Cell Surface ICAM-1 Research Articles

T7.2 Deported apoptotic syncytial knots have a role in the maternal vascular adaptation to normal pregnancy

Background: Preeclampsia is characterised by an exaggerated inflammatory response and maternal endothelial cell activation. Syncytial knots are multinucleated fetal cells shed from the placenta in large numbers during all pregnancies which may be phagocytosed by endothelial cells. Our previous studies showed that phagocytosis of necrotic but not apoptotic syncytial knots led to endothelial cell activation. It is known that phagocytosis of apoptotic cells leads to active tolerance of immune responses and in this study we questioned whether phagocytosis of apoptotic syncytial knots leads to suppression of the endothelial cells ability to be activated. Methods: Syncytial knots were harvested from 1st trimester placental explants. Monolayers of endothelial cells were pre-treated with apoptotic syncytial knots for 24 hours. After washing, the endothelial cells were treated with the endothelial cell activators LPS, PMA, IL-6, or necrotic syncytial knots for 24 hours. In some experiments the inhibitor of phagocytosis, cytochalasin D, was added into the cultures along with apoptotic syncytial knots. Endothelial cell-surface ICAM-1 was measured using cell based ELISAs. Results: Expression of ICAM-1 by endothelial cells that had phagocytosed apoptotic syncytial knots prior to treatment with LPS, PMA, IL-6, or necrotic syncytial knots was significantly (p≤0.003) reduced, compared to control endothelial cells that had not phagocytosed apoptotic syncytial knots. Inhibiting phagocytosis of apoptotic syncytial knots with cytochalasin D abolished this protective effect. Conclusions: Our data suggest phagocytosis of apoptotic syncytial knots results in the suppression of the ability of endothelial cells to be activated by a number of potent chemical activators, as well as by the physiologically relevant activator, necrotic syncytial knots. This work suggests that the release of apoptotic syncytial knots from the placenta during normal pregnancy may be a mechanism by which the fetus attempts to protect the maternal vasculature against activation.

A phosphodiesterase 4 inhibitor, roflumilast N-oxide, inhibits human lung fibroblast functions in vitro

The PDE4 inhibitor roflumilast mitigates bleomycin-induced lung fibrotic remodeling in rodents. In the current study it was explored whether roflumilast N-oxide, the active metabolite of roflumilast influences functions of cultured lung fibroblasts. Cells of the human foetal lung fibroblast strain GM06114 were stimulated with TNF-α (5 ng ml −1) and cell surface ICAM-1 and eotaxin release were assessed. [ methyl- 3H] thymidine incorporation was measured following stimulation with bFGF (10 ng ml −1). α-Smooth muscle actin (protein), CTGF (mRNA) and fibronectin (mRNA) were determined secondary to TGFß1 (1 ng ml −1). In the presence of PGE 2 (1 nM), roflumilast N-oxide reduced TNF-α-induced ICAM-1 and eotaxin by about 70% and >90% with half-maximum inhibition at 0.9 nM and 0.5 nM, respectively. Roflumilast N-oxide also attenuated [ methyl- 3H] thymidine incorporation secondary to bFGF by about 75% with half-maximum inhibition at 0.7 nM when cells were co-incubated with IL-1ß (10 pg ml −1). In the presence of this cytokine roflumilast N-oxide (1 μM) diminished TGFß1-induced expression of α-smooth muscle actin and transcripts of CTGF and fibronectin. In addition, IL-1ß up-regulated PDE4 activity in the lung fibroblasts. Taken together, these findings indicate that roflumilast N-oxide directly targets human lung fibroblasts, which may at least partially explain the efficacy of roflumilast to mitigate a pulmonary fibrotic response in vivo.

160. THE ROLE OF PHAGOCYTOSIS OF APOPTOTIC SYNCYTIAL KNOTS IN THE PREVENTION OF ENDOTHELIAL CELL ACTIVATION: AN IMPORTANT ADAPTATION FOR NORMAL PREGNANCY

Preeclampsia is characterised by an exaggerated inflammatory response and maternal endothelial cell activation. Syncytial knots, dead multinucleated fetal cells shed from the placenta in large numbers during all pregnancies, may be phagocytosed by maternal endothelial cells. Our previous studies showed that phagocytosis of necrotic but not apoptotic syncytial knots led to endothelial cell activation. It is known that phagocytosis of apoptotic cells leads to active tolerance of immune responses and in this study we questioned whether phagocytosis of apoptotic syncytial knots leads to suppression of the endothelial cells ability to be activated. Syncytial knots were harvested from 1st trimester placental explants. Monolayers of endothelial cells were pre-treated with apoptotic syncytial knots for 24 h. After washing, the endothelial cells were treated with the endothelial cell activators LPS, PMA, IL-6, or necrotic syncytial knots for 24 h. In some experiments the inhibitor of phagocytosis, cytochalasin D, was added into the cultures along with apoptotic syncytial knots. Endothelial cell-surface ICAM-1 was measured using cell based ELISAs. Expression of ICAM-1 by endothelial cells that had phagocytosed apoptotic syncytial knots prior to treatment with LPS, PMA, IL-6, or necrotic syncytial knots was significantly (P =/<0.003) reduced, compared to control endothelial cells that had not phagocytosed apoptotic syncytial knots. Inhibiting phagocytosis of apoptotic syncytial knots with cytochalasin D abolished this protective effect. Our data suggest phagocytosis of apoptotic syncytial knots results in the suppression of the ability of endothelial cells to be activated by a number of potent chemical activators, as well as by the physiologically relevant activator, necrotic syncytial knots. This work suggests that the release of apoptotic syncytial knots from the placenta during normal pregnancy may be a mechanism by which the fetus attempts to protect the maternal vasculature against activation.

Anti-phospholipid Antibodies Increase Non-apoptotic Trophoblast Shedding: A Contribution to the Pathogenesis of Pre-eclampsia in Affected Women?

Pre-eclampsia is associated with trophoblast shedding–deportation and endothelial cell dysfunction. Anti-phospholipid autoantibodies increase a women's risk factor of developing pre-eclampsia. In this study we examined the hypothesis that anti-phospholipid antibodies alter the number and nature of trophoblasts shed from the placenta, and that phagocytosis of these altered trophoblasts results in endothelial cell activation. To investigate this we used a placental explant model in which explants were treated with anti-phospholipid antibodies. This treatment resulted in a doubling of the amount of trophoblast shed from the explants. Furthermore, the trophoblasts shed from anti-phospholipid antibody-treated explants were more readily phagocytosed by endothelial cells and subsequently caused the activation of the endothelial cells, as indicated by increased expression of endothelial cell surface ICAM-1 determined by cell-based ELISA, and monocyte adhesion as determined by flow cytometry. Confocal microscopy analysis of trophoblasts shed from anti-phospholipid antibody-treated or control explants demonstrated that anti-phospholipid antibodies, but not control antibodies, were internalised within trophoblasts shed from the explants, and this was accompanied by a reduction in the activity of caspases 3 and 7 in the shed trophoblasts as indicated by FLICA. These results suggest that anti-phospholipid antibodies are selectively transported into trophoblasts where they affect the regulation of the cell cycle leading to excess and aberrant death (necrotic or aponecrotic) and shedding of trophoblasts. If reflected in vivo this might explain, at least in part, how anti-phospholipid antibodies contribute to the pathogenesis of pre-eclampsia.

Odoroside A and ouabain inhibit Na +/K +-ATPase and prevent NF-κB-inducible protein expression by blocking Na +-dependent amino acid transport

Inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin-1 (IL-1), trigger the activation of transcription factor NF-κB that induces the expression of a variety of genes, including intercellular adhesion molecule (ICAM)-1. Odoroside A [3 β-O-( β- d-diginosyl)-14-hydroxy-5 β,14 β-card-20(22)-enolide] was found to inhibit the cell-surface expression of ICAM-1 induced by TNF-α and IL-1 at comparable concentrations in human lung carcinoma A549 cells. In this study, the molecular mechanism underlying the inhibition of TNF-α-induced cell-surface ICAM-1 expression by odoroside A together with the specific Na +/K +-ATPase inhibitor ouabain was further investigated. Odoroside A and ouabain neither prevented IκBα degradation nor NF-κB translocation to the nucleus upon TNF-α stimulation. While odoroside A and ouabain had no inhibitory effect on the induction of ICAM-1 mRNA, they inhibited the TNF-α-induced ICAM-1 expression at the protein level. Consistent with these results, odoroside A and ouabain potently reduced de novo protein synthesis, largely due to its ability to block Na +-dependent transport of amino acids across the plasma membrane, but not to interfering with the translation machinery. As a direct molecular target, odoroside A was found to inhibit the ATP-hydrolyzing activity of Na +/K +-ATPase as potently as ouabain. These results clearly demonstrate that odoroside A and ouabain prevent NF-κB-inducible protein expression by blocking the Na +-dependent amino acid transport.

Microfilaments Facilitate TLR4-Mediated ICAM-1 Expression in Human Aortic Valve Interstitial Cells

Inflammation is involved in the pathogenesis of calcific aortic valve stenosis, and aortic valve interstitial cells (AVICs) play an important role in valve disease and remodeling. We have previously shown that human AVICs express functional toll-like receptor 4 (TLR4), and express increased levels of ICAM-1 in response to stimulation with bacterial lipopolysaccharide (LPS). This study examines (1) the role of TLR4 in regulating ICAM-1 expression in human AVICs, (2) the interaction of ICAM-1 with microfilaments, and (3) the influence of microfilament integrity on ICAM-1 expression. AVICs isolated from explanted human hearts were stimulated with LPS (0.2 μg/mL) to induce ICAM-1 expression, as shown by Western blot. TLR4 activity was influenced with a neutralizing antibody or by gene knockdown with siRNA. Flow cytometry and immunofluorescence were used to characterize membrane localization of ICAM-1. Immunoprecipitation and immunofluorescence were employed to show colocalization of ICAM-1 with microfilaments. Microfilament function was influenced with an actin polymerization inhibitor as well as siRNA. Cellular ICAM-1 levels increased 10-fold after 24 h of LPS stimulation, and this increase was significantly attenuated by prior treatment with TLR4-neutralizing antibody or TLR4 siRNA. ICAM-1 exhibited a filamentous distribution pattern after 4 h LPS stimulation, which was associated with increased total and cell-surface ICAM-1 expression. ICAM-1 colocalized and coprecipitated with the microfilament proteins F-actin and α-actinin 1. Pretreatment with the actin polymerization inhibitor latrunculin B and silencing α-actinin 1 with siRNA reduced LPS-induced total and cell-surface ICAM-1 expression. This study demonstrates that (1) LPS-stimulated ICAM-1 expression in human AVICs is mediated by TLR4, (2) ICAM-1 interacts with microfilaments soon after LPS stimulation, and (3) microfilament disruption reduces ICAM-1 expression. These results suggest that the interaction between ICAM-1 and microfilaments facilitates LPS-induced ICAM-1 expression in human AVICs.

106. IL-6 INCREASES THE SHEDDING OF NECROTIC TROPHOBLASTS FROM PLACENTAL EXPLANTS

Preeclampsia (PE) is characterised by elevated maternal blood pressure, preceded by endothelial cell dysfunction. Dead trophoblasts, shed from the placenta may be one of the factors that trigger PE. Women with PE frequently have elevated serum levels of inflammatory markers such as, IL-6 and TNF a but their functional significance is unclear. In this study we investigated whether these or other cytokines can alter trophoblast shedding from placental explants. Placental explants were treated with 9 different cytokines for 72 hours. Shed trophoblasts then were harvested using our published method1. The numbers of trophoblasts shed were quantified by automated cell counter. Expression of active of caspases 3&7 by the shed trophoblasts was determined using a FLICA kit. The trophoblasts shed from cytokine-treated or control explants were exposed to endothelial cell monolayers and endothelial activation determined by ELISA for cell surface ICAM-1. Treatment of explants with IL-6 caused a 50% increase (p=0.001), while TNF a and TGF b 1, caused smaller significant increases in the numbers of trophoblasts shed. Trophoblasts shed from explants treated with IL-6, TGF b 1, or TGF b 3 expressed significantly less active caspases 3&7 than controls or trophoblasts shed from explants treated with other cytokines. Exposing trophoblasts shed from IL-6- or TGF b 1-treated explants to endothelial cells caused a significant (P<0.001) increase in endothelial activation. Normally trophoblasts shed from the placenta die by an apoptosis-like process and their phagocytosis by endothelial cells is silent but a shift to shedding of necrotic trophoblasts can lead to endothelial cell activation 2. However, it remains unclear what might trigger a shift from apoptotic to necrotic trophoblast death. This study suggests that IL-6 and possibly other cytokines can alter both the number and the nature of shed trophoblasts such that the trophoblast are more necrotic and their phagocytosis by maternal endothelial cells could contribute to the pathogenesis of preeclampsia.

Expression of endothelial intercellular adhesion molecule-1 is determined by genotype: Effects on efficiency of leukocyte adhesion to human endothelial cells

Two biallelic polymorphisms, previously described in the human intercellular adhesion molecule (ICAM)-1 gene at codon 241 (glycine [G] to arginine [R] substitution) and codon 469 (glutamic acid [E] to lysine [K] substitution) have been associated with a number of diseases including myocardial infarction, transplant rejection, and diabetes. However, the functional significance of these polymorphisms has not been determined. ICAM-1 cell surface expression and ICAM-1-mediated leukocyte adhesion were investigated using Cos7 transfected with ICAM-1 polymorphic variants or human umbilical vein endothelial cells (HUVEC) of different ICAM-1 genotypes. There was significantly higher expression of surface ICAM-1 on Cos7 transfected with a plasmid encoding the GE (G241/E469) ICAM-1 variant or untreated HUVEC of GEGE (G241/E469 homozygous genotype). ICAM-1-mediated adhesion of peripheral blood mononuclear cells (PBMC) to GE-Cos7 cells or TNF-treated GEGE HUVEC was significantly increased. However, there was no significant difference in adhesion of PBMC to recombinant ICAM-1 of each polymorphic variant plated onto plastic wells. We conclude that the GE genotype of ICAM-1 is associated with greater cell surface expression of ICAM-1, which in turn leads to greater adhesion of leukocytes. This may explain the previously described associations of ICAM-1 polymorphisms with chronic inflammatory disease.

Lysophosphatidic acid regulates inflammation-related genes in human endothelial cells through LPA 1 and LPA 3

Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid (LPL), which regulates endothelial cells participating in inflammation processes via interactions with endothelial differentiation gene (Edg) family G protein-coupled receptors. In this study, we attempted to determine which LPA receptors mediate the inflammatory response in human endothelial cells. Introduction of siRNA against LPA 1 significantly suppressed LPA-induced ICAM-1 mRNA, total protein, and cell surface expressions, and subsequent U937 monocyte adhesion to LPA-treated human umbilical endothelial cells (HUVECs). By knock down of LPA 1 and LPA 3 in HUVECs, LPA-enhanced IL-1β mRNA expression was significantly attenuated. Moreover, LPA 1 and LPA 3 siRNA also inhibited LPA-enhanced IL-1-dependent long-term IL-8 and MCP-1 mRNA expression, and subsequent THP-1 cell chemotaxis toward LPA-treated HUVEC-conditioned media. These results suggest that the expression of LPA-induced inflammatory response genes is mediated by LPA 1 and LPA 3. Our findings suggest the possible utilization of LPA 1 or LPA 3 as drug targets to treat severe inflammation.

Hypoxia and antioxidants enhance soluble ICAM-1 release from cardiac fibroblasts

Intercellular adhesion molecule-1 plays a key role in mediating inflammatory and immune responses. There is also increasing appreciation of the role of its soluble form, sICAM-1, in regulating inflammation. This study evaluated the effects of hypoxia and N-acetyl-L-cysteine on sICAM-1 production by adult rat cardiac fibroblasts. By ELISA, hypoxia was found to cause a 61% increase in sICAM-1 in cardiac fibroblast culture supernates. However, RT-PCR did not reveal a concomitant increase in cell surface ICAM-1 transcript levels, suggesting that the increase in sICAM-1 may involve post-transcriptional and/or post-translational mechanisms. Using pharmacological inhibitors, it was observed that p42/44 MAPK and PKC mediate the stimulatory effect of hypoxia on sICAM-1 production. Remarkably, N-acetyl-L-cysteine caused a 3-fold increase in sICAM-1 by p42/44 MAPK-, p38 MAPK- and PKC-independent mechanisms. Pyrrolidine dithiocarbamate, another potent antioxidant, also augmented sICAM-1. The findings presented in this communication underscore the link between redox status and sICAM-1 release from cardiac fibroblasts. Further, because hypoxia is a major component of myocardial ischemia and is pro-inflammatory, and both N-acetylcysteine and pyrrolidine dithiocarbamate are clinically used antioxidants, the observations may have clinical significance.

Blockade of natural killer cell‐mediated lysis by NCAM140 expressed on tumor cells

Expression of the neural cell adhesion molecule (NCAM) on malignant cells of neuroendocrine, epithelial and hematopoeitic origin has been reported, but its role for tumor cell recognition by the immune system remained uncertain so far. We have studied the cytotoxicity of the natural killer (NK) cell line NK92 and polyclonal NK cells from different donors, against NCAM-deficient and NCAM-transfected tumors. While the pancreatic carcinoma PANC-1 and the glioblastoma T98G showed no enhanced susceptibility to NK lysis after NCAM transfection, de novo NCAM expression in HeLa cervical carcinoma, SHEP neuroblastoma and the multiple myeloma lines RPMI-8226 and LP-1 was associated with significantly decreased lysis by NK cells. Binding of an NCAM-specific monoclonal antibody to NCAM-positive target cells was able to reverse the reduced lysis susceptibility. Conjugate formation of NCAM-expressing tumor cells with NK cells was blocked and could be restored by anti-NCAM. NK cell-expressed NCAM molecules which might engage in homotypic cis- or trans-interactions had no apparent inhibitory function. The known cis-ligands of NCAM, heparan sulfate proteoglycan and L1-CAM, were also not directly involved in NK inhibition. ICAM-1 mRNA and cell surface expression was downmodulated in NCAM-transfected HeLa cells. ICAM-1 is involved in killer cell immune synapse formation. Its downmodulation may therefore contribute to the reduced lysis of NCAM-expressing target cells. We conclude that aberrant expression of NCAM on tumor cells of different histogenetic origin can lead to inhibition of target cell recognition and lysis by NK cells.

Sphingosine 1-phosphate regulates inflammation-related genes in human endothelial cells through S1P 1 and S1P 3

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid (LPL) ligand that binds endothelial differentiation gene (Edg) family G-protein-coupled receptors and has been implicated as an important regulator in endothelial cells during inflammation processes. In this study, we attempt to determine which S1P receptors mediating the inflammatory response in human endothelial cells. Our results indicated that introduction of siRNA against S1P 1 significantly suppressed S1P-induced ICAM-1 mRNA, total protein, and cell surface expressions in human umbilical vein endothelial cells (HUVECs). Moreover, U937 cells adhesion to S1P-treated HUVECs was profoundly reduced by knock-down of S1P 1 in HUVECs. By knock-down of S1P 1 or S1P 3 in HUVECs, S1P-enhanced IL-8, MCP-1 mRNA expression, and THP-1 cell chemotaxis toward S1P-treated HUVEC-conditioned media was profoundly reduced. These results suggested that S1P-induced inflammatory response genes expression is mediated through S1P 1 and S1P 3. Our findings suggest the possible utilization of S1P 1 or S1P 3 as drug targets to treat severe inflammation.

Expression and Function of the Lysophospholipids Receptors, S1P1 and LPA1, in Human Endothelial Cells and the Regulation of Inflammation‐Related Genes

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive lysophospholipid (LPL) ligands that bind endothelial differentiation gene (Edg) family G protein-coupled receptors and have been implicated as important regulators in endothelial cells during inflammation processes. In this study, introduction of siRNA against S1P1 or LPA1 significantly suppressed S1P- or LPA-upregulated ICAM-1, IL-8, and MCP-1 mRNA expressions, and ICAM-1 total protein and cell surface expressions in human umbilical vein endothelial cells (HUVECs). Moreover, U937 cells adhesion to S1P- or LPA-treated HUVECs and THP-1 cell chemotaxis toward S1P- or LPA-treated HUVEC-conditioned media was profoundly reduced by knocking down S1P1 or LPA1 in HUVECs. These results suggest that S1P1 or LPA1 mediates S1P- or LPA- induced ICAM-1, IL-8, and MCP-1 gene expressions, which contribute to monocyte adhesion and chemoattraction toward the endothelium, thus facilitating inflammation processes. Our findings suggest the possible utilization of S1P1 or LPA1 as drug targets to treat severe inflammation.

Phosphatidylinositol 3-Kinase γ Signaling through Protein Kinase Cζ Induces NADPH Oxidase-mediated Oxidant Generation and NF-κB Activation in Endothelial Cells

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kgamma in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kgamma-mediated oxidant signaling in the mechanism of NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110gamma catalytic subunit of PI3Kgamma. Tumor necrosis factor (TNF) alpha challenge of wild type ECs caused p110gamma translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110gamma-/- ECs. ROS production was the result of TNFalpha activation of Ser phosphorylation of NADPH oxidase subunit p47(phox) and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110gamma-/- ECs. Additionally, the TNFalpha-activated NF-kappaB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kgamma. TNFalpha challenge of p110gamma-/- ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCzeta. Thus, PI3Kgamma lies upstream of PKCzeta in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-kappaB activation and ICAM-1 expression.

Increased proinflammatory responses from asthmatic human airway smooth muscle cells in response to rhinovirus infection

BackgroundExacerbations of asthma are associated with viral respiratory tract infections, of which rhinoviruses (RV) are the predominant virus type. Airway smooth muscle is important in asthma pathogenesis, however little is known about the potential interaction of RV and human airway smooth muscle cells (HASM). We hypothesised that rhinovirus induction of inflammatory cytokine release from airway smooth muscle is augmented and differentially regulated in asthmatic compared to normal HASM cells.MethodsHASM cells, isolated from either asthmatic or non-asthmatic subjects, were infected with rhinovirus. Cytokine production was assayed by ELISA, ICAM-1 cell surface expression was assessed by FACS, and the transcription regulation of IL-6 was measured by luciferase activity.ResultsRV-induced IL-6 release was significantly greater in HASM cells derived from asthmatic subjects compared to non-asthmatic subjects. This response was RV specific, as 5% serum- induced IL-6 release was not different in the two cell types. Whilst serum stimulated IL-8 production in cells from both subject groups, RV induced IL-8 production in only asthmatic derived HASM cells. The transcriptional induction of IL-6 was differentially regulated via C/EBP in the asthmatic and NF-κB + AP-1 in the non-asthmatic HASM cells.ConclusionThis study demonstrates augmentation and differential transcriptional regulation of RV specific innate immune response in HASM cells derived from asthmatic and non-asthmatics, and may give valuable insight into the mechanisms of RV-induced asthma exacerbations.

Thrombin induces ICAM‐1 expression via store‐operated Ca2+ influx signaling in endothelial cells

Thrombin-induced endothelial cell surface ICAM-1 expression plays a critical role in the pathogenesis of lung vascular injury. NF-κB activation is required for stable thrombin-induced ICAM-1 expression. We explore the role of Ca2+ signaling in the mechanism of NF-κB-dependent ICAM-1 expression human pulmonary artery endothelial cells (HPAEC). We show that IP3-receptor antagonist, 2-APB inhibits thrombin-induced increase in intracellular Ca2+ ([Ca2+]i), NF-κB activation and ICAM-1 expression in HPAEC. 2-APB failed to inhibit TNF-α-induced ICAM-1 mRNA. Thrombin-induced increase in [Ca2+]i is dependent on both ER stored-Ca2+ release and Ca2+-store depletion-mediated Ca2+ influx occurring via TRP channels in endothelial cells. To address the importance of Ca2+ influx signal, we measured thrombin-induced NF-κB activation and ICAM-1 transcript expression in the absence of Ca2+ influx in HPAEC. Thrombin-induced p65 nuclear translocation and ICAM-1 mRNA expression were reduced by preventing Ca2+ influx. Since TRPC4 is the essential component of SOC in mouse endothelial cells, we measured NF-κB activation and ICAM-1 expression in TRPC4 null (TRPC4−/ −) mice. Isolated perfused mouse lung studies showed impaired thrombin-induced NF-κB activation in TRPC4−/ − compared to wild type (WT). Thrombin-induced ICAM-1 mRNA expression was reduced in lung endothelial cells (LEC) from TRPC4−/ − compared to WT LEC. Thus, thrombin-induced store-operated Ca2+ influx signaling is critical in the mechanism of NF- B-dependent ICAM-1 expression in endothelial cells.

Respiratory Syncytial Virus Infection of Human Lung Endothelial Cells Enhances Selectively Intercellular Adhesion Molecule-1 Expression

Respiratory syncytial virus (RSV) is worldwide the most frequent cause of bronchiolitis and pneumonia in infants requiring hospitalization. In the present study, we supply evidence that human lung microvascular endothelial cells, human pulmonary lung aorta endothelial cells, and HUVEC are target cells for productive RSV infection. All three RSV-infected endothelial cell types showed an enhanced cell surface expression of ICAM-1 (CD54), which increased in a time- and RSV-dose-dependent manner. By using noninfectious RSV particles we verified that replication of RSV is a prerequisite for the increase of ICAM-1 cell surface expression. The up-regulated ICAM-1 expression pattern correlated with an increased cellular ICAM-1 mRNA amount. In contrast to ICAM-1, a de novo expression of VCAM-1 (CD106) was only observed on RSV-infected HUVEC. Neither P-selectin (CD62P) nor E-selectin (CD62E) was up-regulated by RSV on human endothelial cells. Additional experiments performed with neutralizing Abs specific for IL-1alpha, IL-1beta, IL-6, and TNF-alpha, respectively, excluded an autocrine mechanism responsible for the observed ICAM-1 up-regulation. The virus-induced ICAM-1 up-regulation was dependent on protein kinase C and A, PI3K, and p38 MAPK activity. Adhesion experiments using polymorphonuclear neutrophil granulocytes (PMN) verified an increased ICAM-1-dependent adhesion rate of PMN cocultured with RSV-infected endothelial cells. Furthermore, the increased adhesiveness resulted in an enhanced transmigration rate of PMN. Our in vitro data suggest that human lung endothelial cells are target cells for RSV infection and that ICAM-1 up-regulated on RSV-infected endothelial cells might contribute to the enhanced accumulation of PMN into the bronchoalveolar space.

Effect of activin A on tumor necrosis factor-α/intercellular adhesion molecule-1 pathway in endometrial stromal cells

Objective[s] Activin A and inhibin A are growth factors expressed by human endometrium involved in the control of endometrial functions. In the present study we investigated the effects of activin A and inhibin A in modulating the tumor necrosis factor (TNF)-α/intercellular adhesion molecule (ICAM)-1 system in cultured human endometrial stromal cells. Study design Endometrial samples were obtained from 34 reproductive age women undergoing laparoscopy for benign ovarian cysts or infertility. Endometrial stromal cells were cultured and soluble ICAM-1 and TNF-α were measured in cell-free supernatants following treatment with or without activin A or inhibin A. Cell surface ICAM-1 was assayed by flow cytometry by staining endometrial cells with specific monoclonal antibodies. Results Activin A and inhibin A did not influence either the expression of cell surface ICAM-1 or soluble ICAM-1 shedding by cultured endometrial cells. On the other hand, TNF-α secretion significantly increased in presence of activin A but not of inhibin A. Conclusions Since TNF-α modulates several endometrial processes such as menstruation, proliferation, apoptosis, implantation and decidualization, an effect of activin A in the physiological control of endometrium is further supported by the present data.

W10-O-001 Endothelial lipase deficiency increases plasma cholesterol but reduces susceptibility to atherosclerosis in apolipoprotein E-deficient mice

High density lipoprotein (HDL) as well as annexin A1 have been reported to be associated with cardiovascular protection. However, the correlation between HDL and annexin A1 was still unknown. In this study, HDL increased endothelial annexin A1 and prevented the decrease of annexin A1 in TNF-α-activated endothelial cells in vitro and in vivo, and above effects were attenuated after knockdown of annexin A1. Annexin A1 modulation affected HDL-mediated inhibition of monocyte adhesion to TNF-α-activated endothelium (45.2 ± 13.7% decrease for annexin A1 RNA interference; 78.7 ± 16.3% decrease for anti-Annexin A1 antibody blocking; 11.2 ± 6.9% increase for Ad-ANXA1 transfection). Additionally, HDL up-regulated annexin A1 through scavenger receptor class B type I, involving ERK, p38MAPK, Akt and PKC signaling pathways, and respective inhibitors of these pathways attenuated HDL-induced annexin A1 expression as well as impaired HDL-mediated inhibition of monocyte–endothelial cell adhesion. Apolipoprotein AI also increased annexin A1 and activated similar signaling pathways. Endothelial annexin A1 from apolipoprotein AI knockout mice was decreased in comparison to that from wild type mice. Finally, HDL-induced annexin A1 inhibited cell surface VCAM-1, ICAM-1 and E-selectin, and secretion of MCP-1, IL-8, VCAM-1 and E-selectin, thereby inhibiting monocyte adhesion.

Proinflammatory properties of murine aortic endothelial cells exclusively expressing a non cleavable form of TNFα

SummarySoluble (sTNF) and transmembrane (tmTNF) forms of TNFα (TNF) have distinct proinflammatory effects. We investigated whether tmTNF altered the synthesis of some proinflammatory proteins involved in atherothrombosis, in murine aortas and aortic endothelial cells (MAEC). Samples were obtained from wild-type (WT) mice and TNF-deficient mice that express a mutated non cleavable tmTNF transgene (tmTNFnc).The levels of secreted MCP-1, RANTES, IL-6, PAI-1, soluble ICAM-1, and soluble TNF receptor type 1 (TNFR1; CD120a) antigens, MMP-9 activity and of cell surface ICAM-1 were not significantly different between the two types of MAEC.The magnitude of endotoxin-stimulated production of RANTES, MCP-1 and IL-6 was similar in the two types of cells. Of note, the amount of synthesized TNF receptor type 2 (TNFR2; CD120b), measured by its secreted (in aorta and MAEC), intracellular and mRNA levels (in MAEC), was significantly 4-fold lower in tmTNFnc than in WT mice, both in basal and endotoxin-stimulated conditions. A neutralizing anti-TNF antibody or the recombinant murine TNF did not modify the magnitude of the difference in TNFR2 production between the two types of cells, suggesting a preponderant role of tmTNF in the down-regulation of TNFR2 synthesis. Macrophages of tmTNFnc mice also produced less TNFR2 than WT macrophages (−30%). Plasmas of tmTNFnc mice contained significantly less sTNFR2 than WT mice (−75%). In conclusion, an increase in tmTNF levels, rather than the lack of sTNF, significantly down-modulated TNFR2 synthesis in aortic endothelial cells, but had no major influence on the synthesis of some major pro-inflammatory and pro-atherothrombotic proteins.