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Statins suppress THP-1 cell migration and secretion of matrix metalloproteinase 9 by inhibiting geranylgeranylation

Macrophages secrete matrix metalloproteinase 9 (MMP-9), an enzyme that weakens the fibrous cap of atherosclerotic plaques, predisposing them to plaque rupture and subsequent ischemic events. Recent work indicates that statins strongly reduce the possibility of heart attack. Furthermore, these compounds appear to exert beneficial effects not only by lowering plasma low-density-lipoprotein cholesterol but also by directly affecting the artery wall. To evaluate whether statins influence the proinflammatory responses of monocytic cells, we studied their effects on the chemotactic migration and MMP-9 secretion of human monocytic cell line THP-1. Simvastatin dose dependently inhibited THP-1 cell migration mediated by monocyte chemoattractant protein 1, with a 50% inhibitory concentration of about 50 nM. It also inhibited bacterial lipopolysaccharide-stimulated secretion of MMP-9. The effects of simvastatin were completely reversed by mevalonate and its derivatives, farnesylpyrophosphate and geranylgeranyl pyrophosphate, but not by ubiquinone. Additional studies revealed similar but more profound inhibitory effects with L-839,867, a specific inhibitor of geranylgeranyl transferase. However, α-hydroxyfarnesyl phosphonic acid, an inhibitor of farnesyl transferase, had no effect. C3 exoenzyme, a specific inhibitor of the prenylated small signaling Rho proteins, mimicked the inhibitory effects of simvastatin and L-839,867. These data supported the role of geranylgeranylation in the migration and MMP-9 secretion of monocytes.

http://www.jleukbio.org/content/69/6/959.long

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Human Bone Marrow Stromal Cells Express a Distinct Set of Biologically Functional Chemokine Receptors

Stromal cells isolated from bone marrow (BMSCs), often referred to as mesenchymal stem cells, are currently under investigation for a variety of therapeutic applications. However, limited data are available regarding receptors that can influence their homing to and positioning within the bone marrow. In the present study, we found that second passage BMSCs express a unique set of chemokine receptors: three CC chemokine receptors (CCR1, CCR7, and CCR9) and three CXC chemokine receptors (CXCR4, CXCR5, and CXCR6). BMSCs cultured in serum-free medium secrete several chemokine ligands (CCL2, CCL4, CCL5, CCL20, CXCL12, CXCL8, and CX3CL1). The surface-expressed chemokine receptors were functional by several criteria. Stimulation of BMSCs with chemokine ligands triggers phosphorylation of the mitogen-activated protein kinase (e.g., extracellular signal–related kinase [ERK]-1 and ERK-2) and focal adhesion kinase signaling pathways. In addition, CXCL12 selectively activates signal transducer and activator of transcription (STAT)-5 whereas CCL5 activates STAT-1. In cell biologic assays, all of the chemokines tested stimulate chemotaxis of BMSCs, and CXCL12 induces cytoskeleton F-actin polymerization. Studies of culture-expanded BMSCs, for example, 12–16 passages, indicate loss of surface expression of all chemokine receptors and lack of chemotactic response to chemokines. The loss in chemokine receptor expression is accompanied by a decrease in expression of adhesion molecules (ICAM-1, ICAM-2, and vascular cell adhesion molecule 1) and CD157, while expression of CD90 and CD105 is maintained. The change in BMSC phenotype is associated with slowing of cell growth and increased spontaneous apoptosis. These findings suggest that several chemokine axes may operate in BMSC biology and may be important parameters in the validation of cultured BMSCs intended for cell therapy.

http://onlinelibrary.wiley.com/doi/10.1634/stemcells.2005-0319/full

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Differential Effects of Two Fluorescent Probes on Macrophage Migration as Assessed by Manual and Automated Methods]

Fluorescent probes have been utilized to label leukocytes for both in vivo and in vitro studies of cell migration; however, the effects of such probes on migration have not been determined. The aim of this study was to examine the effects of two commonly used fluorescent probes on leukocyte chemotaxis. J774 macrophages were labeled with either calcein-acetoxymethyl ester (calcein-AM) or 2′,7′-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, acetomethyl ester (BCECF-AM), then assayed for their ability to migrate to zymosan-activated serum (ZAS). Cell migration was quantified by two methods: visual counting of cells and measuring cell fluorescence. Using the cell counts, comparison of unlabeled and fluorescently labeled macrophages demonstrated that BCECF-AM decreased the number of cells responding to ZAS, while calcein-AM had essentially no effect. Neither probe significantly affected the number of cells migrating to medium alone. The inhibitory effects of BCECF-AM on cell migration increased with probe concentration (0.1-1.0 microM) and cell fluorescence. Cell viability was unaffected by either probe. In contrast to the results obtained by visual counting, measuring fluorescence of migrated cells did not reveal a significant difference between the chemotactic response of macrophages labeled with BCECF-AM and those labeled with calcein-AM. These experiments indicated that fluorescent probes can affect the chemotactic response and that inhibitory activity of these probes may not be detected when chemotaxis is quantified solely by automated methods.

pdf at: http://onlinelibrary.wiley.com/doi/10.1002/cyto.990190412/pdf

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Rapid and quantitative in vitro measurement of cellular chemotaxis and invasion

An in vitro, fluorimetric method for cellular chemotaxis and invasion has been developed using a commercially available, disposable, 96-well chamber. This 4–18 hour microtiter chamber assay has a number of important advantages over existing methods. It does not require prior labeling of cells or radioactivity, and is rapid, automatable and quantitative. Cells are quantitated by a novel actin-based fluorescence tag as reported previously (Methods in Cell Science 17: 263–270, 1995). Following quantitation, cells are easily detectable by fluorescence microscopy. In addition, this assay conserves reagents due to its low volumes in the upper and lower chambers. The assay has been optimized using cultured human lung cancer cells to identify inhibitors or activators of directed cell migration. The effects of antibodies to agrVbeta3, agrVbeta5, and CD44 on the chemotaxis and invasion of A549 cultured lung tumor cells are reported.

pdf available at: http://www.springerlink.com/content/wg1v6175p40wm3x0/

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An improved method for the quantitation of cellular migration: Role of αvβ3 integrin in endothelial and smooth muscle cell migration

The present study was undertaken to develop a simple and improvedmethod for the accurate quantitation of cellular migration and to examinethe role of agrvbeta3 integrins in different cellular migration. Using our newly developed micro-volume chemotaxis assay, we developed an improved quantitative method to measure in vitro chemotaxis of smooth muscle or endothelial cells toward different extracellular matrix proteins. The convenience in setup and counting of migrated cells using this method allows for large capacity screening and for various research applications with other cells as well. The signal to noise ratios were in the range of 10/1, along with about 10–20% intra- or inter-assay variabilities. Using this method, we have determined that either vitronectin at 0.4 µg/well or osteopontin at 0.4 µg/well are selective agrvbeta3 chemoattractants for endothelial or smooth muscle cells (0.5 × 105 cells/well). Additionally, a selective agrvbeta3 small molecule peptiddomimetic, monoclonal antibody LM609, or an anti-beta3 (agrvbeta3/agrIIbeta3) anti-body, c7E3 demonstrated maximal inhibition of cellular migration toward vitronectin or osteopontin. These data suggest the potential utility of this method in assessing the role of various mechanisms in cellular migration and also suggests the potential implication of an agrvbeta3 antagonist in blocking pathological processes involving endothelial or smooth muscle cell adhesion/migration.

link to pdf at: http://www.springerlink.com/content/k60v0373k4962116/

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Septic Shock and Acute Lung Injury in Rabbits with pertonitis

The major goal of this study was to investigate the mechanisms that link the host response to a local infection in the peritoneal cavity with the development of sepsis and lung injury. Rabbits were infected by intraperitoneal inoculation of fibrin clots containing Escherichia coli at 108, 109, or 1010 cfu/clot. Physiologic, bacteriologic, and inflammatory responses were monitored, and the lungs were examined postmortem. At a dose of 108 cfu/clot the animals had resolving infection, and a dose of 109 cfu/clot resulted in persistent infection at 24 h, with minimal systemic manifestations. In contrast, inoculation of 1010 cfu/clot resulted in rapidly lethal local infection, with septic shock and lung injury. The onset of septic shock was associated with a paradoxical lack of identifiable polymorphonuclear leukocytes (PMN; neutrophils) in the peritoneal cavity. The absence of PMN in the peritoneum was due in part to lysis of intraperitoneal PMN, because the peritoneal fluids contained free myeloperoxidase and induced rapid death of normal rabbit PMN in vitro. Although most animals became bacteremic, only those with a severe systemic inflammation response developed lung injury. These data show that control of an infection in the first compartment in which bacteria enter the host is a critical determinant of the systemic response. Above a threshold dose of bacteria, failure of the local neutrophil response is a key mechanism associated with deleterious systemic responses. Bacteremia alone is not sufficient to cause lung injury. Lung injury occurs only in the setting of a severe systemic inflammatory response and an inadequate leukocyte response at the primary site of infection.

http://ajrccm.atsjournals.org/content/163/1/234.long

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Roles for C-X-C chemokines and C5a in lung injury after hindlimb ischemia-reperfusion

We evaluated the roles of the C-X-C chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) as well as the complement activation product C5a in development of lung injury after hindlimb ischemia-reperfusion in rats. During reperfusion, CD11b and CD18, but not CD11a, were upregulated on neutrophils [bronchoalveolar lavage (BAL) and blood] and lung macrophages. BAL levels of CINC and MIP-2 were increased during the ischemic and reperfusion periods. Treatment with either anti-CINC or anti-MIP-2 IgG significantly reduced lung vascular permeability and decreased lung myeloperoxidase content by 93 and 68%, respectively (P < 0.05). During the same period, there were significant increases in serum C5a-related neutrophil chemotactic activity. Treatment with anti-C5a decreased lung vascular permeability, lung myeloperoxidase, and BAL CINC by 51, 58, and 23%, respectively (P < 0.05). The data suggest that the C-X-C chemokines CINC and MIP-2 as well as the complement activation product C5a are required for lung neutrophil recruitment and full induction of lung injury after hindlimb ischemia-reperfusion in rats.

http://ajplung.physiology.org/content/276/1/L57.long

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Neutrophil-mediated epithelial injury during transmigration: role of elastase

Neutrophil-mediated injury to gut epithelium may lead to disruption of the epithelial barrier function with consequent organ dysfunction, but the mechanisms of this are incompletely characterized. Because the epithelial apical junctional complex, comprised of tight and adherens junctions, is responsible in part for this barrier function, we investigated the effects of neutrophil transmigration on these structures. Using a colonic epithelial cell line, we observed that neutrophils migrating across cell monolayers formed clusters that were associated with focal epithelial cell loss and the creation of circular defects within the monolayer. The loss of epithelial cells was partly attributable to neutrophil-derived proteases, likely elastase, because it was prevented by elastase inhibitors. Spatially delimited disruption of epithelial junctional complexes with focal loss of E-cadherin, β-catenin, and zonula occludens 1 was observed adjacent to clusters of transmigrating neutrophils. During neutrophil transmigration, fragments of E-cadherin were released into the apical supernatant, and inhibitors of neutrophil elastase prevented this proteolytic degradation. Addition of purified leukocyte elastase also resulted in release of E-cadherin fragments, but only after opening of tight junctions. Taken together, these data demonstrate that neutrophil-derived proteases can mediate spatially delimited disruption of epithelial apical junctions during transmigration. These processes may contribute to epithelial loss and disruption of epithelial barrier function in inflammatory diseases.

http://ajpgi.physiology.org/content/281/3/G705.long

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Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis

Leukocyte recruitment in inflammation is critical for host defense, but excessive accumulation of inflammatory cells can lead to tissue damage. Neutrophil-derived serine proteases (cathepsin G [CG], neutrophil elastase [NE], and proteinase 3 [PR3]) are expressed specifically in mature neutrophils and are thought to play an important role in inflammation. To investigate the role of these proteases in inflammation, we generated a mouse deficient in dipeptidyl peptidase I (DPPI) and established that DPPI is required for the full activation of CG, NE, and PR3. Although DPPI–/– mice have normal in vitro neutrophil chemotaxis and in vivo neutrophil accumulation during sterile peritonitis, they are protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen. Specifically, there is no accumulation of neutrophils in the joints of DPPI–/– mice. This protective effect correlates with the inactivation of neutrophil-derived serine proteases, since NE–/– × CG–/– mice are equally resistant to arthritis induction by anti-collagen antibodies. In addition, protease-deficient mice have decreased response to zymosan- and immune complex–mediated inflammation in the subcutaneous air pouch. This defect is accompanied by a decrease in local production of TNF-α and IL-1β. These results implicate DPPI and polymorphonuclear neutrophil–derived serine proteases in the regulation of cytokine production at sites of inflammation.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150852/

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Cross reactivity of three T cell attracting murine chemokines stimulating the CXC chemokine receptor CXCR3 and their induction in cultured cells and during allograft rejection

Recent work identified the murine gene homologous to the human T cell attracting chemokine CXC receptor ligand 11 (CXCL11, also termed I-TAC, SCYB11, ss-R1, H174, IP-9). Here, the biological activity and expression patterns of murine CXCL11 relative to CXCL9 (MIG) and CXCL10 (IP-10/crg-2), the other two CXCR3 ligands, were assessed. Calcium mobilization and chemotaxis experiments demonstrated that murine CXCL11 stimulated murine CXCR3 at much lower doses than murine CXCL9 or murine CXCL10. Murine CXCL11 also evoked calcium mobilization in CHO cells transfected with human CXCR3 and was chemotactic for CXCR3-expressing human T lymphocytes as well as for 300–19 pre-B cells transfected with human or murine CXCR3. Moreover, murine CXCL11 blocked the chemotactic effect of human CXCL11 on human CXCR3 transfectants. Depending on cell type (macrophage-like cells RAW264.7, J774A.1, fetal F20 and adult dermal fibroblasts, immature and mature bone marrow-derived dendritic cells) and stimulus (interferons, LPS, IL-1 beta and TNF-alpha), an up to 10,000-fold increase of CXCL9, CXCL10 and CXCL11 mRNA levels, quantified by real-time PCR, was observed. In vivo, the three chemokines are constitutively expressed in various tissues from healthy BALB/c mice and were strongly up-regulated during rejection of allogeneic heart transplants. Chemokine mRNA levels exceeded those of CXCR3 and IFN-gamma which were induced with similar kinetics by several orders of magnitude.

full text available by subscription at: http://onlinelibrary.wiley.com/doi/10.1002/1521-4141%28200108%2931:8%3C2521::AID-IMMU2521%3E3.0.CO;2-Q/abstract;jsessionid=7CC3E7F4171070E371E5507EFC332EE3.d03t02