injury. In this study, the role of reactive oxygen species (ROS) in the activation of RANTES and ICAM-1 in murine MC was examined. Tumor necrosis factor alpha (TNF-alpha) and aggregated immunoglobulin (aggr. Ig) G, which enhance ROS formation in MC, increased mRNA transcripts of both RANTES and ICAM-1. Thiol-containing free-radical scavengers N-acetyl cysteine, dimethyl- and tetramethylthiourea, or pyrrolidinedithiocarbamate abrogated the increase in mRNA for RANTES and ICAM-1 in response to TNF-alpha or IgG. Hydroxy-methoxy acetophenone, an inhibitor of NADPH-dependent oxidase, also attenuated RANTES and ICAM-1 in response to TNF-alpha or IgG. ROS generated by addition of xanthine oxidase and hypoxanthine induced RANTES and ICAM-1 expression, whereas hydrogen peroxide caused no response. Because cAMP can interfere with gene activation in MC, the effects of 8-Br-cAMP, forskolin, and prostaglandin E2 on mRNA levels were examined for RANTES and ICAM-1. These agents attenuated the response to IgG aggregates and also to superoxide generation. Finally, the effect of glucocorticoids, which are frequently used in glomerular immune injury, was examined. Dexamethasone decreased mRNA for both RANTES and ICAM-1 after stimulation with aggr. IgG or TNF-alpha. Both forskolin and dexamethasone also reduced the amount of RANTES protein secreted by MC in response to aggr. IgG. Only dexamethasone decreased RANTES secretion in response to TNF-alpha stimulation. The inhibitory effects of cAMP and dexamethasone may explain the beneficial effects of cAMP mimetics, such as prostaglandin E2 and glucocorticoid administration on glomerular inflammatory processes.

Pervanadate Mimics IFNγ-Mediated Induction of ICAM-1 Expression via Activation of STAT Proteins

Differential expression of intercellular adhesion molecule-1 (ICAM-1) in the epidermis plays a critical role in the regulation of cutaneous inflammation, immunologic reactions, and tissue repair. Transcriptional upregulation of ICAM-1 in response to interferon-gamma (IFNgamma) occurs through a palindromic response element pIgammaRE. pIgammaRE is homologous to IFNgamma-activated sequences, which bind to tyrosine phosphorylated members of the transcription factor family known as signal transducers and activators of transcription (STAT). The importance of tyrosine phosphorylation events in the STAT pathway led us to investigate the effect of the protein tyrosine phosphatase inhibitor, pervanadate, on ICAM-1 expression. We show that treatment of A431 cells and human keratinocytes with pervanadate stimulates protein complex formation on pIgammaRE in a time- and concentration-dependent manner. As demonstrated by mobility supershift assays, the pervanadate-stimulated complex is similar to the IFNgamma-stimulated complex and contains Stat1. Pervanadate treatment also led to an increase in overall protein tyrosine phosphorylation and phosphorylation of Stat1, as well as the subsequent increase in ICAM-1 mRNA and cell surface protein levels. These data show that pervanadate can mimic each step in the IFNgamma-mediated pathway leading to ICAM-1 expression, demonstrate the ability of a pharmacologic agent to bypass the standard cytokine-receptor interaction required for increased ICAM-1 expression, and emphasize the importance of protein tyrosine phosphatases and protein tyrosine kinases in mediating inflammatory responses in the skin.

Contrasting response of lung parenchymal cells to instilled TNF alpha and IFN gamma: the inducibility of specific cell ICAM-1 in vivo.

Induction of intercellular adhesion molecule-1 (ICAM-1) by proinflammatory cytokines during inflammation plays an important role in regulating polymorphonuclear neutrophil (PMN) migration and localization. In this report, we examined the effects of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) on specific lung cell expression of ICAM-1 in vivo and the accompanying morphological changes. Balb-c mice were treated with phosphate-buffered saline (PBS) alone or with PBS containing 5 micrograms TNF alpha or IFN gamma through intranasal instillation. Twenty-four hours after treatment, their lungs were processed for immunoblot analysis and electron microscope immunocytochemistry. In the normal lung, the ICAM-1 level is high on type I alveolar epithelial cells, medium on arterial and venous endothelial cells, low on type II epithelial cells and capillary endothelium, and not detectable on bronchial epithelium. Topical treatment of the lung with either TNF alpha or IFN gamma induced a 50-60% increase in total lung and alveolar ICAM-1. A dramatic increase of alveolar type II cell surface ICAM-1 was observed (> 20-fold). Both cytokines caused 2-3-fold higher ICAM-1 expression on capillary endothelial cells and a 40% increase of ICAM-1 on alveolar type I cells that was not uniform. However, due to the large total surface area of type I epithelium, type I cells contribute 70-86% of total alveolar septal ICAM-1 and > 90% of alveolar surface ICAM-1 in either treated or normal mouse lungs. Increased ICAM-1 expression was also observed on nonparenchymal endothelial and epithelial cells. Margination and sequestration of PMN in cytokine-treated lungs were observed by histologic examination, measurements of total lung myloperoxidase activity, and number of neutrophils recovered in bronchoalveolar lavage fluid. These results showed that TNF alpha and IFN gamma induce ICAM-1 expression and infiltration of neutrophils in the lung. The response of specific lung cells in terms of induction of ICAM-1 in response to cytokine stimulation varied significantly, particularly between type I and type II epithelial cells.

Failure to detect type 1 interferon production in human umbilical cord vein endothelial cells after viral exposure.

Cytokine production by endothelial cells is known to occur after the reception of signals such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). In the present study, we demonstrate cytokine release and upregulation of adhesion molecule expression of human endothelial cells derived from umbilical cord veins in response to stimulants. The cells were exposed to the plant lectin phytohemagglutinin A (PHA), the viral inducers Newcastle disease virus (NDV) and Sendai virus, and the nucleic acid analog polyinosinic/polycytidylic acid (polyI:C). All stimulants induced expression of IL-1 beta and IL-6. The titers of these cytokines were comparable to those obtained in human leukocytes, whereas no TNF-alpha release was detectable. A further feature of activation was upregulation of intercellular adhesion molecule 1 (ICAM-1), which was analyzed after contact with these stimulants. These experiments revealed an increased expression of ICAM-1 in response to all stimulants tested. A major part of our studies was devoted to the ability of endothelial cells to produce interferons as an important function of a number of cell types in response to viral infections. To ensure stimulation of the cells, the inducers NDV, Sendai virus, and polyI:C were analyzed. Expression of interferon (IFN) was not detected, although viral inducers mediated the release of IL-1 beta and IL-6, suggesting that endothelial cells are quite responsive to these stimulants. In conclusion, these findings show that the ability of endothelial cells to secrete cytokines is not restricted to mediators of the immune system, such as IL-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in human vascular smooth muscle cells.

Vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin are inducible proteins involved in cell-cell adhesion. Immunohistochemical studies have indicated that human atherosclerotic plaques contain smooth muscle cells (SMCs) that express ICAM-1 and VCAM-1. Recently, we demonstrated that SMCs in culture express a functionally active cytokine-inducible ICAM-1. SMCs and mononuclear cells participate in the local accumulation of cytokines and related growth factors in atherosclerotic lesions. Therefore, we determined the effects of different cytokines and growth factors on mRNA content and cell surface expression of VCAM-1, ICAM-1, and E-selectin in cultured human aortic SMCs by Northern blotting, quantitative polymerase chain reaction amplification, and immunofluorescence flow cytometry. Under basal conditions of cultivation, both VCAM-1 mRNA and membrane expression of VCAM-1 were low and were induced very little by interleukin-1 beta (100 U/mL). Platelet-derived growth factor or transforming growth factor-beta decreased VCAM-1 mRNA basal expression. Treatment of SMCs with tumor necrosis factor-alpha (TNF-alpha) led to an increase in both VCAM-1 mRNA and cell surface expression for VCAM-1 in a dose- and time-dependent manner. Interferon-gamma induced a weak increase in VCAM-1 mRNA expression, with no synergistic effect on the stimulation by TNF-alpha. Various differences were noted between the expression of ICAM-1 and VCAM-1 genes, because interleukin-1 beta induced substantial amounts of ICAM-1 but not VCAM-1. The addition of interferon-gamma delays the time at which peak expression of ICAM-1 in response to TNF-alpha stimulation occurs. Under our conditions, we did not detect any expression of E-selectin by SMCs. These results suggest that cytokines regulate VCAM-1 and ICAM-1 expression on arterial SMCs and could play an important role in the pathophysiology of inflammatory and immune processes in atherosclerosis.

In vitro cytokine modulation of intercellular adhesion molecule-1 expression on systemic sclerosis dermal fibroblasts.

Our objective was to study the regulation of intercellular adhesion molecule-1 (ICAM-1) expression by cytokines on cultured fibroblasts obtained from systemic sclerosis and normal skin. ICAM-1 expression on dermal fibroblasts obtained from diffuse systemic sclerosis patients with early disease (< or = 2 years) and normal dermal fibroblasts incubated with and without cytokines was measured by radioimmunoassay and flow cytometry. Systemic sclerosis dermal fibroblasts expressed lower basal levels of ICAM-1 than did normal dermal fibroblasts. Both the normal and systemic sclerosis dermal fibroblasts increased their cell surface expression of ICAM-1 in response to interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in a dose-dependent fashion. Systemic sclerosis dermal fibroblasts appeared to be hyperresponsive to IL-1 beta, TNF-alpha, and IFN-gamma. ICAM-1 expression in response to cytokine stimulation increased to a greater degree on systemic sclerosis compared to normal dermal fibroblasts. The enhanced ICAM-1 expression may play a role in the retention of leukocytes involved in systemic sclerosis skin lesions.

Adherence of neutrophils to canine cardiac myocytes in vitro is dependent on intercellular adhesion molecule-1.

The adhesiveness of isolated canine cardiac myocytes for neutrophils is greatly increased by stimulation with cytokines such as tumor necrosis factor alpha (TNF alpha). Since this adhesion is significantly inhibited by an anti-CD18 MAb, experiments were performed to test the hypothesis that the newly expressed adhesion molecule on the cardiac myocytes was intercellular adhesion molecule-1 (ICAM-1). A newly developed MAb, CL18/6, was found to exhibit the functional and binding characteristics with canine neutrophils and canine jugular vein endothelial cells expected of an antibody recognizing ICAM-1. MAb CL18/6 also bound to isolated cardiac myocytes after stimulation of the myocytes with cytokines, and it blocked by greater than 90% the adhesion of neutrophils to stimulated myocytes. A partial cDNA clone for canine ICAM-1 was isolated, and ICAM-1 mRNA was found to be increased in both endothelial cells and cardiac myocytes after cytokine stimulation. Cytokines that both increased the CL18/6-inhibitable adhesion of neutrophils to myocytes and induced expression of ICAM-1 were IL-1 beta, TNF alpha, and LPS. These results are consistent with the conclusion that canine endothelial cells and cardiac myocytes express ICAM-1 in response to cytokine stimulation, and that ICAM-1 functions as an adhesive molecule for neutrophils on both cell types.

The induction of intercellular adhesion molecule 1 (ICAM-1) expression on human fetal astrocytes by interferon-λ, tumor necrosis factor α, lymphotoxin, and interleukin-1: relevance to intracerebral antigen presentation

Antigen presentation reactions are dependent upon the expression of the class II major histocompatibility antigens (MHC), the T-cell receptor, and the presented antigen. Recent studies demonstrate that such processes also require the presence of adhesion molecules such as lymphocyte functional antigen 1 (LFA-1) and its cell surface ligand, intercellular adhesion molecule 1 (ICAM-1). It has been suggested that the brain astrocyte can function as a facultative antigen presenting cell (APC). This hypothesis is based upon th ability to induce the expression of the class II MHC antigens on astrocytes, and on their ability to present myelin basic protein on encephalitogenic T-cells in vitro. The best in vivo data showing that astrocytes serve as intracerebral APCs is the finding that astrocytes in multiple sclerosis plaques are DR + (class II MHC in human). However, it still remains to be resolved whether the in vivo expression of the MHC antigens in disease states is instrumental to antigen presentation mechanisms or whether these cell suface glycoproteins are expressed secondary to brain immune responses. If astrocytes function as immunocompetent APCs within the brain, it would seem that they would also be able to express molecules important for intercellular adhesion. Here, we present the first data that indicates that human astrocytes are capable of expressing ICAM-1 in response to cytokines that either induce or upregulate the expression of DR. In essence, cytokines derived from different cell types seem to exert similar pleiotropic effects on the modulation of MHC and ICAM-1 expression on astrocytes. This suggests that mechanisms critical to the initiation of brain immune responses involve a dynamic molecular interface between a variety of resident central nervous system and peripheral cell types.