The multiple faces of leukocyte interstitial migration

Abstract

Spatiotemporal control of leukocyte dynamics within tissues is critical for successful innate and adaptive immune responses. Homeostatic trafficking and coordinated infiltration into and within sites of inflammation and infection rely on signaling in response to extracellular cues that in turn controls a variety of intracellular protein networks regulating leukocyte motility, migration, chemotaxis, positioning, and cell-cell interaction. In contrast to mesenchymal cells, leukocytes migrate in an amoeboid fashion by rapid cycles of actin polymerization and actomyosin contraction, and their migration in tissues is generally referred to as low adhesive and nonproteolytic. The interplay of actin network expansion, contraction, and adhesion shapes the exact mode of amoeboid migration, and in this review, we explore how leukocyte subsets potentially harness the same basic biomechanical mechanisms in a cell-type-specific manner. Most of our detailed understanding of these processes derives from in vitro migration studies in three-dimensional gels and confined spaces that mimic geometrical aspects of physiological tissues. We summarize these in vitro results and then critically compare them to data from intravital imaging of leukocyte interstitial migration in mouse tissues. We outline the technical challenges of obtaining conclusive mechanistic results from intravital studies, discuss leukocyte migration strategies in vivo, and present examples of mode switching during physiological interstitial migration. These findings are also placed in the context of leukocyte migration defects in primary immunodeficiencies. This overview of both in vitro and in vivo studies highlights recent progress in understanding the molecular and biophysical mechanisms that shape robust leukocyte migration responses in physiologically complex and heterogeneous environments.

Fig. 1

(A) Environmental signals induce shape changes, motility patterns, and migration modes. In physiological environments, leukocytes perceive multiple signals and integrate them into intracellular signaling cascades to induce shape changes, cell polarization, or cell migration. In a homogeneous field of soluble ligands, leukocytes can (1) increase cytoskeletal activity, leading to morphological shape changes without cell movement or (2) induce self-polarization as prerequisite for nondirected cell migration (chemokinesis). (3) Soluble gradients of external ligands can polarize cells along the gradient and stimulate directed migration (chemotaxis). (4) Upon perceiving homogeneous surface-bound chemokines, leukocytes can increase cell adhesion, self-polarize, and confine their migration in a nondirected manner along the surface (haptokinesis). For inherently adhesive immune cell types, haptokinesis might not require the presence of an activating chemokine (not shown). Leukocytes can migrate by two forms of haptotaxis: (5) along a gradient of extracellular matrix and cellular adhesion sites and (6) along a gradient of substrate-bound chemoattractants. (B) The environmental geometry supports different leukocyte migration strategies. (1) Movement along 2D surfaces requires actin polymerization to push the leading edge membrane forward and surface anchorage to the substrate. Myosin II (red ellipses)-based contractions behind the leading edge detach very adhesive cells from the substrate and are not necessarily required when cells migrate on low adhesive substrates. (Insert below) Integrin-mediated adhesions not only confine migration to the surface, but also inhibit the retrograde actin flow (green), converting actin polymerization into forward protrusion at the leading edge. (2) In confined environments, the leukocyte body can exert lateral pushing forces on the substrate, which is not possible on 2D surfaces. Intracellular pressure gradients increase friction against the walls and potentially compensate for adhesion-dependent anchorage. Friction against the walls can be generated by actin polymerization or increase in hydrostatic pressure by actomyosin contractions. (Insert below) Model of leading edge actin flow during adhesive migration in microchannels (adapted from [20]): Two F-actin networks form at the leading edge and interact with each other. The “free” network (dark green) polymerizes from the membrane at the cell front. The “adherent” network (light green) polymerizes perpendicular to the walls and compresses the retrograde flow of the “free” network, converting new polymerization against the membrane and enabling forward protrusions (see text for molecular details). (3) In 3D porous environments, leukocytes can potentially exert the same lateral forces as in confined spaces, but require additional mechanisms to navigate through meshes, which are not necessary when moving in the open space of a microchannel. Depending on the leukocyte subtype, multiple frontal protrusions require coordinated cell shape changes for efficient movement. (4, 5) In physiological tissues, leukocytes navigate in a very heterogeneous environment with confined spaces, fibrillar 3D meshes, and 2D surfaces in close proximity, most likely adapting to the tissue geometry by constant switching of migration modes. Fibrillar (4) and cell-rich (5) tissues differ in their structure, composition, and texture, potentially favoring distinct migration strategies.

Fig. 2. Leukocyte subtypes cover a broad spectrum of morphological amoeboid phenotypes

The force-relationship between the three migration modules—actin polymerization (“P”), contraction (“C”) and cell adhesion (“A”)—determines the shape of leukocytes and the exact mode of migration. This overview of selected leukocyte subtypes presents their presumptive force balances (displayed as force triangle) based on cell morphologies in suspension (upper row), 2D adhesive surfaces (middle row), and 3D porous scaffolds (lower row). Tissue macrophages are illustrated in an adhesive 3D porous network (e.g., fibronectin) (top) and nonadhesive 3D porous network (below). Assuming slight differences in the balanced force interplay between leukocyte subsets, pharmacological or genetic interference with actin flow, contraction or cell adhesion might affect leukocyte interstitial migration in a cell-type-specific manner.

Fig. 3. Nonadhesive neutrophil migration in the dermal interstitium and mode switching to adherent crawling at wound centers

(A) Subcutaneously injected talin-deficient (red) and control neutrophils (green) were imaged side-by-side in the same tissue volume to eliminate problematic quantitative comparisons based on measurements of cells imaged in different experiments and animals. This experimental design minimizes the influence of tissue heterogeneity between experiments and ensures the same imaging conditions and nutrient availability for both cell types. Collagen fibers are visualized by second harmonic generation (SHG) signals (white), cell tracks are indicated in turquoise. Neutrophils perform chemotaxis toward sites of sterile tissue damage and migrate in a nonadhesive mode through the dermal interstitium. (B) When neutrophils congregate at sites of local wounding, the developing cell aggregates remodel the interstitial fiber architecture and form a collagen-free wound center. At the transition zone between 3D fibrillar interstitium and the cell-rich, collagen-free wound zone, neutrophils switch to adhesion-dependent crawling. Unlike control cells (red), talin-deficient neutrophils (green) accumulate exactly at the transition zone. Scale bars = 20 µm.

Fig. 4. Optimized analysis of interstitial leukocyte migration in physiological settings

To dissect cell-intrinsic aspects of leukocyte motility from external guidance, simultaneous imaging of cell dynamics and tissue structures is recommended. (A) 2P-IVM was applied to murine ear skin to visualize the dermis of a transgenic DsRed^+/+ CD11c-YFP^+/− B6.Albino mouse. Ubiquitously expressed DsRed illuminates all stromal elements (red), YFP-signals highlight endogenous dendritic cells/macrophages (yellow), and SHG signals reflect collagen bundles (white). The mouse was crossed to a B6.Albino background to avoid cell death of light-sensitive melanophages, avoiding laser light-induced artifactual effect in the tissue. Scale bar = 40 µm. (B) The movement of a wild-type dendritic cell, presumably not activated, is followed over 14 min in relation to tissue structures. Simultaneous visualization of cells, stroma, and collagen gives a closer representation of the 3D tissue geometry and reveals channel-like structures and dense networks and surfaces. Still, several other tissue determinants that might influence leukocyte behavior are unknown, e.g., the potential presence of cytokines and chemokines that can induce cell shape changes and upregulation of cell adhesion. Scale bar = 10 µm. CD11c-YFP mice were a kind gift of Michel Nussenzweig.

Fig. 5. Correlation of neutrophil migration paths and velocities with connective tissue structure

(A) 2P-IVM was applied to murine ear skin to visualize the dermis of a transgenic DsRed^+/− Lyz2-GFP^+/− B6.Albino mouse. Ubiquitously expressed DsRed illuminates all stromal elements (red), GFP-bright cells represent endogenous neutrophils (green), and SHG signals reflect collagen bundles. Neutrophil motility was recorded simultaneously with stromal elements over 30 min. Scale bar = 50 µm. (B) Cell tracking analysis in relation to tissue structures. (Left) Tracks over the entire imaging session were plotted for all cells in the tissue volume and related to the vascular network; 15 % of migrating neutrophils crawled along vessels (yellow tracks), whereas the remaining cells migrated in the interstitial spaces between vessels (blue tracks). (Middle) Dragontails indicate the migration paths covered by individual cells in the last 5 min. The colors of the dragontails indicate instantaneous velocities (red: fast, green: slow). This particular short time frame suggests that neutrophils have high instantaneous velocities preferentially when migrating close to vessels. (Right) In contrast to the 5-min interval analysis, plotting the velocity distributions over the entire imaging session of 30 min (purple: fast, dark blue: slow) revealed that most neutrophils migrated fastest in interstitial spaces with a loose local collagen network. (C) Depending on the graphic representation, neutrophils close to vessels appear to migrate along the vascular surface (middle left) or along a 2D surface of collagen matrix (middle right), which might actually be a channel-like confined space (outer right). Scale bar = 50 µm. Lyz2-GFP mice were a kind gift of Thomas Graf.