Leukocyte extravasation

During chemotaxis, cell movement is facilitated by the binding of β1 integrins to components of the extracellular matrix: VLA-3, VLA-4 and VLA-5 to fibronectin and VLA-2 and VLA-3 to collagen and other extracellular matrix components.

Cytokines

Extravasation is regulated by the background cytokine environment produced by the

• IL-1 activates resident lymphocytes
  and vascular endothelia
• TNFα
  increases vascular permeability and activates vascular endothelia
• CXCL8 (IL-8) forms a chemotactic gradient that directs leukocytes towards site of tissue injury/infection (CCL2
  has a similar function to CXCL8, inducing monocyte extravasation and development into macrophages); also activates leukocyte integrins

In 1976, SEM images showed that there were homing receptors on microvilli-like tips on leukocytes that would allow white blood cells to get out of the blood vessel and get into tissue.[7] Since the 1990s the identity of ligands involved in leukocyte extravasation have been studied heavily. This topic was finally able to be studied thoroughly under physiological shear stress conditions using a typical flow chamber.^[8] Since the first experiments, a strange phenomenon was observed. Binding interactions between the white blood cells and the vessel walls were observed to become stronger under higher force. Selectins (E-selectin, L-selectin, and P-selectin) were found to be involved in this phenomenon. The shear threshold requirement seems counterintuitive because increasing shear elevates the force applied to adhesive bonds and it would seem that this should increase the dislodging ability. Nevertheless, cells roll more slowly and more regularly until an optimal shear is reached where rolling velocity is minimal. This paradoxical phenomenon has not been satisfactorily explained despite the widespread interest.

One initially dismissed hypothesis that has been gaining interest is the catch bond hypothesis, where the increased force on the cell slows off-rates and lengthen the bond lifetimes and stabilizing the rolling step of leukocyte extravasation.^[9] Flow-enhanced cell adhesion is still an unexplained phenomenon that could result from a transport-dependent increase in on-rates or a force-dependent decrease in off-rates of adhesive bonds. L-selectin requires a particular minimum of shear to sustain leukocyte rolling on P-selectin glycoprotein ligand-1 (PSGL-1) and other vascular ligands. It has been hypothesized that low forces decrease L-selectin–PSGL-1 off-rates (catch bonds), whereas higher forces increase off-rates (slip bonds). Experiments have found that a force-dependent decrease in off-rates dictated flow-enhanced rolling of L-selectin–bearing microspheres or neutrophils on PSGL-1. ^[5] Catch bonds enable increasing force to convert short bond lifetimes into long bond lifetimes, which decrease rolling velocities and increase the regularity of rolling steps as shear rose from the threshold to an optimal value. As shear increases, transitions to slip bonds shorten their bond lifetimes and increase rolling velocities and decrease rolling regularity. It is hypothesized that force-dependent alterations of bond lifetimes govern L-selectin–dependent cell adhesion below and above the shear optimum. These findings establish a biological function for catch bonds as a mechanism for flow-enhanced cell adhesion.^[10] While leukocytes seem to undergo a catch bond behavior with increasing flow leading to the tethering and rolling steps in leukocyte extravasation, firm adhesion is achieved through another mechanism, integrin activation.

Other biological examples of a catch bond mechanism is seen in bacteria that tightly cling to urinary tract walls in response to high fluid velocities and large shear forces exerted on the cells and bacteria with adhesive tips of fimbria.^[9][11] Schematic mechanisms of how increased shear force is proposed to cause stronger binding interactions between bacteria and target cells show that the catch bond acts very similar to a Chinese finger trap. For a catch-bond, the force on the cell pulls the adhesive tip of a fimbria to close tighter on its target cell. As the strength of the forces increases, the stronger the bond between the fimbria and the cell-receptor on the surface of the target cell.^[11] For a cryptic-bond, the force causes the fimbria to swivel toward the target cell and have more binding sites able to attach to the target cell ligands, mainly sugar molecules. This creates a stronger bonding interaction between the bacteria and the target cell.

Advent of microfluidic devices

Parallel plate flow chambers are among the most popular flow chambers used to study the leukocyte-endothelial interaction in vitro. They have been used for investigation since the later 1980s.^[12] Although flow chambers have been an important tool to study leukocyte rolling, there are several limitations when it comes to studying the physiological in vivo conditions, as they lack correspondence with in vivo geometry, including scale/aspect ratio (microvasculature vs large vessel models), flow conditions (e.g. converging vs diverging flows at bifurcations), and require large reagent volumes (~ ml) due to their large size (height > 250 µm and width > 1mm).^[13] With the advent of microfluidic-based devices, these limitations have been overcome. A new in vitro model, called SynVivo Synthetic microvascular network (SMN) was produced by the CFD Research Corporation (CFDRC) and developed using the polydimethylsiloxane (PDMS) based soft-lithography process. The SMN can recreate the complex in vivo vasculature, including geometrical features, flow conditions, and reagent volumes, thereby providing a biologically realistic environment for studying the extravasation cellular behavior, but also for drug delivery and drug discovery. ^[14][15]

Leukocyte adhesion deficiency

Leukocyte adhesion deficiency (LAD) is a genetic disease associated with a defect in the leukocyte extravasation process, caused by a defective integrin β2 chain (found in LFA-1 and Mac-1). This impairs the ability of the leukocytes to stop and undergo diapedesis. People with LAD suffer from recurrent bacterial infections and impaired wound healing. Neutrophilia is a hallmark of LAD.

Neutrophil dysfunction

In widespread diseases such as sepsis, leukocyte extravasation enters an uncontrolled stage, where white blood neutrophils begin destroying host tissues at unprecedented rates, claiming the lives of about 200,000 people in the United States alone.^[16] Neutrophil dysfunction is usually preceded by an infection of some sort, which triggers pathogen-associated molecular patterns (PAMP). As leukocyte extravasation intensifies, more tissues are damaged by neutrophils, which release oxygen radicals and proteases.^[16]

Recent studies with SynVivo Synthetic microvascular network (SMN) made it possible to study anti-inflammatory therapeutics to treat pathologies caused by neutrophil dysfunction. The SMN enables the thorough analysis of each stage of leukocyte extravasation, thereby providing a methodology to quantify the effect of the drug in impeding leukocyte extravasation. Some of the recent findings demonstrate the effect of hydrodynamics on neutrophil-endothelial interactions. In other words, adhesion of neutrophils is heavily impacted by shear forces as well as molecular interactions. Moreover, as shear rate decreases (e.g., in post-capillary venules), immobilization of the leukocytes becomes easier and thus, more prevalent. The opposite is also true; vessels in which shear forces are high render the immobilization of the leukocytes more difficult. This has high implications in various diseases, where disruptions in blood flow gravely impact immune system response by impeding or expediting the immobilization of the leukocytes. Having this knowledge allows for better studies of the effect of drugs on leukocyte extravasation.^[13][16][14]

Footnotes