Complex motions of immune cells are an integral part of diapedesis, chemotaxis, phagocytosis, and other vital processes. To better understand how immune cells execute such motions, we present a detailed analysis of phagocytic spreading of human neutrophils on flat surfaces functionalized with different densities of immunoglobulin G (IgG) antibodies. We visualize the cell-substrate contact region at high resolution and without labels using reflection interference contrast microscopy (RICM) and quantify how the area, shape, and position of the contact region evolves over time. We find that the likelihood of the cell commitment to spreading strongly depends on the surface density of IgG, but the rate at which the substrate-contact area of spreading cells increases does not. Validated by a theoretical companion study, our results resolve controversial notions about the mechanisms controlling cell spreading, establishing that active forces generated by the cytoskeleton rather than cell-substrate adhesion primarily drive cellular protrusion. Adhesion, on the other hand, aids phagocytic spreading by regulating the cell commitment to spreading, the maximum cell-substrate contact area, and the directional movement of the contact region.
The ability of motile immune cells to detect and follow gradients of chemoattractant is critical to numerous vital functions, including their recruitment to sites of infection and—in emerging immunotherapeutic applications—to malignant tumors. Facilitated by a multitude of chemotactic receptors, the cells navigate a maze of stimuli to home in on their target. Distinct chemotactic processes direct this navigation at particular times and cell-target distances. The expedient coordination of this spatiotemporal hierarchy of chemotactic stages is the central element of a key paradigm of immunotaxis. Understanding this hierarchy is an enormous interdisciplinary challenge that requires, among others, quantitative insight into the shape, range, and dynamics of the profiles of chemoattractants around their sources. We here present a closed-form solution to a diffusion–reaction problem that describes the evolution of the concentration gradient of chemoattractant under various conditions. Our ready-to-use mathematical prescription captures many biological situations reasonably well and can be explored with standard graphing software, making it a valuable resource for every researcher studying chemotaxis. We here apply this mathematical model to characterize the chemoattractant cloud of anaphylatoxins that forms around bacterial and fungal pathogens in the presence of host serum. We analyze the spatial reach, rate of formation, and rate of dispersal of this locator cloud under realistic physiological conditions. Our analysis predicts that simply being small is an effective protective strategy of pathogens against complement-mediated discovery by host immune cells over moderate-to-large distances. Leveraging our predictions against single-cell, pure-chemotaxis experiments that use human immune cells as biosensors, we are able to explain the limited distance over which the cells recognize microbes. We conclude that complement-mediated chemotaxis is a universal, but short-range, homing mechanism by which chemotaxing immune cells can implement a last-minute course correction toward pathogenic microbes. Thus, the integration of theory and experiments provides a sound mechanistic explanation of the primary role of complement-mediated chemotaxis within the hierarchy of immunotaxis, and why other chemotactic processes are required for the successful recruitment of immune cells over large distances.
The efficient recruitment of immune cells is a vital cornerstone of our defense against infections and a key challenge of immunotherapeutic applications. It relies on the ability of chemotaxing cells to prioritize their responses to different stimuli. For example, immune cells are known to abandon gradients of host-cell-produced cytokines in favor of complement-derived anaphylatoxins, which then guide the cells toward nearby pathogen surfaces. The aptitude to triage stimuli depends on the cells' specific sensitivities to different chemoattractants. We here use human neutrophils as uniquely capable biodetectors to map out the anaphylatoxic cloud that surrounds microbes in the presence of host serum. We quantify the neutrophil sensitivity in terms of the ratio between the chemoattractant concentration c and the production rate j 0 of the chemoattractant at the source surface. An integrative experimental/theoretical approach allows us to estimate the c/j 0 -threshold at which human neutrophils first detect nearby b-glucan surfaces as c/j 0 z 0.0044 s/mm.How does an immune cell cope with situations in which it faces multiple chemotactic stimuli? How does the cell decide on a particular response? Such questions touch on the core of our mechanistic understanding of immune-cell behavior, and have inspired the paradigm that immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes (1-6). The systematic dissection of this hierarchy is an enormous interdisciplinary challenge that requires, among others, quantitative analyses of the stimulus-specific sensitivity of the responding cells.Complement-mediated chemotaxis has emerged as a universal, short-range homing mechanism by which chemotaxing immune cells can implement a last-minute course correction toward pathogenic bacteria and fungi. Recent single-cell experiments have validated human neutrophils as uniquely capable biodetectors of minuscule amounts of complement-derived anaphylatoxins in the proximity of microbial and model pathogens (Fig. 1) (6-8). But the question just how sensitive these immune cells are was not addressed by earlier studies.We here use an integrative theoretical/experimental strategy to tackle this difficult question. A recently found closed-form solution of the appropriate reaction-diffusion problem (V.H., E.A.F., and W.D. Simpson, unpublished data) predicts the spatiotemporal distribution of anaphylatoxins as a function of the time t and the radial distance from the source, Dr ¼ r À R (Fig. 1 B; Supporting Material). In the considered scenario, the source of chemoattractant is a sphere of radius R that, at time t ¼ 0, starts releasing anaphylatoxins at a constant rate given by the boundary flux j 0 . The chemoattractant is redistributed in the surrounding infinite space by diffusion. A realistic estimate gives a diffusion coefficient of D z 130 mm 2 /s for the dominant anaphylatoxin C5a (V.H., E.A.F., and W.D. Simpson, unpublished data). We further model the deactivation of chemoattractant by carboxype...
Global bursts in free intracellular calcium (Ca) are among the most conspicuous signaling events in immune cells. To test the common view that Ca bursts mediate rearrangement of the actin cytoskeleton in response to the activation of G protein-coupled receptors, we combined single-cell manipulation with fluorescence imaging and monitored the Ca concentration in individual human neutrophils during complement-mediated chemotaxis. By decoupling purely chemotactic pseudopod formation from cell-substrate adhesion, we showed that physiological concentrations of anaphylatoxins, such as C5a, induced nonadherent human neutrophils to form chemotactic pseudopods but did not elicit Ca bursts. By contrast, pathological or supraphysiological concentrations of C5a often triggered Ca bursts, but pseudopod protrusion stalled or reversed in such cases, effectively halting chemotaxis, similar to sepsis-associated neutrophil paralysis. The maximum increase in cell surface area during pseudopod extension in pure chemotaxis was much smaller-by a factor of 8-than the known capacity of adherent human neutrophils to expand their surface. Because the measured rise in cortical tension was not sufficient to account for this difference, we attribute the limited deformability to a reduced ability of the cytoskeleton to generate protrusive force in the absence of cell adhesion. Thus, we hypothesize that Ca bursts in neutrophils control a mechanistic switch between two distinct modes of cytoskeletal organization and dynamics. A key element of this switch appears to be the expedient coordination of adhesion-dependent lock or release events of cytoskeletal membrane anchors.
The dynamic interplay between cell adhesion and protrusion is a critical determinant of many forms of cell motility. When modeling cell spreading on adhesive surfaces, traditional mathematical treatments often consider passive cell adhesion as the primary, if not exclusive, mechanistic driving force of this cellular motion. To better assess the contribution of active cytoskeletal protrusion to immune-cell spreading during phagocytosis, we here develop a computational framework that allows us to optionally investigate both purely adhesive spreading (“Brownian zipper hypothesis”) as well as protrusion-dominated spreading (“protrusive zipper hypothesis”). We model the cell as an axisymmetric body of highly viscous fluid surrounded by a cortex with uniform surface tension and incorporate as potential driving forces of cell spreading an attractive stress due to receptor-ligand binding and an outward normal stress representing cytoskeletal protrusion, both acting on the cell boundary. We leverage various model predictions against the results of a directly related experimental companion study of human neutrophil phagocytic spreading on substrates coated with different densities of antibodies. We find that the concept of adhesion-driven spreading is incompatible with experimental results such as the independence of the cell-spreading speed on the density of immobilized antibodies. In contrast, the protrusive zipper model agrees well with experimental findings and, when adapted to simulate cell spreading on discrete adhesion sites, it also reproduces the observed positive correlation between antibody density and maximum cell-substrate contact area. Together, our integrative experimental/computational approach shows that phagocytic spreading is driven by cellular protrusion, and that the extent of spreading is limited by the density of adhesion sites.
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