Daniel H. Sturn

The immune modulator FTY720 targets sphingosine–kinase-dependent migration of human monocytes in response to amyloid beta-protein and its precursor

Accumulation of inflammatory mononuclear phagocytes in Alzheimers senile plaques, a hallmark of the innate immune response to β‐amyloid fibrils, can initiate and propagate neurodegeneration characteristic of Alzheimers disease. Phagocytes migrate toward amyloid β‐ protein involving formyl peptide receptor like‐1‐dependent signaling. Using human peripheral blood monocytes in Boyden chamber micropore filter assays, we show that the amyloid β‐ protein‐ and amyloid β‐precursor protein‐induced migration was abrogated by dimethylsphingosine, a sphingosine kinase inhibitor. Amyloid β‐protein stimulated in monocytes the gene expression for sphingosine‐1‐phosphate receptors 2 and 5, but not 1, 3, and 4. FTY720 that acts as a sphingosine‐1‐phosphate receptor agonist after endogenous phosphorylation by sphingosine kinase, as well as various neuropeptides that are known to be monocyte chemoattractants, dose‐dependently inhibited amyloid β‐protein‐induced migration. These data demonstrate that the migratory effects of β‐amyloid in human monocytes involve spingosine‐1‐ phosphate signaling. Whereas endogenous neuropeptides may arrest and activate monocytes at sites of high β‐amyloid concentrations, interference with the amyloid β‐protein‐dependent sphingosine‐1‐phosphate pathway in monocytes by FTY720, a novel immunomodulatory drug, suggests that FTY720 may be efficacious in β‐amyloid‐related inflammatory diseases.

Syndecan-4-dependent signaling in the inhibition of endotoxin-induced endothelial adherence of neutrophils by antithrombin

Circulating endotoxin is elevated in sepsis and plays a role in endothelial dysfunction whereas antithrombin is decreased by virtue of its consumption during complex formation with clotting factors and by proteolytic degradation by granulocyte elastase. Dysfunction of endothelium results in enhanced leukocyte rolling and diapedesis into tissues leading to edema formation and injury. Antithrombin exerts beneficial effects on endothelial function in sepsis. A direct anti-inflammatory action of anti-thrombin in inflammatory cells is exerted via heparan sulfate proteoglycans. In this study, we investigated whether antithrombin affects endotoxin-induced adhesion of neutrophils to human endothelial cells in vitro and whether glycosaminoglycans are involved in its signaling. Adhesion of human neutrophils to monolayers of umbilical vein endothelial cells was tested under static conditions. Endothelial cells were pretreated with endotoxin, interleukin-1, heparinase-I, chondroitinase-ABC or anti-syndecan-4-antibody. Endotoxin and interleukin-1 increased neutrophil adherence to human umbilical vein endothelial cells which was inhibited by antithrombin. Concomitant incubation with pentasaccharide abolished this effect of antithrombin. Treatment of endothelial cells with heparinase or chondroitinase led to higher adhesion and prevented effects of antithrombin. With antibodies to syndecan-4, enhanced adhesion of neutrophils was observed. As studied by Western blotting, endotoxin-induced signaling was diminished by antithrombin and the effect was reversible by chondroitinase or heparinase. From our results, we can conclude that endotoxin-induced adhesion of leukocytes to endothelium can be reversed by ligation of syndecan-4 with antithrombins heparin-binding site and interferences with stress response signaling events in endothelium.

Endothelial Protein C Receptor-Dependent Inhibition of Migration of Human Lymphocytes by Protein C Involves Epidermal Growth Factor Receptor

The protein C pathway is an important regulator of the blood coagulation system. Protein C may also play a role in inflammatory and immunomodulatory processes. Whether protein C or activated protein C affects lymphocyte migration and possible mechanisms involved was tested. Lymphocyte migration was studied by micropore filter assays. Lymphocytes that were pretreated with protein C (Ceprotin) or activated protein C (Xigris) significantly reduced their migration toward IL-8, RANTES, MCP-1, and substance P, but not toward sphingosine-1-phosphate. The inhibitory effects of protein C or activated protein C were reversed by Abs against endothelial protein C receptor and epidermal growth factor receptor. Evidence for the synthesis of endothelial protein C receptor by lymphocytes is shown by demonstration of receptor mRNA expression and detection of endothelial protein C receptor immunoreactivity on the cells’ surface. Data suggest that an endothelial protein C receptor is expressed by lymphocytes whose activation with protein C or activated protein C arrests directed migration. Exposure of lymphocytes to protein C or activated protein C stimulates phosphorylation of Tyr845 of epidermal growth factor receptor, which may be relevant for cytoprotective effects of the protein C pathway.

Angiopoietin Affects Neutrophil Migration

Objective: After an ischemic event vascular growth factors are involved in regulating leukocyte infiltration in inflammatory processes. This study focused on effects of 2 other angiogenic growth factors, angiopoietin‐1 and angiopoietin‐2, on human neutrophils and on the involvement of the angiopoietin receptor Tie‐2.

Mechanisms of Neutrophil Transmigration Across Renal Proximal Tubular HK-2 Cells

Background: Adhesion of intratubular leukocytes to proximal tubules in biopsies of patients with rapidly progressive glomerulonephritis and the appearance of leukocytes in the urine in interstitial nephritis suggest interactions between leukocytes and tubular epithelia in renal diseases. The aim of this study was to investigate the effect of cytokines and endotoxin on leukocyte migration through proximal tubular epithelial cells and also to determine the role of the transmembrane adhesion molecules ICAM-1 and CD47 in this process. Methods: Experiments determined transepithelial migration (TEM) of PMN (polymorphonuclear) leukocytes through monolayers of HK-2. Expression of ICAM-1 and CD47 was assessed via confocal immunofluorescence, FACS analysis and western blotting. The effect of antibodies against ICAM-1 and CD47 on TEM was examined. Furthermore measurements of cytokine release (IL- 6 and IL-8) were performed. Results: Preincubation of HK-2 cells with either TNFα or LPS resulted in stimulation of PMN migration through monolayers of HK-2 cells. There was no preferred direction of transmigration. ICAM-1 was expressed by HK-2 cells and expression was increased after 4 h stimulation with TNFα or LPS. Application of ICAM-1 antibodies inhibited TEM. CD47 was expressed in both HK-2 cells and PMN. CD47 antibodies inhibited predominantly basolateral-to-apical TEM. HK-2 cells released IL-8 and IL-6 preferably into the apical compartment. Additionally, we showed that fMLP induced transmigration through monolayers of HK-2 cells was associated with significant increased CD47 expression on PMN cell surfaces. Conclusions: Inflammatory mediators stimulate TEM of PMN through monolayers of HK-2 cells without a clearly discernible preference of direction. Mechanisms involved in TEM stimulated by cytokines or endotoxin appear to be mainly changes in surface receptor densities of HK-2 cells with ICAM-1 and CD47 playing an essential role.

Cd40-ligand-dependent induction of Cox-2 gene expression in endothelial cells by activated platelets: inhibitory effects of atorvastatin

Increasing evidence shows the importance of platelet–endothelial cell interactions in the progression of atherosclerosis. Platelets contribute to coronary events both as major components of thrombi and as a triggering factor in inflammation that leads to plaque vulnerability. Recent data suggest that statins, besides their lipid-lowering properties, exert pleiotropic effects that may be beneficial in atherosclerosis. Whether activated platelets influence cyclooxygenase-2 (COX-2) expression in human umbilical vein endothelial cells (HUVEC), the effect of atorvastatin, and possible mechanisms were investigated. COX-2 gene expression in HUVEC was studied using real-time polymerase chain reaction. CD40 ligand surface expression of platelets was tested by fluorescence-activated cell sorting analyses. Activated platelets significantly up-regulated COX-2 gene expression in HUVEC. Co-incubation of platelets with atorvastatin was shown to reverse this up-regulation via reduction of CD40 ligand surface expression on platelets. Data suggest that atorvastatin influences CD40–CD40-ligand-dependent platelet–endothelial interaction and that this influence affects platelet-induced COX-2 expression in HUVEC.