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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.

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Activation of CD8 T cells induces expression of CD4, which functions as a chemotactic receptor

It was previously shown that costimulation of CD8+ lymphocytes results in de novo expression of CD4. This study expanded on this observation to investigate the function of CD4 on CD8 cells. The ability of costimulated CD8 cells to respond to interleukin 16 (IL-16), a ligand that binds CD4 and induces cellular chemotaxis, was examined. IL-16–mediated ligation of CD4 expressed on CD8 T cells was found to induce an intracellular signal that directs migration of these cells in vitro. Thus, expression of CD4 on a CD8 lymphocyte has functional importance and may serve to control distribution of newly activated CD8 T cells in vivo.

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A Bovine Whey Protein Extract Can Enhance Innate Immunity by Priming Normal Human Blood Neutrophils

Bovine milk-derived products, in particular whey proteins, exhibit beneficial properties for human health, including the acquired immune response. However, their effects on innate immunity have received little attention. Neutrophils are key cells of innate defenses through their primary functions of chemotaxis, phagocytosis, oxidative burst, and degranulation.
A whey protein extract (WPE) purified from bovine lactoserum was evaluated for its direct and indirect effects on these primary functions of normal human blood neutrophils in vitro. Although WPE had no direct effects on primary functions, a 24-h pretreatment of neutrophils with WPE was associated with a significant and dose-dependent increase of their chemotaxis, superoxide production, and degranulation in response to N-formyl-methionine-leucine-phenylalanine, as well as of their phagocytosis of bioparticles. The pretreatment increased the surface expression of CD11b, CD16B, and CD32A receptors. The major WPE protein components b-lactoglobulin (b-LG) and a-lactalbumin (a-LA) were the main active fractions having an additive effect on human neutrophils that became more responsive to a subsequent stimulation. This effect on NADPH oxidase activity was associated with translocation of p47phox to plasma membrane. Glycomacropeptide, a peptide present in measurable amounts in WPE products, was able to enhance the individual effect of b-LG or a-LA on neutrophils. The present data suggest that WPE, through b-LG and a-LA, has the capacity to enhance or ‘‘prime’’ human neutrophil responses to a subsequent stimulation, an effect that could be associated with increased innate defenses in vivo.

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Rapid fluorescence-based measurement of neutrophil migration in vitro

We have standardized a new chemotaxis chamber that uses fluorescence as the cellular marker for the measurement of leukocyte migration in vitro in disposable 96-well microplates. This new fluorescence-based assay is a robust assay because filter pore size, cell density, filter composition, and filter thickness do not affect PMN migration towards interleukin-8 or the complement fragment, C5a. When compared to two separate chemotaxis assays in which the migrated cells are counted
visually, the fluorescence-based assay was more rapid, less labor intensive, and more sensitive. This new assay is a significant advance in the measurement of leukocyte migration in vitro.

Frevert, Wong, Goodman, Goodwin, and Martin. “Rapid Fluorescence-based Measurement of Neutrophil Migration in Vitro.” 1998, Journal of Immunological Methods, 213, 41-52.

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Slit2 involvement in glioma cell migration is mediated by Robo1 receptor

Slit and Robo proteins are evolutionarily conserved molecules whose interaction underlies axon guidance and neuronal precursor cell migration. During development secreted Slit proteins mediate chemorepulsive signals on cells expressing Robo receptors. Because similar molecular mechanisms may be utilized in glioma cell invasion and neuroblast migration, we studied the expression of Slit2 and its transmembrane receptor Robo1 as well as their functional role in migration in glioma cells. qRTPCR and immunohistochemistry of human specimens revealed that Slit2 was distinctly expressed by non-neoplastic neurons, but at only very low levels in fibrillary astrocytoma and glioblastoma. Robo1 also was mainly restricted to neurons in the normal brain, whereas astrocytic tumor cells in situ as well as glioblastoma cell lines overexpressed Robo1 at mRNA and protein levels.
Recombinant human Slit2 in a concentration of 0.45 nM was repulsive for glioma cell lines in a modified Boyden chamber assay. RNAi-mediated knockdown of Robo1 in glioma cell lines neutralized the repulsive effect of Slit2, demonstrating that Robo1 served as the major Slit2
receptor. Our findings suggest that a chemorepulsive effect mediated by interaction of Slit2 and Robo1 participates in glioma cell guidance in the brain.

available as a pdf from

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Ramatroban (BAY u3405), Inhibits Prostaglandin D2-Induced Eosinophil Migration in Vitro

Ramatroban (Baynas, BAY u3405), a thromboxane A2(TxA2) antagonist marketed for allergic rhinitis, has been shown to partially attenuate prostaglandin (PG)D2-induced bronchial hyperresponsiveness in humans, as well as reduce antigen-induced early- and late-phase inflammatory responses in mice, guinea pigs, and rats. PGD2 is known to induce eosinophilia following intranasal administration, and to induce eosinophil activation in vitro. In addition to the TxA2 receptor, PGD2 is known as a ligand for the PGD2receptor, and the newly identified G-protein-coupled chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). To fully characterize PGD2-mediated inflammatory responses relevant to eosinophil activation, further analysis of the mechanism of action of ramatroban has now been performed. PGD2-stimulated human eosinophil migration was shown to be mediated exclusively through activation of CRTH2, and surprisingly, these effects were completely inhibited by ramatroban. This is also the first report detailing an orally bioavailable small molecule CRTH2 antagonist. Our findings suggest that clinical efficacy of ramatroban may be in part mediated through its action on this Th2-, eosinophil-, and basophil-specific chemoattractant receptor.

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Rapid, multiwell colorimetric assay for measuring neutrophil chemoattractant activity in bronchoalveolar lavage fluid of horses with recurrent airway obstruction

The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter are collected into the bottom well of a disposable chemotaxis–cell migration chamber. The number of viable cells collected in the
bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT), which is reduced by dehydrogenase in mitochondria of live cells. The number of migrating cells corresponds to the amount of MTT reduced, which is measured with an enzyme-linked immunosorbent assay plate reader. Fourteen adult horses were enrolled in this study, 7 of which had owner
histories consistent with RAO. Each horse was sedated, a bronchoalveolar lavage tube was passed, and saline was infused and immediately aspirated. An aliquot of BALF was used for differential cell count, and BALF supernatant was harvested to assess neutrophil chemoattractant activity. Normal control horses and RAOaffected
horses were distinguished according to clinical signs and percent neutrophils in BALF. Neutrophil chemoattractant activity of BALF was significantly greater in RAO-affected horses (P 5 0.001) compared with control horses. This assay may be useful in future studies for monitoring response to therapy in RAO affected horses. (link to pdf)

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Human B Cells Immortalized with Epstein-Barr Virus Upregulate CCR6 and CCR10 and Downregulate CXCR4 and CXCR5

Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.

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CXCR3 chemokine receptor-induced chemotaxis in human airway epithelial cells: role of p38 MAPK and PI3K signaling pathways

Human airway epithelial cells (HAEC) constitutively express the CXC chemokine receptor CXCR3, which regulates epithelial cell movement. In diseases such as chronic obstructive pulmonary disease and asthma, characterized by denudation of the epithelial lining, epithelial cell migration may contribute to airway repair and reconstitution. This study compared the potency and efficacy of three CXCR3 ligands, I-TAC/CXCL11, IP-10/CXCL10, and Mig/CXCL9, as inducers of chemotaxis in HAEC and examined the underlying signaling pathways involved. Studies were performed in cultured HAEC from normal subjects and the 16-HBE cell line. In normal HAEC, the efficacy of I-TAC-induced chemotaxis was 349 ± 88% (mean ± SE) of the medium control and approximately one-half the response to epidermal growth factor, a highly potent chemoattractant. In normal HAEC, Mig, IP-10, and I-TAC induced chemotaxis with similar potency and a rank order of efficacy of I-TAC = IP-10 > Mig. Preincubation with pertussis toxin completely blocked CXCR3-induced migration. Of interest, intracellular [Ca2+] did not rise in response to I-TAC, IP-10, or Mig. I-TAC induced a rapid phosphorylation (5–10 min) of two of the three MAPKs, i.e., p38 and ERK1/2. Pretreatment of HAEC with the p38 inhibitor SB 20358 or the PI3K inhibitor wortmannin dose-dependently inhibited the chemotactic response to I-TAC. In contrast, the ERK1/2 inhibitor U0126 had no effect on chemotaxis. These data indicate that in HAEC, CXCR3-mediated chemotaxis involves a G protein, which activates both the p38 MAPK and PI3K pathways in a calcium-independent fashion.

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Chemotaxis Assay

The purpose of a chemotaxis assay is to determine whether your protein or small molecule of interest has chemotactic activity on a specific cell type. Chemotaxis is the ability of a protein to direct the migration of a specific cell. This assay is based on the premise of creating a gradient of the chemotactic agent and allowing cells to migrate through a membrane towards the chemotactic agent. If the agent is not chemotactic for the cell, then the majority of the cells will remain on the membrane. If the agent is chemotactic, then the cells will migrate through the membrane and settle on the bottom of the well of the chemotaxis plate.