Mechanisms involved in human eosinophil chemotaxis induced by the newly cloned C-C chemokine eotaxin
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The present study was performed in order to investigate the mechanism(s) involved in eotaxin-induced normal human eosinophil chemotaxis using a 48-well micro-chemotaxis chamber assay. Eotaxin, at a wide range of doses, induced eosinophil chemotaxis with optimal activity at 100 ng/mL. To elucidate the role of Ca2 + as a second messenger, eosinophils were depleted of intracellular Ca2 + which, per se, did not modify eosinophil chemotaxis. To gain insight of the possible intracellular signal transduction, we blocked pertussis toxin (PTX)-sensitive Gj proteins as well as several protein kinases. It was found that the inhibition of tyrosine kinase with herbimycin A and the inhibition of mitogen-activated protein kinase (MAPK) with MEK-1 inhibitor (PD98059) significantly blocked chemotaxis; however, inhibition of protein kinase C with staurosporine, protein kinase A with H-89 and Gi proteins with PTX did not affect chemotaxis. These results suggest a signal transduction pathway(s) involving Ca2 +-independent tyrosine kinase and MAPK activities.Keywords:
Chemotaxis assay
Staurosporine
A wide range of basic cellular process depends on cell motility that is fundamental for all eukaryotes. The ability of cells to migrate, adhere, and change shape requires most of the time external signals, although few cells respond primarily to internal cues. One of the most interesting and important response to external stimuli is chemotaxis. Chemotaxis, the directional movement of cells according to a concentration gradient of chemicals, is implicated in physiologically relevant phenomena such as inflammatory response, homeostatic circulation, and development and several disorders including infectious and allergic diseases, wound healing, angiogenesis, atherosclerosis, and tumor metastasis. However, despite the ubiquity and importance of chemotaxis, it remains a difficult process to study in vitro. The work carried out in this thesis present a novel chemotaxis assay in 3-D collagen gels in a direct-viewing chamber. Chemotaxis studies require a way to deliver chemicals to cells in a controlled gradient because cells need to be able to sense an increase in concentration of chemokine to direct their motion. In this chemotaxis assay a chemoattractant concentration gradient in the collagen gel sample seeded with cells is generated by diffusion trough a porous membrane. The diffusion process is monitored by fluorescence microscopy of FITC labelled dextran. Cell motion under the action of the chemoattractant gradient is followed by time-lapse video microscopy. Cell tracking is performed off-line by image analysis and the results are expressed in terms of a chemotactic index and velocity. The assay has been tested by using human neutrophils as a model.
Chemotaxis assay
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The directed motility of cells toward the source of a soluble chemical (chemotaxis) plays a role in events ranging from immune function to cancer progression. Numerous chemotaxis assays are commonly employed, yet none provides an optimal combination of the relevant parameters. The ideal chemotaxis assay for use in the analysis of cells crawling across a planar surface should be cost-effective, simple to perform, and suitable for high-throughput multiplexing, as well as permit alteration of experimental conditions during cell motility. Here we describe a novel chemotaxis assay based upon the invasion of cells into agarose spots into which chemoattractants are suspended. Our studies demonstrate that this system assays chemotaxis and not chemokinesis, and provide proof-of-principle for drug screening studies as well as analysis through high-resolution cellular imaging.
Chemokinesis
Chemotaxis assay
Agarose
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Parallel tests were conducted utilizing, the capillary tube migration test and the Boyden chamber assay, in order to determine whether the decrease in leukocyte chemotaxis that occurs if overoptimal cytotaxin concentrations are applied is due to migration inhibition. Overoptimal doses of casein produced decreased chemotactic response and migration inhibition for both rabbit macrophages and neutrophils. However, guinea pig neutrophils exhibited no decrease in chemotaxis despite high casein doses. Overoptimal doses of acid-denatured anaphylatoxin produced a decreased chemotactic response and migration inhibition of neutrophils. In both assays, this agent showed no effect upon macrophages. It is concluded that a chemotactic signal at different concentrations can elicit unidirectional migration or migration inhibition. Accordingly, chemotactic leukocyte attraction could be antagonistically regulated not only by serum-derived and lymphocyte-derived migration inhibitory factors but also by high doses of the chemotactic factor itself. Thus, the Boyden chamber technique can measure both chemotactic migration and migration inhibition phenomena.
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Abstract Studies of in vitro chemotaxis and spontaneous migration of human leukocytes using the accepted method with the Boyden‐chamber‐filter are troublesome, because of the need for specially constructed vessels as well as the difficulties caused by the use of membrane filters. We describe a new and simplified method for measuring human leucocyte chemotaxis, which is a modification of the recently described underagarose migration method and which is based upon spontaneous migration of cells from a soft agarose droplet and in response to a chemotactic gradient. We examined suspensions of leukocytes, purified granulocytes, and mononuclear cells from 10 healthy normal adults and from 10 samples of cord blood using E Coli O 111 B 4 endotoxin‐activated human serum as attractant. Our results showed that the mean chemotactic indices (C.I.‐chemotaxis/migration) for purified granulocytes and for mononuclear cells from normal individuals were 3.0 ± 1.2 and 2.7 ± 1.5, respectively. Chemotaxis was significantly reduced when unwashed leukocytes were studied, indicating a detrimental effect of autologous plasma on leukocytic response to a chemotactic stimulus in this system. Cord blood cells showed normal spontaneous migration, but significantly decreased chemotaxis. This preliminary report shows that the technique is simple, rapid, and reproducible, and can detect abnormalities of chemotaxis in both granulocytes and mononuclear cells.
Chemotaxis assay
Cord blood
Agarose
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Considering the essential role of chemotaxis of adherent, slow-moving cells in processes such as tumor metastasis or wound healing, a detailed understanding of the mechanisms and cues that direct migration of cells through tissues is highly desirable. The state-of-the-art chemotaxis instruments (e.g. microfluidic-based devices, bridge assays) can generate well-defined, long-term stable chemical gradients, crucial for quantitative investigation of chemotaxis in slow-moving cells. However, the majority of chemotaxis tools are designed for the purpose of an in-depth, but labor-intensive analysis of migratory behavior of single cells. This is rather inefficient for applications requiring higher experimental throughput, as it is the case of e.g. clinical examinations, chemoattractant screening or studies of the chemotaxis-related signaling pathways based on subcellular perturbations. Here, we present an advanced migration assay for accelerated and facilitated evaluation of the chemotactic response of slow-moving cells. The revised chemotaxis chamber contains a hydrogel microstructure–the migration arena, designed to enable identification of chemotactic behavior of a cell population in respect to the end-point of the experiment. At the same time, the assay in form of a microscopy slide enables direct visualization of the cells in either 2D or 3D environment, and provides a stable and linear gradient of chemoattractant. We demonstrate the correctness of the assay on the model study of HT-1080 chemotaxis in 3D and on 2D surface. Finally, we apply the migration arena chemotaxis assay to screen for a chemoattractant of primary keratinocytes, cells that play a major role in wound healing, being responsible for skin re-epithelialization and a successful wound closure. In direction of new therapeutic strategies to promote wound repair, we identified the chemotactic activity of the epithelial growth factor receptor (EGFR) ligands EGF and TGFα (transforming growth factor α).
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Abstract Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems.
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Effective tools for measurement of chemotaxis are desirable since cell migration towards given stimuli plays a crucial role in tumour metastasis, angiogenesis, inflammation, and wound healing. As for now, the Boyden chamber assay is the longstanding "gold-standard" for in vitro chemotaxis measurements. However, support for live cell microscopy is weak, concentration gradients are rather steep and poorly defined, and chemotaxis cannot be distinguished from migration in a single experiment.Here, we describe a novel all-in-one chamber system for long-term analysis of chemotaxis in vitro that improves upon many of the shortcomings of the Boyden chamber assay. This chemotaxis chamber was developed to provide high quality microscopy, linear concentration gradients, support for long-term assays, and observation of slowly migrating cells via video microscopy. AlexaFluor 488 dye was used to demonstrate the establishment, shape and time development of linear chemical gradients. Human fibrosarcoma cell line HT1080 and freshly isolated human umbilical vein endothelial cells (HUVEC) were used to assess chemotaxis towards 10% fetal calf serum (FCS) and FaDu cells' supernatant. Time-lapse video microscopy was conducted for 48 hours, and cell tracking and analysis was performed using ImageJ plugins. The results disclosed a linear steady-state gradient that was reached after approximately 8 hours and remained stable for at least 48 hours. Both cell types were chemotactically active and cell movement as well as cell-to-cell interaction was assessable.Compared to the Boyden chamber assay, this innovative system allows for the generation of a stable gradient for a much longer time period as well as for the tracking of cell locomotion along this gradient and over long distances. Finally, random migration can be distinguished from primed and directed migration along chemotactic gradients in the same experiment, a feature, which can be qualified via cell morphology imaging.
Chemotaxis assay
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Chemokinesis
Phagocyte
Chemotaxis assay
Mononuclear phagocyte system
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Polycarbonate (Nuclepore) filters were used to develop a rapid, reproducible technique to measure simultaneously mononuclear and polymorphonuclear leukocyte chemotaxis in vitro . Both cell types, obtained from guinea pig peritoneal exudates, showed a chemotactic response to an Escherichia coli filtrate. Neutrophils showed a faster, stronger response than did mononuclear cells and seemed to inhibit mononuclear chemotactic activity. Peritoneal exudate stored for 4 days did not lose its chemotactic responsiveness. This technique may be used to detect chemotactic activity generated by humoral and cellular immune reactions and to study the relative responsiveness of various leukocytes to the same agent.
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Decreased Mononuclear and Polymorphonuclear Chemotaxis in Human Newborns,Infants, and Young Children
A new method, chemotaxis under agarose gel, was used to assess the directed motility of polymorphonuclear (PMN) and mononuclear (MN) phagocytes of 21 newborns, 71 infants and children, and 50 adults. This assay requires only small quantities of cells and is rapid, easy, reproducible, and provides a permanent record. The chemotactic substance was zymosan-activated human serum. Monocyte chemotaxis in the newborn was approximately 50% of adult control values, using the Boyden chamber (8.1 ± 2 cells per high-power field[HPF] [SE] in newborns compared to 17 ± 3 cells per HPF in adults), and 25% of adult control values, using the agarose method (50 ± 10 cells in newborns compared to 216 ± 15 cells in adults). Both are significant differences (P < .005). MN chemotaxis remains extremely low through age 5, and remains moderately reduced until age 10. PMN chemotaxis in the newborn was 82 ± 21 cells compared to adult controls of 300± 42 cells, also a significant difference (P < .05). PMN chemotaxis remains markedly depressed through age 2. Thereafter, PMN chemotaxis increases but remains significantly less than in adults until age 16. These chemotactic defects may play an important role in depressed delayed hypersensitivity skin tests, diminished inflammatory reactions, and increased susceptibility to infection present in the newborn and young infant.
Chemotaxis assay
Agarose
Monocyte
Zymosan
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