The inhibition of locomotion of the polymorphonuclear leucocyte by organophosphorus compounds

The effects of diisopropylphosphorofluoridate (DFP) and other organophosphorus compounds on the locomotion of rabbit polymorphonuclear leucocytes have been investigated in vitro using time-lapse cinémicrography. Both phosphorylating and non-phosphorylating compounds were observed to inhibit cell locomotion, not only increasing the proportion of stationary cells, but also decreasing the velocity of those cells whose movement continued. This inhibition of locomotion occurred over the same concentration range of organophosphorus compound which was previously found to enhance the effect of leucocidin on the leucocyte. Although the inhibitory effects of low concentrations of organophosphorus compounds were partly reversible, higher concentrations produced effects which continued to increase even after the cells had been returned to normal medium. It is suggested that the supposed effect of organophosphorus compounds on chemotaxis may actually be due to the inhibition of locomotion per se, probably through the detergent properties of these compounds rather than their properties as enzyme inhibitors.     

How do leucocytes perceive chemical gradients?

Abstract Chemoattractants determine not only the direction of leucocyte locomotion (chemotaxis) but also its speed (chemokinesis). Various mechanisms by which leucocytes may detect chemotactic gradients, including spatial and temporal detection, are briefly reviewed. These mechanisms as originally proposed did not address the question how attractants cause leucocytes to migrate in persistent random paths in the absence of a gradient. Stochastic models have recently been presented in which leucocytes either respond by polarizing and migrating in the direction from which they receive their first signal, or respond to random flucuations in the perceived attractant concentration. Stochastic models allow an explanation for the persistent random walk shown by cells in uniform concentrations of attractant as well as for directional locomotion in gradients. They suggest that, at the biochemical level, the mechanisms by which attractants stimulate chemotaxis and chemokinesis are probably the same.     

How do leucocytes perceive chemical gradients?

Chemoattractants determine not only the direction of leucocyte locomotion (chemotaxis) but also its speed (chemokinesis). Various mechanisms by which leucocytes may detect chemotactic gradients, including spatial and temporal detection, are briefly reviewed. These mechanisms as originally proposed did not address the question how attractants cause leucocytes to migrate in persistent random paths in the absence of a gradient. Stochastic models have recently been presented in which leucocytes either respond by polarizing and migrating in the direction from which they receive their first signal, or respond to random fluctuations in the perceived attractant concentration. Stochastic models allow an explanation for the persistent random walk shown by cells in uniform concentrations of attractant as well as for directional locomotion in gradients. They suggest that, at the biochemical level, the mechanisms by which attractants stimulate chemotaxis and chemokinesis are probably the same.     

The difference between random movement and chemotaxis. Effects of antitubulins on neutrophil granulocyte locomotion

The antitubulins demecolcine and podophyllic acid ethylhydrazide (SPI) were used in experiments designed to elucidate the role of centriole-associated cytoplasmic microtubules in the locomotion of human neutrophil granulocytes (PMNs). The PMN locomotion was studied as chemotaxis and as the velocity of random movement. The PMN chemotaxis was inhibited by demecolcine (0.01 μg/ml) and SPI (0.1 μg/ml), i.e. concentrations below the reported threshold ones for mitotic arrest in metaphase. The velocity of single PMNs during random movement was only slightly reduced by treatment with 0.5 μg/ml of SPI. PMN locomotion was not appreciably inhibited by SPI, 0.5 μg/ml. The discrepancies mentioned suggest that centriole-associated microtubules are essential structures in the PMN direction-finding or PMN directional movement of chemotaxis but not in the mechanism of PMN locomotion. The present observations might, at least in part, explain the beneficial effects of antitubulins on acute gout.     

The control of chemotactic cell movement during Dictyostelium morphogenesis

Differential cell movement is an important mechanism in the development and morphogenesis of many organisms. In many cases there are indications that chemotaxis is a key mechanism controlling differential cell movement. This can be particularly well studied in the starvation-induced multicellular development of the social amoeba Dictyostelium discoideum. Upon starvation, up to 10(5) individual amoebae aggregate to form a fruiting body The cells aggregate by chemotaxis in response to propagating waves of cAMP, initiated by an aggregation centre. During their chemotactic aggregation the cells start to differentiate into prestalk and prespore cells, precursors to the stalk and spores that form the fruiting body. These cells enter the aggregate in a random order but then sort out to form a simple axial pattern in the slug. Our experiments strongly suggest that the multicellular aggregates (mounds) and slugs are also organized by propagating cAMP waves and, furthermore, that cell-type-specific differences in signalling and chemotaxis result in cell sorting, slug formation and movement.     

Cell traction in collective cell migration and morphogenesis: The chase and run mechanism

Directional collective cell migration plays an important role in development, physiology, and disease. An increasing number of studies revealed key aspects of how cells coordinate their movement through distances surpassing several cell diameters. While physical modeling and measurements of forces during collective cell movements helped to reveal key mechanisms, most of these studies focus on tightly connected epithelial cultures. Less is known about collective migration of mesenchymal cells. A typical example of such behavior is the migration of the neural crest cells, which migrate large distances as a group. A recent study revealed that this persistent migration is aided by the interaction between the neural crest and the neighboring placode cells, whereby neural crest chase the placodes via chemotaxis, but upon contact both populations undergo contact inhibition of locomotion and a rapid reorganization of cellular traction. The resulting asymmetric traction field of the placodes forces them to run away from the chasers. We argue that this chase and run interaction may not be specific only to the neural crest system, but could serve as the underlying mechanism for several morphogenetic processes involving collective cell migration.     

Chemoattractant stimulation of polymorphonuclear leucocyte locomotion

Chemoattractants stimulate both cell locomotion and the orientation of this locomotion (chemotaxis) in polymorphonuclear leukocytes. Cell locomotion is a complex process which includes the coordinated protrusion of cell processes, formation of attachments to the substrate and contraction of the rear of the cell. To understand how chemoattractants regulate this process, it is helpful to dissect the process into components that can be examined separately. Comparison of these components in cells before and after stimulation with chemoattractant provides information about their regulation. In this review we focus on three components: how chemoattractants induce the development of cell polarity; how chemoattractants modulate cytoskeletal components (especially actin) to cause pseudopod protrusion; and how chemoattractant modulation of cell adhesions might contribute to cell locomotion. Spatial and temporal coordination of these and other components of locomotion result in efficient and directed cell movement. Our treatment of these questions is speculative and not comprehensive. We propose simple hypothetical models which can provide the reader with a conceptual framework that integrates the information available.     

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and--unexpectedly--lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractants.     

Relationship of Human Neutrophil Morphology and Actin Distribution to Dispersive Locomotion caused by a steroid induced factor

A monocyte-derived steroid-induced factor has been shown previously to induce dispersive locomotion in human neutrophils and to lower adhesion to an albumin-coated glass surface. In this paper we show that this factor inhibits adhesion of neutrophils to bovine aorta and human endothelial cells by an undetermined mechanism. It induces unique changes in neutrophil shape with a characteristic monopolar pattern of F-actin distribution, which may correlate with the dispersive locomotion observed in the absence of a concentration gradient. This factor also inhibits N-formyl-methionyl-leucyl-phenylalanine-induced chemotaxis of neutrophils in a modified Boyden chamber assay. The reduction of adhesion and the inhibition of chemotaxis by the factor in vitro indicate a possible in vivo anti-inflammatory role.     

Chemotaxis in densely populated tissue determines germinal center anatomy and cell motility: a new paradigm for the development of complex tissues

Germinal centers (GCs) are complex dynamic structures that form within lymph nodes as an essential process in the humoral immune response. They represent a paradigm for studying the regulation of cell movement in the development of complex anatomical structures. We have developed a simulation of a modified cyclic re-entry model of GC dynamics which successfully employs chemotaxis to recapitulate the anatomy of the primary follicle and the development of a mature GC, including correctly structured mantle, dark and light zones. We then show that correct single cell movement dynamics (including persistent random walk and inter-zonal crossing) arise from this simulation as purely emergent properties. The major insight of our study is that chemotaxis can only achieve this when constrained by the known biological properties that cells are incompressible, exist in a densely packed environment, and must therefore compete for space. It is this interplay of chemotaxis and competition for limited space that generates all the complex and biologically accurate behaviors described here. Thus, from a single simple mechanism that is well documented in the biological literature, we can explain both higher level structure and single cell movement behaviors. To our knowledge this is the first GC model that is able to recapitulate both correctly detailed anatomy and single cell movement. This mechanism may have wide application for modeling other biological systems where cells undergo complex patterns of movement to produce defined anatomical structures with sharp tissue boundaries.     

Fibroblast chemotaxis and prolidase activity modulation by insulin-like growth factor II and mannose 6-phosphate

Chemotactic locomotion of fibroblasts requires extensive degradation of extracellular matrix components. The degradation is provided by a variety of proteases, including lysosomal enzymes. The process is regulated by cytokines. The present study shows that mannose 6-phosphate and insulin-like growth factor II (IGF-II) enhance fibroblast chemotaxis toward platelet-derived growth factor (PDGF). It is suggested that lysosomal enzymes (bearing mannose 6-phosphate molecules) are involved in chemotactic activity of the cells. The suggestion is supported by the observation that a-mannosidase and cathepsin D inhibitor - pepstatin are very potent inhibitors of fibroblast chemotaxis. Simultaneously, mannose 6-phosphate stimulates extracellular collagen degradation. The final step in collagen degradation is catalyzed by the cytosolic enzyme - prolidase. It has been found that mannose 6-phosphate stimulates also fibroblast prolidase activity with a concomitant increase in lysosomal enzymes activity. The present study demonstrates that the prolidase activity in fibroblasts may reflect the chemotactic activity of the cells and suggests that the mechanism of cell locomotion may involve lysosomal enzyme targeting, probably through IGF-II/mannose 6-phosphate receptor.     

Phospholipid directed motility of surface-motile bacteria

Myxococcus xanthus is a surface-motile bacterium that has adapted at least one chemosensory system to allow directed movement towards the slowly diffusible lipid phosphatidylethanolamine (PE). The Dif chemosensory pathway is remarkable because it has at least three inputs coupled to outputs that control extracellular matrix (ECM) production and lipid chemotaxis. The methyl-accepting chemotaxis protein, DifA, has two different sensor inputs that have been localized by mutagenesis. The Dif chemosensory pathway employs a novel protein that slows adaptation. Lipid chemotaxis may play important roles in the M. xanthus life cycle where prey-specific and development-specific attractants have been identified. Lipid chemotaxis may also be an important mechanism for locating nutrients by lung pathogens such as Pseudomonas aeruginosa.     

Sequential expression of cell surface C3bi receptors during neutrophil locomotion

Fluorescence microscopy has been used to study the cell surface distribution of the complement receptor for C3bi (CR3) on human neutrophils during locomotion. CR3 is an integral membrane protein that participates in cell attachment phenomena including chemotaxis. Fluorescein- and rhodamine-conjugated monoclonal IgG or Fab fragments were used to label CR3. We have previously shown that CR3 is uniformly distributed on unstimulated cells. During cell locomotion the fluorescent labels redistribute to the uropod and retraction fibers. To better understand the role of CR3 in chemotaxis, we have performed sequential two-color labeling experiments in conjunction with fluorescence microscopy. Double-labeling experiments were conducted by labeling adherent neutrophils with fluorescein-conjugated anti-CR3 followed by chemotaxis in a gradient of FMLP (10(-7) M). The cells were then labeled again with rhodamine-conjugated anti-CR3. The uropod and distal training filopodia were labeled with fluorescein, whereas the cell body and occasionally proximal filopodia near the uropod were labeled with rhodamine. When neutrophils were fixed and permeabilized prior to the second CR3 labeling, the second fluorescent label was localized to a granule-like compartment(s), often near the lamellipodium. The results suggest a flow of CR3 from intracellular granules----lamellipodia and cell body----uropod----trailing filopodia during chemotaxis.     

Shaking 3T3 cells: further studies on diffusion boundary effects

Confluent quiescent cultures of 3T3 cells can be stimulated to grow—with or without a medium change—simply by shaking (20 mm excursions, 3 per sec). Shaking without medium change, or with low serum concentration in fresh medium (4%), has the greatest effect on thymidine labeling and mitotic index compared with unshaken cultures, but high serum concentration (20%) stimulates both shaken and unshaken cultures equally. Even without medium change, the majority of cells can be stimulated to incorporate thymidine and to divide over 3 days, though initial shaking for 7 hr is sufficient to initiate growth. We conclude that growth in confluent 3T3 cell cultures is limited by a diffusion barrier in the microenvironment, probably affecting uptake of a serum component. The limitation can be overcome by increased fluid velocity as an alternative to raising the serum concentration.Shaking also causes increased cell movement and a change in cell morphology (which may be partly due to alterations in serum caused by shaking). Despite increased movement in the cell sheet, emigration from the border of a wound is diminished by shaking, suggesting that chemotaxis may be involved.To test whether loss of contact between the migrating cells is essential for growth stimulation at the border of a confluent layer, wounds were made in unshaken cultures in the presence of cytochalasin B, which prevents locomotion of cells and maintains the original topography. Nevertheless, the thymidine labeling index was still increased along the wound boundary, and included cells which were near but not on the edge. This supports the view that short range effects on cell growth at discontinuities in cell populations can be due to modification of a diffusion barrier and not simply alteration in cell contacts.     

The role of cytoplasmic microtubules in polymorphonuclear leukocyte chemotaxis : Evidence for the release hypothesis by means of time-lapse analysis of PMN movement relative to dot-like attractants

The present experiments were designed to elucidate the role of cytoplasmic microtubules in the chemotaxis of human polymorphonuclear leukocytes (PMNs) by means of the Boyden chamber technique and by means of analysis of PMN locomotion around a dot-like attractant.Casein induced positive chemotaxis in a small and variable fraction of the PMNs in the Boyden chamber.The movements of individual PMNs in coverslip preparations of clotted autoplasma were analysed as regards velocity of locomotion, locomotive index and net radial dislocation relative to the cell centre, with or without a yeast-phagocytosing leukocyte as a dot-like attractant.PMNs without obvious attractants tended to leave the visual field, i.e. they had a negative net radial dislocation relative to the centre of the visual field. Their locomotive indices suggested that their disappearance from the visual field was due to random movement. In contrast, the locomotive indices of PMNs influenced by attractants suggested the presence of both positive and negative chemotaxis in the population of moving PMNs.Yeast-phagocytosing leukocytes attracted wandering PMNs isolated by the Isopaque-Ficoll method (IF-PMNs) with a force which approximately balanced the basic tendency of the IF-PMNs to leave the visual field. Selective pretreatment of the moving IF-PMNs with podophyllic acid ethylhydrazide (SPI), 0.5 μg/ml (1.05 × 10⁻⁶ M), did not inhibit their attraction towards the central yeast phagocyte. The attraction of wandering IF-PMNs towards the central yeast phagocyte was inhibited by selective pretreatment of the phagocytes with SPI, 0.5 μg/ml. These observations indicate that cytoplasmic microtubules have an essential role in the release of chemotactic substances from phagocytosing leukocytes but not in the direction-finding of attractant-approaching PMNs.From the present observations by means of SPI, it is suggested that antitubulin inhibition of the release of chemotactic substances from phagocytosing leukocytes is the mechanism of inhibition of PMN chemotaxis by sub-antimitotic antitubulin concentrations in vitro. The latter phenomenon is thought to reflect the cellular basis of the anti-inflammatory action of the antitubulins.     

Moving forward moving backward: directional sorting of chemotactic cells due to size and adhesion differences

Differential movement of individual cells within tissues is an important yet poorly understood process in biological development. Here we present a computational study of cell sorting caused by a combination of cell adhesion and chemotaxis, where we assume that all cells respond equally to the chemotactic signal. To capture in our model mesoscopic properties of biological cells, such as their size and deformability, we use the Cellular Potts Model, a multiscale, cell-based Monte Carlo model. We demonstrate a rich array of cell-sorting phenomena, which depend on a combination of mescoscopic cell properties and tissue level constraints. Under the conditions studied, cell sorting is a fast process, which scales linearly with tissue size. We demonstrate the occurrence of “absolute negative mobility”, which means that cells may move in the direction opposite to the applied force (here chemotaxis). Moreover, during the sorting, cells may even reverse the direction of motion. Another interesting phenomenon is “minority sorting”, where the direction of movement does not depend on cell type, but on the frequency of the cell type in the tissue. A special case is the cAMP-wave-driven chemotaxis of Dictyostelium cells, which generates pressure waves that guide the sorting. The mechanisms we describe can easily be overlooked in studies of differential cell movement, hence certain experimental observations may be misinterpreted.     

Polymorphonuclear Leucocyte Chemotaxis in Patients with Bacterial Infections

A new in vitro method of measuring the chemotaxis of polymorphonuclear leucocytes from peripheral blood has been used to calculate a chemotactic index. The mean chemotactic index in 15 patients with bacterial infection (434) was significantly less (P <0·0005) than in 15 normal controls (553) matched for age and sex. The reduction in chemotaxis could be correlated with the duration of the infection, with the greatest impairment being found in those patients with the shortest duration of infection. In five patients studied before and after appropriate therapy the chemotactic index returned to normal values with clearing of the infection. It is suggested that the impairment in chemotaxis may be due to prior phagocytosis of antibody-antigen complexes by the polymorphonuclear leucocytes.     

Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes from a patient with leukocyte adhesion deficiency-1: normal displacement in close quarters via chimneying

The beta2 integrins are known to be important in the motile function of leukocytes in general and in the adhesive response to inflammatory stimuli in particular. In the current study, under direct microscopic observation with concomitant time-lapse video recording, we examined the locomotion of human blood PMN from a patient with Leukocyte Adhesion Deficiency-1 (LAD), a disorder in which beta2 integrins on the cell surface are markedly deficient in number or function. In thin slide preparations such that the leukocytes were somewhat compressed between slide and cover slip, PMNLAD exhibited normal random locomotion and chemotaxis, apparently by using the opposing surfaces to generate the force for locomotion (chimneying). In thicker preparations, an adherence deficit was evident, but chemotaxis still occurred, even by PMNLAD anticoagulated in EDTA. Consistent with the paucity of beta2 integrins on the surface of the PMNLAD was their failure to aggregate in the presence of antibodies to beta2 integrins, even when they had been brought together by chemotaxis. We relate these findings to the reported independence from integrins of PMN in the lung vasculature in LAD, as well as in certain experimental conditions.     

A stochastic model for leukocyte random motility and chemotaxis based on receptor binding fluctuations

Two central features of polymorphonuclear leukocyte chemosensory movement behavior demand fundamental theoretical understanding. In uniform concentrations of chemoattractant, these cells exhibit a persistent random walk, with a characteristic "persistence time" between significant changes in direction. In chemoattractant concentration gradients, they demonstrate a biased random walk, with an "orientation bias" characterizing the fraction of cells moving up the gradient. A coherent picture of cell movement responses to chemoattractant requires that both the persistence time and the orientation bias be explained within a unifying framework. In this paper, we offer the possibility that "noise" in the cellular signal perception/response mechanism can simultaneously account for these two key phenomena. In particular, we develop a stochastic mathematical model for cell locomotion based on kinetic fluctuations in chemoattractant/receptor binding. This model can simulate cell paths similar to those observed experimentally, under conditions of uniform chemoattractant concentrations as well as chemoattractant concentration gradients. Furthermore, this model can quantitatively predict both cell persistence time and dependence of orientation bias on gradient size. Thus, the concept of signal "noise" can quantitatively unify the major characteristics of leukocyte random motility and chemotaxis. The same level of noise large enough to account for the observed frequency of turning in uniform environments is simultaneously small enough to allow for the observed degree of directional bias in gradients.     

A 'chemotactic dipole' mechanism for large-scale vortex motion during primitive streak formation in the chick embryo

Primitive streak formation in the chick embryo involves significant coordinated cell movement lateral to the streak, in addition to the posterior-anterior movement of cells in the streak proper. Cells lateral to the streak are observed to undergo 'polonaise movements', i.e. two large counter-rotating vortices, reminiscent of eddies in a fluid. In this paper, we propose a mechanism for these movement patterns which relies on chemotactic signals emitted by a dipolar configuration of cells in the posterior region of the epiblast. The 'chemotactic dipole' consists of adjacent regions of cells emitting chemo-attractants and chemo-repellents. We motivate this idea using a mathematical analogy between chemotaxis and electrostatics, and test this idea using large-scale computer simulations. We implement active cell response to both neighboring mechanical interactions and chemotactic gradients using the Subcellular Element Model. Simulations show the emergence of large-scale vortices of cell movement. The length and time scales of vortex formation are in reasonable agreement with experimental data. We also provide quantitative estimates for the robustness of the chemotaxis dipole mechanism, which indicate that the mechanism has an error tolerance of about 10% to variation in chemotactic parameters, assuming that only 1% of the cell population is involved in emitting signals. This tolerance increases for larger populations of cells emitting signals.