Neutrophil adhesion and chemotaxis depend on substrate mechanics

Abstract

Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma, and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micro-machined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the K(D) of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against beta(2)-integrins leads to a significant reduction but not an elimination of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.

Figure 1. Fabrication of a microfluidic system to study neutrophil responses to gradients on hydrogel substrates

A schematic of the photolithography process used to prepare microfluidic devices that generate gradients over the surface of polyacrylamide hydrogels as described in Materials and Methods.

Figure 2. Schematic representation of an adherent neutrophil on a hydrogel within a microfluidic gradient chamber

A single cell is shown interacting with a ligand-coated surface oriented in the direction of increasing chemical gradient. Ligands are immobilized on the surface of the hydrogel via covalent bonds as described in Materials and Methods. The direction of fluid flow as well as chemical, and mechanical parameters within the system that can be varied independently of each other are also illustrated.

Figure 3. Neutrophil adhesion and spreading on hydrogels depends on substrate mechanical properties

(a) Neutrophils were activated with 10 nM fMLP and observed during adhesion to hydrogel substrates. The area of individual cells attached to a hydrogel during a 15 minute time interval was averaged and plotted against hydrogel shear modulus to show the influence of substrate mechanics on cell spreading. (b,c) Phase contrast images of neutrophils adhering to hydrogel surfaces in uniform solutions of 10 nM fMLP on soft (300 Pa) and stiff (12 kPa) gels as a function of time. (d) Cell area of neutrophils activated with uniform solutions of 10 nM fMLP on hydrogels at the indicated stiffnesses were measured and plotted as a function of time. (e) The time at which cell spreading area for a given hydrogel stiffness had decreased by 50%.

Figure 4. Neutrophil Trajectories During Chemokinesis or Chemotaxis on Hydrogels with Varying Mechanical Properties

Representative 2D Wind-Rose plots showing individual cell trajectories for a range of gel stiffnesses in both uniform (a) and gradient (b) solutions of fMLP. After cells are imaged using time-lapse microscopy, centroids are computed and individual cell tracks are generated. Trajectories were obtained by tracking cell centroids for a total time of 15 minutes per condition. Lines begin at the zero-centered initial position of each cell and end at the final position to display net cell dispersion. For each graph with a gradient, the spatial gradient of fMLP increases from top to bottom.

Figure 5. Relationship between cell speed and directionality as a function of hydrogel stiffness

Migration parameters for neutrophils obtained on hydrogels functionalized with ICAM-1/E-Selectin in the presence of uniform or gradient solutions of fMLP. (a) Migration speed, (b) chemotactic index in a uniform concentration of 10 nM fMLP. (c) Migration speed, (d) chemotactic index in a gradient of 0-1125nM fMLP or 10nM/cell diameter (10nM/10μm).

Figure 6. Traction Stress Maps of Neutrophils Migrating on Hydrogels of Varying Stiffness in Response to a Gradient of fMLP

Neutrophil traction stress maps and corresponding phase contrast images during migration on stiff hydrogels (12,000 Pa) in response to a gradient of fMLP (10 nM/cell diameter). Arrow pointing downwards indicates the direction of the fMLP gradient from low to high concentration within the microfluidic chamber. Color map indicates the magnitude of stress in different regions of the cell. Horizontal arrow indicates the magnitude of the force vectors drawn within each cell.

Figure 7. Chemotaxis in linear fMLP gradients with varying mean concentrations as a function of hydrogel stiffness

Neutrophil chemotaxis in linear fMLP gradients with mean concentrations ranging between 7-49nM and constant slope was measured on hydrogels with shear moduli of 300 Pa, 2000 Pa, 7000 Pa, 12000 Pa, and 19000 Pa. For each line, shear modulus increases from left to right.

Figure 8. Chemotactic index values for neutrophils on hydrogels of varying stiffness normalized to observed cell radius

To account for differences in spatial gradient sensing that would result from changes in cell spreading, chemotactic index values were replotted after normalization to average cell radii observed on different gel stiffnesses. The cell radii was used as the characteristic length for normalization because spatial sensing occurs across a cell’s diameter.

Figure 9. Effect of β 2 integrin blocking antibody

Neutrophils were incubated with 10 μg/mL TS1/18 antibody and allowed to attach and migrate on either soft (300 Pa) or stiff (12,000 Pa) hydrogels in the presence of a 0-1125 nM gradient (10nM/μm). (a) Cell spreading, (b) migration, (c) chemotactic index were measured for individual cells tracked for a total of 15 minutes.