β2-Integrins - Regulatory and Executive Bridges in the Signaling Network Controlling Leukocyte Trafficking and Migration

Abstract

Leukocyte trafficking is an essential process of immunity, occurring as leukocytes travel within the bloodstream and as leukocyte migration within tissues. While it is now established that leukocytes can utilize the mesenchymal migration mode or amoeboid migration mode, differences in the migratory behavior of leukocyte subclasses and how these are realized on a molecular level in each subclass is not fully understood. To outline these differences, first migration modes and their dependence on parameters of the extracellular environments will be explained, as well as the intracellular molecular machinery that powers migration in general. Extracellular parameters are detected by adhesion receptors such as integrins. β2-integrins are surface receptors exclusively expressed on leukocytes and are essential for leukocytes exiting the bloodstream, as well as in mesenchymal migration modes, however, integrins are dispensable for the amoeboid migration mode. Additionally, the balance of different RhoGTPases - which are downstream of surface receptor signaling, including integrins - mediate formation of membrane structures as well as actin dynamics. Individual leukocyte subpopulations have been shown to express distinct RhoGTPase profiles along with their differences in migration behavior, which will be outlined. Emerging aspects of leukocyte migration include signal transduction from integrins via actin to the nucleus that regulates DNA status, gene expression profiles and ultimately leukocyte migratory phenotypes, as well as altered leukocyte migration in tumors, which will be touched upon.

Keywords: LFA-1; RhoGTPases; actin tread milling; amoeboid migration; epigenetics; leukocyte migration; β2-integrins.

Figure 1

The cell adhesion cascade. During adhesion to and extravasation through the endothelium, leukocytes undergo several steps, namely fast rolling and tethering, slow rolling and arrest, spreading and crawling and lastly transmigration. These steps are mediated by several different adhesion receptors expressed by leukocytes, indicated by underscore, and the endothelium (2). P-selectins and E-selectins expressed by the endothelium bind to their carbohydrate ligands on leukocytes during fast rolling. Leukocytes express L-selectins, which bind to ligands on the endothelium during fast rolling. The most well known selectin ligand is P-selectin glycoprotein ligand-1 (PSGL-1). Selectin-ligand binding results in integrin activation in leukocytes, which then mediates slow rolling, arrest, spreading and crawling. During transmigration, several homophilic adhesion receptors, including VE-cadherins important for tight junctions between endothelial cells, and CD99 and PECAM-1 are expressed by endothelial cells and leukocytes. Additionally, leukocytes express α6β1 integrins that bind to laminin.

Figure 2

Leukocyte integrins. Leukocyte integrins arranged around their β chains. α and β chains marked with * are specific for leukocytes (27), while platelets also express β2-integrins (28).

Figure 3

Integrin conformation. In leukocytes traveling through the bloodstream, in absence of ligands, 90% of integrins are in the bent inactive conformation, depicted on the left. Inside-out signaling as a result of chemokine receptor/ligand and selectin/ligand binding during fast rolling results in talin and kindlin-3 binding to β chains which results in unbending and changing to the active conformation (right). The unbending results in ligand binding sites being revealed.

Figure 4

Mesenchymal and amoeboid migration modes. The mesenchymal migration mode, depicted on top, is mediated by strong focal adhesions including integrin–ligand bonds to the substrate. Actin polymerization at the cell’s leading edge and actin retrograde flow powers this migration mode, which requires the counterforce of the adhesion sites. In more detail (77): (left) Actin is polymerized at the leading edge while it is depolymerized at the rear. In this depiction adhesome/clutch proteins have not bound integrin cytoplasmic tails and are instead distributed in the cytoplasm. This means the integrin-actin clutch is disengaged. (middle) Actin polymerization at the front necessitates actin monomers being moved to the front. The growing actin polymer at the front pushes against the elastic fibers at the cell membrane and generates an actin retrograde flow relative to actin’s position in the cell. If the integrin-actin clutch is not engaged this flow is fast. Integrin–ligand bonds are reinforced by adhesome protein binding, which results in focal adhesions, along with the metaphorical clutch, being formed. The clutch is still disengaged if the clutch does not bind actin. (right) The adhesome proteins within the clutch mediate binding to polymerized actin moving towards the rear. This results in slowing of the retrograde actin flow, traction force generation and ultimately protrusion. At the cell rear, focal adhesions are broken and proteins, including integrins recycled. During amoeboid migration (bottom), leukocytes exert weak adhesive forces on the extracellular matrix. This mode is powered by myosin II sliding along actin filaments at the cell’s rear, which translates into contraction and squeezing forces that push the cytoplasm and cell body forwards. Leukocytes find their paths through complex 3D environments and navigating these involves rapid cell shape changes within minutes and shorter, as depicted on the right. The reference pictures were taken during a timelapse 3D dendritic cell migration experiment through a collagen matrix and are of a single dendritic cell, with most cell shapes not being depicted.

Figure 5

Membrane structures in migrating leukocytes. Actively migrating leukocytes on 2D surfaces form distinct membrane structures. Depicted is the lamellipodia, the actin-rich leading cell edge, the lamella behind it (containing myosin II), as well as the uropod or the cell rear of migrating leukocytes, which are membrane structures that need to be formed during polarization to achieve directional migration. Pseudopodial protrusions are thick, arm-like membrane protrusions in the direction of migration that mediate material uptake. Podosomes are small membrane protrusions which are associated with ECM degradation using proteases. Finally, filopodia are thin finger-like membrane protrusions used to probe the surrounding environment. During amoeboid migration in 3D, leukocytes form small membrane blebs to enact low adhesive forces on the ECM.

Figure 6

Simplified RhoGTPase signaling to their downstream effectors. RhoA signals to mDia2 and, via ROCK, can inhibit actin depolymerization via LIMK/cofilin. Cdc42 can activate Rac proteins, which activates PAK proteins that phosphorylate LIMK family proteins, which phosphorylate and thereby deactivate cofilin. Deactivated phosphorylated cofilin is reactivated by dephosphorylation mediated by slingshot proteins such as SSHL1 (not shown). Cdc42 also initiates actin polymerization via mDia2 and actin network formation (typically in lamellipodia/lamella) via WASp, which activates Arp2/3. Arp2/3 is also activated by the WAVE complex of which hem1 is an associated protein. WAVE is activated by Rac proteins such as Rac1 and Rac2. Rac proteins also activate mDia2 as well as the PAK/LIMK/cofilin axis.