Migration-inducing Activity Research Articles

Chemotactic activity of products of Elizabethkingia anophelis derived from Aedes albopictus against RAW264 murine macrophage cell line.

Dengue viruses enter dermal macrophages which are derived from other tissues after mosquito bites. We examined chemotactic factors derived from the dengue vector mosquito, Aedes albopictus, toward a RAW264 murine macrophage cell line. We found that Elizabethkingia anophelis that was isolated from mosquitoes exhibited migration-inducing activity toward RAW264 cells. The active substances were extracted using ethyl acetate to induce chemotactic movements. Chemotactic activity was eluted in the several fractions using the reversed-phase chromatography, suggesting that multiple substances were responsible for the activity. We isolated three bacterial colonies from the wild A. albopictus mosquitoes collected in Toyama Park, Tokyo prefecture, Japan. The bacterial 16S rRNA gene sequences were the most similar to those of Lonsdalea quercina. These bacteria also exhibited migration-inducing activity toward RAW264 cells. The migration-inducing activity of mosquito bacteria might be a new aspect of mosquito-mediated viral infections.

Migration of breast cancer cell lines in response to pulmonary laminin 332.

Because tumor cell motility is a requirement for metastasis, we hypothesized that lung tissue harbors substances that induce tumor cell migration. MCF‐7 breast carcinoma cells exposed to small airway epithelial cells and conditioned medium exhibited dose‐dependent tumor cell migration. Among the extracellular matrix proteins in the conditioned medium identified by mass spectrometry, laminin 332 (LM332) had the greatest contribution to the migration of MCF‐7 cells. Immunoblotting and immunohistochemistry for LM332‐specific chains identified LM332 in the lung and in pulmonary epithelial cells. Antibodies to either LM332 or its integrin receptor inhibited MCF‐7 motility, and knockdown of LM332 chains also reduced its migration‐inducing activity. Taken together, these findings implicate LM332 as a component of lung tissue that can induce motility in breast carcinoma cells that have been transported to lung during metastasis. Earlier studies on LM332 in tumor progression have examined LM332 expression in tumor cells. This investigation, in comparison, provides evidence that the tumor promoting potential of LM332 may originate in the lung microenvironment rather than in tumor cells alone. Furthermore, this study provides evidence that the motility‐inducing properties of the microenvironment can reside in epithelial cells. The findings raise the possibility that LM332 plays a role in the pulmonary metastases of breast carcinoma and may provide a target for antimetastasis therapy.

Abstract 1293: Neutrophil gelatinase-associated lipocalin regulates the expression of angiogenic cytokines and VEGF in human breast and ovarian cancer cells

Abstract Neutrophil gelatinase-associated lipocalin (NGAL; lipocalin 2) is a small extracellular protein (25 kDa) that is a member of the lipocalin protein family. It has been shown to bind ferric siderophores and to play an important role in iron metabolism. Increased NGAL levels have been reported in a variety of epithelial cancers, including breast, ovary, pancreas, colon, esophagus and thyroid cancer. We have recently reported that NGAL promotes breast cancer progression by inducing the epithelial to mesenchymal transition. In the course of these studies, we observed an increase in microvessel density in NGAL-expressing tumors. In the current study, we explored the potential role of NGAL in tumor angiogenesis by studying its effects on the expression of angiogenic factors. We first determined the levels of vascular endothelial growth factor (VEGF) in NGAL-expressing cells derived from the MCF-7 human breast cancer cell line. Using reverse-transcriptase PCR and ELISA, VEGF was found to be induced by NGAL at both the transcript and protein levels. Consistently, HIF-1α, one of the most important transcriptional regulators of VEGF, was observed to be accumulated in the nuclei of NGAL-expressing cells. Silencing of HIF-1α in NGAL-expressing cells significantly reduced VEGF expression. Consistent with this NGAL-induced VEGF production, there was a significant increase in endothelial cell migration towards the conditioned media collected from NGAL-expressing cells. Interestingly, treatment with a VEGF-neutralizing antibody only partially inhibited the migration-inducing activity of the conditioned media, suggesting that NGAL might induce the secretion of other angiogenic factors. To investigate this possibility, a panel of angiogenic factors was compared between the conditioned media collected from NGAL-expressing cells and control MCF-7 cells using antibody arrays. Several angiogenic cytokines, including IL-6, MCP-1 and GM-CSF, were found to be upregulated with NGAL expression. The effects of NGAL on VEGF and angiogenic cytokine production were also studied in SKOV-3 ovarian cancer cells. In this study, NGAL was silenced in SKOV-3 cells using specific siRNAs. The levels of both VEGF and IL-6 were found to be reduced after NGAL silencing, suggesting that NGAL's control of VEGF and cytokine production may be common among cancer cell types. These results suggest a novel mechanism through which NGAL may promote tumor angiogenesis and progression. (This work is supported by NIH R01 CA118764, P01 CA045548 and The Advanced Medical Foundation.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1293.

Bis Is Involved in Glial Differentiation of P19 Cells Induced by Retinoic Acid

Previous observations suggest that Bis, a Bcl-2-binding protein, may play a role the neuronal and glial differentiation in vivo. To examine this further, we investigated Bis expression during the in vitro differentiation of P19 embryonic carcinoma cells induced by retinoic acid (RA). Western blotting and RT-PCR assays showed that Bis expression was temporarily decreased during the free floating stage and then began to increase on day 6 after the induction of differentiation. Double immunostaining indicated that Bis-expressing cells do not express several markers of differentiation, including NeuN, MAP-2 and Tuj-1. However, some of the Bis-expressing cells also were stained with GFAP-antibodies, indicating that Bis is involved glial differentiation. Using an shRNA strategy, we developed bis-knock down P19 cells and compared them with control P19 cells for the expression of NeuroD, Mash-1 and GFAP during RA-induced differentiation. Among these, only GFAP induction was significantly attenuated in P19-dnbis cells and the population showing GFAP immunoreactivity was also decreased. It is noteworthy that distribution of mature neurons and migrating neurons was disorganized, and the close association of migrating neuroblasts with astrocytes was not observed in P19-dnbis cells. These results suggest that Bis is involved in the migration-inducing activity of glial cells.

CD137 is expressed on blood vessel walls at sites of inflammation and enhances monocyte migratory activity

The cytokine receptor CD137 is a member of the TNF receptor family and a potent T cell costimulatory molecule. Its ligand is expressed on antigen presenting cells as a transmembrane protein and it too can deliver signals into the cells it is expressed on (reverse signaling). In monocytes, immobilized CD137 protein induces activation, prolongation of survival and proliferation. Here we show that recombinant immobilized CD137 protein enhances migration of monocytes in vitro. Further, CD137 expression on spheroids leads to a significantly enhanced infiltration by monocytes. The migration-inducing activity of CD137 could be confirmed in vivo. Matrigel, which was coated with recombinant CD137 protein and was inserted into the flanks of mice attracted large numbers of monocytes and was heavily infiltrated by these cells. In vivo, expression of CD137 by blood vessel walls at sites of inflammation was detectable by immunohistochemistry. CD137 expression is inducible by proinflammatory cytokines in endothelial cells, suggesting that a physiological function of CD137 may be the facilitation of monocyte extravasation in inflammatory tissues.

Toxoplasma gondiiregulates recruitment and migration of human dendritic cells via different soluble secreted factors

We investigated in vitro the properties of soluble factors produced by Toxoplasma gondii on the recruitment, maturation and migration of human dendritic cells (DC) derived from CD34+ progenitor cells. We used soluble factors including excreted secreted antigens (ESA) produced under various conditions by the virulent type I RH strain (ESA-RH) and the less virulent PRU type II strain (ESA-PRU). Soluble factors of both T. gondii strains appeared to possess a chemokine-like activity that attracted immature DC. This recruitment activity required the presence of functional CCR5 molecules on the cell membrane. Incubation of DC for 24 h with ESA triggered the migration of a large percentage of these cells towards the chemokine MIP-3beta; ESA-PRU was more efficient than ESA-RH. ESA produced in absence of exogenous protein and crude extract did not induce DC migration but retained recruitment activity. These data indicate that recruitment activity and migration-inducing activity are not governed by the same factors. Moreover, incubation of DC for 48 h with ESA did not modify the expression of costimulation or maturation markers (CD83, CD40, CD80, CD86 or HLA-DR), but induced a decrease in CCR6 expression associated with an increased expression of CCR7. Taken together, these results suggest that T. gondii controls recruitment and migration of immature DC by different soluble factors and may induce a dysfunction in the host-specific immune response.

Spermadhesin PSP-I/PSP-II heterodimer and its isolated subunits induced neutrophil migration into the peritoneal cavity of rats.

Spermadhesins are a group of (glyco)proteins from seminal fluid involved in various aspects of porcine fertilization. PSP-I/PSP-II, a heterodimer of glycosylated spermadhesins, is the major component of porcine seminal fluid. Its biological function remains, however, enigmatic. Using an in vitro chemotaxis assay, we showed that PSP-I/PSP-II and its isolated subunits induced migration of purified neutrophils. A possible proinflammatory activity of PSP-I/PSP-II induced upon injection of the spermadhesin heterodimer and its isolated subunits into the peritoneal cavity of rats was investigated. Lavage of peritoneal cavities, thioglycolate treatment, and mast cell depletion were done before spermadhesin administration, and neutrophil migration was evaluated 4 h after injections. Pharmacological modulation was also investigated. Resident cell depletion by lavage reduced the neutrophil migration induced by PSP-I/PSP-II and the PSP-II subunit but had no effect on that induced by isolated PSP-I. Both an increase of macrophage population by thioglycolate treatment and mast cell depletion potentiated the neutrophil migration induced by PSP-I/PSP-II and by PSP-II. The glucocorticoid dexamethasone but not indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and BN50739 (platelet activation factor [PAF] antagonist) inhibited neutrophil migration induced by PSP-I/PSP-II. Coincubation with mannose-6-phosphate (a PSP-II-specific ligand) inhibited neutrophil recruitment induced by PSP-II but did not alter the PSP-I activity. As a whole, the data suggested that enhancement of the neutrophil migration-inducing activity of PSP-I/PSP-II and PSP-II involved an indirect mechanism, i.e., via activation of resident cells, probably macrophages. On the other hand, PSP-I appeared to act directly on neutrophils. We hypothesize that the neutrophil migration-inducing effect displayed by PSP-II might be due to interaction of its lectin domain with cellular receptors and that neutrophil recruitment induced by PSP-I may involve protein-protein interactions.

Extracellular Signals That Regulate the Tangential Migration of Olfactory Bulb Neuronal Precursors: Inducers, Inhibitors, and Repellents

Neuronal migration is an essential developmental step in the construction of the vertebrate nervous system, but the extracellular signals involved in initiating and regulating neuronal movement remain unclear. Here we report the identification of a novel astrocyte-derived migration-inducing activity (MIA). Using an in vitro assay, we show that MIA induces the migration of olfactory bulb interneuron precursors, increasing the number of migrating cells and the distance they move. We established quantitative criteria to distinguish between the biological effects of inducers, inhibitors, repellents, and attractants on migrating cells and used them to compare the effects of MIA with those of Slit, a putative repulsive guidance cue. Our analysis demonstrates that, by themselves, MIA induces and Slit inhibits migration from subventricular zone explants. However, when presented together with MIA, Slit acts as a repellent. This study shows that glial cells play a critical role in initiating and modulating the movement of neuronal precursors through the release of a diffusible protein. Moreover, this study provides evidence that the guidance of migrating neuronal precursors is an integrative process, resulting from the cooperation of distinct extracellular factors, and that the function of Slit is context dependent.

Neutrophil migration induced in vivo and in vitro by marine algal lectins.

Since some plant and mammalian lectins specific for monosaccharides are able to induce neutrophil migration, we studied the neutrophil migration-inducing activities of marine algal lectins, specific for complex oligosaccharides from glycoproteins, from Amansia multifida (AM), Bryothamnion seaforthii (BS), Bryothamnion triquetrum (BT) and Gracilaria caudata (GC). The neutrophil migration-inducing activity of AM, BS, BT and GC was assayed in vitro and in vivo in the peritoneal cavity or dorsal air pouch of rats or mice, and was inhibited by glycans. AM, BS, BT and GC induced neutrophil migration in vivo and in vitro, determining bell-shaped dose-dependent curves. Maximal neutrophil influx was determined by BT in rats and by AM in mice. Maximal human neutrophil chemotaxis was obtained with GC. These activities were not inhibited by glycoproteins previously identified as being recognized by these lectins. D-mannose was a strong inhibitor, especially of BT activity both in vitro and in vivo. Algal lectins induced neutrophil migration, which was inhibited by a monosaccharide, contrasting with the view that they only recognize complex oligosaccharides. Neutrophil chemotaxis assays are appropriate to study low molecular mass lectins containing a single carbohydrate recognition domain, as is the case of some lectins from algae and mammals.

Fibroblast migratory factor derived from mouse colon carcinoma cells: Potential roles of fibronectin in tumor stroma formation

Mouse colon carcinoma cell line colon 38 was used to investigate migratory factor for fibroblasts because marked fibrotic tissue was associated with these cells growing at transplanted sites and liver metastases in vivo. Migration-inducing activity was detected in the serum-free culture supernatants of colon 38 cells, as shown by the Boyden chamber assays using NIH3T3 cells as target cells. The active substance was partially purified by a combination of anion-exchange, hydrophobic, and gel-permeation chromatography. An approximate relative molecular mass of the active substance was estimated to be between 100,000 and 400,000, judging from the eluting position in the gel-permeation chromatography. The migratory activity in the partially purified preparation was removed by incubation with beads coated with an anti-mouse fibronectin antibody. NIH3T3 cells incubated in the presence of culture supernatants of colon 38 cells exhibited higher growth rate, organized actin filaments, and increased chondroitin sulfate and hyaluronan. Fibronectin did not elicit such effects and partially purified migratory factor showed relatively low activity in these regards. Thus, colon 38 cells seem to secrete fibronectin and other soluble substances, which induce tumor stroma formation through migration and activation of host fibroblasts.

A Receptor-like Protein-tyrosine Phosphatase PTPζ/RPTPβ Binds a Heparin-binding Growth Factor Midkine: INVOLVEMENT OF ARGININE 78 OF MIDKINE IN THE HIGH AFFINITY BINDING TO PTPζ

Midkine is a 13-kDa heparin-binding growth factor with 45% sequence identity to pleiotrophin. Pleiotrophin has been demonstrated to bind to protein-tyrosine phosphatase zeta (PTPzeta) with high affinity. In this study, we examined the binding of midkine to PTPzeta by solid-phase binding assay. Midkine and pleiotrophin binding to PTPzeta were equally inhibited by soluble pleiotrophin and also by some specific glycosaminoglycans. For both bindings, Scatchard analysis revealed low (3.0 nM) and high (0.58 nM) affinity binding sites. These results suggested that PTPzeta is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the N- and C-terminal halves, and the latter exhibited full activity for PTPzeta binding and neuronal migration induction. The C-terminal half contains two heparin-binding sites consisting of clusters of basic amino acids, Clusters I and II. A mutation at Arg78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys83 and Lys84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTPzeta exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg78. These results suggested that Arg78 in midkine plays an essential role in high affinity binding to PTPzeta by interacting with the chondroitin sulfate portion of this receptor.

A newly identified chemotactic sexual pheromone from Closterium ehrenbergii

In sexual reproduction of Closterium ehrenbergii, pairing with the sexual partner cells is the first process observed. A cell migration-inducing activity, specific for mating-type plus (mt+; NIES-228) cells, was detected in the culture medium of mating-type minus (mt–; NIES-229) cells. Light was necessary for production of the active substance by mt– cells and for migration of mt+ cells. The active substance was heat-labile and had an apparent molecular mass of 20 kDa, as determined by gel filtration. A protein of 20 kDa was detected in the active fraction of gel filtration after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on these results, it is proposed that a chemotactic sexual pheromone involved in the formation of sexual pairs of cells is secreted by mt– cells of C. ehrenbergii and is proteinaceous, like other sexual pheromones secreted by Closterium species.

LPS induces migration of bone marrow cells in LPS-nonresponsive C3H/HeJ mice.

To determine possible migratory responses to LPS of haemopoietic stem cells (CFUs) in LPS-nonresponsive (C3H/HeJ) mice, changes in the number of CFUs and granulocyte-macrophage progenitor cells (CFUc) after LPS-treatment where studied in splenectomized C3H/ HeJ mice. Splenectomy abrogated the decrease in bone marrow stem cells (CFUs and CFUc) induced by LPS in both LPS-responsive and nonresponsive mice. Recovery of bone marrow stem cells after irradiation was greatly enhanced by splenectomy in C3H/slc mice, but such enhancement was not observed in C3H/HeJ mice. These data suggest that the increase in the number of haemopoietic stem cells in the spleen in C3H/HeJ mice depends on the migration of haemopoietic stem cells from marrow to the spleen. Thus, the expansion of CFUs does not correlate with increase in the number of whole body haemopoietic stem cells. The migrationinducing activity of LPS seems to differ from its haemopoietic stimulatory activity, since the LPS-nonresponsive C3H/HeJ mice respond to the former activity of LPS.

Stimulation of locomotion of peripheral blood monocytes by human plasma fibronectin.

The motility of human peripheral blood granulocytes and monocytes in response to human plasma fibronectin was quantified by an in vitro assay using blind-well chemotaxis chambers. Purified fibronectin under nondenaturing conditions produced increased migration of granulocytes only at concentrations higher than 100 nM, and induced increased chemotactic and random locomotion of monocytes at concentrations higher than 0.1 nM. The monocyte migration-inducing activity of fibronectin was concentration dependent, and was strongly inhibited by low concentrations of colchicine (100 nM-100 microM). These findings suggest the possibility that plasma fibronectin serves as a chemotactic stimulus for monocytes in vivo and attracts these cells to sites of microscopic tissue injury where plasma fibronectin is deposited.