Quantification of random motility and chemotaxis bacterial transport coefficients using individual-cell and population-scale assays

A number of individual-cell and population-scale assays have been introduced to quantify bacterial motility and chemotaxis. The transport coefficients reported in the literature, however, span several orders of magnitude, making it difficult to ascertain to what degree variations in bacterial species/strain, growth medium, growth and experimental conditions, and experiment type contribute to the reported differences in coefficient values. We quantified the random motility of Escherichia coli AW405 using the capillary assay, stopped-flow diffusion chamber (SFDC), and tracking microscope. We obtained good agreement for the random motility coefficient between these assays when using the same bacterial strain and consistent growth and experimental conditions. Chemotaxis of E. coli toward the attractant alpha-methylaspartate was quantified using the SFDC and capillary assay. Good agreement for the chemotactic sensitivity coefficient between the SFDC and the capillary assay was obtained across a limited attractant concentration range. Three different mathematical models were considered for analyzing capillary assay data to obtain a chemotactic sensitivity coefficient. These models differed by their treatment of the bacterial concentration in the chamber and the attractant concentration at the mouth. Results from our study indicate that the capillary assay, the most commonly used bacterial random motility and chemotaxis assay, can be used to accurately quantify bacterial transport coefficients over a limited range of attractant concentrations, provided experiments are performed carefully and appropriate mathematical models are used to interpret the experimental data.     

Measurement of bacterial random motility and chemotaxis coefficients: I. Stopped-flow diffusion chamber assay

Bacterial chemotaxis, the directed movement of a cell population in response to a chemical gradient, plays a critical role in the distribution and dynamic interaction of bacterial populations in nonmixed systems. Therefore, in order to make reliable predictions about the migratory behavior of bacteria within the environment, a quantitative characterization of the chemotactic response in terms of intrinsic cell properties is needed.The design of the stopped-flow diffusion chamber (SFDC) provides a well-characterized chemical gradient and reliable method for measuring bacterial migration behavior. During flow through the chamber, a step change in chemical concentration is imposed on a uniform suspension of bacteria. Once flow is stopped, diffusion causes a transient chemical gradient to develop, and bacteria respond by forming a band of high cell density which travels toward higher concentrations of the attractant. Changes in bacterial spatial distributions observed through light scattering are recorded on photomicrographs during a 10-min period. Computer-aided image analysis converts absorbance of the photographic negatives to a digital representation of bacterial density profiles. A mathematical model (part II) is used to quantitatively characterize these observations in terms of intrinsic cell parameters: a chemotactic sensitivity coefficient, mu(0), from the aggregate cell density accumulated in the band and a random motility coefficient, mu, from population dispersion in the absence of a chemical gradient.Using the SFDC assay and an individual-cell-based mathematical model, we successfully determined values for both of these population parameters for Escherichia coli K12 responding to fucose. The values obtained were mu = 1.1 +/- 0. 4 x 10(-5) cm(2)/s and chi(o) = 8 +/- 3 +/- 10(-5) cm(2)/s. We have demonstrated a method capable of determining these parameter values from the now validated mathematical model which will be useful for predicting bacterial migration in application systems.     

Stopped-flow chamber and image analysis system for quantitative characterization of bacterial population migration: Motility and chemotaxis ofEscherichia coli K12 to fucose

The directed movement of a bacterial population in response to a chemical gradient is known as bacterial chemotaxis and plays a critical role in the distribution and dynamic interaction of bacterial populations. A quantitative characterization of the chemotactic response in terms of intrinsic cell properties is necessary for making reliable predictions about the migratory behavior of bacterial populations within the environment. The design of the stopped-flow diffusion chamber (SFDC) provides a well-characterized chemical gradient and reliable method for measuring bacterial migration behavior. During flow through the chamber a step change in the chemical concentration is imposed on a uniform suspension of bacteria. Once flow is stopped a transient chemical gradient forms due to diffusion; bacteria respond by forming a band of high cell density that travels toward higher concentrations of the attractant. Sequential observations of bacterial spatial distributions over a period of about ten minutes are recorded on photomicrographs. Computer-aided image analysis of the photographic negatives converts light-scattering information to a digital representation of the bacterial density profiles. A mathematical model is used to quantitatively characterize these observations in terms of intrinsic cell parameters: a chemotactic sensitivity coefficient, χ₀, from the aggregate cell density accumulated in the band and a random motility coefficient, μ₀, from population dispersion in the absence of a chemical gradient. Using the SFDC assay and an individual cell-based mathematical model we successfully determined values for both of these population parameters forEscherichia coli K12 responding to fucose. The values we obtained were μ₀=1.1 ± 0.4 x 10^-5 cm²/sec and χ₀=8 ± 3 x 10^-5 cm²/sec. These parameters will be useful for predicting population behavior in application systems such as biofilm development, population dynamics of genetically-engineered bacteria released into the environment, and in situ bioremediation technologies.     

Analysis of chemotactic bacterial distributions in population migration assays using a mathematical model applicable to steep or shallow attractant gradients

The mathematical model developed by Riveroet al. (1989,Chem. Engng Sci. 44, 2881–2897) is applied to literature data measuring chemotactic bacterial population distributions in response to steep as well as shallow attractant gradients. This model is based on a fundamental picture of the sensing and response mechanisms of individual bacterial cells, and thus relates individual cell properties such as swimming speed and tumbling frequency to population parameters such as the random motility coefficient and the chemotactic sensitivity coefficient. Numerical solution of the model equations generates predicted bacterial density and attractant concentration profiles for any given experimental assay. We have previously validated the mathematical model from experimental work involving a step-change in the attractant gradient (Fordet al., 1991Biotechnol. Bioengng.37, 647–660; For and Lauffenburger, 1991,Biotechnol. Bioengng,37, 661–672). Within the context of this experimental assay, effects of attractant diffusion and consumption, random motility, and chemotactic sensitivity on the shape of the profiles are explored to enhance our understanding of this complex phenomenon. We have applied this model to various other types of gradients with successful intepretation of data reported by Dalquistet al. (1972,Nature New Biol. 236, 120–123) forSalmonella typhimurum validating the mathematical model and supportin the involvement of high and low affinity receptors for serine chemotaxis by these cells.     

A simple expression for quantifying bacterial chemotaxis using capillary assay data: application to the analysis of enhanced chemotactic responses from growth-limited cultures.

An individual cell-based mathematical model of Rivero et al. provides a framework for determining values of the chemotactic sensitivity coefficient chi 0, an intrinsic cell population parameter that characterizes the chemotactic response of bacterial populations. This coefficient can theoretically relate the swimming behavior of individual cells to the resulting migration of a bacterial population. When this model is applied to the commonly used capillary assay, an approximate solution can be obtained for a particular range of chemotactic strengths yielding a very simple analytical expression for estimating the value of chi 0, [formula: see text] from measurements of cell accumulation in the capillary, N, when attractant uptake is negligible. A0 and A infinity are the dimensionless attractant concentrations initially present at the mouth of the capillary and far into the capillary, respectively, which are scaled by Kd, the effective dissociation constant for receptor-attractant binding. D is the attractant diffusivity, and mu is the cell random motility coefficient. NRM is the cell accumulation in the capillary in the absence of an attractant gradient, from which mu can be determined independently as mu = (pi/4t)(NRM/pi r2bc)2, with r the capillary tube radius and bc the bacterial density initially in the chamber. When attractant uptake is significant, a slightly more involved procedure requiring a simple numerical integration becomes necessary. As an example, we apply this approach to quantitatively characterize, in terms of the chemotactic sensitivity coefficient chi 0, data from Terracciano indicating enhanced chemotactic responses of Escherichia coli to galactose when cultured under growth-limiting galactose levels in a chemostat.     

Measurement of the chemotaxis coefficient for human neutrophils in the under-agarose migration assay

Clinical and scientific investigations of leukocyte chemotaxis will be greatly aided by an ability to measure quantitative parameters characterizing the intrinsic random motility, chemokinetic, and chemotactic properties of cell populations responding to a given attractant. Quantities typically used at present, such as leading front distances, migrating cell numbers, etc., are unsatisfactory in this regard because their values are affected by many aspects of the assay system unrelated to cell behavioral properties. In this paper we demonstrate the measurement of cell migration parameters that do, in fact, characterize the intrinsic cell chemosensory movement responses using cell density profiles obtained in the linear under-agarose assay. These parameters are the random motility coefficient, mu, and the chemotaxis coefficient, chi, which appear in a theoretical expression for cell population migration. We propose a priori the dependence of chi on attractant concentration, based on an independent experimental correlation of individual cell orientation bias in an attractant gradient with a spatial difference in receptor occupancy. Our under-agarose population migration results are consistent with this proposition, allowing chemotaxis to be reliably characterized by a chemotactic sensitivity constant, chi 0, to which chi is directly proportional. Further, chi 0 has fundamental significance; it represents the reciprocal of the difference in number of bound receptors across cell dimensions required for directional orientation bias. In particular, for the system of human peripheral blood polymorphonuclear neutrophil leukocytes responding to FNLLP, we find that the chemotaxis coefficient is a function of attractant concentration, a following the expression: chi = chi 0NT0 f(a) S(a) Kd/(Kd + a)2 where Kd is the FNLLP-receptor equilibrium dissociation constant and NT0 is the total number of cell surface receptors for FNLLP. f(a) is the fraction of surface receptors remaining after down-regulation, and S(a) is the cell movement speed, both known functions of FNLLP concentration. We find that chi 0NT0 = 0.2 cm; according to a theoretical argument outlined in the Appendix this means that these cells exhibit 75% orientation toward higher attractant concentration when the absolute spatial difference in bound receptors is 0.0025NT0 over 10 micron. (For example, if NT0 = 50,000 this would correspond to a spatial difference of 125 bound receptors over 10 micron.) This result can be compared with estimates obtained from visual studies of individual neutrophils.     

Quantitative relationships between single-cell and cell-population model parameters for chemosensory migration responses of alveolar macrophages to C5a

Phenomenological parameters from a mathematical model of cell motility are used to quantitatively characterize chemosensory migration responses of rat alveolar macrophages migrating to C5a in the linear under-agarose assay, simultaneously at the levels of both single cells and cell populations. This model provides theoretical relationships between single-cell and cell-population motility parameters. Our experiments offer a critical test of these theoretical linking relationships, by comparison of results obtained at the cell population level to results obtained at the single-cell level. Random motility of a cell population is characterized by the random motility coefficient, mu (analogous to a particle diffusion coefficient), whereas single-cell random motility is described by cell speed, s, and persistence time, P (related to the period of time that a cell moves in one direction before changing direction). Population chemotaxis is quantified by the chemotactic sensitivity, chi 0, which provides a measure of the minimum attractant gradient necessary to elicit a specified chemotactic response. Single-cell chemotaxis is characterized by the chemotactic index, CI, which ranges from 0 for purely random motility to 1 for perfectly directed motility. Measurements of cell number versus migration distance were analyzed in conjunction with the phenomenological model to determine the population parameters while paths of individual cells in the same experiment were analyzed in order to determine the single-cell parameters. The parameter mu shows a biphasic dependence on C5a concentration with a maximum of 1.9 x 10(-8) cm2/sec at 10(-11) M C5a and relative minima of 0.86 x 10(-8) cm2/sec at 10(-7) M C5a and 1.1 x 10(-8) cm2/sec in the absence of Ca; s and P remain fairly constant with C5a concentration, with s ranging from 2.1 to 2.5 microns/min and P varying from 22 to 32 min. chi 0 is equal to 1.0 x 10(-6) cm/receptor for all C5a concentrations tested, corresponding to 60% correct orientation for a difference of 500 bound C5a receptors across a 20 microns cell length. The maximum CI measured was 0.2. Values for the population parameters mu and chi 0 were calculated from single-cell parameter values using the aforementioned theoretical linking relationships. The values of mu and chi 0 calculated from single-cell parameters agreed with values of mu and chi 0 determined independently from population migrations, over the full range of C5a concentrations, confirming the validity of the linking equations. Experimental confirmation of such relationships between single-cell and cell-population parameters has not previously been reported.     

Determination of Effective Transport Coefficients for Bacterial Migration in Sand Columns

A well-characterized experimental system was designed to evaluate the effect of porous media on macroscopic transport coefficients which are used to characterize the migration of bacterial populations. Bacterial density profiles of Pseudomonas putida PRS2000 were determined in the presence and absence of a chemical attractant (3-chlorobenzoate) gradient within sand columns having a narrow distribution of particle diameters. These experimental profiles were compared with theoretical predictions to evaluate the macroscopic transport coefficients. The effective random motility coefficient, used to quantify migration due to a random process in a porous medium, decreased nearly 20-fold as grain size in the columns decreased from 800 to 80 (mu)m. The effective random motility coefficient (mu)(infeff) was related to the random motility coefficient (mu), measured in a bulk aqueous system, according to (mu)(infeff) = ((epsilon)/(tau))(mu) with porosity (epsilon) and tortuosity (tau). Over the times and distances examined in these experiments, bacterial density profiles were unaffected by the presence of an attractant gradient. Theoretical profiles with the aqueous phase value of the chemotactic sensitivity coefficient (used to quantify migration due to a directed process) were consistent with this result and suggested that any chemotactic effect on bacterial migration was below the detection limits of our assay.     

Measurement of leukocyte motility and chemotaxis parameters with a linear under-agarose migration assay

The interpretation of quantitative assays for leukocyte chemotactic migration is usually made in terms of measurements such as leading front distance, total migrating cells, and leukotactic index. These quantities allow comparison of cellular migration behavior under specified conditions. They are not useful; however, for comparisons between systems or for correlation with in vivo performance, because they depend upon specific physical aspects of the assay system, such as the geometry, chemoattractant concentration and diffusivity, and observation time. It would be more helpful to measure intrinsic properties of cell movement that could be used for comparison between systems, for correlation with in vivo studies, and to increase our understanding of the cell physiology. In this paper we demonstrate a means of quantitating leukocyte random motility, chemokinesis, and chemotaxis in terms of parameters that do characterize intrinsic cell properties. These parameters are the random motility coefficient and the chemotaxis coefficient, which appear in theoretical models of cell migration. We examine how well such a model describes the leukocyte density profile data observed in a modified under-agarose assay having a linear geometry. Furthermore, we obtain values for the random motility coefficient (and its dependence upon the concentration of the attractant peptide FNLLP) and for the chemotaxis coefficient for leukocytes responding to FNLLP.     

Mathematical model for characterization of bacterial migration through sand cores

The migration of chemotactic bacteria in liquid media has previously been characterized in terms of two fundamental transport coefficients-the random motility coefficient and the chemotactic sensitivity coefficient. For modeling migration in porous media, we have shown that these coefficients which appear in macroscopic balance equations can be replaced by effective values that reflect the impact of the porous media on the swimming behavior of individual bacteria. Explicit relationships between values of the coefficients in porous and liquid media were derived. This type of quantitative analysis of bacterial migration is necessary for predicting bacterial population distributions in subsurface environments for applications such as in situ bioremediation in which bacteria respond chemotactically to the pollutants that they degrade.We analyzed bacterial penetration times through sand columns from two different experimental studies reported in the literature within the context of our mathematical model to evaluate the effective transport coefficients. Our results indicated that the presence of the porous medium reduced the random motility of the bacterial population by a factor comparable to the theoretical prediction. We were unable to determine the effect of the porous medium on the chemotactic sensitivity coefficient because no chemotactic response was observed in the experimental studies. However, the mathematical model was instrumental in developing a plausible explanation for why no chemotactic response was observed. The chemical gradients may have been too shallow over most of the sand core to elicit a measurable response. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 487-496, 1997.     

Quantitative analysis of experiments on bacterial chemotaxis to naphthalene

A mathematical model was developed to quantify chemotaxis to naphthalene by Pseudomonas putida G7 (PpG7) and its influence on naphthalene degradation. The model was first used to estimate the three transport parameters (coefficients for naphthalene diffusion, random motility, and chemotactic sensitivity) by fitting it to experimental data on naphthalene removal from a discrete source in an aqueous system. The best-fit value of naphthalene diffusivity was close to the value estimated from molecular properties with the Wilke-Chang equation. Simulations applied to a non-chemotactic mutant strain only fit the experimental data well if random motility was negligible, suggesting that motility may be lost rapidly in the absence of substrate or that gravity may influence net random motion in a vertically oriented experimental system. For the chemotactic wild-type strain, random motility and gravity were predicted to have a negligible impact on naphthalene removal relative to the impact of chemotaxis. Based on simulations using the best-fit value of the chemotactic sensitivity coefficient, initial cell concentrations for a non-chemotactic strain would have to be several orders of magnitude higher than for a chemotactic strain to achieve similar rates of naphthalene removal under the experimental conditions we evaluated. The model was also applied to an experimental system representing an adaptation of the conventional capillary assay to evaluate chemotaxis in porous media. Our analysis suggests that it may be possible to quantify chemotaxis in porous media systems by simply adjusting the model's transport parameters to account for tortuosity, as has been suggested by others.     

Chemotactic transport theory for neutrophil leukocytes

The simplest admissible phenomenological transport theory for the chemotactic migration of a population of neutrophil leukocytes is formulated along the lines of the original Keller-Segel model for bacterial chemotaxis, but with appropriate specialization of the motility and chemotactic flux coefficient to reflect their dependence on the local cytotaxin (chemoattractant) concentration, as observed experimentally by Wilkinson and other workers. By supplementing deductions from the governing transport equation with inferences from measurements and then reasoning both forwards and backwards, the functional forms of the motility and chemotactic flux coefficient can be established for any prescribed cytotaxin. This analysis is performed here with numerical details for casein, a cytotaxin which gives rise to a motility function with an increasing-then-decreasing form of dependence on the concentration and a chemotactic flux coefficient that is essentially constant with variations in the concentration. Three dimensionless numbers are associated with the chemotactic response of neutrophil leukocytes to casein.     

Studies of bacterial chemotaxis in defined concentration gradients. A model for chemotaxis toward L-serine

The details of the chemotactic response of Salmonella typhimurium to gradients of L-serine have been examined in some detail. Two relatively macroscopic techniques have been employed to measure the bacterial response. These include measurements of the average velocity as the bacterial population moves toward attractants, and measurement of the upward-to-downward flux ratio, R, in the stable preformed attractant gradients. The dependence of the average velocity on gradient appears to be hyperbolic in nature, while the flux ratio depends linearly on the gradient. These data suggest a microscopic model for the dependence of bacterial behavior on the serine gradient. The model involves a linear dependence of the mean lifetime of a bacterial trajectory on the gradient for those bacteria moving toward higher attractant concentration. Those moving toward low concentrations of attractant do not change the mean duration of their trajectories, or the speed at which a given bacterium swims through the solution. This model generates the observed dependences of the average velocity and flux ratio on gradient. Interpretation of the experimental data suggests that a gradient which increases serine concentration by a factor of 2 in 10 mm is sufficient to double the average duration of a trajectory for a bacterium moving directly up the gradient. The concentration dependence of the chemotactic response to serine is more complicated. It suggests that more than one receptor of serine may be involved in determining chemotactic behavior to this attractant.     

Growth rate effects on fundamental transport properties of bacterial populations

In many natural environments, bacterial populations experience suboptimal growth due to the competition with other microorganisms for limited resources. The chemotactic response provides a mechanism by which bacterial populations can improve their situation by migrating toward more favorable growth conditions. For bacteria cultured under suboptimal growth conditions, evidence for an enhanced chemotactic response has been observed previously. In this article, for the first time, we have quantitatively characterized this behavior in terms of two macroscopic transport coefficients, the random motility and chemotactic sensitivity coefficients, measured in the stopped-flow diffusion chamber assay. Escherichia coli cultured over a range of growth rates in a chemostat exhibits a dramatic increase in the chemotactic sensitivity coefficient for D-fucose at low growth rates, while the random motility coefficient remains relatively constant by comparison. The change in the chemotactic sensitivity coefficient is accounted for by an independently measured increase in the number of galactose-binding proteins which mediate the chemotactic signal. This result is consistent with the relationship between macroscopic and microscopic parameters for chemotaxis, which was proposed in the mathematical model of Rivero and co-workers. (c) 1993 John Wiley & Sons, Inc.     

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.     

Effects of leukocyte random motility and chemotaxis in tissue inflammatory response.

A rapidly growing body of experimental evidence indicates that defects in leukocyte motility and chemotactic response correlate with increased susceptibility to and severity of bacterial infection in tissue. While this is understandable in qualitative terms, the sensitivity of the correlation is remarkable.In the present study, a theoretical analysis has been developed to relate the dynamics of bacterial growth to the growth and transport parameters of bacteria and leukocytes in tissue. The model considers a local tissue region in the vicinity of a venule and applies continuum unsteady state species conservation equations to the bacterial population, the phagocytic leukocytes, and a chemotactically active chemical mediator assumed to be produced by the bacteria. The analysis quantifies the effects of key parameters, such as leukocyte random motility and chemotactic coefficients, phagocytic and growth rate constants, and leukocyte vessel wall permeability, upon host ability to eliminate the bacteria.As an example, the model's predictions are compared to experimental results correlating inhibition of leukocyte chemotaxis by hemoglobin with its adjuvant action in experimental peritoneal infection by E. coli.     

Effect of bacterial chemotaxis on dynamics of microbial competition

Although the dynamic behavior of microbial populations in nonmixed systems is a central aspect of many problems in biochemical engineering and microbiology, the factors that govern this behavior are not well understood. In particular, the effects of bacterial chemotaxis (biased migration of cells in the direction of chemical concentration gradients) have been the subject of much speculation but very little quantitative investigation. In this paper, we provide the first theoretical analysis of the effects of bacterial chemotaxis on the dynamics of competition between two microbial populations for a single rate-limiting nutrient in a confined nonmixed system. We use a simple unstructured model for cell growth and death, and the most soundly based current model for cell population migration. Using numerical finite element techniques, we examine both transient and steady-state behavior of the competing populations, focusing primarily on the influence of the cell random motility coefficient,, and the cell chemotaxis coefficient, . We find that, in general, there are four possible steady-state outcomes: both populations die out, population 1 exists alone, population 2 exists alone, and the two populations coexist. We find that, in contrast to well-mixed systems, the slower-growing population can coexist and even exist alone if it possesses sufficiently superior motility and chemotaxis properties. Our results allow estimation of the value of necessary to allow coexistence and predominance for reasonable values of growth and random motility parameters in common systems. An especially intriguing finding is that there is a minimum value of necessary for a chemotactic population to have a competitive advantage over an immotile population in a confined nonmixed system. Further, for typical system parameter values, this minimum value of is the range of values that can be estimated from independent experimental assays for chemotaxis.Thus, in typical nonmixed systems, cell motility and chemotaxis properties can be the determining factors in governing population dynamics.     

“Pseudochemotaxis” by micro-organisms in an attractant gradient

The random behavior of microorganisms in defined attractant gradients may exhibit apparent chemotaxis, or pseudochemotaxis, if the random motility of the population is a function of the attractant concentration. Several examples are studied using computer techniques to solve the differential equations of motion for populations of micro-organisms responding chemokinetically to variable attractant concentrations. The chemokinetic coefficient is a convenient parameter to characterize the time course of experiments in which micro-organisms move in attractant gradients. Measurements of the chemokinetic coefficient as a function of time may be used to directly determine the random mobility as a function of attractant concentration.     

Regulation of human neutrophil chemotaxis by intracellular pH

The relationship of N-formyl-methionyl-leucyl-phenylalanine-stimulated Na+/H+ exchange to the chemotactic responsiveness of human neutrophils was investigated. The pHi changes, measured from the equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione, were correlated with the migratory behavior of the cells as assessed by the leading front method. Exposure of cells to 10 nM FMLP caused activation of Na+/H+ exchange, leading to a rise in pHi from approximately 7.25 to approximately 7.75. This intracellular alkalinization was inhibited by amiloride and by three more potent analogues. All four compounds reduced the chemotactic response to FMLP with apparent Ki values similar to those for inhibition of the pHi transients, thereby suggesting that the blocking effect of the drugs on directed cell migration was related to inhibition of Na+/H+ exchange. The effect was specific for stimulated cell locomotion: FMLP-induced chemotaxis and chemokinesis were inhibited in parallel, whereas random motility was unimpaired. The relationship of pHi to function was also studied as the pHi of FMLP-activated cells was varied between 6.8 and 8.6 by altering the chemical gradients for Na+ and H+ across the cell membrane. There was a direct, positive correlation between the pHi value attained following FMLP-stimulation and the locomotor response to a chemotactic gradient. These results indicate that the motile functions of human neutrophils can be regulated by their pHi.