What drives immune cells to engulf pathogens?

BPJ_111_12.c1.inddMacrophages and neutrophils (phagocytes) are the front-line defenders in your body’s immune system. They seek out, ingest, and destroy pathogens and other debris through a process called phagocytosis.  Typically, phagocytosis is initiated when receptors on the immune cell surface bind to ligands which have coated a pathogen particle. Once the cell’s receptors have found their target ligands, they initiate a chemical cascade within the cell which recruits the biochemical machinery necessary to drive the cell to envelop its target, forming a vacuole in which the pathogen can be degraded.

On a small scale, nearly every cell type in your body internalizes nutrients and various signals through a similar engulfment process called endocytosis What makes immune cells and phagocytosis unique is the relative size of the internalized particle. During endocytosis, cells internalize small objects, typically no larger than 100 nanometers, a fraction of the cell’s size (usually 10-30 micrometers). However, during phagocytosis, immune cells need to be able to internalize very large particles such as bacteria, which could be several microns long, and debris like dead cells, which could be larger than the immune cell itself.

Phagocytes accomplish this seemingly heroic feat by leveraging the biomechanical machinery typically involved with cellular migration, specifically the actin cytoskeleton and myosin molecular motors. Once phagocytosis has been initiated, actin monomers within the cell begin to polymerize near the location of the bound particle. As the polymerized network forms, it pushes the cell’s membrane around the particle, forming what is called a phagocytic cup. Interestingly, as a particle is internalized, the leading edge of the phagocytic cup constricts, pinching down on the particle.

Earlier studies have shown that actin tends to accumulate in a dense ring at the point of constriction. Unfortunately, due to limitations of the microscopy techniques available at the time, the precise structural organization of actin filaments within this ring could not be resolved. Consequently, exactly how the actin ring facilitates constriction remained elusive.

Using super-resolution fluorescent imaging (Structured Illumination Microscopy) we sought to illuminate how actin is reorganized during phagocytosis, with the goal of providing insight as to how phagocytes constrict around their targets.  One of the challenges in using microscopy to study phagocytosis is that particle internalization is three-dimensional, yet nearly all microscopy techniques are inherently two-dimensional. To side-step this issue, we turned to a planar technique called Frustrated Phagocytosis. Instead of presenting immune cells with pathogen particles, we deposited cells onto glass coverslips functionalized with antibodies. When the immune cells contact the surface, they perceive it as a giant pathogen and begin to flatten and spread as if trying to phagocytose the entire plane, yielding an unfolded view of what’s happening at the cell-target interface.

The cover image in the December 20th issue of the Biophysical Journal shows several macrophage cells at various stages of the frustrated phagocytic process. In the image, each cell’s actin cytoskeleton is shown in green (using Atto488-phalloidin) and the nuclei are shown in blue (using DAPI). During the early stages of phagocytosis (top left), actin polymerizes at the leading edge of the cell, forming a dense zone. This is similar to the structure formed at the leading edge of migrating cells. As actin polymerizes at the edge, it pushes the membrane outward causing the cell to spread. As the cell nears its maximum contact area, the actin zone begins to dissociate (top center) and actin-filaments throughout the body of the cell bundle to form fibers (middle right). As the cell enters the later stages of phagocytosis those bundles reorient, surrounding the perimeter of the cell (bottom center and middle left). With actin bundles surrounding the cell, myosin motor proteins exert tension between adjacent bundles. This tension causes the network to contract, forcing the cell to pinch down on the substrate. For frustrated phagocytosis, this constriction drives the cell to retract from the surface, leaving fragments of actin and tethered membrane in its trail (spinney protrusions around bottom center and middle left cells).

The mechanism that triggers the bundles to form and reorganize around the cell perimeter remains a mystery; although, there is mounting evidence that mechanical factors such as the cell’s membrane tension are involved in signaling transitions to late-stage phagocytic behavior.  These images, along with other studies of phagocyte mechanics, illustrate the robust and dynamical processes that unfold when immune cells carry out their essential task of clearing debris and eliminating pathogens.

– Wenbin Wei, Patrick Chang, Jan-Simon Toro, Ruth Fogg Beach, Dwight Chambers, Karen Porter, Doyeon Koo, Jennifer Curtis, Daniel Kovari

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