Blood pressure and TNF-α act synergistically to increase leucocyte CD11b adhesion molecule expression in the BELFAST study: implications for better blood pressure control in ageing

Hypertension, a key risk factor for stroke, cardiovascular disease and dementia, is associated with chronic vascular inflammation, and although poorly understood, putative mechanisms include proinflammatory responses induced by mechanical stretching, with cytokine release and associated upregulated expression of adhesion molecules. Because blood pressure increases with age, we measured baseline and tumour necrosis alpha (TNF-a)-stimulated CD11b/CD18 adhesion molecule expression on leucocytes to assess any association between the two. In 38 subjects (mean age 85 years), consecutively enrolled from Belfast Elderly Longitudinal Free-Living Aging Study (BELFAST), baseline and TNF-a-stimulated CD11b/CD18 expression on separated monocytes and neutrophils increased with systolic blood pressure >120 mmHg (p=0.05) and for lymphocytes, with diastolic blood pressure >80 mmHg (p<0.05).These findings show increased potential stickiness of intravascular cells with increasing blood pressure which is accentuated by TNF-a, and suggest mechanistic reasons why better hypertension control is important. 

Tumor necrosis factor-α induces adhesion molecule expression through the sphingosine kinase pathway

The signaling pathways that couple tumor necrosis factor-α (TNFα) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFα induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFα resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFα on endothelial cells leading to extracellular signal-regulated kinases and NF-κB activation, whereas ceramide or sphingosine was not. Furthermore, N,N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFα-induced extracellular signal-regulated kinases and NF-κB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFα-induced endothelial cell activation.

CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression.

Cultured human umbilical vein endothelial cells (EC) constitutively express a low level of CD40 antigen as detected by monoclonal antibody binding and fluorescence flow cytometric quantitation. The level of expression on EC is increased about 3-fold following 24 h treatment with optimal concentrations of tumor necrosis factor, interleukin 1, interferon beta, or interferon gamma; both interferons show greater than additive induction of CD40 when combined with tumor necrosis factor or interleukin 1. Expression of CD40 increases within 8 h of cytokine treatment and continues to increase through 72 h. A trimeric form of recombinant murine CD40 ligand acts on human EC to increase expression of leukocyte adhesion molecules, including E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1. CD40 may be detected immunocytochemically on human microvascular EC in normal skin. We conclude that endothelial CD40 may play a role as a signaling receptor in the development of T-cell-mediated inflammatory reactions.

Up-regulation of the endothelial cell adhesion molecule intercellular adhesion molecule-1 (ICAM-1) by autoantibodies in autoimmune vasculitis

Autoimmune vasculitis is characterized by the presence of autoantibodies, particularly anti-neutrophil cytoplasmic antibodies (ANCA) and anti-nuclear antibodies (ANA), in patient sera. These autoantibodies have an incompletely understood role in development of vascular injury. The expression or up-regulation of cell adhesion molecules is an early phase in the development of an inflammatory vascular lesion. Autoantibody-positive sera from patients with vasculitis were assessed for their ability to modulate adhesion molecule expression by human umbilical vein endothelial cells (HUVEC). Autoantibody-positive serum samples from 11 out of 21 patients with primary vasculitis produced substantial up-regulation of ICAM-1 on HUVEC. Autoantibody-negative samples did not produce adhesion molecule up-regulation. Up-regulation of adhesion molecules on HUVEC was observed with samples positive for ANA, a phenomenon not previously reported. Preincubation of the sera with purified antigens recognized by ANCA failed to block this activation. In addition, MoAbs to ANCA antigens were ineffective at inducing ICAM-1 up-regulation, suggesting that activation is independent of the molecular specificity of the antibody. This capacity of ANCA- and ANA-positive sera to up-regulate adhesion molecules on endothelial cells may be a factor in the vessel wall inflammation seen in ANCA-associated vasculitis.

Cell adhesion molecule uvomorulin expression in human breast cancer cell lines : relationship to morphology and invasive capacities

Loss of cell-cell adhesion in carcinoma cells may be an important step in the acquisition of an invasive, metastatic phenotype. We have examined the expression of the epithelial-specific cell adhesion molecule uvomorulin (E-cadherin, cell-CAM 120/80, L-CAM) in human breast cancer cell lines. We find that fibroblastoid, highly invasive, vimentin-expressing breast cancer cell lines do not express uvomorulin. Of the more epithelial-appearing, less invasive, keratin-expressing breast cancer cell lines, some express uvomorulin, and some do not. We examined the morphologies of the cell lines in the reconstituted basement membrane matrix Matrigel and measured the ability of the cells to traverse a Matrigel-coated filter as in vitro models for detachment of carcinoma cells from neighboring cells and invasion through basement membrane into surrounding tissue. Colonies of uvomorulin-positive cells have a characteristic fused appearance in Matrigel, whereas uvomorulin-negative cells appear detached. Cells which are uvomorulin negative and vimentin positive have a stellate morphology in Matrigel. We show that uvomorulin is responsible for the fused colony morphology in Matrigel since treatment of uvomorulin-positive MCF-7 cells with an antibody to uvomorulin caused the cells to detach from one another but did not induce invasiveness in these cells, as measured by their ability to cross a Matrigel-coated polycarbonate filter in a modified Boyden chamber assay. Two uvomorulin-negative, vimentin-negative cell lines are also not highly invasive as measured by this assay. We suggest that loss of uvomorulin-mediated cell-cell adhesion may be one of many changes involved in the progression of a carcinoma cell to an invasive phenotype.

Differences in expression of junctional adhesion molecule-A and beta catenin in multiple sclerosis brain tissue: increasing evidence for the role of tight junction pathology

Previously we have employed antibodies to the tight junction (TJ)-associated proteins ZO-1 and occludin to describe endothelial tight junction abnormalities, in lesional and normal appearing white matter, in primary and secondary progressive multiple sclerosis (MS). This work is extended here by use of antibodies to the independent TJ-specific proteins and junctional adhesion molecule A & B (JAM-A, JAM-B). We have also assessed the expression in MS of ß-catenin, a protein specific to the TJ-associated adherens junction. Immunocytochemistry and semiquantitative confocal microscopy for JAM-A and ß-catenin was performed on snap-frozen sections from MS cases (n = 11) and controls (n = 6). Data on 1,443 blood vessels was acquired from active lesions (n = 13), inactive lesions (n = 13), NAWM (n = 20) and control white matter (n = 13). In MS abnormal JAM-A expression was found in active (46%) and inactive lesions (21%), comparable to previous data using ZO-1. However, a lower level of TJ abnormality was found in MS NAWM using JAM-A (3%) compared to ZO-1 (13%). JAM-B was strongly expressed on a small number of large blood vessels in control and MS tissues but at too low a level for quantitative analysis. By comparison with the high levels of abnormality observed with the TJ proteins, the adherens junction protein ß-catenin was normally expressed in all MS and control tissue categories. These results confirm, by use of the independent marker JAM-A, that TJ abnormalities are most frequent in active white matter lesions. Altered expression of JAM-A, in addition to affecting junctional tightness may also both reflect and affect leukocyte trafficking, with implications for immune status within the diseased CNS. Conversely, the adherens junction component of the TJ, as indicated by ß-catenin expression is normally expressed in all MS and control tissue categories.

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-kappa B ACTIVATION IN LPS-INDUCED ACUTE LUNG INJURY IN RATS

Diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment. The goal of this study was to investigate whether insulin regulates transcription of cytokines and intercellular adhesion molecule 1 (ICAM-1) via nuclear factor-kappa B (NF-kappa B) signaling pathway in Escherichia coli LIPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, iv., 10 days) and controls were instilled intratracheally with saline containing LPS (750 mu g/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LIPS. Analyses performed 6 h after LPS included: (a) lung and mesenteric lymph node IL-1 beta, TNF-alpha, IL-10, and ICAM-1 messenger RNA (mRNA) were quantified by real-time reverse transcriptase-polymerase chain reaction; (b) number of neutrophils in the bronchoalveolar lavage (BAL) fluid, and concentrations of IL-1 beta, TNF-alpha, and IL-10 in the BAL were determined by the enzyme-linked immunosorbent assay; and (c) activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were quantified by Western blot analysis. Relative to controls, diabetic rats exhibited a reduction in lung and mesenteric lymph node IL-1 beta (40%), TNF-alpha (similar to 30%), and IL-10 (similar to 40%) mRNA levels and reduced concentrations of IL-1 beta (52%), TNF-alpha (62%), IL-10 (43%), and neutrophil counts (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were almost suppressed in diabetic rats. Treatment of diabetic rats with insulin completely restored mRNA and protein levels of these cytokines and potentiated lung ICAM-1 mRNA levels (30%) and number of neutrophils (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were partially restored by insulin treatment. In conclusion, data presented suggest that insulin regulates transcription of proinflammatory (IL-1 beta, TNF-alpha) and anti-inflammatory (IL-10) cytokines, and expression of ICAM-1 via the NF-kappa B signaling pathway.

Vascular cell adhesion molecule 1 expression by biliary epithelium promotes persistence of inflammation by inhibiting effector T-cell apoptosis

Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM-1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding a4b1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM-1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM-1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM-1 promotes the survival of intrahepatic a4b1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova-bil antigen-driven murine model of biliary inflammation. VCAM-1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM-1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin-17. Liver-derived T cells adhered to cholangiocytes in vitro by a4b1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen-activated protein kinase phosphorylation. This led to increased mitochondrial B-cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM-1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 Tcell survival. Conclusions: VCAM-1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to a4b1 expressing proinflammatory T lymphocytes in CLD.

Optimal selectin-mediated rolling of leukocytes during inflammation in vivo requires intercellular adhesion molecule-1 expression

Leukocyte interactions with vascular endothelium during inflammation occur through discrete steps involving selectin-mediated leukocyte rolling and subsequent firm adhesion mediated by members of the integrin and Ig families of adhesion molecules. To identify functional synergy between selectin and Ig family members, mice deficient in both L-selectin and intercellular adhesion molecule 1 (ICAM-1) were generated. Leukocyte rolling velocities in cremaster muscle venules were increased significantly in ICAM-1-deficient mice during both trauma- and tumor necrosis factor α-induced inflammation, but rolling leukocyte flux was not reduced. Elimination of ICAM-1 expression in L-selectin-deficient mice resulted in a sharp reduction in the flux of rolling leukocytes during tumor necrosis factor α-induced inflammation. The observed differences in leukocyte rolling behavior demonstrated that ICAM-1 expression was required for optimal P- and L-selectin-mediated rolling. Increased leukocyte rolling velocities presumably translated into decreased tissue emigration because circulating neutrophil, monocyte, and lymphocyte numbers were increased markedly in L-selectin/ICAM-1-deficient mice. Furthermore, neutrophil emigration during acute peritonitis was reduced by 80% in the double-deficient mice compared with either L-selectin or ICAM-1-deficient mice. Thus, members of the selectin and Ig families function synergistically to mediate optimal leukocyte rolling in vivo, which is essential for the generation of effective inflammatory responses.

Down-Regulation of Platelet Endothelial Cell Adhesion Molecule-1 Results in Thrombospondin-1 Expression and Concerted Regulation of Endothelial Cell Phenotype

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell–cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND.3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a “switch” that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.

Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene

UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing ICAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation- and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and/or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response.

A binding site for homeodomain and Pax proteins is necessary for L1 cell adhesion molecule gene expression by Pax-6 and bone morphogenetic proteins

The cell adhesion molecule L1 regulates axonal guidance and fasciculation during development. We previously identified the regulatory region of the L1 gene and showed that it was sufficient for establishing the neural pattern of L1 expression in transgenic mice. In the present study, we characterize a DNA element within this region called the HPD that contains binding motifs for both homeodomain and Pax proteins and responds to signals from bone morphogenetic proteins (BMPs). An ATTA sequence within the core of the HPD was required for binding to the homeodomain protein Barx2 while a separate paired domain recognition motif was necessary for binding to Pax-6. In cellular transfection experiments, L1-luciferase reporter constructs containing the HPD were activated an average of 4-fold by Pax-6 in N2A cells and 5-fold by BMP-2 and BMP-4 in Ng108 cells. Both of these responses were eliminated on deletion of the HPD from L1 constructs. In transgenic mice, deletion of the HPD from an L1-lacZ reporter resulted in a loss of β-galactosidase expression in the telencephalon and mesencephalon. Collectively, our experiments indicate that the HPD regulates L1 expression in neural tissues via homeodomain and Pax proteins and is likely to be a target of BMP signaling during development.

Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.