Increased Expression Of ICAM-1 Research Articles

Autocrine regulation of endothelial barrier integrity by CAP37

BACKGROUNDCAP37 is a peptide released by neutrophils upon stimulation of β2 integrins. Its effects include increased ICAM‐1 expression and endothelial cell cytoskeleton reorganization. Previous work has shown that endothelial cells may produce CAP37. We sought to determine the effect of neutralization of CAP37 on endothelial monolayer resistance.METHODSHuman umbilical vein endothelial cells (HUVECs) were grown to confluence and activated with TNFα for six hours. Two mouse‐derived monoclonal anti‐CAP37 antibodies (B1B5 and D5F10) were applied to the monolayer. The effect on endothelial barrier function was determined using Electric Cell‐substrate Impedance Sensing (ECIS). Barrier function of the monolayer was expressed as a fraction of pre‐treatment resistance values.RESULTSBoth antibodies caused a biphasic change in monolayer resistance. Within 30 minutes, resistance increased to a peak of 1.16+/−0.04 (B1B5 [p<0.0001 vs. IgG control]) and 1.12+/−0.11 (D5F10 [p=0.03 vs. IgG control]). After this initial peak, resistance progressively decreased, with endothelial barrier integrity falling below 50% of the pre‐treatment value within 5 hours.CONCLUSIONSNeutralization of CAP37 causes a change in endothelial barrier function. This occurs in the absence of leukocytes, demonstrating the role of CAP37 in the autocrine regulation of endothelial monolayer integrity, and suggesting a potential therapeutic target. Future work will include further characterization of endothelial cell CAP37. (NIH GM066194)

Uncovering the neuroenteric–pulmonary axis: Vagal nerve stimulation prevents acute lung injury following hemorrhagic shock

AimsTrauma/hemorrhagic shock (T/HS) induced gut injury is known to initiate a systemic inflammatory response which can lead to secondary lung injury. We have shown that vagal nerve stimulation (VNS) protects intestinal epithelial integrity after a severe burn insult. We hypothesize that VNS will protect the lung from injury following T/HS by preventing intestinal barrier failure. Main methodsMale Balb/c mice were subjected to a T/HS model with and without cervical VNS. Intestinal injury was evaluated by measuring changes in gut barrier function and tight junction protein localization. Lung injury was evaluated using histology and markers of lung inflammation. Using NF-kB-luciferase (NF-kB-luc) transgenic mice, NF-kb–DNA binding was measured by photon emission analysis at 4 after injury. Key findingsT/HS is associated gut injury characterized by histologic injury, increased epithelial permeability, and altered localization of gut tight junction proteins. Cervical VNS prevented the T/HS-induced changes in gut barrier integrity. Gut injury after T/HS was associated with acute lung injury at 24h characterized by histologic injury, increased number of MPO positive stained cells and MPO enzymatic activity, and increased ICAM-1 expression in lung endothelium. VNS decreased T/HS-induced lung injury with a marked decrease in lung inflammation compared to T/HS alone. Lungs harvested from NF-kB-luc mice at 4h post VNS+T/HS demonstrated decreased DNA binding of NF-kB compared to T/HS alone as measured by changes in bioluminescence. SignificanceVNS is effective in protecting against acute lung injury caused by hemorrhagic shock through its ability to prevent gut barrier dysfunction.

Inflammatory factors and leucocytes are involved in the pathogenesis of malaria associated acute lung injury/acute respiratory distress syndrome in murine model.

Event Abstract Back to Event Inflammatory factors and leucocytes are involved in the pathogenesis of malaria associated acute lung injury/acute respiratory distress syndrome in murine model. Michelle K. Sercundes1*, Luana D. Ortolan2, Daniela Debone1, Elizabeth H. Aitken3, José M. Alvarez3, Momtchilo Russo3, Cláudio R. Marinho4 and Sabrina Epiphanio2, 3 1 University of São Paulo, Tropical Medicine, Brazil 2 Federal University of São Paulo, Brazil 3 University of São Paulo, Immunology, Brazil 4 University of São Paulo, Parasitology, Brazil INTRODUCTION: Malaria is a huge burden on global health and caused 2.4 million deaths in 2012. Severe malaria can manifest in the lungs, an illness known as acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Using an experimental model previously established by our group we evaluated the immune cell profile and inflammatory response in malaria associated ALI/ARDS. METHODS AND RESULTS: DBA/2 mice were infected with Plasmodium berghei ANKA. Lungs and bronchoalveolar lavage (BAL) were collected 7 days post infection (7dpi). Using the respiratory patterns (enhanced pause and respiratory frequency) and parasitemia from a survival group, we established ROC curves and were able to classify experimental animals euthanized 7dpi as suffering ALI/ARDS or HP (hyperparasitemia). There was an increase in neutrophils, seen by increased Ncf2 mRNA expression (qRTPCR) and increased numbers of Gr-1high CD11b+ cells (flow cytometry), in the lungs of ALI/ARDS mice compared to HP mice. In the lung we observed increased numbers of alveolar macrophages (F4/80+/CD11C+) and fewer interstitial macrophages (F480+) (flow cytometry) and in the BAL we observed an increase in macrophages and neutrophils (cytospin) in the ALI/ARDS mice. We also found that mRNA expression of iCAM-1, HO-1, IL-10 (anti-inflammatory) and IFNγ (pro-inflammatory) genes was elevated (qRTPCR) in the lungs of the ALI/ARDS mice. CONCLUSION: Our data suggests that neutrophils and alveolar macrophages play a role in ALI/ARDS development. We hypothesize that the neutrophil recruitment is partly dependent on increased ICAM-1 expression and that the high IL-10 mRNA expression is an attempt to re-establish homeostasis. Keywords: Acute Lung Injury, DBA/2 mice, Neutrophils, Inflammatory factors, Acute Respiratory Distress Syndrome Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Sercundes MK, Ortolan LD, Debone D, Aitken EH, Alvarez JM, Russo M, Marinho CR and Epiphanio S (2013). Inflammatory factors and leucocytes are involved in the pathogenesis of malaria associated acute lung injury/acute respiratory distress syndrome in murine model.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00004 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Ms. Michelle K Sercundes, University of São Paulo, Tropical Medicine, São Paulo, Brazil, michellesercundes@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Michelle K Sercundes Luana D Ortolan Daniela Debone Elizabeth H Aitken José M Alvarez Momtchilo Russo Cláudio R Marinho Sabrina Epiphanio Google Michelle K Sercundes Luana D Ortolan Daniela Debone Elizabeth H Aitken José M Alvarez Momtchilo Russo Cláudio R Marinho Sabrina Epiphanio Google Scholar Michelle K Sercundes Luana D Ortolan Daniela Debone Elizabeth H Aitken José M Alvarez Momtchilo Russo Cláudio R Marinho Sabrina Epiphanio PubMed Michelle K Sercundes Luana D Ortolan Daniela Debone Elizabeth H Aitken José M Alvarez Momtchilo Russo Cláudio R Marinho Sabrina Epiphanio Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Cross-linking of IgGs bound on circulating neutrophils leads to an activation of endothelial cells: possible role of rheumatoid factors in rheumatoid arthritis-associated vascular dysfunction.

BackgroundRheumatoid arthritis is characterized by the presence of circulating auto-antibodies, including rheumatoid factors, which recognize the Fc portion of IgGs. The neutrophil is the most abundant circulating leukocyte and it expresses high levels of FcγRs on its surface. The aim of the present study was to examine the capacity of circulating human neutrophils to be activated by rheumatoid factors and the consequences of these events on endothelium.MethodsNeutrophil-bound IgGs were cross-linked with anti-human IgGs to mimick the presence of circulating rheumatoid factors and FcγRs-dependent signalling events and functions were examined. The IgG and IgM composition of rheumatoid factors isolated from the serum of RA patients was characterized. Adhesion of neutrophils to endothelial cells was quantified in response to the addition of rheumatoid factors.ResultsCross-linking of IgGs bound on neutrophils leads to FcγRs-dependent tyrosine phosphorylation, mobilisation of intracellular calcium and the extracellular release of superoxide anions and lysozyme. Incubation of endothelial cells with the supernatant of activated neutrophils increases ICAM-1 expression and IL-8 production by endothelial cells. Finally, rheumatoid factors enhance neutrophil adhesion to endothelial cells.ConclusionsOur results show that activation of neutrophils’ FcγRs by rheumatoid factors could participate in rheumatoid arthritis-associated vascular damage.

Angiotensin II Receptor Blocker Improves Tumor Necrosis Factor-α-Induced Cytotoxicity via Antioxidative Effect in Human Glomerular Endothelial Cells

Background/Aims: Tumor necrosis factor-α (TNF-α) is known to involve the progression of renal dysfunction through its cytotoxicity and proinflammatory effects such as the induction of intercellular adhesion molecule (ICAM)-1 expression in vascular endothelial cells (ECs). Olmesartan, one of the angiotensin II type 1 receptor blockers (ARBs), has been reported to show protective effects on injured ECs by some causal factors of renal disorder other than angiotensin II. However, the effects of olmesartan on TNF-α-induced glomerular EC damage have not been investigated. In the present study, we investigated the effects of RNH-6270, an active metabolite of olmesartan, on TNF-α-induced human glomerular EC (HGEC) damage to clarify the renoprotective mechanisms of ARBs. Methods: Cultured HGECs were stimulated by TNF-α, and then cell viability and cytotoxicity were measured by MTT assay and lactate dehydrogenase release assay, respectively. TNF-α-induced oxidative stress was estimated by dihydroethidium assay and lucigenin chemiluminescence assay. ICAM-1 expression and the phosphorylations of mitogen-activated protein kinases were measured using Western blotting assay. Results: RNH-6270 suppressed cell death and the increase in ICAM-1 expression induced by TNF-α via the inhibition of reactive oxygen species in HGECs. Conclusion: Our findings suggested that olmesartan might have protective effects against TNF-α-induced glomerular EC dysfunction.

Interleukin‐11 Increases Cell Motility and Up‐Regulates Intercellular Adhesion Molecule‐1 Expression in Human Chondrosarcoma Cells

Interleukin-11 (IL-11) was originally identified as the cytokine that could induce the proliferation of human cells. Recent studies have shown that IL-11 plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of IL-11 on human chondrosarcoma cells are largely unknown. Here, we found that IL-11 increased the migration and expression of intercellular adhesion molecule-1 (ICAM)-1 in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significant expression of the IL-11 which was higher than that in primary chondrocytes. The phosphatidylinositol 3-kinase (PI3K), Akt, and NF-κB pathways were activated by IL-11 treatment, and the IL-11-induced expression of ICAM-1 and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF-κB cascades. Taken together, our results indicate that IL-11 enhanced the migration of the chondrosarcoma cells by increasing ICAM-1 expression through the IL-11Rα receptor, PI3K, Akt, and NF-κB signal transduction pathway.

Direct Pro-Inflammatory Effect of C-Reactive Protein on Human Pulmonary Artery Endothelial Cells

C-reactive protein (CRP) has a prognostic role in cardiovascular and pulmonary diseases. Recent data suggest its pro-inflammatory effects in atherosclerotic lesion formation. This raises the hypothesis of whether or not CRP has pro-inflammatory effects on pulmonary vasculature by inducing the production of endothelin-1 (ET)-1, a potent vasoconstrictor and proliferative cytokine, and expression of adhesion molecules which could culminate in inflammatory cell recruitment and vascular injury. Human pulmonary artery endothelial cells (HPAECs) were cultured and incubated with 25μg/ml of human recombinant CRP and with interleukin (IL)-1β 10ng/ml, a well-known activator of endothelial cells, which served as a positive control for 24 hours. Expression of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 was assessed by flow cytometry. Secretion of ET-1 from HPAECs was also evaluated. In this study we show that incubation of HPAECs with human recombinant CRP for 24 hours induced a significant increase in ICAM-1 expression (from 610 to 6553 mean fluorescence intensity, p < 0.005) and VCAM-1 expression (from 212 to 303 mean fluorescence intensity, p < 0.05), as compared to control. Adhesion molecule induction was similar to that observed in endothelial cells activated with IL-1β. Likewise, CRP potentiated the ET-1 production by HPAECs. The levels of ET-1 were significantly higher at 24 hours (control 19.94±3 vs CRP 46.54±18 pg/ml, p < 0.05). In conclusion, this study makes a novel observation that CRP induces expression of adhesion molecules and secretion of ET-1 in HPAECs. Our study provides the first evidence that CRP exerts direct proinflammatory effects on pulmonary artery endothelial cells.

ICAM-1 Upregulation in Ethanol-Induced Fatty Murine Livers Promotes Injury and Sinusoidal Leukocyte Adherence after Transplantation

Background. Transplantation of ethanol-induced steatotic livers causes increased graft injury. We hypothesized that upregulation of hepatic ICAM-1 after ethanol produces increased leukocyte adherence, resulting in increased generation of reactive oxygen species (ROS) and injury after liver transplantation (LT). Methods. C57BL/6 wildtype (WT) and ICAM-1 knockout (KO) mice were gavaged with ethanol (6 g/kg) or water. LT was then performed into WT recipients. Necrosis and apoptosis, 4-hydroxynonenal (4-HNE) immunostaining, and sinusoidal leukocyte movement by intravital microscopy were assessed. Results. Ethanol gavage of WT mice increased hepatic triglycerides 10-fold compared to water treatment (P < 0.05). ICAM-1 also increased, but ALT was normal. At 8 h after LT of WT grafts, ALT increased 2-fold more with ethanol than water treatment (P < 0.05). Compared to ethanol-treated WT grafts, ALT from ethanol-treated KO grafts was 78% less (P < 0.05). Apoptosis also decreased by 75% (P < 0.05), and 4-HNE staining after LT was also decreased in ethanol-treated KO grafts compared to WT. Intravital microscopy demonstrated a 2-fold decrease in leukocyte adhesion in KO grafts compared to WT grafts. Conclusions. Increased ICAM-1 expression in ethanol-treated fatty livers predisposes to leukocyte adherence after LT, which leads to a disturbed microcirculation, oxidative stress and graft injury.

Manganese downregulates ICAM‐1 expression in endothelial cells treated with high glucose

Diabetes is associated with low levels of manganese (Mn2+), an important component of many enzyme systems. Previous studies in our laboratory have shown that Zucker diabetic fatty rats (ZDF) supplemented with Mn2+ lower cholesterol, triglycerides, MCP‐1, ICAM‐1 and higher adiponectin levels in blood. This study examined the potential mechanisms by which Mn2+ supplementation decreases blood cholesterol and triglycerides and atherosclerosis associated with diabetes. Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of Mn2+ (0, 5, 10 and 20μM) for 24 hours, then exposed to high glucose (25mM) or MCP‐1 (250pg/mL) for another 24 hrs. ICAM‐1 expression was determined by Western blotting. Our results demonstrate that HG increases ICAM‐1 expression by two fold compared with controls, and that pre‐treatment with 5μM Mn2+ decreased ICAM‐1 expression back to control levels. Similar experiments with 3T3‐L1 adipocytes showed that pre‐treatment with 10μM Mn2+ inhibited HG‐induced secretion of MCP‐1 levels to control levels. ICAM‐1 and MCP‐1 are key players in the development of atherosclerosis and CVD in diabetes. This study suggests that Mn2+ supplementation inhibits MCP‐1 secretion and ICAM‐1 expression, which in turn may lower circulating triglycerides and cholesterol and risk of development of atherosclerosis in diabetes. Supported by NIDDK (RO1 DK072433), Office of Dietary Supplements and Malcolm Feist Chair in Diabetes.

Leukocyte rolling and adhesion via ICAM-1 signals to endothelial permeability. Focus on “Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1”

THE VASCULAR ENDOTHELIUM plays a vital role in the inflammatory response by induction and surface expression of adhesion molecules and chemokines. At sites of acute inflammation, blood flow is increased and postcapillary venules exhibit increased permeability (leakiness) and support the influx of blood leukocytes, primarily neutrophils. The relationship between neutrophil influx and increased vessel permeability has been a topic of investigation for decades. Many investigations have reported a direct neutrophil-dependent increase in vessel permeability during inflammation using in vivo and in vitro models (reviewed in Ref. 4). One aspect that has received increased attention is whether endothelial cell adhesion molecules contribute to the regulation of vessel permeability, in addition to their well-characterized role in neutrophil rolling and adhesion. Intercellular adhesion molecule-1 (ICAM-1, also known as CD54) is an immunoglobulin superfamily member that is abundantly expressed on the endothelial cell surface and is enriched at endothelial cell borders in vivo and in vitro (2, 12, 14, 17). ICAM-1 surface expression is upregulated in endothelial cells by proinflammatory cytokines like TNF-, IL-1, IFN-, or bacterial endotoxins, and ICAM-1 serves as a receptor for leukocyte 2-integrins (LFA-1 and Mac-1) (reviewed in Ref. 15). In an experiment of nature, patients with leukocyte adhesion deficiency-1 (LAD-1) have a severe primary immune deficiency in which blood neutrophils fail to localize to sites of inflammation or injury. This defect occurs because patients’ neutrophils either lack 2-integrins or contain mutations in these molecules. Studies using neutrophils from LAD-1 patients or 2-integrin-deficient mice clearly demonstrate that these cells have normal selectin-dependent rolling on activated endothelium but fail to stably arrest and transmigrate. Additional studies have demonstrated that endothelial-expressed ICAM-1 contributes to both kinetics of leukocyte rolling and arrest in vivo as determined by intravital microscopy of the cremaster muscle microcirculation in ICAM-1 / mice (16). In this issue of American Journal of Physiology-Cell Physiology, Sumagin and colleagues (18) use elegant in vivo studies to demonstrate that microvascular permeability in the cremaster muscle model is regulated by leukocyte engagement of ICAM-1. The authors monitored permeability (Ps) in cremaster microcirculation by efflux of luminal fluorescent-tagged albumin by fluorescence confocal intravital microscopy. Prior studies by Sarelius and colleagues (19) in the same model demonstrated that ICAM-1 engagement induced a localized increase in vessel permeability in unstimulated arterioles and in TNF-stimulated venules. In agreement with this finding ,4ho f TNF- stimulation of arterioles and venules in ICAM-1 knockout (KO) animals did not result in increased vessel permeability as compared with wild-type (WT) mice under identical conditions. Similar results were seen with 2-integrin KO mice. In the current study, the authors provide further evidence linking ICAM-1 expression to regulation of endothelial permeability. Comparisons between unstimulated venules and TNF--treated venules reveal that neutrophil rolling regulates venule permeability in unstimulated venules while leukocyte adhesion regulates venule permeability in TNF--stimulated venules. The 2-integrin-blocking antibodies, which inhibit neutrophil arrest on endothelium, reduced the vessel permeability, and this was further reduced by treatment with a combination of 2-integrin and P-selectin blocking antibodies that inhibits both rolling and arrest. Interestingly, the reduction in venule permeability observed by blockade of P-selectin and 2-integrin function were suggested to be due to insufficient ICAM-1 engagement due to the absence of rolling leukocytes. To examine the role that neutrophils play, circulating neutrophils were depleted by antineutrophil GR-1 antibody treatment. Notably, depletion of neutrophils caused a marked decrease in

Constitutive Expression and Enzymatic Cleavage of ICAM-1 in the Spontaneously Hypertensive Rat

Background/Aims: Leukocyte adhesion to the endothelium is abnormal in hypertension. We have recently shown that spontaneously hypertensive rats (SHRs) have circulating leukocytes with enhanced CD18 receptor cleavage. In the current study, we investigate expression levels of its counter receptor, intercellular adhesion molecule (ICAM-1), and its possible proteolytic cleavage in the SHR and control Wistar rat. Methods: ICAM-1 was labeled on tissue sections with two antibodies targeting its extracellular and intracellular domains and evaluated by light absorption measurements. The in situ cleavage of ICAM-1 was assessed by treating vessel sections with matrix metalloproteinase (MMP)-7, MMP-9 and elastase. Results: SHRs showed a significant increase in ICAM-1 expression in liver and kidney compared with Wistar rats. The liver and kidney glomeruli exhibit a discrepancy in label density between intra- and extracellular antibodies, which suggests that enzymatic cleavage may be a factor determining ICAM-1 distribution. MMP-7 and MMP-9, which are elevated in SHR plasma, and elastase, which has elevated activity in SHR neutrophils, cleave the extracellular domain of ICAM-1 when applied to the tissue. Conclusion: ICAM-1 expression in SHRs is upregulated in a tissue-specific manner. Proteolytic cleavage of the extracellular domain of ICAM-1 and accumulation in kidney glomeruli may play a role in the renal involvement of inflammation.

Real‐time fluorescence microscopy reveals ICAM‐1‐dependent leukocyte margination in microvessels of LPS‐treated lungs

Increased leukocyte migration from lung vessels into airspaces is a hallmark of sepsis. However, specific migration sites and mechanisms remain unclear. Toward this, we prepared isolated blood‐perfused rat lungs and imaged surface vessels by real‐time fluorescence microscopy. Via a left atrial catheter, we infused Ringer's solution or LPS (100 μg/ml) for 30 min into microvessels. After 1hr we infused leukocytes (100000 cells/ml) labeled with Rhodamine 6G and imaged their transit through microvessels. Then we quantified the number of marginated leukocytes in microvessels in 5 distinct 62500 μm2 regions for each lung. Ringer's infusion resulted in margination of 9.2±0.8 (mean±SE, n=3) leukocytes. In contrast, LPS infusion doubled the number of marginated leukocytes to 21.5±3.7 (n=3; p&lt;0.05). Margination increased in both capillaries and venules, suggesting that LPS increased leukocyte accumulation in both vessel segments. To define mechanisms, we infused an ICAM‐1 antibody into LPS‐treated microvessels prior to leukocyte infusion. The antibody blunted the LPS response to control levels (6.5±0.5; n=3) in both venules and capillaries, indicating that ICAM‐1 mediates LPS‐induced leukocyte margination in these microvessels. We interpret that in lung, LPS increases ICAM‐1 expression in capillaries and venules; and both microvessel segments may be sites of leukocyte migration during sepsis. (NIH HL75503)

RANKL increases migration of human lung cancer cells through intercellular adhesion molecule‐1 up‐regulation

Invasion of distant tissues by tumor cells is the primary cause of therapeutic failure in the treatment of malignant lung cancer cells. Receptor activator of nuclear factor-κB ligand (RANKL) and its receptor, RANK, play a key role in osteoclastogenesis and tumor metastasis. Intercellular adhesion molecule-1 (ICAM-1, also called CD54), a member of the immunoglobulin supergene family, is an inducible surface glycoprotein that mediates adhesion-dependent cell-to-cell interactions. The effects of RANKL on cell migration and ICAM-1 expression in human lung cancer cells are largely unknown. We found that RANKL directed the migration and increased ICAM-1 expression in human lung cancer (A549) cells. Pretreatment of A549 cells with the MAPK kinase (MEK) inhibitor PD98059 or U0126 inhibited RANKL-mediated migration and ICAM-1 expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, an NF-κB inhibitor (PDTC) and IκB protease inhibitor (TPCK) also inhibited RANKL-mediated cell migration and ICAM-1 up-regulation. Taken together, these results suggest that the RANKL and RANK interaction acts through MEK/ERK, which in turn activates NF-κB, resulting in the activation of ICAM-1 and contributing to the migration of human lung cancer cells.

The effect of physiologically relevant dynamic shear stress on platelet and endothelial cell activation

IntroductionBlood flow induced shear stress plays an important role in platelet and endothelial cell functions. The goal of this study was to investigate the effect of physiologically relevant dynamic shear stress on platelet and endothelial cells. Materials and MethodsPulsatile shear stress waveforms mimicking the flow in a normal left coronary artery (0.1-1Pa), at a 60% stenosis (0.2 - 6Pa) and in the recirculation zone (0.01 - 0.5Pa) behind a stenosis were used to stimulate platelets and endothelial cells in a cone and plate shearing device. Platelet activation was measured by CD62P expression and thrombogenicity. Meanwhile, endothelial cell activation and damage was measured by cell surface ICAM-1 and tissue factor expression using fluorescence microscopy. Endothelial tissue factor activity was measured using a commercial kit. ResultsResults showed that for platelets, a short exposure to elevated shear stress at the stenosis throat did not induce significant increase in platelet activation or thrombogenicity. While the low pulsatile shear stress had a potential for enhanced thrombosis. Both low and high pulsatile shear stress led to a significant increase in ICAM-1 expression on endothelial cell surface, but only low shear stress caused tissue factor over expression and enhanced tissue factor activity. ConclusionThese results suggest that low pulsatile shear stress may be more atherogenic, compared to elevated shear stress induced by stenosis.

Interleukin‐17 synergizes with IFNγ or TNFα to promote inflammatory mediator release and intercellular adhesion molecule‐1 (ICAM‐1) expression in human intervertebral disc cells

Interleukin-17 (IL-17) is a cytokine recently shown to be elevated, along with interferon-γ (IFNγ) and tumor necrosis factor (TNFα), in degenerated and herniated intervertebral disc (IVD) tissues, suggesting a role for these cytokines in intervertebral disc disease. The objective of our study was to investigate the involvement of IL-17 and costimulants IFNγ and TNFα in intervertebral disc pathology. Cells were isolated from anulus fibrosus and nucleus pulposus tissues of patients undergoing surgery for intervertebral disc degeneration or scoliosis. The production of inflammatory mediators, nitric oxide (NOx), prostaglandin E2 (PGE2) and interleukin-6 (IL-6), as well as intercellular adhesion molecule (ICAM-1) expression, were quantified for cultured cells following exposure to IL-17, IFNγ, and TNFα. Intervertebral disc cells exposed to IL-17, IFNγ, or TNFα showed a remarkable increase in inflammatory mediator release and ICAM-1 expression (GLM and ANOVA, p < 0.05). Addition of IFNγ or TNFα to IL-17 demonstrated a synergistic increase in inflammatory mediator release, and a marked increase in ICAM-1 expression. These findings suggest that IVD cells not only respond with a catabolic phenotype to IL-17 and costimulants IFNγ and TNFα, but also express surface ligands with consequent potential to recruit additional lymphocytes and immune cells to the IVD microenvironment. IL-17 may be an important regulator of inflammation in the IVD pathologies.

Modulation by Flunisolide of Tumor Necrosis Factor-α–Induced Stimulation of Airway Epithelial Cell Activities Related to Eosinophil Inflammation

Background: Tumor necrosis factor (TNF)-α, a proinflammatory cytokine involved in the pathogenesis of asthma, displays multiple functions on a variety of cells, including bronchial epithelial cells (BECs). Objective: To characterize in vitro changes induced by TNF-α on the function of BECs that may be related to eosinophilic inflammation and to evaluate their modulation by an inhaled corticosteroid, flunisolide. Methods: A normal human bronchial epithelial cell line (BEAS-2B) was incubated with TNF-α (10 ng/ml) to evaluate (a) intercellular adhesion molecule (ICAM)-1 expression and granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-5 release by BEAS-2B; (b) eosinophil adhesion to BEAS-2B; and (c) the modulation of these activities by flunisolide (0.1 to 10 μM). Results: Stimulation of BEAS-2 with TNF-α generated an increase in ICAM-1 expression (p = .0012), in GM-CSF and IL-5 release (p < .01), and in eosinophil adhesion to BEAS-2B, but this latter effect did not reach statistical significance. Flunisolide at all the tested concentrations effectively inhibited ICAM-1 expression and GM-CSF and IL-5 release (p < .05). The percent inhibition induced by the highest flunisolide concentration (10 μM) for the various BEAS-2B functions was 30%, 60%, and 70%, respectively. The effect of flunisolide appeared to be related to an inhibition of “TNF-α–induced” ICAM-1 expression and cytokine release with little or no involvement of the “constitutive” expression and release. Conclusion: An increase in ICAM-1 expression in BECs was found to be induced by TNF-α and associated with enhancement of the constitutive secretion of GM-CSF and IL-5, cytokines related to eosinophilic inflammation. The ability of flunisolide to modulate these BECs activities appears to be mostly related to the inhibition of the “TNF-α–induced” responses.

Interaction between nanoparticles and cytokine proteins: impact on protein and particlefunctionality

There is increased use of nanomaterials in many applications due to their uniqueproperties, such as their high surface area and surface reactivity. However, thepotential health effects to workers, consumers and the environment exposed tonanoparticles (NPs) is unknown. The aim of this study was to investigate whether NPswhich may enter the body could adsorb proteins and whether this interactionaffects both the particle and the protein function. The cytokines IL-8 and TNF-α were adsorbed significantly more by 14 nm carbon black (CB) compared with a similar dose of260 nm CB. Uncoated 14 nm CB particles produced a significant increase in intracellular calcium[Ca2 + ]i which was greater than a similar mass dose of 260 nm CB. The 260 nmCB produced an increase in ICAM-1 expression in A549 epithelialcells at a comparable dose of 14 nm CB, and after coating with TNF-α 260 nm CB produced significantly more ICAM-1 expression compared with control cells. TNF-α bound to 14 nm CB induced a level of ICAM-1 expression thatwas no greater than the control level, suggesting that the TNF-α activity may be inhibited. These results suggest that NP–protein interaction results both ina decrease in protein function and particle activity in the cellular assays tested and this iscurrently being investigated.

Reduction of ICAM-1 expression by carbon monoxide via soluble guanylate cyclase activation accounts for modulation of neutrophil migration

Previously, it was demonstrated that the heme/heme oxygenase (HO)/carbon monoxide (CO) pathway inhibits neutrophil recruitment during the inflammatory response. Herein, we addressed whether the inhibitory effect of the HO pathway on neutrophil adhesion and migration involves the reduction of intracellular adhesion molecule type (ICAM)-1 and beta(2)-integrin expression. Mice pretreated with a specific inhibitor of inducible HO (HO-1), zinc protoporphyrin (ZnPP) IX, exhibit enhanced neutrophil adhesion and migration induced by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS). These findings are associated with an increase in ICAM-1 expression on mesentery venular endothelium. In accordance, HO-1 inhibition did not enhance LPS-induced neutrophil migration and adhesion in ICAM-1-deficient mice. Furthermore, the treatment with a CO donor (dimanganese decacarbonyl, DMDC) that inhibits adhesion and migration of the neutrophils, reduced LPS-induced ICAM-1 expression. Moreover, neither DMDC nor ZnPP IX treatments changed LPS-induced beta(2)-integrin expression on neutrophils. The effect of CO on ICAM-1 expression seems to be dependent on soluble guanylate cyclase (sGC) activation, since 1H-(1,2,4)oxadiazolo (4,3-a)quinoxalin-1-one (sGC inhibitor) prevented the observed CO effects. Finally, it was observed that the nitric oxide (NO) anti-inflammatory effects on ICAM-1 expression appear to be indirectly mediated by HO-1 activation, since the inhibition of HO-1 prevented the inhibitory effect of the NO donor (S-nitroso-N-acetylpenicillamine) on LPS-induced ICAM-1 expression. Taken together, these results suggest that CO inhibits ICAM-1 expression on endothelium by a mechanism dependent on sGC activation. Thus, our findings identify the HO-1/CO/guanosine 3'5'-cyclic monophosphate pathway as a potential target for the development of novel pharmacotherapy to control neutrophil migration in inflammatory diseases.

Corticosteroids and Montelukast: Effects on Airway Epithelial and Human Umbilical Vein Endothelial Cells

Our primary objective was to investigate the possible contribution of controller medications to asthmatic airway remodeling, by (1) comparing the apoptotic and necrotic effects of several corticosteroids and montelukast on cultured airway human bronchial surface epithelial (16HBE) and submucosal (Calu3) cells; (2) measuring epithelial shedding potential and desmosome length in response to a cytokine challenge, with or without co-administered corticosteroids; and (3) studying corticosteroids and montelukast effects on inter-cellular adhesion molecule (ICAM) expression in both 16HBE and human umbilical vein endothelial cells (HUVEC). For this purpose, apoptosis, necrosis, and ICAM expression were quantified by flow cytometry, with 16HBE cells sensitive to both the apoptotic and necrotic effects of dexamethasone and montelukast; Calu3 cells sensitive only to budesonide. Transmission electron microscopy revealed decreased desmosome length in the presence of cytokines (TNF-alpha and INF-gamma), with corticosteroids counteracting this reduction. Dexamethasone, beclomethasone, and montelukast decreased versus increased ICAM-1 expression in airway epithelial cells and HUVEC, respectively. For conclusions, bronchial surface epithelial and submucosal cells exhibit a different sensitivity profile toward dexamethasone, budesonide, and montelukast, with corticosteroids preventing cytokineinduced desmosomal damage in 16HBE cells. The studied drugs led to increased ICAM-1 expression in endothelium, potentially facilitating inflammatory cell migration into lung tissue.

Degraded Carrageenan Causing Colitis in Rats Induces TNF Secretion and ICAM-1 Upregulation in Monocytes through NF-κB Activation

Carrageenan (CGN) is a high molecular weight sulphated polysaccharide derived from red seaweeds. In rodents, its degraded forms (dCGN) can induce intestinal inflammation associated with macrophage recruitment and activation. The aim of this study was: 1) to analyze the size-dependent effects of dCGN on colon inflammation in vivo, and 2) to correlate these effects with monocyte/macrophage proliferation, cytokine production and expression of various cell surface antigens including ICAM-1 adhesion molecule. Peripheral blood monocytes (PBM) and THP-1 monocytic cells were cultured in the presence of either 10 or 40 kDa, dCGN. The 40 kDa, but not the 10 kDa dCGN, induced colitis in in vivo. Degraded CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype.