Mechanisms of frustrated phagocytic spreading of human neutrophils on antibody-coated surfaces

Abstract

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.

Integrative experimental/computational approach establishes active cellular protrusion as the primary driving force of phagocytic spreading by immune cells

“…Reflecting the above-mentioned contrasting mechanistic notions about the driving force of generic cell spreading, two hypotheses have been put forward to explain phagocytic spreading, i.e., the “Brownian zipper” and “protrusive zipper” models [ 23 ]. Both models concur that fresh contact between the cell membrane and target surface results in a zipper-like adhesive attachment that is essentially irreversible, in agreement with experimental observations [ 16 , 24 , 25 ]. However, regarding the driving force of cellular advancement, the Brownian zipper model postulates that strong adhesion alone is responsible for pulling the cell membrane onto the target surface.…”

Integrative experimental/computational approach establishes active cellular protrusion as the primary driving force of phagocytic spreading by immune cells

“…Error bars in both (D) and (E) indicate standard deviation. The IgG density was quantified for each condition as described in [19].…”

“…The computational framework presented in the previous section can be adopted to simulate interactions by various cell types with targets of different axisymmetric geometries. Our present focus on human neutrophils spreading on flat substrates is motivated by a wealth of experimental results obtained for this particular scenario and presented in a companion paper [19]. In brief, we exposed cells to glass coverslips coated with IgG antibodies at densities ( ρ IgG ) spanning ~3 orders of magnitude.…”

“…These experimental results provide useful quantitative benchmarks for comparison with computer simulations and will be used in the following sections to evaluate the validity of different mechanistic notions about cell spreading. The IgG density was quantified for each condition as described in [24].…”

“…We seek to establish which of these hypotheses is more realistic by developing mathematical models of the mechanics of isotropic cell spreading and comparing our theoretical predictions with experimental observations of human neutrophils spreading on flat, IgG-coated surfaces. As reported in detail in a companion paper, our experimental design allowed us to examine the spreading behavior of neutrophils on surfaces coated with different densities of IgG [19]. Because higher IgG densities correspond to stronger adhesive forces, we reasoned that the Brownian zipper model should predict a much stronger correlation between IgG density and rate of contact-area growth (“spreading speed”) than the protrusive zipper model.…”