Neutrophil Cell Shape Change: Mechanism and Signalling during Cell Spreading and Phagocytosis

Abstract

Perhaps the most important feature of neutrophils is their ability to rapidly change shape. In the bloodstream, the neutrophils circulate as almost spherical cells, with the ability to deform in order to pass along narrower capillaries. Upon receiving the signal to extravasate, they are able to transform their morphology and flatten onto the endothelium surface. This transition, from a spherical to a flattened morphology, is the first key step which neutrophils undergo before moving out of the blood and into the extravascular tissue space. Once they have migrated through tissues towards sites of infection, neutrophils carry out their primary role-killing infecting microbes by performing phagocytosis and producing toxic reactive oxygen species within the microbe-containing phagosome. Phagocytosis involves the second key morphology change that neutrophils undergo, with the formation of pseudopodia which capture the microbe within an internal vesicle. Both the spherical to flattened stage and the phagocytic capture stage are rapid, each being completed within 100 s. Knowing how these rapid cell shape changes occur in neutrophils is thus fundamental to understanding neutrophil behaviour. This article will discuss advances in our current knowledge of this process, and also identify an important regulated molecular event which may represent an important target for anti-inflammatory therapy.

Keywords: Ca2+; calpain; cell spreading; cortical actin; ezrin; membrane expansion; membrane tension; neutrophils; phagocytosis.

Figure 1

The role of the cell surface reservoir of membrane. The surface reservoir (wrinkles) permits cell spreading onto a surface (lower sequence) or for phagocytosis (upper sequence) in three steps. The first step (disconnecting surface wrinkles from underlying cortical actin) is the result of the release of the ‘molecular Velcro’ initially holding the wrinkles in place. The second step is the unfolding of the wrinkles as the result of Brownian ratchet driven actin polymerisation initiated by the slackening of membrane tension. The last step results in the final cell configuration with the additional membrane employed to form the phagosomal.

Figure 2

The Brownian ratchet for actin polymerisation near the plasma membrane. From the cortical actin network, branch points are formed by insertion of WASP protein which allows an additional point for another actin polymer to grow towards the plasma membrane. This actin polymer continues to grow until it encounters the plasma membrane and the gap between the polymer tip and the plasma membrane is less that the diameter of a single actin monomer. However, if there is slack in the membrane, Brownian fluctuations in the position of the plasma membrane will occur (as the membrane moves back and forth randomly) and the gap may transiently be greater than the diameter of an actin monomer. In which case, the actin polymer will grow and ‘push’ the membrane. The Brownian ratchet will continue until the tension in the plasma membrane increases to a point when Brownian fluctuations are so lessened that no gap which is greater than the actin monomer size can form between the actin polymer tip and the plasma membrane.

Figure 3

The molecular anatomy of the neutrophil cell surface wrinkles. The relative locations of ezrin (within the wrinkles) and the cortical actin network are shown. As in Figure 3, the ezrin crosslinks polymerised actin to the plasma membrane and prevents the operation of the Brownian ratchet, which drives actin polymerisation to push out the plasma membrane. The wrinkle is consequently a stable structure on the cell surface, which is little affected by Brownian effects.

Figure 4

Ezrin crosslinking actin and the plasma membrane. The upper cartoon of an ezrin molecule shows the three main features—the phospholipid binding domain, the actin-binding domain and the linker region between the two. Beneath this is an illustration depicting the effect of ezrin crosslinking on the ability of actin to polymerise. With ezrin binding the cortical actin network to the plasma membrane, Brownian fluctuations in the plasma membrane are locally prevented and actin polymerisation cannot occur. In this way, ezrin binding stabilises cell surface structures such as villi and neutrophil wrinkles.

Figure 5

How ezrin cleavage initiates actin polymerisation. In (a), the position of the membrane in relation to the plasma membrane is stable and is also shown in Figure 2. In (b), the ezrin linkage is broken by the action of activated µ-calpain, which results in (c) the establishment of the Brownian ratchet and the growth of actin polymers ‘pushing out’ the plasma membrane. In (d), the actin polymer is sufficiently long enough for branch points to be added actin polymerisation continues to push out the plasma membrane. The arrows at the side of a, b, c and d show the distance from the plasma membrane to the cortical actin network increasing.

Figure 6

Intra-wrinkle Ca²⁺ reaches a concentration high enough to activate µ-calpain. Mathematical modelling [78] shows that within the folded region of the plasma membrane, Ca²⁺ concentrations reach very high levels, at least as high as that required for calpain activation. This is because the limited volume of the wrinkles has a relatively large surface area, so the effect of Ca²⁺ influx is exaggerated within the wrinkle as compared to the whole cell. Once local Ca²⁺ buffers within the cytosol of the wrinkles are saturated, the elevation of Ca²⁺ is limited only by the rate of diffusion of new mobile Ca²⁺ buffers into the wrinkle. The figure shows a pseudo-coloured representation of the Ca²⁺ concentration, together with examples of the expected Ca²⁺ transients within the wrinkle and within the cell.

Figure 7

The sequence of intracellular molecular events leading to ”membrane expansion”. In (a) the wrinkles are held in place (more detail in Figure 4). Following cleavage of ezin by activated µ-calpain (as shown in Figure 4), the tension in the membrane is relaxed and (c) shows that result on actin polymerisation, which can now proceed via the Brownian ratchet mechanism. In (d), actin polymerisation has progressed and branch points added such that the membrane made available by wrinkle detachment is pushed out. During phagocytosis, this would be coordinated to form a phagocytic cup, as a result of localised adhesion via integrin or antibody on the particle and during cell spreading coordinated to spread onto the contacting substrate.