The effect of cell sedimentation on measuring chondrocyte population migration using a Boyden chamber

The Boyden chamber assay provides a convenient method of assessing cell migration and measuring cell motility coefficients at the population level. Previous models of this assay completely ignore cell sedimentation in the suspension, assuming that all cells have already settled on the filter surface before commencing migration within the filter. However, ignoring cell sedimentation could lead to poor data interpretation because the time required for cells to settle through the suspension is close to the incubation period of only a few hours. This study models the Boyden chamber assay by incorporating the cell settling process to account for the cells remaining in the upper well when other cells migrate in the filter. The simulations in this study elucidate the experiments in the literature that test the haptotactic and chemotactic responses of rabbit chondrocytes to type II collagen. This study determines the cell population random motility, as well as the haptotaxis and chemotaxis coefficients, by fitting the experimental data. Results show that the chemotactic motility coefficient is 100 times greater than the haptotactic coefficient, and the equilibrium collagen-receptor dissociation constant is about 10-fold the haptotactic counterpart. Diffusion causes the soluble collagen gradients in the chemotactic case to decline over time, while the coated collagen gradients in the haptotactic assay are likely to remain fixed. As a result, the chemotactic case exhibits a lower number of migrated cells than the haptotactic assay. This study also demonstrates the influences of the dimensionless parameters that control cell behavior in the Boyden assay, providing a reference for future experiment designs.     

Cell migration in tumors

Invasion of cancer cells into surrounding tissue and the vasculature is an initial step in tumor metastasis. This requires chemotactic migration of cancer cells, steered by protrusive activity of the cell membrane and its attachment to the extracellular matrix. Recent advances in intravital imaging and the development of an in vivo invasion assay have provided new insights into how cancer cell migration is regulated by elements of the local microenvironment, including the extracellular matrix architecture and other cell types found in primary tumors. These results, combined with new findings from in vitro studies, have led to new insights into the molecular mechanisms of cell protrusive activity and chemotactic migration during invasion and metastasis.     

Cellular changes in bone marrow of malaria-infected mice. III. Chemotaxis of granulocytes

The number of bone marrow cells and their chemotactic activity was studied during malaria infection. Two days after infection of Balb/c mice with Plasmodium berghei, an increase in granulocyte number was observed in the blood. A modified Boyden chamber chemotaxis assay was employed to investigate the mechanism of granulocyte accumulation in the blood. Bone marrow cells from normal mice, from mice during a primary lethal infection and from immune mice after challenge were compared. The complement factor C5a showed chemotactic activity for bone marrow cells; a significant decrease of chemotaxis was only observed after 6 days of primary infection. Extracts of spleen, liver and infected erythrocytes lacked chemotactic activity, or caused inhibition of cell migration. Serum from mice with a 2-day primary infection contained chemotactic activity. The active component was heat labile, protease sensitive and had an estimated molecular weight of 250,000.     

Automated real-time measurement of chemotactic cell motility.

We have developed a novel method, (ECIS/taxis), for monitoring cell movement in response to chemotactic and chemokinetic factors. In this system, cells migrate in an under-agarose environment, and their positions are monitored using the electric cell-substrate impedance sensor technology to measure the impedance change at a target electrode, that is lithographed onto the substrate, as the cells arrive at the target. In the studies reported here, Dictyostelium discoideum was used as a prototypical, motile eukaryotic cell. Using the ECIS/taxis system, the arrival of cells at the target electrode was proportional to the dose offolate used to stimulate the cells and could be assessed by changes in resistance at the electrode. ECIS/taxis was readily able to distinguish between wild-type cells and a mutant that is deficient in its chemotactic response. Finally, we have shown that an agent that interferes with chemotactic motility leads to the delayed arrival of cells at the target electrode. The multi-well assay configuration allows for simultaneous automated screening of many samples for chemotactic or anti-chemotactic activity. This assay system is compatible with measurements of mammalian cell movement and should be valuable in the assessment of both agonists and antagonists of cell movement.     

Dual role of respiratory syncytial virus glycoprotein fragment as a mucosal immunogen and chemotactic adjuvant

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.     

Chemotactic response of splenic mononuclear cells to angiotensin II in murine schistosomiasis

Murine Schistosomiasis mansoni is a parasitic infection associated with a delayed-type hypersensitivity granulomatous reaction to the schistosome eggs. This reaction is characterized by the accumulation of mononuclear cells and other inflammatory cell types around the eggs. Granuloma macrophages produce angiotensin II (AII), which appears to have immunoregulatory function. By using an in vitro chemotaxis assay, this study demonstrated that AII is a chemotactic factor for splenic mononuclear cells derived from infected mice. The response was bimodal, with peak activities occurring at 10(-10) and 10(-6) M. AII was chemotactic for T lymphocytes, B lymphocytes, and a large population of unidentified mononuclear cells at the optimal chemotactic concentrations of the peptide. At high concentrations, AII was also chemotactic for phagocytic mononuclear cells. Sar1, ala8-AII, an analog of AII with antagonist activity, completely blocked AII-induced chemotaxis. A [3H]-AII binding assay revealed high-affinity specific binding on spleen cells. The binding was rapid, was dependent on radioligand concentration, and was reversible. These latter observations suggest that the chemotactic activity of AII for subpopulations of splenic mononuclear cells is mediated via AII receptors.     

Eosinophils and immune mechanisms: V. Demonstration of mouse spleen cell-derived chemotactic activities for eosinophils and mononuclear cells and comparisons with eosinophil stimulation promoter

The chemotactic response of mouse eosinophil-rich peritoneal exudative cells to the lymphokine eosinophil stimulation promoter (ESP) was examined. Both eosinophils and monocytes are chemotactically attracted across a 3-μm-pore size polycarbonate filter toward a concentration gradient of ESP-containing culture supernatant fluids. Deactivation of both cell types occurs following preincubation of the responding cells in culture supernates containing ESP activity. The chemotactic activity for both eosinophils and mononuclear cells is stable when incubated at 60 °C for 30 min but is labile at 80 °C, is nondialyzable, and at peak activity exhibits an apparent molecular weight of approximately 25,700 daltons, based on Sephadex G-100 gel chromatography. Production conditions required for the generation of chemotactic and ESP activities are identical, and fractions of culture supernatant fluids possessing one activity are also positive for the other. Preliminary results therefore indicate that the lymphokine ESP attracts both eosinophils and monocytes in a gradient-induced chemotaxis assay.     

Guanylate Cyclase Activity in Dictyostelium discoideum and Its Increase during Cell Development

Following consumption of the food supply, cells of the cellular slime mould Dictyostelium discoideum aggregate and form a multicellular organism. The mechanism for cell aggregation is chemotaxis. The chemotactic signal in D. discoideum is released periodically from aggregation centers and propagated from cell to cell. cAMP mediates cell aggregation by acting as chemotactic attractant and as propagator of the signal. cAMP signals are measured by cell-surface receptors. Recent evidence indicates a role for cGMP during cAMP-mediated cell aggregation in D. discoideum .
During cell differentiation to aggregation competence, cAMP binding sites appear at the cell surface, and the activity of the enzymes adenylate cyclase and phosphodiesterase increases several-fold. In the present work we investigate the synthesis of cGMP in D. discoideum . Conditions for the assay of guanylate cyclase in cell homogenates are described. Guanylate cyclase activity was followed during cell differentiation to aggregation competence and found to increase fourfold. These results indicate that cGMP is involved in cell differentiation of D. discoideum . In contrast to adenylate cyclase, which is activated by cAMP, guanylate cyclase was under our conditions activated neither by cAMP, nor by folic acid.     

Development of a new method of analysing chemotactic deactivation of human neutrophil granulocytes

Measurement of chemotactic migration of human neutrophil granulocytes (PMN) induced by chemotaxins serves as a simple and reliable method for assessing the expression of chemotaxin receptors. Incubation of PMN with a certain chemotaxin leads to a diminished chemotactic migration towards this chemotaxin. This is called chemotactic deactivation. We developed a new deactivation chamber to determine chemotaxis and chemotactic deactivation of human PMN. This novel chamber is a modification of the commercially available acrylic 48-well microchemotaxis chamber consisting of an upper block with wells drilled all the way through the block and a blind-well lower block. Both blocks are separated by a polycarbonate membrane. PMN from the wells in the upper block migrate through the pores of the membrane into the wells of the lower block containing the chemoattractants. Migrated PMN on the lower side of the PC membrane were quantified after staining by measuring specific light absorbance. The chemotactic activity is quantified as a ratio of stimulated migration and random migration (chemotactic index=CI). For our novel chamber, only the upper blocks of this commercial chamber were connected like a sandwich, including a polyvinylpyrrolidone-free polycarbonate membrane with a pore size of 3 microm. The wells in the upper compartment were filled with 5 x 10(4) PMN and deactivating chemotaxin. The lower block was then filled with the chemotactic stimulus and the chamber was then incubated in humidified air with 5% CO2 atmosphere at 37 degrees C. The influence of cell concentration, incubation time, chemotactic factor concentration, pore size and alkaline treatment of polycarbonate membranes on migrational activity of PMN have been investigated. The technique was rigorously standardized in order to optimize the assay conditions. The method is relatively simple, sensitive and fast. The determination of chemotaxis and deactivation are performed in the same chamber, thus avoiding cell loss due to nonspecific adherence in other incubation tubes. The chamber can be used to characterize the chemotactic activity of chemoattractants of unknown structure via known and unknown receptors. This new chamber can be very helpful in detecting unknown chemotactic stimuli, which are not detectable by, for example, antibodies.     

Inhibition of chemotactic motility and trans-endothelial migration of human neutrophils by sphingosine 1-phosphate

In previous studies, we reported that sphingosine 1-phosphate (Sph-1-P) inhibits the chemotactic motility of some cancer cell lines such as mouse melanoma cells, as well as human smooth muscle cells, at a very low concentration, as demonstrated by a transwell migration assay method (Proc. Natl. Acad. Sci. USA 89, 9698, 1992; J. Cell Biol. 130, 193, 1995). In this study, we investigated the effect of Sph-1-P on the chemotactic motility and invasiveness of human neutrophils, utilizing three different assay systems: (a) a transwell migration assay where IL-8 or fLMP was added as a chemotactic factor, (b) a phagokinetic assay with gold colloids, and (c) a trans-endothelial migration assay with human umbilical vein endothelial cells (HUVECs) plated on collagen layers. We found that among various sphingosine derivatives, Sph-1-P specifically inhibited the IL-8- or fLMP-induced chemotactic migration of neutrophils at concentrations below 1 μM. Phagokinetic activity of neutrophils was also suppressed by Sph-1-P, but more moderately than by the PKC inhibitory sphingosine analog, trimethylsphingosine. Finally, Sph-1-P inhibited trans-endothelial migration and invasiveness of neutrophils into HUVEC-covered collagen layers, whereas no effect on their adhesion to HUVECs was observed. These observations strongly suggest that Sph-1-P can act as a specific and effective motility regulator of human neutrophils, raising the possibility of future applications of Sph-1-P, or its analogs, as anti-inflammatory agents regulating invasive migration of neutrophils through endothelial layers at injured vascular sites.     

A chemotactic factor for rat thymocytes may regulate T-lymphocyte migration toward the thymic microenvironment

Using a modified Boyden chamber assay, extracts or culture supernatants of rat thymic stromal cells, or thymocytes were examined by chemotactic activity to rat leukocytes. Rat thymocytes responded chemotactically to the aqueous extract as well as to culture supernatants of thymic stromal cells. However, neither the extract and culture medium from concanavalin A-stimulated thymocytes nor any component of rat serum has shown such an activity. The thymic extract was fractionated into three molecular species with chemotactic activity for thymocytes. The thymocyte chemotactic factor(s) (TCFs) in the extract was distinct from known lymphocytic chemotactic factors, such as interleukin-1 (IL-1), IL-2, C5a, and the culture supernatant of stimulated thymocytes. In vitro, TCFs could attract, in addition to thymocytes, bone marrow cells, fetal liver cells, and nylon-wool nonadherent lymphocytes from peripheral blood and spleen. Lymph node cells, neutrophils, macrophages, and B cells from peripheral blood could not respond to TCFs. Thymocytes also responded to the extract of splenic stromal cells. Unlike the thymic extract, however, the splenic extract was chemotactically active for lymphocytes from lymph nodes but not for bone marrow cells. These results indicate that thymic stromal cells secrete a chemotactic factor(s) for a relatively immature type of T-lineage cells, which may by a thymus-homing progenitor T cell, while spleen may contain an attractant for a relatively mature type of T-lineage cells.     

Characterization of f-Met-Leu-Phe-stimulated fluid pinocytosis in human polymorphonuclear leukocytes by flow cytometry

N-formylated chemotactic peptide stimulation of human neutrophils initiates a number of cellular processes, such as lysosomal enzyme release and superoxide anion production, that are indicative of the events of neutrophil activation during the acute inflammatory response in disease. This study characterizes a newly recognized neutrophil activation event, N-formylated chemotactic peptide-stimulated fluid pinocytosis in human neutrophils, using a novel flow cytometric assay for this activity. Fluid pinocytosis was found to be inhibited by acidic pH and low temperature but could be enhanced by cytochalasin B treatment or surface adherence by neutrophils. The activity measured by this new assay of fluid pinocytosis appears to be separate and distinct from lysosomal enzyme release and receptor-mediated adsorptive endocytosis in neutrophils. The physiologic significance of N-formylated chemotactic peptide-stimulated fluid pinocytosis is not known, but a possible relationship to neutrophil locomotion is discussed.     

Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin 1 (IL 1)

LPS stimulated human blood mononuclear leukocytes to produce a chemotactic factor for human neutrophils. The effect of LPS was dose-dependent; 10 micrograms/ml was optimal for production of chemotactic factor. Chemotactic activity was detected 3 hr after LPS stimulation, and reached its peak at 12 hr. No activity was detected in culture supernatants of unstimulated cells, provided LPS-free media were selected. Isoelectric point of the factor, determined by chromatofocusing, was approximately 8 to 8.5. Molecular weight was approximately 10 kilodaltons by Sephacryl S-200 gel filtration or by HPLC gel filtration on TSK-2000 and -3000 columns in succession. The gel filtration fractions were also assayed for IL 1 activity. The elution position of IL 1 activity corresponded to a m.w. of 18. There was no chemotactic activity in the IL 1 activity peak. Furthermore, highly purified natural Il 1 alpha and -beta and recombinant Il 1 alpha and -beta did not exhibit chemotactic activity for neutrophils in our assay. Among mononuclear leukocytes, the monocyte was the principal producer of neutrophil chemotactic factor. These results suggest that a chemotactic factor for neutrophils, different from IL 1, is produced by LPS-stimulated blood monocytes.     

Chemotactic activity of cerebrospinal fluid in experimental cryptococcal meningitis

Cerebrospinal fluid from rabbits with chronic cryptococcal meningitis was tested for its chemotactic activity towards polymorphonuclear cells and monocytes. CSF chemotactic activity was present; it peaked 5-8 days after infection, coinciding with the time when the number of inflammatory cells in CSF was greatest. However, little chemotactic activity could be found in the early stages of infection, during the initial ingress of inflammatory cells. The chemotactic activity appeared to be host-derived, with characteristics consistent with lymphokine(s) or C5a. Treatment with cortisone significantly reduced the CSF chemotactic activity for both cell types; this reduction may contribute to the severe CSF leukopenia observed in cortisone-treated animals, which are unable to eradicate this yeast infection. Modulation of CSF chemotactic activity may be important to the success or failure of the host central nervous system response to Cryptococcus neoformans.     

Generation of lipid neutrophil chemoattractant by irradiated bovine aortic endothelial cells

Radiation injury to blood vessels is associated with an acute inflammatory process. We investigated the capacity of cultured bovine aortic endothelial cells (BAEC) to produce chemotactic factors after radiation injury. BAEC in serum-free media were irradiated with a cobalt-60 Gammacell 220 and the cell supernatants were assayed for chemotactic activity for human neutrophils in a Boyden chamber. There was a rapid release of chemotactic activity into the BAEC supernatants which was dependent both on the dose of radiation (5 to 40 Gy) and the time between irradiation and sample collection. In contrast, isolation of BAEC lysates by freeze-thawing was not associated with the presence of similar chemotactic activity. The chemotactic activity released from the irradiated BAEC was not destroyed by boiling nor by treatment with trypsin. The release of the chemotactic activity was, however, inhibited by the addition of a lipoxygenase inhibitor but not by the addition of a cyclooxygenase inhibitor before the irradiation. The chemotactic activity was recovered from the cell supernatants in the lipid phase after extraction with chloroform/methanol. Furthermore, the chloroform/methanol extracts co-eluted with authentic leukotriene B4 when the BAEC were prelabeled with [14C] arachidonic acid. However, we were unable to detect endogenous leukotriene B4 with RIA. Instead, the only detectable endogenous lipid present in the supernatants was 13-hydroxyoctadecadienoic acid which is derived from linoleic acid via the lipoxygenase pathway. 13-Hydroxyoctadecadienoic acid, however, had no chemotactic activity. These findings suggest that endothelial cells rapidly release a chemotactic agent after irradiation, the release of which is associated with a lipoxygenase pathway. The release of this chemotactic activity may account in part for the acute inflammatory response that is observed after ionizing irradiation.     

A novel dioxygenation product of arachidonic acid possesses potent chemotactic activity for human polymorphonuclear leukocytes

We have found that a novel dioxygenation product of arachidonic acid, 8(S),15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid (8,15-diHETE), possesses chemotactic activity for human polymorphonuclear leukocytes comparable to that of leukotriene B4. Authentic 8,15-diHETE, identified by gas chromatography-mass spectrometry, was prepared by treating arachidonic acid with soybean lipoxygenase and was purified by reverse-phase high performance liquid chromatography. Using a "leading front" assay, 8,15-diHETE exhibited significant chemotactic activity at a concentration of 5.0 ng/ml. Maximum chemotactic activity was observed at a concentration of 30 ng/ml. The 8,15-diHETE generated by mixed human leukocytes after stimulation with arachidonic acid and the calcium ionophore, A23187, exhibited quantitatively similar chemotactic activity. Two synthetic all-trans conjugated isomers of 8,15-diHETE, however, were not chemotactic at concentrations up to 500 ng/ml. In contrast to its potent chemotactic activity, 8,15-diHETE (at concentrations up to 10 micrograms/ml) was relatively inactive with respect to its ability to provoke either degranulation or generation of superoxide anion radicals by cytochalasin B-treated leukocytes. Both leukotriene B4 and 8,15-diHETE may be important mediators of inflammation.     

Chemotaxis of a Ralstonia sp. SJ98 toward different nitroaromatic compounds and their degradation

A Ralstonia sp. SJ98, isolated by a chemotactic enrichment technique, was capable of utilizing different nitroaromatic compounds (NACs). It utilized p-nitrophenol, 4-nitrocatechol, o-nitrobenzoic acid, and p-nitrobenzoic acid as the sole source of carbon and energy. It was observed that Ralstonia sp. SJ98 was chemotactic to the above-mentioned NACs as tested by the drop assay, swarm plate assay, and capillary assay. However, it failed to show chemotactic behavior toward those compounds which were not degraded by the microorganism. This is the first report which shows the chemotaxis of a microorganism toward different NACs and their subsequent degradation. Some of the intermediates of the NACs' degradative pathways have been identified using TLC, GC, and GC-MS studies. The results presented here indicate a correlation between chemotaxis and biodegradation of NACs.     

Role of B7-CD28/CTLA-4 costimulation and NF-kappa B in allergen-induced T cell chemotaxis by IL-16 and RANTES

The mechanisms that cause T cell recruitment into inflamed airways of asthmatic individuals are poorly understood. It has been shown previously that both natural exposure to allergen and challenge in the laboratory induce T cell accumulation in the bronchial mucosa of sensitized asthmatics. To study the mechanisms involved in this process, we have used an explant model in which bronchial biopsies taken from mild atopic asthmatic volunteers during fiberoptic bronchoscopy were stimulated in culture for 24 h by the common aeroallergen house dust mite (Dermatophagoides pteronyssinus (Der p)). Analysis of culture supernatants showed that stimulation with Der p significantly enhanced both the generation of T cell chemotactic activity by the mucosal tissue, as assayed in microchemotaxis chambers, and the production of IL-16 and RANTES. Neutralization experiments showed that IL-16 contributed more to the chemotactic activity than RANTES. The fusion protein CTLA-4-Ig, blocking B7:CD28 costimulation, and dexamethasone both significantly reduced the ex vivo production of chemotactic activity and release of IL-16 and RANTES. The proteasome inhibitor Cbz-Ile-Glu(OtBu)-Ala-leucinal also had a significant inhibitory effect on T cell chemotactic activity and IL-16 but not RANTES generation, indicating a role for nuclear factor NF kappa B activation. These results indicate that allergen stimulates cells within the bronchial mucosa to increase IL-16 and RANTES release, both of which contribute to T cell accumulation in asthmatic airways. The allergen-induced chemotactic activity is dependent on cell activation via CD28 and involves, at least partly, NF-kappa B.     

A model for cell type localization in the migrating slug of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> based on differential chemotactic sensitivity to cAMP and differential sensitivity to suppression of chemotaxis by ammonia

The three basic cell types in the migrating slug of Dictyostelium discoideum show differential chemotactic response to cyclic AMP (cAMP) and differential sensitivity to suppression of the chemotaxis by ammonia. The values of these parameters indicate a progressive maturation of chemotactic properties during the transdifferentiation of slug cell types. We present a model that explains the localization of the three cell types within the slug based on these chemotactic differences and on the maturation of their chemotactic properties.