Tag: fluorescence reader

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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

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Toll-like receptors stimulate human neutrophil function

The first immune cell to arrive at the site of infection is the neutrophil. Upon arrival, neutrophils quickly initiate microbicidal functions, including the production of antimicrobial products and proinflammatory cytokines that serve to contain infection. This allows the acquired immune system enough time to generate sterilizing immunity and memory. Neutrophils detect the presence of a pathogen through germ line-encoded receptors that recognize microbe-associated molecular patterns. In vertebrates, the best characterized of these receptors are Toll-like receptors (TLRs). We have determined the expression and function of TLRs in freshly isolated human neutrophils. Neutrophils expressed TLR1, 2, 4, 5, 6, 7, 8, 9, and 10—all the TLRs except TLR3. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment increased TLR2 and TLR9 expression levels. The agonists of all TLRs expressed in neutrophils triggered or primed cytokine release, superoxide generation, and L-selectin shedding, while inhibiting chemotaxis to interleukin-8 (IL-8) and increasing phagocytosis of opsonized latex beads. The response to the TLR9 agonist nonmethylated CpG-motif-containing DNA (CpG DNA) required GM-CSF pretreatment, which also enhanced the response to the other TLR agonists. Finally, using quantitative polymerase chain reaction (QPCR), we demonstrate a chemokine expression profile that suggests that TLR-stimulated neutrophils recruit innate, but not acquired, immune cells to sites of infection.

http://bloodjournal.hematologylibrary.org/content/102/7/2660.full.html

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LSP1 modulates leukocyte populations in resting and inflamed peritoneum

Lymphocyte-specific protein 1, recently renamed leukocyte-specific protein 1 (LSP1), is an F-actin binding protein expressed in lymphocytes, macrophages, and neutrophils in mice and humans.

This study examines LSP1-deficient (Lsp1-/-) mice for the development of myeloid and lymphocytic cell populations and their response to the development of peritonitis induced by thioglycollate (TG) and to a T-dependent antigen.

LSP1-/- mice exhibit significantly higher levels of resident macrophages in the peritoneum compared to wild-type (wt) mice, whereas the development of myeloid cells is normal. This increase, which is specific for conventional CD5– macrophages appears to be tissue specific and does not result from differences in adhesion to the peritoneal mesothelium. The level of peritoneal lymphocytes is decreased in LSP1-/- mice without affecting a particular lymphocytic subset. The proportions of precursor and mature lymphocytes in the central and peripheral tissues of LSP1-/- mice are similar to those of wt mice and LSP1-/- mice mount a normal response to the T-dependent antigen, ovalbumin (OVA). On injection of TG, the LSP1-/- mice exhibit an accelerated kinetics of changes in peritoneal macrophage and neutrophil numbers as compared to wt including increased influx of these cells.

LSP1– neutrophils demonstrate an enhanced chemotactic response in vitro to N-formyl methionyl-leucyl-phenylalanine (FMLP) and to the C-X-C chemokine, KC, indicating that their enhanced influx into the peritoneum may be a result of increased motility. Our data demonstrate that LSP1 is a negative regulator of neutrophil chemotaxis.

http://bloodjournal.hematologylibrary.org/content/96/5/1827.full