Intercellular Adhesion Molecule-1 Messenger RNA Research Articles

Methylglyoxal-derived hydroimidazolone-1 evokes inflammatory reactions in endothelial cells via an interaction with receptor for advanced glycation end products.

Glyceraldehyde-derived advanced glycation end products contribute to vascular inflammation in diabetes. However, what advanced glycation end product structure could evoke inflammatory reactions remains unknown. We examined whether and how methylglyoxal-derived hydroimidazolone 1, one of the advanced glycation end products formed from glyceraldehyde, elicits inflammatory reactions in human umbilical vein endothelial cells. Glyceraldehyde-advanced glycation end products-aptamer was prepared using a systemic evolution of ligands by exponential enrichment. The binding affinities of methylglyoxal-derived hydroimidazolone 1 to receptor for advanced glycation end products or advanced glycation end product-aptamer were measured with a quartz crystal microbalance. Intracellular reactive oxygen species generation and THP-1 cell adhesion were evaluated using fluorescent probes. Gene expression was analysed by reverse transcription polymerase chain reaction. Methylglyoxal-derived hydroimidazolone 1 bound to receptor for advanced glycation end products and advanced glycation end product-aptamer with a dissociation constant ( Kd) of 56.7 µM and 1.51 mM, respectively. Methylglyoxal-derived hydroimidazolone 1 at 100 µg/mL significantly increased reactive oxygen species generation in human umbilical vein endothelial cells, which were attenuated by anti-receptor for advanced glycation end products antibody or advanced glycation end product-aptamer. In all, 100 µg/mL methylglyoxal-derived hydroimidazolone 1 significantly increased receptor for advanced glycation end products and intercellular adhesion molecule-1 messenger RNA levels in, and THP-1 cell adhesion to, human umbilical vein endothelial cells, all of which were blocked by anti-receptor for advanced glycation end products antibody. Our present results indicate that methylglyoxal-derived hydroimidazolone 1 evokes inflammatory reactions in human umbilical vein endothelial cells via receptor for advanced glycation end products, although apparently limited to supraphysiological levels of methylglyoxal-derived hydroimidazolone 1. Methylglyoxal-derived hydroimidazolone 1 is a distinct advanced glycation end product structure that could mediate harmful effects of methylglyoxal and glyceraldehyde-mediated glycation processes.

α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke.

Human rhinovirus (HRV) infections target airway epithelium and are the leading cause of acute exacerbations of COPD. Cigarette smoke (CS) increases the severity of viral infections, but there is no effective therapy for HRV infection. We determined whether α1-antitrypsin (A1AT) reduces HRV-16 infection in CS-exposed primary human airway epithelial cells. Brushed bronchial epithelial cells from normal subjects and patients diagnosed with COPD were cultured at air–liquid interface to induce mucociliary differentiation. These cells were treated with A1AT or bovine serum albumin for 2 hours and then exposed to air or whole cigarette smoke (WCS) with or without HRV-16 (5×104 50% Tissue Culture Infective Dose [TCID50]/transwell) infection for 24 hours. WCS exposure significantly increased viral load by an average of fivefold and decreased the expression of antiviral genes interferon-λ1, OAS1, and MX1. When A1AT was added to WCS-exposed cells, viral load significantly decreased by an average of 29-fold. HRV-16 infection significantly increased HRV-16 receptor intercellular adhesion molecule-1 messenger RNA expression in air-exposed cells, which was decreased by A1AT. A1AT-mediated reduction of viral load was not accompanied by increased epithelial antiviral gene expression or by inhibiting the activity of 3C protease involved in viral replication or maturation. Our findings demonstrate that A1AT treatment prevents a WCS-induced increase in viral load and for the first time suggest a therapeutic effect of A1AT on HRV infection.

Rosiglitazone, a Peroxisome Proliferator-Activated Receptor (PPAR)-γ Agonist, Attenuates Inflammation Via NF-κB Inhibition in Lipopolysaccharide-Induced Peritonitis.

We assessed the anti-inflammatory effect of peroxisome proliferator-activated receptor (PPAR)-γ agonist, rosiglitazone, in a lipopolysaccharide (LPS)-induced peritonitis rat model. LPS was intraperitoneally injected into rats to establish peritonitis model. Male Sprague-Dawley (SD) rats were assigned to normal saline (the solvent of LPS), LPS, rosiglitazone plus LPS, and rosiglitazone alone. A simple peritoneal equilibrium test was performed with 20ml 4.25% peritoneal dialysis fluid. We measured the leukocyte count in dialysate and ultrafiltration volume. Peritoneal membrane histochemical staining was performed, and peritoneal thickness was assessed. CD40 and intercellular adhesion molecule-1 messenger RNA (ICAM-1 mRNA) levels in rat visceral peritoneum were detected by reverse transcription (RT)-PCR. IL-6 in rat peritoneal dialysis effluent was measured using enzyme-linked immunosorbent assay. The phosphorylation of NF-κB-p65 and IκBα was analyzed by Western blot. LPS administration resulted in increased peritoneal thickness and decreased ultrafiltration volume. Rosiglitazone pretreatment significantly decreased peritoneal thickness. In addition to CD40 and ICAM-1 mRNA expression, the IL-6, p-p65, and p-IκBα protein expressions were enhanced in LPS-administered animals. Rosiglitazone pretreatment significantly decreased ICAM-1 mRNA upregulation, secretion of IL-6 protein, and phosphorylation of NF-κB-p65 and IκBα without decreasing CD40 mRNA expression. Rosiglitazone has a protective effect in peritonitis, simultaneously decreasing NF-κB phosphorylation, suggesting that NF-κB signaling pathway mediated peritoneal inflammation induced by LPS. PPAR-γ might be considered a potential therapeutic target against peritonitis.

Protective effect of galangin in Concanavalin A-induced hepatitis in mice.

Galangin is an active pharmacological ingredient from propolis and Alpinia officinarum Hance, and has been reported to have anti-inflammatory and antioxidative properties. The present study aims to reveal the effect of galangin on Concanavalin A (ConA)-induced hepatitis (CIH), a well-established animal model of immune-mediated liver injury, and to clarify the related mechanism. C57BL/6 mice were pretreated with galangin followed by ConA challenge. Results indicated that galangin inhibited ConA-induced liver damage. Mice pretreated with galangin showed more reduction of liver damage when compared with control mice pretreated with vehicle solution. In galangin-pretreated mice with induced CIH, increases in serum levels of several inflammatory cytokines, including tumor necrosis factor-α, interferon-γ, and interleukin-12 were dramatically attenuated, and chemokines and adhesion molecules like interferon inducible protein-10, macrophage inflammatory protein-1α, and inter-cellular adhesion molecule-1 messenger RNA expressions in liver were decreased. Moreover, CIH mice pretreated with galangin showed less leukocyte infiltration and T-cell activation in the liver. Further, the mechanism of the anti-inflammatory effects of galangin may be attributed to its modulation of crucial inflammatory signaling pathways, including nuclear factor kappa B and interferon-gamma/signal transducer and activator of transcription 1. Collectively, these findings suggest the preventive and therapeutic potential of galangin in immune-mediated liver injury in vivo.

Effect of interleukin-1β on ICAM-1 expression of dental pulp cells: role of PI3K/Akt, MEK/ERK, and cyclooxygenase

Interleukin-1β (IL-1β) is an important inflammatory mediator of the dental pulp. IL-1β stimulates cyclooxygenase-2 (COX-2) expression and prostanoid production of pulp cells and affects the inflammatory and healing processes of the dental pulp. There are two interleukin-1 (IL-1) receptors, IL-1RI and IL-1RII, with opposing effect after activation. However, the expression of IL-1Rs, the effects of IL-1β on intercellular adhesion molecule-1 (ICAM-1) of dental pulp cells, and its relation to protein kinase B (Akt) signaling and COX activation are not clear. Human dental pulp cells were treated with IL-1β with/without pretreatment and co-incubation by LY294002 (a PI3K/Akt inhibitor), U0126 [a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) inhibitor], aspirin (a COX inhibitor), or eugenol (a COX inhibitor) for different time periods. The expression of ICAM-1, IL-1RI, and IL-1RII messenger RNA (mRNA) was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). ICAM-1 protein expression was examined by western blotting. Soluble ICAM-1 (sICAM-1) level in the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). Activation of Akt and ERK by IL-1β was measured by Pathscan p-Akt ELISA or western blot. Viable cell number was evaluated by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Dental pulp cells expressed IL-1RI, but little IL-1RII. IL-1β stimulated COX-2 and ICAM-1 mRNA and protein expression as well as sICAM-1 production of pulp cells. Aspirin and eugenol enhanced the IL-1β-induced sICAM-1 production and ICAM-1 expression. IL-1β rapidly activated Akt and ERK. LY294002 and U0126 attenuated IL-1β-induced ICAM-1 expression and sICAM-1 production. These results reveal that IL-1β may be involved in the pulpal inflammatory processes by stimulating ICAM-1 expression and secretion. These events are associated with IL-1RI expression and differential activation of PI3K/Akt and MEK/ERK and COX. Pharmacological inhibition of IL-1β, IL-1RI, COX-2, ICAM-1, and related signaling pathways (MEK/ERK and PI3K/Akt) may be useful for the control of pulpal inflammation.

Hydrogen Sulfide Suppresses High Glucose–Induced Expression of Intercellular Adhesion Molecule-1 in Endothelial Cells

Hydrogen sulfide (H₂S) is a newly identified endogenous gasotransmitter that has been implicated in the pathophysiology of several biologic systems. However, the role of H₂S in the pathogenesis of diabetic vascular injury remains unclear. The aims of this study were to determine the effect of H₂S on the high glucose (HG)-induced expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells and to explore the possible underlying mechanisms. Human umbilical vein endothelial cells were exposed either to a normal concentration of D-glucose (5.5 mmol/L) or to HG (16.7 mmol/L) in the absence or presence of NaHS for the indicated periods. The ICAM-1 protein and messenger RNA (mRNA) levels were analyzed by Western blotting and real-time reverse transcriptase-polymerase chain reaction, respectively. Exposure to HG for 48 or 72 hours significantly increased ICAM-1 expression at both the protein and mRNA levels, and these increases correlated with increases in both the production of intracellular reactive oxygen species and the activation of nuclear factor-κB. Pretreatment with NaHS inhibited HG-induced ICAM-1 expression at both the protein and mRNA levels and resulted in a reduction in the intracellular reactive oxygen species level and the suppression of nuclear factor-κB activity. NaHS also inhibited tumor necrosis factor-α-induced ICAM-1 protein expression, which was similar to the effect of antioxidant N-acetyl-L-cysteine. These findings indicate that H₂S might protect against HG-induced vascular damage by down-regulating ICAM-1 expression in endothelial cells.

Future Therapeutic Approaches for Inflammatory Bowel Diseases

In this review, we speculate about future therapeutic approaches for inflammatory bowel diseases (IBDs), focusing on the need for better preclinical and clinical models and approaches beyond small molecules and systemically administered biologics. We offer ideas to change clinical trial programs and to use immunologic and genetic biomarkers to personalize medicine. We attempt to reconcile past therapeutic successes and failures to improve future approaches. Some of our ideas might be provocative, but we hope that the examples we provide will stimulate discussion about what will advance the field of IBD therapy.

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-κB 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-kappaB (NF-kappaB) signaling pathway in Escherichia coli LPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 10 days) and controls were instilled intratracheally with saline containing LPS (750 microg/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LPS. 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-kappaB p65 subunit and phosphorylation of I-kappaB 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 (approximately 30%), and IL-10 (approximately 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-kappaB p65 subunit and phosphorylation of I-kappaB 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-kappaB p65 subunit and phosphorylation of I-kappaB 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-kappaB signaling pathway.

NF-κB Activation, Rather Than TNF, Mediates Hepatic Inflammation in a Murine Dietary Model of Steatohepatitis

We explored the roles of nuclear factor-kappaB (NF-kappaB) and tumor necrosis factor (TNF) alpha (TNF-alpha) as mediators of inflammation in a nutritional model of steatohepatitis. Wild-type (wt), TNF null -/-, and TNF receptor (R)-1-/- mice were fed a methionine- and choline-deficient (MCD) diet for up to 5 weeks. Liver injury (serum alanine aminotransferase [ALT]), hepatic inflammation, triglycerides, and lipid peroxide levels were determined. Hepatic NF-kappaB activation and expression of TNF and intercellular adhesion molecule-1 (ICAM-1) were assayed. Irrespective of genotype, MCD diet-fed mice developed hepatic lipid peroxidation and serum ALT elevation; at day 10, livers from wt, TNF-/-, and TNFR-1-/- mice showed equivalent steatohepatitis. NF-kappaB/DNA binding was enhanced in hepatic nuclear fractions from MCD diet-fed wt mice compared with dietary controls; there were corresponding increases of ICAM-1 and TNF messenger RNA (mRNA). Likewise, NF-kappaB activation and ICAM-1 expression were enhanced by MCD dietary feeding in TNF-/- and TNFR-1-/- mice compared with respective controls. To establish whether NF-kappaB is a primary mediator of inflammation in experimental steatohepatitis, we over-expressed a mutant, nondegradable IkappaB (mIkappaB), delivered by adenovirus in vivo. As expected, hepatic mIkappaB expression reduced NF-kappaB/DNA binding induced by MCD dietary feeding, with resultant abrogation of ICAM-1 and TNF synthesis. Such blockade of NF-kappaB transcriptional activation substantially protected against development of steatohepatitis, with significant reductions in liver injury and hepatic inflammation. In the MCD dietary model of steatohepatitis, NF-kappaB is activated early and is an important proinflammatory mediator of lesion development, but steatohepatitis occurs independently of TNF synthesis and TNFR-1 activation.

Effects of continuous low-dose infusion of lipopolysaccharide on expression of E-selectin and intercellular adhesion molecule-1 messenger RNA and neutrophil accumulation in specific organs in dogs

To determine the effects of continuous low-dose infusion of lipopolysaccharide (LPS) on the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) mRNA and neutrophil accumulation in the lungs, liver, spleen, small intestine, and pancreas in dogs. 11 healthy adult Beagles. Dogs received a continuous infusion of a low dose (10 microg/kg/h, i.v.) of LPS (Escherichia coli 055:B5) or saline (0.9% NaCI) solution (20 mL/kg/h, i.v.) for 8 hours. Activity levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (1L-6) and the number of WBCs in circulation were examined before and 1, 2, 4, and 8 hours after the onset of LPS infusion. Expression of E-selectin and ICAM-1 mRNA and the number of neutrophils in each tissue were examined. After the onset of LPS infusion, serum TNF-alpha and IL-1beta activities transiently increased. Thereafter, IL-6 activity increased, and high IL-6 activity was maintained throughout the experiment. In dogs in the LPS group, expression of E-selectin mRNA increased only in the lungs, and expression of ICAM-1 mRNA increased in the lungs and liver; the number of neutrophils in the tissue increased in the lungs and liver. Results suggested that expression of E-selectin and ICAM-1 mRNA increased during sepsis, particularly in the lungs and liver, and that this increase was associated with neutrophil accumulation. Hence, inhibiting the activation of endothelial cells in the lung and liver may decrease organ damage caused by accumulated neutrophils and help regulate multiple-organ dysfunction.

Association of intercellular adhesion molecule-1 with clinical manifestations and interleukin-18 in patients with active, untreated adult-onset Still's disease

To investigate the association of intercellular adhesion molecule 1 (ICAM-1) with clinical manifestations and interleukin-18 (IL-18) levels in patients with active untreated adult-onset Still's disease (AOSD). We determined serum soluble ICAM-1 (sICAM-1) levels by enzyme-linked immunosorbent assay in 50 patients with active untreated AOSD, 20 patients with active rheumatoid arthritis (RA), and 20 healthy controls. The levels of ICAM-1 messenger RNA expression in IL-18-stimulated peripheral blood mononuclear cells (PBMCs) and in biopsy specimens obtained from AOSD patients with Still's rash or synovitis were investigated using real-time quantitative polymerase chain reaction. Significantly higher serum levels of sICAM-1 were observed in patients with active untreated AOSD compared with those with active RA and healthy controls. Serum sICAM-1 levels were significantly correlated with the clinical activity score (r = 0.565, P < 0.001), ferritin values (r = 0.462, P < 0.005), and IL-18 levels (r = 0.462, P < 0.005) in patients with AOSD. The serum sICAM-1 level was identified as a predictor of hepatic dysfunction (odds ratio [OR] 1.016, P = 0.011) and disseminated intravascular coagulation (DIC) (OR 1.013, P = 0.023). Up-regulation of ICAM-1 gene expression was demonstrated in IL-18-stimulated PBMCs from patients with AOSD. Increased levels of ICAM-1 transcripts were observed in the biopsy specimens obtained from AOSD patients with Still's rash or synovitis compared with healthy skin and patients with osteoarthritis. The serum sICAM-1 level may be used as a clinical marker to assess disease activity and may predict the occurrence of hepatic dysfunction and DIC in AOSD. IL-18-up-regulated gene expression of ICAM-1 may contribute to the inflammatory response in AOSD.

TNFα suppresses human colonic circular smooth muscle cell contractility by SP1- and NF-κB-mediated induction of ICAM-1

Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low levels on human colonic circular smooth muscle cells (HCCSMCs) and their expression is increased in patients with Crohn's disease. We investigated the roles of transcription factors Sp1 and nuclear factor kappa B (NF-kappaB) in the regulation of ICAM-1 expression on HCCSMCs and examined whether ICAM-1 expression mediates the suppression of contractility in response to TNFalpha. Experiments were performed on primary cultures of HCCSMCs and fresh human colonic circular muscle strips. TNFalpha treatment of HCCSMCs induced rapid and prolonged accumulation of ICAM-1 messenger RNA (mRNA) and protein. NF-kappaB inhibition before, but not after, 1 hour of TNFalpha-stimulation blocked the expression of ICAM-1. TNFalpha significantly enhanced Sp1/DNA binding. Sp1 bound to the 3' flanking region of a variant kappaB site in the -192/-172 region of ICAM-1 promoter. Mutation of this region abolished the response to TNFalpha. The treatment of HCCSMCs with Sp1 antisense oligonucleotides (ODNs) blocked the expression of ICAM-1, but sense ODNs had no effect. Protein kinase C zeta (PKCzeta) inhibition before or 3 hours after stimulation with TNFalpha also blocked the expression of ICAM-1. TNFalpha treatment of circular muscle strips pretreated with ICAM-1 sense ODNs or control medium significantly reduced their response to acetylcholine, whereas pretreatment with antisense ODNs blocked this effect. The expression of ICAM-1 on HCCSMCs in response to TNFalpha is regulated by transcription factors Sp1 and NF-kappaB binding independently to the -192/-172 region of the ICAM-1 promoter. The expression of ICAM-1 plays a critical role in the suppression of cell contractility in response to TNFalpha.

Microvascular endothelial cell activation is present in the umbilical placental microcirculation in fetal placental vascular disease

Objective Fetal growth restriction is associated with an abnormal umbilical artery Doppler study. A vascular disease is present in the fetal umbilical placental microcirculation. We hypothesized that the local production of factors that are injurious to microvessel endothelium is responsible for this vascular disease and that endothelial cell activation is a feature of this. Because the expression of the cell adhesion molecules is associated with endothelial cell activation, we isolated endothelial cells from the microvessels of the umbilical placenta and examined them for evidence of gene expression of cell adhesion molecules. Study design Endothelial cells from the microcirculation of human placenta were isolated and purified with collagenase digestion and extraction with superparamagnetic beads that were coated with monoclonal antibody against CD31. Microvessel endothelial cells were isolated from the placentae of 13 women with a normal pregnancy and delivery at term and 10 placentas with umbilical placental vascular disease that was defined by abnormal umbilical artery Doppler study. Total RNA was extracted from isolated endothelial cells. The messenger RNA expressions of cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and platelet endothelial cell adhesion molecule-1) were assessed by semiquantitative reverse transcription–polymerase chain reaction. Results Microvessel endothelial cells from the fetal placentae of pregnancies that were complicated by umbilical placental vascular disease showed an enhanced expression of intercellular adhesion molecule-1 messenger RNA (2.12±0.45 vs 0.92±0.25) and platelet endothelial cell adhesion molecule-1 messenger RNA (4.29±0.87 vs 2.41±0.42) in comparison to normal pregnancies. There was no significant difference in expression of vascular cell adhesion molecule-1 messenger RNA (1.55±0.37 vs 1.68±0.38). Conclusion We have shown that vascular disease in the fetal umbilical placental circulation is associated with an increase in the expression of intercellular adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 by microvessel endothelial cells. We postulate that locally released factors cause injury and activation to microvessel endothelial cells. In this regard, the process in the fetus is similar to that of atherothrombotic vascular disease of later life.

Effects of glucocorticoids on tumor necrosis factor alpha-dependent activation of nuclear factor kappaB and expression of the intercellular adhesion molecule 1 gene in osteoblast-like ROS17/2.8 cells.

Recently, we showed that tumor necrosis factor alpha (TNF-alpha) stimulates expression of the intercellular adhesion molecule 1 (ICAM-1) and interleukin-6 (IL-6) genes through activation of p65-p50 heterodimer nuclear factor KB (NF-kappaB) in rat osteoblast-like ROS17/2.8 cells. In the present study, we investigated effects of a synthetic glucocorticoid, dexamethasone (Dex), on TNF-alpha-dependent activation of NF-kappaB and expression of the ICAM-1 gene. ROS17/2.8 cells were pretreated with Dex for 6 h and then exposed to TNF-alpha. Electrophoretic mobility shift assay (EMSA) revealed that TNF-alpha-dependent activation of NF-kappaB was almost completely suppressed by Dex treatment. Increase in ICAM-1 messenger RNA (mRNA) level by TNF-alpha also was markedly suppressed by Dex. Western blot and immunocytochemical analyses showed that Dex attenuated the TNF-alpha-induced nuclear translocation of p65. Treatment with protein synthesis inhibitor cycloheximide (CHX) reversed the Dex effect, indicating that Dex requires de novo protein synthesis for its action. Northern blot analysis revealed that Dex increased IkappaB-alpha mRNA level synergistically with TNF-alpha, whereas it decreased p65 mRNA level. The p105 and IkappaB-beta mRNA levels were not altered by Dex. Consistent with the mRNA level, Dex increased the amount of IkappaB-alpha protein in the cytoplasm in either the presence or the absence of TNF-alpha. Considering a role of IkappaB to sequester NF-kappaB in the cytoplasm, it was suggested that an increase in IkappaB-alpha protein and the concomitant decrease in p65 synthesis account for the Dex-induced suppression of NF-kappaB activation in osteoblastic cells.

Corticosteroids inhibit rhinovirus-induced intercellular adhesion molecule-1 up-regulation and promoter activation on respiratory epithelial cells

Background: Rhinoviruses are associated with the majority of asthma exacerbations. To date, the pathogenesis of virus-induced asthma exacerbations is still unclear, and no safe effective therapy is available. Intercellular adhesion molecule-1 (ICAM-1) has a central role in inflammatory cell recruitment to the airways in asthma and is the receptor for 90% of rhinoviruses. We have previously shown that rhinovirus infection of lower airway epithelium induces ICAM-1 expression by a transcriptional mechanism that is critically nuclear factor-κB-dependent. Objective: The purpose of this study was to investigate the effect of systemic (hydrocortisone [HC], dexamethasone [DM]) and topical (mometasone furoate [MF]) corticosteroids on rhinovirus-induced ICAM-1 up-regulation. Methods: Cultured primary bronchial or transformed (A549) respiratory epithelial cells were pretreated with corticosteroids for 16 hours and infected with rhinovirus type 16 for 8 hours. ICAM-1 surface expression was evaluated by flow cytometry. In A549 cells ICAM-1 messenger RNA was evaluated by specific reverse transcription–PCR and promoter activation by chloramphenicol acetyltransferase assay. Results: We observed inhibition of rhinovirus-induced ICAM-1 up-regulation with corticosteroid pretreatment in both primary bronchial epithelial and A549 cells. In A549 cells systemic and topical corticosteroids demonstrated a dose-dependent inhibition with similar efficacy (inhibitory concentration 50% 10–10 mol/L, 10–11 mol/L, and 10–11 mol/L for HC, DM, and MF respectively). MF also inhibited ICAM-1 messenger RNA induction by rhinovirus infection in a dose-dependent manner. MF completely inhibited rhinovirus-induced ICAM-1 promoter activation. HC, DM, and MF had no direct effect on rhinovirus infectivity and replication in cultured cells. Conclusion: Corticosteroids decrease rhinovirus-induced ICAM-1 up-regulation in respiratory epithelial cells and modulate pretranscriptional mechanisms. This effect may be important for the therapeutic control of virus-induced asthma exacerbations. (J Allergy Clin Immunol 2000;105:318-26.)

Surgically induced leukocytic infiltrates within the rat intestinal muscularis mediate postoperative ileus

Background & Aims: Postoperative ileus is a poorly understood and common problem. We previously demonstrated an association between a suppression in jejunal circular muscle activity and a massive extravasation of leukocytes into the muscularis after surgical manipulation of the small bowel. This study was pursued to establish a direct causal link between these events. Methods: Reverse-transcription polymerase chain reaction and immunohistochemistry were used to detect and localize expression of adhesion molecules: P-selectin, intercellular adhesion molecule 1 (ICAM-1), and lymphocyte function–associated antigen 1 (LFA-1). Leukocyte infiltration and in vitro jejunal circular muscle function were quantified in controls and manipulated animals with and without antibody treatment (1A29, WT.1, and WT.3). Results: Surgical manipulation caused a significant up-regulation within the muscularis of ICAM-1 and P-selectin messenger RNA. ICAM-1 and P-selectin protein expression was increased within the muscularis microvasculature, and ICAM-1 and LFA-1 were expressed on infiltrating cells. Administration of adhesion molecule antibodies prevented the recruitment of monocytes and neutrophils into the muscularis and also averted jejunal circular muscle dysfunction. Conclusions: The data demonstrate that adhesion molecule antibodies prevent surgically induced suppression of intestinal muscle contractions and therefore suggests that late postoperative ileus is mediated through a leukocytic inflammatory response within the intestinal muscularis externa.

Inhibition of VCAM-1 expression in human bronchial epithelial cells by glucocorticoids.

We have demonstrated previously that cytokines induce surface expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on BEAS-2B bronchial epithelial cells in vitro. The present studies demonstrate glucocorticoid inhibition of cytokine-induced VCAM-1 expression as detected using flow cytometry and Northern blot analysis. Several commonly used inhaled glucocorticoids were tested for their ability to inhibit VCAM-1 and ICAM-1 expression. All glucocorticoids tested inhibited VCAM-1 expression in a dose-dependent manner. No inhibition of ICAM-1 expression was observed. The most potent of the glucocorticoids tested for inhibition of VCAM-1 expression were mometasone furoate and fluticasone propionate (FP), which had IC50 values (i.e., concentrations at which each glucocorticoid produced 50% inhibition) of under 10 pM. Budesonide, triamcinolone acetonide, and beclomethasone dipropionate (BDP) had intermediate potency, and hydrocortisone and the BDP metabolite beclomethasone-17-monopropionate were the least potent of the steroids tested. Kinetic analysis of the ability of FP to inhibit VCAM-1 expression revealed that preincubation with FP for 3 h completely inhibited VCAM-1 expression induced by tumor necrosis factor-alpha (TNF-alpha). FP inhibited VCAM-1 expression by 50% even when added as late as 6 h after stimulation with TNF-alpha. Using Northern blot analysis, we confirmed inhibition of VCAM-1 and ICAM-1 messenger RNA (mRNA) expression by FP. Pretreatment with FP (10(-11) M to about 10(-7) M, 24 h) inhibited TNF-alpha-induced VCAM-1 mRNA expression in BEAS-2B in a dose-dependent manner, but did not inhibit expression of ICAM-1 mRNA. Studies with actinomycin D indicate that FP treatment accelerated the degradation of TNF-alpha-induced VCAM-1 mRNA. FP (10(-7) M) also inhibited VCAM-1 mRNA expression induced by TNF-alpha in primary human bronchial epithelial cells as assessed by reverse transcription-polymerase chain reaction. These results suggest that suppression of epithelial VCAM-1 expression by glucocorticoids may contribute to their anti-inflammatory effects.

Vascular cell adhesion molecule 1 (CD106) on primary human articular chondrocytes: functional regulation of expression by cytokines and comparison with intercellular adhesion molecule 1 (CD54) and very late activation antigen 2.

To investigate the expression of adhesion molecules belonging to the immunoglobulin superfamily on human primary articular chondrocytes and to determine their response pattern to cytokines with respect to the adhesion of lymphocytes. The expression of adhesion molecules was studied by flow cytometry (cultured cells), immunohistochemistry (cartilage), reverse transcription-polymerase chain reaction, and Northern blotting. Adhesion of T cells to chondrocytes was measured using the Jurkat T cell line. Vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) were found to be constitutively expressed on large percentages of unstimulated chondrocytes in culture and in cartilage ex vivo. ICAM-2, ICAM-3, and very late activation antigen 4 (VLA-4; alpha4beta1 integrin), the ligand for VCAM-1, were not detected. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) further induced VCAM-1 and ICAM-1 messenger RNA and protein expression. Transforming growth factor beta (TGFbeta) had no effect on ICAM-1 and decreased the expression of VCAM-1. Another adhesion molecule, VLA-2 alpha2beta1 integrin) that was also expressed on unstimulated chondrocytes, was differentially regulated by cytokines. While neither IL-1beta nor TNFalpha had any effect on expression of VLA-2, TGFbeta markedly increased the alpha2 subunit of VLA-2. Adhesion of Jurkat T cells to chondrocytes was further induced by IL-1beta and TNFalpha. Pretreatment of chondrocytes with monoclonal antibodies to VCAM-1 and ICAM-1 inhibited adhesion of T cells to chondrocytes. VCAM-1, ICAM-1, and VLA-2 are constitutively expressed by human articular chondrocytes. Expression is regulated by cytokines. As shown for other chondrocyte genes, IL-1beta/TNFalpha and TGFbeta antagonistically modulate the expression of adhesion molecules. VCAM-1 and ICAM-1 contribute to adhesion of T lymphocytes to chondrocytes, and may thus participate in host defense mechanisms during inflammatory joint conditions such as rheumatoid arthritis and after cartilage transplantation.

Glutathione depletion prevents lipopolysaccharide-induced local skin inflammation.

We have previously shown that the thiol-oxidizing agent diethyl maleate prevents lipopolysaccharide (LPS)-induced up-regulation of endothelial cell intercellular adhesion molecule-1 (ICAM-1) in vitro. To determine the effect of glutathione depletion on the development of local skin inflammation in vivo, a model known to be dependent on ICAM-1. Swiss Webster mice were injected with intradermal LPS (30 micrograms) or isotonic saline solution. Mice were pretreated for 1 hour with intraperitoneal diethyl maleate (6 mmol/kg) or corn oil vehicle. Injection sites were harvested after 12 and 24 hours and evaluated for changes in vascular permeability and histological characteristics. To determine the mechanism underlying our findings, we evaluated skin ICAM-1 immunohistochemistry, levels of ICAM-1 protein and messenger RNA (mRNA), and neutrophil CD11b expression at the 24-hour point. Diethyl maleate significantly decreased the skin permeability index in a dose-dependent fashion at 24 hours but not at 12 hours. Skin histological examination under light microscopy showed a marked LPS-induced neutrophil infiltration at 24 hours, which was inhibited with diethyl maleate pretreatment. Immunohistochemical examination showed that diethyl maleate reduced ICAM-1 expression. In keeping with the hypothesized mechanism, diethyl maleate attenuated the LPS-induced up-regulation of ICAM-1 mRNA by 44%. Diethyl maleate also slightly but insignificantly reduced CD11b expression in vivo. Diethyl maleate markedly attenuates LPS-induced dermal inflammation, primarily through a reduction in ICAM-1 protein and mRNA expression. These data suggest that manipulation of the intracellular redox state may have a beneficial role in neutrophil-mediated inflammation.

Expression of lung vascular and airway ICAM-1 after exposure to bacterial lipopolysaccharide.

Airway instillation of bacterial lipopolysaccharide (LPS) into rat lungs induces neutrophil accumulation, which is known to be intercellular adhesion molecule-1 (ICAM-1)-dependent. In the present study, ICAM-1 messenger RNA (mRNA) of whole lung was found to increase by 20-fold in this inflammatory model. This increase was reduced by 81% after treatment of animals with anti-tumor necrosis factor-alpha (TNF-alpha) antibody and by 37% after treatment with anti-interleukin-1 (IL-1) antibody. The same interventions reduced whole-lung ICAM-1 protein by 85% and 25%, respectively. The studies were extended to assess the locale in lung of ICAM-I upregulation. Lung vascular ICAM-1 content, which was assessed by vascular fixation of [125I]anti-ICAM-1, rose 4-fold after airway instillation of LPS. This rise was also TNF-alpha-dependent. Under the same experimental conditions, fixation of [125I]anti-ICAM-1 to airway surfaces increased 11-fold in a TNF-alpha-dependent manner. In situ hybridization and immunohistochemical analyses of lung tissue revealed ICAM-1 upregulation in the bronchiolar epithelium and in peribronchiolar smooth muscle. Soluble ICAM-1 could also be detected in bronchoalveolar lavage fluids (BALFs) of animals after intratracheal instillation of LPS. Retrieved alveolar macrophages showed a small, significant, and transient increase in surface expression of ICAM-1. These data indicate, at the very least, a dual compartmentalized (vascular and airway) upregulation of ICAM-1 after airway instillation of LPS. This upregulation requires TNF-alpha and IL-1. The functional significance of upregulated airway ICAM-1 remains to be determined.