Effective neutrophil chemotaxis is strongly influenced by mean IL-8 concentration

Neutrophils need to correctly interpret gradients of chemotactic factors (CFs) such as interleukin 8 (IL-8) to migrate to the site of infection and perform immune functions. Because diffusion-based chemotaxis assays used in previous studies suffer from temporally changing gradients, it is difficult to distinguish the influence of CF gradient steepness from mean CF concentration on chemotaxis. To better understand the roles of mean CF concentration and CF gradient steepness, we developed a microfluidic device that can maintain stable IL-8 gradients. We report that the random motility of neutrophils is a biphasic function of IL-8 concentration and its magnitude plays a decisive role in effective chemotaxis, a quantitative measure of migration. We show that the concentrations for the optimum chemotaxis in linear IL-8 gradients and for the maximum random motility in uniform IL-8 coincide. In contrast, we find that the steepness of IL-8 gradients has no significant effect on effective chemotaxis.     

Dependence of Bacterial Chemotaxis on Gradient Shape and Adaptation Rate

Simulation of cellular behavior on multiple scales requires models that are sufficiently detailed to capture central intracellular processes but at the same time enable the simulation of entire cell populations in a computationally cheap way. In this paper we present RapidCell, a hybrid model of chemotactic Escherichia coli that combines the Monod-Wyman-Changeux signal processing by mixed chemoreceptor clusters, the adaptation dynamics described by ordinary differential equations, and a detailed model of cell tumbling. Our model dramatically reduces computational costs and allows the highly efficient simulation of E. coli chemotaxis. We use the model to investigate chemotaxis in different gradients, and suggest a new, constant-activity type of gradient to systematically study chemotactic behavior of virtual bacteria. Using the unique properties of this gradient, we show that optimal chemotaxis is observed in a narrow range of CheA kinase activity, where concentration of the response regulator CheY-P falls into the operating range of flagellar motors. Our simulations also confirm that the CheB phosphorylation feedback improves chemotactic efficiency by shifting the average CheY-P concentration to fit the motor operating range. Our results suggest that in liquid media the variability in adaptation times among cells may be evolutionary favorable to ensure coexistence of subpopulations that will be optimally tactic in different gradients. However, in a porous medium (agar) such variability appears to be less important, because agar structure poses mainly negative selection against subpopulations with low levels of adaptation enzymes. RapidCell is available from the authors upon request.     

The role of multiple chemotactic mechanisms in a model of chemotaxis in C. elegans: different mechanisms are specialised for different environments

Unlike simpler organisms, C. elegans possesses several distinct chemosensory pathways and chemotactic mechanisms. These mechanisms and pathways are individually capable of driving chemotaxis in a chemical concentration gradient. However, it is not understood if they are redundant or co-operate in more sophisticated ways. Here we examine the specialisation of different chemotactic mechanisms in a model of chemotaxis to NaCl. We explore the performance of different chemotactic mechanisms in a range of chemical gradients and show that, in the model, far from being redundant, the mechanisms are specialised both for different environments and for distinct features within those environments. We also show that the chemotactic drive mediated by the ASE pathway is not robust to the presence of noise in the chemical gradient. This problem cannot be solved along the ASE pathway without destroying its ability to drive chemotaxis. Instead, we show that robustness to noise can be achieved by introducing a second, much slower NaCl-sensing pathway. This secondary pathway is simpler than the ASE pathway, in the sense that it can respond to either up-steps or down-steps in NaCl but not both, and could correspond to one of several candidates in the literature which we identify and evaluate. This work provides one possible explanation of why there are multiple NaCl sensing pathways and chemotactic mechanisms in C. elegans: rather than being redundant the different pathways and mechanism are specialised both for the characteristics of different environments and for distinct features within a single environment.     

Differential effects of EGF gradient profiles on MDA-MB-231 breast cancer cell chemotaxis

Chemotaxis, directed cell migration in a gradient of chemoattractant, is an important biological phenomenon that plays pivotal roles in cancer metastasis. Newly developed microfluidic chemotaxis chambers (MCC) were used to study chemotaxis of metastatic breast cancer cells, MDA-MB-231, in EGF gradients of well-defined profiles. Migration behaviors of MDA-MB-231 cells in uniform concentrations of EGF (0, 25, 50, and 100 ng/ml) and EGF (0-25, 0-50, and 0-100 ng/ml) with linear and nonlinear polynomial profiles were investigated. MDA-MB-231 cells exhibited increased speed and directionality upon stimulation with uniform concentrations of EGF. The cells were viable and motile for over 24 h, confirming the compatibility of MCC with cancer cells. Linear concentration gradients of different ranges were not effective in inducing chemotactic movement as compared to nonlinear gradients. MDA-MB-231 cells migrating in EGF gradient of 0-50 ng/ml nonlinear polynomial profile exhibited marked directional movement toward higher EGF concentration. This result suggests that MDA-MB-231 cancer cell chemotaxis depends on the shape of gradient profile as well as on the range of EGF concentrations.     

The Role of Adaptation in Bacterial Speed Races

Evolution of biological sensory systems is driven by the need for efficient responses to environmental stimuli. A paradigm among prokaryotes is the chemotaxis system, which allows bacteria to navigate gradients of chemoattractants by biasing their run-and-tumble motion. A notable feature of chemotaxis is adaptation: after the application of a step stimulus, the bacterial running time relaxes to its pre-stimulus level. The response to the amino acid aspartate is precisely adapted whilst the response to serine is not, in spite of the same pathway processing the signals preferentially sensed by the two receptors Tar and Tsr, respectively. While the chemotaxis pathway in E. coli is well characterized, the role of adaptation, its functional significance and the ecological conditions where chemotaxis is selected, are largely unknown. Here, we investigate the role of adaptation in the climbing of gradients by E. coli. We first present theoretical arguments that highlight the mechanisms that control the efficiency of the chemotactic up-gradient motion. We discuss then the limitations of linear response theory, which motivate our subsequent experimental investigation of E. coli speed races in gradients of aspartate, serine and combinations thereof. By using microfluidic techniques, we engineer controlled gradients and demonstrate that bacterial fronts progress faster in equal-magnitude gradients of serine than aspartate. The effect is observed over an extended range of concentrations and is not due to differences in swimming velocities. We then show that adding a constant background of serine to gradients of aspartate breaks the adaptation to aspartate, which results in a sped-up progression of the fronts and directly illustrate the role of adaptation in chemotactic gradient-climbing.     

A fibrin or collagen gel assay for tissue cell chemotaxis: assessment of fibroblast chemotaxis to GRGDSP

Fibroblast chemotaxis is implicated in many physiological processes, including wound healing and morphogenesis. We present a novel assay for chemotaxis of fibroblasts (and other slow-moving tissue cells) in a direct-viewing chamber containing a physiologically relevant three-dimensional fibrin or collagen gel in which long-lasting, spatially continuous gradients have been sustained for at least 24 h, long enough for significant fibroblast migration. This combination of features is not available in any alternative assay of comparable setup simplicity. Using a putative fibroblast chemotactic factor, the fibronectin peptide GRGDSP, we measured human foreskin fibroblast alignment in the direction along the gradient, which followed a biphasic dependence on GRGDSP concentration with an optimal concentration of about 10 nM. Time-lapse video microscopy revealed that cell migration was up the soluble GRGDSP gradient, confirming positive chemotaxis to GRGDSP and rejecting the possibility of dominant haptotaxis down the soluble GRGDSP gradient, that is, up a putative gradient of integrin-mediated adhesion induced by the soluble GRGDSP gradient.     

Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors

Polymorphonuclear leukocyte (PMN) chemotaxis has been examined under conditions which allow phase microscope observations of cells responding to controlled gradients of chemotactic factors. With this visual assay, PMNs can be seen to orient rapidly and reversibly to gradients of N-formylmethionyl peptides. The level of orientation depends upon the mean concentration of peptide present as well as the concentration gradient. The response allows an estimation of the binding constant of the peptide to the cell. In optimal gradients, PMNs can detect a 1% difference in the concentration of peptide. At high cell densities, PMNs incubated with active peptides orient their locomotion away from the center of the cell population. This orientation appears to be due to inactivation of the peptides by the cells. Such inactivation in vivo could help to limit an inflammatory response.     

Sensory adaptation of Dictyostelium discoideum cells to chemotactic signals

Postvegetative Dictyostelium discoideum cells react chemotactically to gradients of cAMP, folic acid, and pterin. In the presence of a constant concentration of 10(-5) M cAMP cells move at random. They still are able to respond to superimposed gradients of cAMP, although the response is less efficient than without the high background level of cAMP. Cells which are accommodated to 10(-5) M cAMP do not react to a gradient of cAMP if the mean cAMP concentration is decreasing with time. This indicates the involvement of adaptation in the detection of chemotactic gradients: cells adapt to the mean concentration of chemoattractant and respond to positive deviations from the mean concentration. Cells adapted to high cAMP concentrations react normally to gradients of folic acid or pterin. Adaptation to one of these compounds does not affect the response to the other attractants. This suggests that cAMP, folic acid, and pterin are detected by different receptors, and that adaptation is localized at a step in the transduction process before the signals from these receptors coincide into one pathway. I discuss the implications of adaptation for chemotaxis and cell aggregation.     

Chemotaxis of large granular lymphocytes

The hypothesis that large granular lymphocytes (LGL) are capable of directed locomotion (chemotaxis) was tested. A population of LGL isolated from discontinuous Percoll gradients migrated along concentration gradients of N-formyl-methionyl-leucyl-phenylalanine (f-MLP), casein, and C5a, well known chemoattractants for polymorphonuclear leukocytes and monocytes, as well as interferon-beta and colony-stimulating factor. Interleukin 2, tuftsin, platelet-derived growth factor, and fibronectin were inactive. Migratory responses were greater in Percoll fractions with the highest lytic activity and HNK-1+ cells. The chemotactic response to f-MLP, casein, and C5a was always greater when the chemoattractant was present in greater concentration in the lower compartment of the Boyden chamber. Optimum chemotaxis was observed after a 1 hr incubation that made use of 12 micron nitrocellulose filters. LGL exhibited a high degree of nondirected locomotion when allowed to migrate for longer periods (greater than 2 hr), and when cultured in vitro for 24 to 72 hr in the presence or absence of IL 2 containing phytohemagluttinin-conditioned medium. The chemotactic LGL was HNK-1+, OKT11+ or HNK-1+, OKT11- on the basis of monoclonal antibody and complement depletion. They did not bear either T cell or monocyte cell surface markers, exhibiting an OKT3-, OKT4-, OKT8-, OKM1-, and MO2- phenotype, and did not form E rosettes at 29 degrees C, which is characteristic of lytic NK cells in contrast to T cells. Furthermore, a rat LGL leukemia (RNK) exhibited a chemotactic response to both f-MLP and casein. LGL chemotaxis to f-MLP could be inhibited in a dose-dependent manner by the inactive structural analog CBZ-phe-met, and the RNK tumor line specifically bound f-ML[3H]P, suggesting that LGL bear receptors for the chemotactic peptide.     

Locomotory characteristics of human lymphocytes undergoing negative chemotaxis to oral carcinomas

Using a continuous-time Markov chain theory technique to quantitate the interaction of patients' lymphocytes with their tumours, we have shown that negative chemotaxis is brought about by stationary state lymphocytes showing a high probability of moving initially in the appropriate direction. Analyses of the data strongly suggest that lymphocytes sense chemotactic gradients using a spatial receptor mechanism analogous to that proposed for polymorphonuclear leukocytes.     

Limits of Feedback Control in Bacterial Chemotaxis

Inputs to signaling pathways can have complex statistics that depend on the environment and on the behavioral response to previous stimuli. Such behavioral feedback is particularly important in navigation. Successful navigation relies on proper coupling between sensors, which gather information during motion, and actuators, which control behavior. Because reorientation conditions future inputs, behavioral feedback can place sensors and actuators in an operational regime different from the resting state. How then can organisms maintain proper information transfer through the pathway while navigating diverse environments? In bacterial chemotaxis, robust performance is often attributed to the zero integral feedback control of the sensor, which guarantees that activity returns to resting state when the input remains constant. While this property provides sensitivity over a wide range of signal intensities, it remains unclear how other parameters such as adaptation rate and adapted activity affect chemotactic performance, especially when considering that the swimming behavior of the cell determines the input signal. We examine this issue using analytical models and simulations that incorporate recent experimental evidences about behavioral feedback and flagellar motor adaptation. By focusing on how sensory information carried by the response regulator is best utilized by the motor, we identify an operational regime that maximizes drift velocity along chemical concentration gradients for a wide range of environments and sensor adaptation rates. This optimal regime is outside the dynamic range of the motor response, but maximizes the contrast between run duration up and down gradients. In steep gradients, the feedback from chemotactic drift can push the system through a bifurcation. This creates a non-chemotactic state that traps cells unless the motor is allowed to adapt. Although motor adaptation helps, we find that as the strength of the feedback increases individual phenotypes cannot maintain the optimal operational regime in all environments, suggesting that diversity could be beneficial.     

Migration of connective tissue-derived cells is mediated by ultra-low concentration gradient fields of EGF

The directed migration of cells towards chemical stimuli incorporates simultaneous changes in both the concentration of a chemotactic agent and its concentration gradient, each of which may influence cell migratory response. In this study, we utilized a microfluidic system to examine the interactions between epidermal growth factor (EGF) concentration and EGF gradient in stimulating the chemotaxis of connective tissue-derived fibroblast cells. Cells seeded within microfluidic devices were exposed to concentration gradients established by EGF concentrations that matched or exceeded those required for maximum chemotactic responses seen in transfilter migration assays. The migration of individual cells within the device was measured optically after steady-state gradients had been experimentally established. Results illustrate that motility was maximal at EGF concentration gradients between .01- and 0.1-ng/(mL.mm) for all concentrations used. In contrast, the number of motile cells continually increased with increasing gradient steepness for all concentrations examined. Microfluidics-based experiments exposed cells to minute changes in EGF concentration and gradient that were in line with the acute EGFR phosphorylation measured. Correlation of experimental data with established mathematical models illustrated that the fibroblasts studied exhibit an unreported chemosensitivity to minute changes in EGF concentration, similar to that reported for highly motile cells, such as macrophages. Our results demonstrate that shallow chemotactic gradients, while previously unexplored, are necessary to induce the rate of directed cellular migration and the number of motile cells in the connective tissue-derived cells examined.     

Ammonia differentially suppresses the cAMP chemotaxis of anterior-like cells and prestalk cells indictyostelium discoideum

A drop assay for chemotaxis to cAMP confirms that both anterior-like cells (ALC) and prestalk cells (pst cells) respond to cAMP gradients. We present evidence that the chemotactic response of both ALC and pst cells is suppressed by ammonia, but a higher concentration of ammonia is required to suppress the response in pst cells. ALC show a chemotactic response to cAMP when moving on a substratum of prespore cells in isolated slug posteriors incubated under oxygen. ALC chemotaxis on a prespore cell substratum is suppressed by the same concentration of ammonia that suppresses ALC chemotaxis on the agar substratum in drop assays. Chemotaxis suppression is mediated by the unprotonated (NH₃) species of ammonia. The observed suppression, by ammonia, of ALC chemotaxis to cAMP supports our earlier hypothesis that ammonia is the tip-produced suppressor of such chemotaxis. We discuss implications of ammonia sensitivity of pst cells and ALC with regard to the movement and localization of ALC and pst cells in the slug and to the roles played by ALC in fruiting body formation. In addition, we suggest that a progressive decrease in sensitivity to ammonia is an important part of the maturation of ALC into pst cells.     

Different Migration Patterns of Sea Urchin and Mouse Sperm Revealed by a Microfluidic Chemotaxis Device

Chemotaxis refers to a process whereby cells move up or down a chemical gradient. Sperm chemotaxis is known to be a strategy exploited by marine invertebrates such as sea urchins to reach eggs efficiently in moving water. Less is understood about how or whether chemotaxis is used by mammalian sperm to reach eggs, where fertilization takes place within the confinement of a reproductive tract. In this report, we quantitatively assessed sea urchin and mouse sperm chemotaxis using a recently developed microfluidic model and high-speed imaging. Results demonstrated that sea urchin Arbacia punctulata sperm were chemotactic toward the peptide resact with high chemotactic sensitivity, with an average velocity V_x up the chemical gradient as high as 20% of its average speed (238 μm/s), while mouse sperm displayed no statistically significant chemotactic behavior in progesterone gradients, which had been proposed to guide mammalian sperm toward eggs. This work demonstrates the validity of a microfluidic model for quantitative sperm chemotaxis studies, and reveals a biological insight that chemotaxis up a progesterone gradient may not be a universal strategy for mammalian sperm to reach eggs.     

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.     

Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device

Although a wealth of knowledge about chemotaxis has accumulated in the past 40 years, these studies have been hampered by the inability of researchers to generate simple linear gradients instantaneously and to maintain them at steady state. Here we describe a device microfabricated by soft lithography and consisting of a network of microfluidic channels that can generate spatially and temporally controlled gradients of chemotactic factors. When human neutrophils are positioned within a microchannel, their migration in simple and complex interleukin-8 (IL-8) gradients can be tested. The cells exhibit strong directional migration toward increasing concentrations of IL-8 in linear gradients. Neutrophil migration halts abruptly when cells encounter a sudden drop in the chemoattractant concentration to zero ("cliff" gradient). When neutrophils are challenged with a gradual increase and decrease in chemoattractant ("hill" gradient), however, the cells traverse the crest of maximum concentration and migrate further before reversing direction. The technique described in this paper provides a robust method to investigate migratory cells under a variety of conditions not accessible to study by earlier techniques.     

The chemokinetic and chemotactic behavior of Rhodobacter sphaeroides: two independent responses.

Rhodobacter sphaeroides exhibits two behavioral responses when exposed to some compounds: (i) a chemotactic response that results in accumulation and (ii) a sustained increase in swimming speed. This latter chemokinetic response occurs without any apparent long-term change in the size of the electrochemical proton gradient. The results presented here show that the chemokinetic response is separate from the chemotactic response, although some compounds can induce both responses. Compounds that caused only chemokinesis induced a sustained increase in the rate of flagellar rotation, but chemoeffectors which were also chemotactic caused an additional short-term change in both the stopping frequency and the duration of stops and runs. The response to a change in chemoattractant concentration was a transient increase in the stopping frequency when the concentration was reduced, with adaptation taking between 10 and 60 s. There was also a decrease in the stopping frequency when the concentration was increased, but adaptation took up to 60 min. The nature and duration of both the chemotactic and chemokinetic responses were concentration dependent. Weak organic acids elicited the strongest chemokinetic responses, and although many also caused chemotaxis, there were conditions under which chemokinesis occurred in the absence of chemotaxis. The transportable succinate analog malonate caused chemokinesis but not chemotaxis, as did acetate when added to a mutant able to transport but not grow on acetate. Chemokinesis also occurred after incubation with arsenate, conditions under which chemotaxis was lost, indicating that phosphorylation at some level may have a role in chemotaxis. Aspartate was the only chemoattractant amino acid to cause chemokinesis. Glutamate caused chemotaxis but not chemokinesis. These data suggest that (i) chemotaxis and chemokinesis are separate responses, (ii) metabolism is required for chemotaxis but not chemokinesis, (iii) a reduction in chemoattractant concentration may cause the major chemotactic signal, and (iv) a specific transport pathway(s) may be involved in chemokinetic signalling in R. sphaeroides.     

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.     

Behavioral responses of Rhodobacter sphaeroides to linear gradients of the nutrients succinate and acetate

Rhodobacter sphaeroides cells were tethered by their flagella and subjected to increasing and decreasing nutrient gradients. Using motion analysis, changes in flagellar motor rotation were measured and the responses of the cells to the chemotactic gradients were determined. The steepness and concentration ranges of increasing and decreasing gradients were varied, and the bacterial responses were measured. This allowed the limits of gradients that would invoke changes in flagellar behavior to be determined and thus predicts the nature of gradients that would evoke chemotaxis in the environment. The sensory threshold was measured at 30 nM, and the response showed saturation at 150 microM. The study determined that cells detected and responded to changing concentration rates as low as 1 nM/s for acetate and 5 nM/s for succinate. The complex sensory system of R. sphaeroides responded to both increasing and decreasing concentration gradients of attractant with different sensitivities. In addition, transition phases involving changes in the motor speed and the smoothness of motor rotation were found.     

Antagonists of chemoattractants reveal separate receptors for cAMP, folk acid and pterin in Dictyostelium

Adenosine 3′,5′-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3′-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3′-amino-cAMP on the chemotactic activity of cAMP is competitive, which suggests that 3′-amino-cAMP antagonizes cAMP via the chemotactic receptor for cAMP. 3′-Amino-cAMP does not antagonize folic acid or pterin. The binding of folic acid to post-vegetative Dictyostelium discoideum cells is inhibited by low concentrations of 2-deamino-2-hydro folic acid (DAFA [7]). DAFA is neither chemotactically active, nor does it inhibit a chemotactic reaction to folic acid. This questions the involvement of the main folic acid cell surface-binding sites in the chemotactic response to folic acid. The pterin analog, 6-aminopterin, is an antagonist of pterin, but not of cAMP or folic acid. Our results show that cAMP, folic acid and pterin are detected by different receptors. Furthermore, they suggest that the antagonistic action of 3′-amino-cAMP and 6-aminopterin is localized in the signal transduction pathway at a step before the signals from the separate receptors have arrived at a single pathway.