Evidence for lymphocyte chemotaxis toward monocytes during PHA-induced aggregation in vitro

An important, early phenomenon during the development of immune cell interactions in vitro is the formation of multicellular aggregates. We have developed a quantitative assay to determine the kinetics of multicellular aggregate formation within a heterotypic population of cells on a flat surface. This assay follows the time rate of change in the value of an aggregation index for cells in undisturbed culture. For an initial, well-separated population of cells, the index is a minimum and remains at this value if the cells do not move and interact. By contrast, for conditions that promote active cell movement followed by interaction, the index value increases with time. The index, which reflects cells’ relative spatial distributions, is an “indirect enumeration” of the number of cells within aggregates as a function of time. We used this index to follow the aggregative behavior of a population of freshly isolated human peripheral lymphocytes and monocytes. Previous studies have shown that monocytes are centrally located within aggregates and that lymphocytes move to surround monocytes. In order to test if lymphocyte movements are random or directed prior to interactions with monocytes, we formulated a simple model to describe changes in the expected number of cells in an “idealized aggregate” as a function of time. A comparison of the model curves with curves generated from the changes in the aggregation index shows that the best fit derives from a model that involves directed movement of lymphocytes toward monocytes. These results suggest that monocytes produce a chemoattracting agent for lymphocytes for these experimental conditions.     

Monocyte requirement for mitogen-induced aggregation of human peripheral mononuclear leukocytes in vitro

Formation of distinct multicellular aggregates is one of the phenomena associated with activation of quiescent human mononuclear leukocytes in vitro. Aggregate formation involves active cell motility and enhances cell-cell interactions required for an optimal proliferative response of T-cells stimulated with agents like phytohemagglutinin. We have developed an assay to quantitate the rate at which motile cells form aggregates on a flat surface. This assay follows the time rate of deviation of cells in undisturbed culture away from an initial random distribution using an "aggregation index." We used this assay to establish minimal culturing conditions required to observe an aggregation response for a partially purified mononuclear leukocyte population. We also studied the ability to aggregate of various subpopulations enriched for T- and B-lymphocytes and monocytes and found evidence for a monocyte requirement for lymphocyte aggregation. In a second assay, we followed the rate of entry of esterase positive monocytes into aggregates and compared this to the rate of entry of mononuclear cells in toto. We found that monocytes are preferentially associated with non-esterase positive cells within one hour of PHA stimulation. The results support the conclusion that monocytes play a central role in directing the motility of human T-lymphocytes leading to their aggregation response in tissue culture.     

Antigen-specific T lymphocytes efficiently cluster with dendritic cells in the human primary mixed-leukocyte reaction

Experimental conditions have been developed to detect the efficient interaction of antigen-presenting cells and antigen-specific CD4+ T lymphocytes early in the human primary mixed-leukocyte reaction (MLR). When monocytes are depleted from the stimulator population, it is evident that small numbers of allogeneic dendritic cells form multicellular aggregates with responsive T cells. B cells and monocytes in allogeneic stimulator populations do not appear to form aggregates in the first 2 days of the MLR. Upon return to culture, most of the lymphocytes that have clustered with dendritic cells become IL-2 responsive, proliferating lymphoblasts. The nonclustered cells exhibit little growth, while mixtures of clusters and nonclusters proliferate comparably to clusters alone. Cluster-derived lymphocytes respond rapidly to rechallenge with foreign leukocytes from the original donor but are greater than 90% depleted of reactivity to other "third party" donors. Nonclustered lymphocytes, in contrast, are greater than 90% depleted in specific reactivity but respond normally to third party. Therefore antigen-specific (alloreactive) resting CD4+ lymphocytes efficiently and selectively aggregate with dendritic cells. Dendritic-T-cell aggregates represent a stable microenvironment in which the MLR begins and might be useful in the experimental analysis of early events in the sensitization phase of cell-mediated immunity in man.     

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.     

Locomotion of white blood cells: a biophysical analysis

We determined some biophysical properties of human granulocytes, monocytes, and lymphocytes in respect to their locomotion. Granulocytes were exposed to plasma and were allowed to crawl on uncoated or glycol methacrylate coated glass plates. Monocytes did not migrate on uncoated glass, but did so on glycol methacrylated glass. Lymphocytes did not move on glass or glycol methacrylated glass, but moved on plexiglas coverslips. Granulocytes and monocytes showed a pronounced, directed movement towards a lysed erythrocyte (necrotaxis), lymphocytes showed no necrotactic response. The information collected by the granulocytes and monocytes in the necrotactic gradient was between 1 and 2 bits. This small amount of information indicated that the cellular decision in favor of a new direction of migration is based on a mechanism involving instability. We showed that the necrotactic response of granulocytes and monocytes is the product of the chemokinetic activity and the polar order parameter (= McCutcheon index) indicating that the cellular decision for a new direction of migration is independent of the speed of the cell movement. The movement of monocytes can be characterized in a similar way to that of granulocytes: the angle of deviation from a straight line path is nearly a fixed value (+/- 35 degrees). Lymphocytes stay in a restricted area after straight line movement. Particular attention was focused on cellular properties involved in locomotion. The characteristic time of the internal clock controlling the locomotion was 0.9 minutes for granulocytes and 2 minutes for monocytes. We were not able to determine the characteristic time of lymphocytes. We were able to determine the internal program responsible for the change in direction of movement. The directional memory time for granulocytes was 0.9 minutes. Monocytes had two directional memory times, short (2 minutes) and long (greater than 18 minutes). Lymphocytes had a very short directional memory time of 40 seconds. The distribution of the track velocities of migrating granulocytes and monocytes was described by bell shaped curves indicating homogeneous populations of cells. The distribution for lymphocytes had two maxima.     

Evolution of living organisms as a multilevel process

There is inherent capacity to increase the degree of aggregation within each of the levels of structural organization of living matter. At the macromolecular level (MML), this is an increase in the gene number in the genomes of evolving organisms; at the cellular level (CL), an increase in cell size; and at the multicellular level (MCL), an increase in the number of cells in the multicellular aggregate. However, the increase in the degree of aggregation causes gene incompatibility in case of genome evolution and instability in case of large cells and multicellular aggregates with simple structure. Gene incompatibility may be neutralized by spacio-temporal disconnection of the products of incompatible genes at the cellular and multicellular levels. The larger cells and multicellular aggregates are stabilized by increased structural complexity which is a consequence of the origin of new genes. There is a feedback between the processes of evolution at different levels MML→CL→ MCL.The processes of evolutionary development at different levels of structural organization are also relatively independent. The coincidence of these processes gives rise to stable organisms of higher complexity, which are then subjected to natural selection and population processes to establish a new step in progressive biological evolution. In all of the normal organisms of newly evolved species there is a correspondence between the different levels of structural organization, i.e. in their degree of aggregation, their complexity and functional organization. The form of correspondence for multicellular organisms is presented.     

Plasma membrane lipid packing and leukocyte function-associated antigen-1-dependent aggregation of lymphocytes

A simple model system for study of adhesion mediated by leukocyte function-associated antigen-1 (LFA-1) is aggregation of lymphocytes stimulated in vitro. Although aggregation is blocked by monoclonal antibodies to LFA-1, not all lymphocytes expressing LFA-1 aggregate, indicating that LFA-1 is necessary but not sufficient for aggregation. To investigate whether the lipid bilayer plays a role in the functional activation of LFA-1, human peripheral blood lymphocytes and murine splenic lymphocytes were stimulated in culture, and measurements made of aggregation vs. packing of plasma membrane lipids. Progression of cells into aggregates was paralleled by a decrease in lipid packing of the population as a whole, as monitored by increased staining with the fluorescent probe merocyanine 540. Cells from aggregates stained more intensely than nonaggregated cells from the same population, indicating that aggregates are preferentially formed from cells in the population with the loosest packed membrane. In contrast, aggregated cells were found to express equivalent or even lower amounts of LFA-1 than nonaggregated cells. Looser lipid packing is therefore associated with the development of LFA-1-dependent aggregation, and might be involved in the functional activation of this cell adhesion molecule. © 1993 Wiley-Liss, Inc.     

Short-Range Guiding Can Result in the Formation of Circular Aggregates in Myxobacteria Populations

Myxobacteria are social bacteria that upon starvation form multicellular fruiting bodies whose shape in different species can range from simple mounds to elaborate tree-like structures. The formation of fruiting bodies is a result of collective cell movement on a solid surface. In the course of development, groups of flexible rod-shaped cells form streams and move in circular or spiral patterns to form aggregation centers that can become sites of fruiting body formation. The mechanisms of such cell movement patterns are not well understood. It has been suggested that myxobacterial development depends on short-range contact-mediated interactions between individual cells, i.e. cell aggregation does not require long-range signaling in the population. In this study, by means of a computational mass-spring model, we investigate what types of short-range interactions between cells can result in the formation of streams and circular aggregates during myxobacterial development. We consider short-range head-to-tail guiding between individual cells, whereby movement direction of the head of one cell is affected by the nearby presence of the tail of another cell. We demonstrate that stable streams and circular aggregates can arise only when the trailing cell, in addition to being steered by the tail of the leading cell, is able to speed up to catch up with it. It is suggested that necessary head-to-tail interactions between cells can arise from physical adhesion, response to a diffusible substance or slime extruded by cells, or pulling by motility engine pili. Finally, we consider a case of long-range guiding between cells and show that circular aggregates are able to form without cells increasing speed. These findings present a possibility to discriminate between short-range and long-range guiding mechanisms in myxobacteria by experimentally measuring distribution of cell speeds in circular aggregates.     

Flow cytometric analysis and modeling of cell-cell adhesive interactions: the neutrophil as a model

The immune function of granulocytes, monocytes, lymphocytes, and other specialized cells depends upon intercellular adhesion. In many cases the molecules mediating leukocyte cell adhesion belong to the Leu-CAM superfamily of adhesive molecules. To elucidate the events of homotypic aggregation in a quantitative fashion, we have examined the aggregation of neutrophils stimulated with formyl peptides, where aggregate formation is a transient reversible cell function. We have mathematically modeled the kinetics of aggregation using a linear model based on particle geometry and rates of aggregate formation and breakup. The time course was modeled as a three-phase process, each phase with distinct rate constants. Aggregate formation was measured on the flow cytometer; singlets and larger particles were distinguished using the intravital stain LDS-751. Aggregation proceeded rapidly after stimulation with formyl peptide (CHO-nle-leu-phe-nle-tyr-lys). The first phase lasted 30-60 s; this was modeled with the largest aggregation rate and smallest rate of disaggregation. Aggregate formation plateaued during the second phase which lasted up to 2.5 min. This phase was modeled with an aggregation rate nearly an order of magnitude less than that of the initial fast phase, whereas the disaggregation rate for this phase did not change significantly. A third phase where disaggregation predominated, lasted the remaining 2-3 min and was modeled with a four to fivefold increase of the disaggregation rate. The mechanism of cell-cell adhesion in the plateau phase was probed with the monoclonal antibody IB4 to the CD18 subunit of the adhesive receptor CR3. Based on these studies it appears that new aggregates do not form to a large degree after the first phase of aggregate formation is complete. However, new adhesive contact sites may form within the contact region of these adherent cells to keep the aggregates together.     

Establishment of a peritubular myoid-like cell line and interactions between established testicular cell lines in culture

Established cell lines and primary cultures derived from somatic cells of the testis have been used to study cell-cell interactions. Primary cultures of Sertoli cells or Sertoli-derived cell lines from the mouse (TM4) and rat (TR-ST) will aggregate when plated on monolayers of primary cultures of peritubular myoid cells or a rat (TR-M) cell line which has many properties of peritubular myoid cells. Time-lapse cinematography and scanning and transmission electron microscopy reveal that Sertoli cells formed aggregates after 1 day in coculture, display surface activity and move on the monolayer. When these aggregates touch one another, they rapidly combine. By the 4th day of culture, spherical aggregates are composed of 50 to 200 cells. They do not display surface activity or movement on the myoid monolayer. On the 5th and 6th day of culture most spherical aggregates have flattened to form dome-shaped aggregates in close association with the monolayer. Cells in the aggregates are characterized by long microvilli and some ruffles. In large aggregates, cells sometimes form close associations within the aggregates although junctions are seldom observed. Sertoli-derived cell lines will not aggregate on monolayers of Leydig-derived (TM3) or testicular endothelial-derived (TR-1) cell lines. Neither TM3 nor TR-1 cells will aggregate when plated on myoid monolayers. The TR-M cells produced an extensive extracellular matrix beneath the cells which contains collagen, an amorphous globular material resembling elastin and a fibrous noncollagenous component. Sertoli cells plated on this matrix will not aggregate. Thus the aggregation of Sertoli cells on myoid cell monolayers is cell type, but not species dependent and not determined solely by extracellular matrix components produced by TR-M cells.     

Keratinization and the effect of vitamin A in aggregates of a squamous carcinoma cell line NBT II

The terminal differentiation, keratinization, of a rat bladder tumor cell line, NBT II, occurred in multicellular aggregates. After aggregation, these cells did not undergo a round of mitosis before keratinization. 5-Bromodeoxyuridine added to the monolayer cell culture 2 days before aggregation completely prevented this differentiation; it was ineffective when added at the time of cell aggregation. Vitamin A prevented the keratinization of NBT II cells in aggregates but did not inhibit aggregate formation; it enhanced the number of cells engaged in DNA synthesis. This model appears to be very useful for analyzing the mechanisms of terminal differentiation and its modulation by vitamin A in tumor cells.     

Establishment and maintenance of stable spatial patterns in lacZ fusion transformants of Polysphondylium pallidum.

Polysphondylium pallidum cells were transformed with a construct containing the Dictyostelium discoideum ecmA promoter fused to a lacZ reporter gene. Two stably transformed lines, one in which beta-galactosidase (beta-gal) is expressed in apical cells of the fruiting body (p63/2.1), and one in which it is expressed in basal cells (p63/D), have enabled us to infer how cells move during aggregation and culmination. Several types of cell movement proposed to occur during slime mold culmination, such as random cell mixing and global cell circulation, can be ruled out on the basis of our observations. Cells of the two transformant lines express beta-gal very early in development. In both cases, stained cells are randomly scattered in a starving population. By mid to late aggregation, characteristic spatial patterns emerge. Marked cells of p63/2.1 are found predominantly at tips of tight aggregates; those of p63/D accumulate at the periphery. These patterns are conserved throughout culmination, showing that marked cells maintain their relative positions within the multicellular mass following aggregation. Neither the apical nor the basal pattern appears to be regulated within the primary sorogen by de novo gene expression or by cell sorting as whorls are formed. However, marked cells within a whorl re-establish the original pattern in secondary sorogens. This must be achieved by cell migration, since beta-gal is not re-expressed.     

Role of streams in myxobacteria aggregate formation

Cell contact, movement and directionality are important factors in biological development (morphogenesis), and myxobacteria are a model system for studying cell-cell interaction and cell organization preceding differentiation. When starved, thousands of myxobacteria cells align, stream and form aggregates which later develop into round, non-motile spores. Canonically, cell aggregation has been attributed to attractive chemotaxis, a long range interaction, but there is growing evidence that myxobacteria organization depends on contact-mediated cell-cell communication. We present a discrete stochastic model based on contact-mediated signaling that suggests an explanation for the initialization of early aggregates, aggregation dynamics and final aggregate distribution. Our model qualitatively reproduces the unique structures of myxobacteria aggregates and detailed stages which occur during myxobacteria aggregation: first, aggregates initialize in random positions and cells join aggregates by random walk; second, cells redistribute by moving within transient streams connecting aggregates. Streams play a critical role in final aggregate size distribution by redistributing cells among fewer, larger aggregates. The mechanism by which streams redistribute cells depends on aggregate sizes and is enhanced by noise. Our model predicts that with increased internal noise, more streams would form and streams would last longer. Simulation results suggest a series of new experiments.     

Keratinization and the effect of vitamin A in aggregates of a squamous carcinoma cell line NBT II

Summary The terminal differentiation, keratinization, of a rat bladder tumor cell line, NBT II, occurred in multicellular aggregates. After aggregation, these cells did not undergo a round of mitosis before keratinization. 5-Bromodeoxyuridine added to the monolayer cell culture 2 days before aggregation completely prevented this differentiation; it was ineffective when added at the time of cell aggregation. Vitamin A prevented the keratinization of NBT II cells in aggregates but did not inhibit aggregate formation; it enhanced the number of cells engaged in DNA synthesis. This model appears to be very useful for analyzing the mechanisms of terminal differentiation and its modulation by vitamin A in tumor cells. This research was supported by Institutional Research Grant 731-01-E from the American Cancer Society and in part by Research Grant CA 14137 from the National Cancer Institute to Dr. J. Leighton.     

A population balance equation model of aggregation dynamics in Taxus suspension cell cultures

The nature of plant cells to grow as multicellular aggregates in suspension culture has profound effects on bioprocess performance. Recent advances in the measurement of plant cell aggregate size allow for routine process monitoring of this property. We have exploited this capability to develop a conceptual model to describe changes in the aggregate size distribution that are observed over the course of a Taxus cell suspension batch culture. We utilized the population balance equation framework to describe plant cell aggregates as a particulate system, accounting for the relevant phenomenological processes underlying aggregation, such as growth and breakage. We compared model predictions to experimental data to select appropriate kernel functions, and found that larger aggregates had a higher breakage rate, biomass was partitioned asymmetrically following a breakage event, and aggregates grew exponentially. Our model was then validated against several datasets with different initial aggregate size distributions and was able to quantitatively predict changes in total biomass and mean aggregate size, as well as actual size distributions. We proposed a breakage mechanism where a fraction of biomass was lost upon each breakage event, and demonstrated that even though smaller aggregates have been shown to produce more paclitaxel, an optimum breakage rate was predicted for maximum paclitaxel accumulation. We believe this is the first model to use a segregated, corpuscular approach to describe changes in the size distribution of plant cell aggregates, and represents an important first step in the design of rational strategies to control aggregation and optimize process performance.     

Aggregative cycles evolve as a solution to conflicts in social investment

Multicellular organization is particularly vulnerable to conflicts between different cell types when the body forms from initially isolated cells, as in aggregative multicellular microbes. Like other functions of the multicellular phase, coordinated collective movement can be undermined by conflicts between cells that spend energy in fuelling motion and ‘cheaters’ that get carried along. The evolutionary stability of collective behaviours against such conflicts is typically addressed in populations that undergo extrinsically imposed phases of aggregation and dispersal. Here, via a shift in perspective, we propose that aggregative multicellular cycles may have emerged as a way to temporally compartmentalize social conflicts. Through an eco-evolutionary mathematical model that accounts for individual and collective strategies of resource acquisition, we address regimes where different motility types coexist. Particularly interesting is the oscillatory regime that, similarly to life cycles of aggregative multicellular organisms, alternates on the timescale of several cell generations phases of prevalent solitary living and starvation-triggered aggregation. Crucially, such self-organized oscillations emerge as a result of evolution of cell traits associated to conflict escalation within multicellular aggregates.     

Quantifying aggregation dynamics during Myxococcus xanthus development

Under starvation conditions, a swarm of Myxococcus xanthus cells will undergo development, a multicellular process culminating in the formation of many aggregates called fruiting bodies, each of which contains up to 100,000 spores. The mechanics of symmetry breaking and the self-organization of cells into fruiting bodies is an active area of research. Here we use microcinematography and automated image processing to quantify several transient features of developmental dynamics. An analysis of experimental data indicates that aggregation reaches its steady state in a highly nonmonotonic fashion. The number of aggregates rapidly peaks at a value 2- to 3-fold higher than the final value and then decreases before reaching a steady state. The time dependence of aggregate size is also nonmonotonic, but to a lesser extent: average aggregate size increases from the onset of aggregation to between 10 and 15 h and then gradually decreases thereafter. During this process, the distribution of aggregates transitions from a nearly random state early in development to a more ordered state later in development. A comparison of experimental results to a mathematical model based on the traffic jam hypothesis indicates that the model fails to reproduce these dynamic features of aggregation, even though it accurately describes its final outcome. The dynamic features of M. xanthus aggregation uncovered in this study impose severe constraints on its underlying mechanisms.     

Modeling cell apoptosis for simulating three-dimensional multicellular morphogenesis based on a reversible network reconnection framework

Morphogenesis in multicellular organisms is accompanied by apoptotic cell behaviors: cell shrinkage and cell disappearance. The mechanical effects of these behaviors are spatiotemporally regulated within multicellular dynamics to achieve proper tissue sizes and shapes in three-dimensional (3D) space. To analyze 3D multicellular dynamics, 3D vertex models have been suggested, in which a reversible network reconnection (RNR) model has successfully expressed 3D cell rearrangements during large deformations. To analyze the effects of apoptotic cell behaviors on 3D multicellular morphogenesis, we modeled cell apoptosis based on the RNR model framework. Cell shrinkage was modeled by the potential energy as a function of individual cell times during the apoptotic phase. Cell disappearance was modeled by merging neighboring polyhedrons at their boundary surface according to the topological rules of the RNR model. To establish that the apoptotic cell behaviors could be expressed as modeled, we simulated morphogenesis driven by cell apoptosis in two types of tissue topology: 3D monolayer cell sheet and 3D compacted cell aggregate. In both types of tissue topology, the numerical simulations successfully illustrated that cell aggregates gradually shrank because of successive cell apoptosis. During tissue shrinkage, the number of cells in aggregates decreased while maintaining individual cell size and shape. Moreover, in case of localizing apoptotic cells within a part of the 3D monolayer cell aggregate, the cell apoptosis caused the global tissue bending by pulling on surrounding cells. In case of localizing apoptotic cells on the surface of the 3D compacted cell aggregate, the cell apoptosis caused successive, directional cell rearrangements from the inside to the surface. Thus, the proposed model successfully provided a basis for expressing apoptotic cell behaviors during 3D multicellular morphogenesis based on an RNR model framework.     

Exploiting differential effects of actomyosin contractility to control cell sorting among breast cancer cells

In order to gain a greater understanding of the factors that drive spatial organization in multicellular aggregates of cancer cells, we investigate the segregation patterns of 6 breast cell lines of varying degree of mesenchymal character during formation of mixed aggregates. Cell sorting is considered in the context of available adhesion proteins and cellular contractility. It is found that the primary compaction mediator (cadherins or integrins) for a given cell type in isolation plays an important role in compaction speed, which in turn is the major factor dictating preference for interior or exterior position within mixed aggregates. In particular, cadherin-deficient, invasion-competent cells tend to position towards the outside of aggregates, facilitating access to extracellular matrix. Reducing actomyosin contractility is found to have a differential effect on spheroid formation depending on compaction mechanism. Inhibition of contractility has a significant stabilizing effect on cell-cell adhesions in integrin-driven aggregation and a mildly destabilizing effect in cadherin-based aggregation. This differential response is exploited to statically control aggregate organization and dynamically rearrange cells in pre-formed aggregates. Sequestration of invasive cells in the interior of spheroids provides a physical barrier that reduces invasion in three-dimensional culture, revealing a potential strategy for containment of invasive cell types.