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 TNFalpha 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 TNFalpha 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.

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. METHODS: Neutrophils were from venous blood of healthy donors and cell migration was studied by micropore filter assays. Receptor expression was investigated by reverse transcriptase-polymerase chain reaction (PCR) for mRNA and fluorescence-activated cell-sorter scanner (FACS) analysis. Signaling mechanisms required for angiopoietin-dependent effects were tested functionally by using signaling enzyme blockers. RESULTS: The angiopoietins were chemotactic for neutrophils. They showed antagonistic effects on each other and both inhibited VEGF-directed migration of neutrophils. The effects of both angiopoietins were Tie-2 dependent. Tie-2 receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by Tie-2 receptor mRNA expression as demonstrated by reverse transcriptase PCR. CONCLUSIONS: Data suggest that a Tie-2 receptor is expressed by human neutrophils whose active site ligation with either angiopoietin-1 or angiopoietin-2 exerts migratory effects on the one hand and arrests VEGF-mediated chemotaxis on the other. These effects suggest a role of angiopoietins in modulating neutrophilic inflammation.

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.

Syndecan-1 is involved in osteoprotegerin-induced chemotaxis in human peripheral blood monocytes.

Chronic inflammation is characterized by tissue infiltration with monocytes/macrophages, which possess broad proinflammatory, destructive, and remodeling capacities. Elevated levels of osteoprotegerin, an important regulator of differentiation and activation of osteoclasts that also affects different cells of the immune system, were found in the serum of patients with chronic inflammatory diseases. The study of whether osteoprotegerin affects monocyte locomotion in vitro and the possible mechanisms and pathways involved was investigated using Boyden microchemotaxis chambers and Western blot analyses. Osteoprotegerin significantly stimulated monocyte chemotaxis, whereas preincubation of monocytes with osteoprotegerin inhibited monocyte migration toward optimal concentrations of regulated upon activation normal T cell expressed and secreted, monocyte chemotactic protein -1, and procalcitonin. The effects of osteoprotegerin were abolished by pretreating cells with heparinase I and chondroitinase or antibodies against the ectodomain of syndecan-1. Osteoprotegerin signaling was shown to involve protein kinase C, phosphatidylinositol 3-kinase/Akt, and tyrosine kinase. Data suggest that osteoprotegerin affects monocyte mi-gration and protein kinase C and phosphatidylinositol 3-kinase/Akt activation via syndecan-1. Osteoprotegerin-induced deactivation of monocyte chemotaxis toward different chemokines is due to interaction of osteoprotegerin with heparan sulfate and chondroitin sulfate.

Expression and function of the angiopoietin receptor Tie-2 in human eosinophils.

BACKGROUND: Increased vascularity of bronchial mucosa is closely related to the expression of angiogenic factors, which contribute to the pathogenesis of diseases such as asthma bronchiale. OBJECTIVE: Here we examine the effects of the angiogenic growth factors angiopoietin 1 and angiopoietin 2 on eosinophil function in vitro and possible involvement of the angiopoietin receptor Tie-2. METHODS: Eosinophil migration was studied by micropore filter assays. Signaling mechanisms required for angiopoietin-dependent migration were tested by using signaling enzyme blockers. Tie-2 mRNA and receptor expression on the cell surface of eosinophils was demonstrated in RT-PCR and by fluorescence-activated cell sorting analysis. RESULTS: Angiopoietin 1 significantly stimulated eosinophil chemotaxis via activation of phosphodiesterase, phosphatidylinositol 3'-kinase, and tyrosine kinases. The effect on eosinophil migration of angiopoietin 1 was reversed by an antibody against the Tie-2 receptor and by angiopoietin 2. Incubation of eosinophils with angiopoietin 1 abolished the chemotactic effects of vascular endothelial growth factor on human eosinophils via the Tie-2 receptor. Finally, Tie-2 expression by human eosinophils was demonstrated on the transcriptional and protein level. CONCLUSIONS: Data suggest that angiopoietin 1 stimulates directed migration and possibly inhibits vascular endothelial growth factor-induced eosinophil chemotaxis via its Tie-2 receptor, which is expressed by eosinophils. Thus, angiopoietin 1 may play an important role in the modulation of eosinophilic inflammation.

Expression and function of RANK in human monocyte chemotaxis.

OBJECTIVE: RANKL, a member of the tumor necrosis factor superfamily, is a central regulator of osteoclast recruitment and activation. Whether RANKL affects monocyte locomotion in vitro via RANK and a possible signaling pathway were investigated. METHODS: Monocytes were obtained from venous blood of healthy donors. Cell migration was studied by micropore filter assays. The signaling mechanisms required for RANKL-dependent migration were tested using signaling enzyme blockers and Western blot analyses. Expression of RANK messenger RNA (mRNA) in monocytes was demonstrated by reverse transcriptase-polymerase chain reaction, and receptor expression on cell surface was investigated by fluorescence-activated cell sorting analyses. RESULTS: RANKL significantly stimulated monocyte chemotaxis via activation of phosphatidylinositol 3-kinase, phosphodiesterase, and Src kinase. The effect on migration was inhibited by osteoprotegerin, which is the decoy receptor for RANKL. Expression of RANK receptor mRNA was shown, and synthesis of RANK in monocytes was suggested by the detection of RANK immunoreactivity on the cell surface. CONCLUSION: These data suggest that RANK is expressed by monocytes whose activation by RANKL stimulates directed migration involving phosphatidylinositol 3-kinase, phosphodiesterase, and Src kinases.

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 Alzheimer's senile plaques, a hallmark of the innate immune response to beta-amyloid fibrils, can initiate and propagate neurodegeneration characteristic of Alzheimer's disease. Phagocytes migrate toward amyloid beta-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 beta-protein- and amyloid beta-precursor protein-induced migration was abrogated by dimethylsphingosine, a sphingosine kinase inhibitor. Amyloid beta-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 beta-protein-induced migration. These data demonstrate that the migratory effects of beta-amyloid in human monocytes involve spingosine-1-phosphate signaling. Whereas endogenous neuropeptides may arrest and activate monocytes at sites of high beta-amyloid concentrations, interference with the amyloid beta-protein-dependent sphingosine-1-phosphate pathway in monocytes by FTY720, a novel immunomodulatory drug, suggests that FTY720 may be efficacious in beta-amyloid-related inflammatory diseases.

Expression and function of the vascular endothelial growth factor receptor FLT-1 in human eosinophils.

Vascular endothelial growth factor (VEGF) is highly expressed in the airway of patients with asthma. Whether VEGF affects eosinophil function in vitro and if VEGF receptors are involved was tested. Eosinophils were from venous blood of healthy donors. Cell migration was studied by micropore filter assays. Signaling mechanisms required for VEGF-dependent migration were tested using signaling enzyme blockers. Expression of flt-1 and KDR/flk-1 mRNA in eosinophils was demonstrated in reverse transcriptase-polymerase chain reaction, and receptor expression was investigated by fluorescence-activated cell sorting analysis. Eosinophil cationic protein release was measured in eosinophil supernatants by enzyme-linked immunosorbent assay. VEGF significantly stimulated eosinophil chemotaxis via activation of protein kinase C and phosphatidylinositol 3'-kinase. The effect on migration was reversed by an antibody against VEGF receptor flt-1, but not by an antibody against KDR/flk-1. Expression of VEGF receptor flt-1 mRNA was shown and synthesis of VEGF receptor in eosinophils is suggested by detection of VEGF receptor immunoreactivity on the cell surface. Data suggest that VEGF receptor flt-1 is expressed by eosinophils whose activation with VEGF stimulates directed migration and release of eosinophil cationic protein. Thus, VEGF may play an important role in the modulation of eosinophilic inflammation.

Natural killer cell functions mediated by the neuropeptide substance P.

The neuropeptide substance P (SP) can modulate a number of immunological functions in vitro and in vivo. Here, we investigated if SP boosts migration and cytotoxicity of natural killer cells, thus providing a further link between "innate immunity" and neurogenic inflammatory processes like asthma bronchiale. We demonstrate a dose-dependent effect of SP on natural killer cell migration with a maximal response at 10(-8) M SP. SP was shown to stimulate unstimulated as well as interleukin-2 (IL-2)-activated natural killer cells. Stimulation of natural killer cell migration was neurokinin-1 receptor dependent. Furthermore, mRNA encoding the neurokinin-1 receptor was demonstrated as being present in natural killer cells using RT-PCR while mRNA of the neurokinin-2 receptor was not detectable. Additionally, SP seems to influence specific cytotoxicity against Raji and K567 effector cells by a receptor-independent mechanism. In conclusion, our data indicate that functionally active neurokinin-1 receptors can be expressed by human natural killer cells. Substance P might therefore be a novel link between neural structures and innate immunity.