leukocyte membrane "expansion": a central mechanism for leukocyte extravasation

8/8/2019 Leukocyte membrane "expansion": a central mechanism for leukocyte extravasation

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Leukocyte membrane expansion: a central mechanism for

leukocyte extravasation

Sharon Dewitt and Maurice Hallett1

Neutrophil Signalling Group, School of Medicine, Cardiff University, Cardiff, United Kingdom

Abstract: The infiltration of inflamed tissues by leukocytes is a key event in the development andprogression of inflammation. Although individualcytokines, which coordinate extravasation, havebecome the targets for therapy, a mechanism thatis common to white cell extravasation, regardlessof the specific molecular mechanism involved,would represent a more attractive therapeutic tar-get. Such a target may be represented by the eventsunderlying the spreading of leukocytes on the en-dothelium, which is a necessary prelude to extrav-

asation. This leukocyte spreading involves an ap-parent increase in the cell surface area. The aim ofthis review is to examine whether the mechanismunderlying the apparent expansion of plasma mem-brane surface area during leukocyte extravasationcould be an Achilles heel, which is amenable totherapeutic intervention. In this short review, weevaluate the models proposed for the mechanismof membrane expansion and discuss recent data,which point to a mechanism of membrane unwrin-kling. The molecular pathway for the unwrinklingof the leukocyte plasma membrane may involveCa2 activation of -calpain and cleavage of cy-

toskeletal linkage molecules such as talin and ezrin.This route could be common to all extravasationsignals and thus, represents a potential target foranti-inflammatory therapy. J. Leukoc. Biol. 81:11601164; 2007.

Key Words: adhesion neutrophil calcium

INTRODUCTION

The infiltration of inflamed tissues by leukocytes, especially

lymphocytes and neutrophils, is a key event in the develop-ment and progression of inflammation. This is signaled by an

array of cytokines, which have become the targets for therapy

individually, such as in the case of anti-TNF therapy. However,

the identification of a mechanism common to white cell extrav-

asation, regardless of the specific molecular mechanism in-

volved, may represent a susceptible target, which could be

attacked beneficially. Neutrophils, macrophage/monocytes,

and lymphocytes adhere to endothelium and undergo a change

from a spherical to a flattened morphological. This is required

for firm adhesion and is the necessary prelude for transmigra-

tion through the endothelium, whether by migration between

the endothelial cells or through an endothelial cell [1]. The

morphology change requires a large expansion of the surface

area of the leukocytes. The molecular pathway involved in this

unwrinkling, which would be common to all extravasation

signals, would thus be an Achilles heel and represent potential

targets for anti-inflammatory therapy. In this short review, we

evaluate the models proposed for the mechanism of membrane

expansion and discuss recent data, which point to a mechanism

of membrane unwrinkling.

LEUKOCYTE PLASMA MEMBRANEEXPANSION DURING EXTRAVASATION

When neutrophils flatten out onto endothelial cells, the trans-

formation from the spherical to the flattened (spread) morphol-

ogy is dramatic. As the spherical geometry is the minimum

surface area required to enclose a certain volume, it is obvious

that the volume of the cell must decrease, or its surface area

must increase during this transformation. In fact, it is the

surface area of the cell that increases during flattening onto the

endothelium. For cells like neutrophils, where the intracellular

organelles such as the nucleus and granules occupy a large

percentage of its volume, there is little possibility of a volume

change in any case. The increase in surface area, however, is

surprisingly large, with an increase by more than 100% (see

Fig. 1a). This doubling of surface area must be explained.

Could the lipid bilayer of the plasma membrane stretch this

far, or are additional sources of membrane (membrane reser-

voirs) required? If additional membrane is required, the mech-

anism by which it is called on must also be explained.

METHODS OF MEMBRANE EXPANSION

The first possibility is that the plasma membrane is able to

stretch this far, as if it were elastic. However, the structure of

the plasma membrane makes this impossible. The plasma

membrane is essentially a phospholipid bilayer with inserted

proteins held together as a sheet by laterally cohesive forces

between the lipid molecules. The cohesion of the sheet, there-

1 Correspondence: Neutrophil Signalling Group, School of Medicine, Cardiff

University, Heath Park, Cardiff, CF14 4XN, UK. E-mail: [email protected]

Received November 30, 2006; revised February 23, 2007; accepted Feb-

ruary 26, 2007.

doi: 10.1189/jlb.1106710

1160 Journal of Leukocyte Biology Volume 81, May 2007 0741-5400/07/0081-1160 Society for Leukocyte Biology


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fore, depends on the hydrophobic interactions of the fatty acid

chains excluding water. Any stretching effect on the bilayer,

which laterally separates the lipid molecules and allows watermolecules between the lipid, will cause rupture of the bilayer.

The proteins in a biological membrane may permit a little

additional stretch, but it has been shown experimentally (and

theoretically calculated) that biological membrane and simple

phospholipid bilayers can expand by no more than 4% before

rupturing [3]. There is thus far too little stretch in the plasma

membrane to account for the expansion of surface area re-

quired for cell flattening during adhesion to the endothelium.

This lack of stretchiness of lipid bilayers means that there must

be a reservoir of membrane, which is made available for the

plasma membrane to expand during adhesion.

A source of membrane could be provided by intracellularmembranes, which enclose secretory granules within the cell.

Exocytosis (or degranulation) of these granules would insert

additional membrane into the plasma membrane. There are two

problems with this source of membrane. The first problem is

that fusion of the membrane of the granule with the plasma

membrane would also release the contents of the vesicle into

the extracellular space. In neutrophils, for example, the gran-

ules contain hydrolases and other degradative enzymes, which

could be disastrous for the underlying endothelium. However,

release of granular content [4] and the localized release of

myeloperoxidase, a major intragranular protein in neutrophils,

have been reported to occur during extravasation [5]. This

would presumably add extra membrane to the neutrophil sur-face. However, this brings the second major problem for pro-

posing intracellular vesicles as the membrane reservoir,

namely, that it would be insufficient to account for the required

membrane expansion. A single granule with a diameter of 0.2

m (1/50th the diameter of a neutrophilic phagocyte) would

contribute only 0.04% additional membrane area. Doubling the

surface area would require 2500 vesicle fusion events/cell to

occur, which would account for all the granules within the

granulocyte. The increase in membrane capacitance of neutro-

phils during exocytosis is directly proportional to its additional

surface area and so can be used as experimental support for

this simple calculation [6]. Such a massive release of granular

material does not occur during extravasation. In fact, it is

difficult to release more than one-third of the vesicle contentexperimentally, even when artificial strategies are used such

as adding cytochalasin B. The release of all the granular

content in the vicinity of the endothelium could also be cata-

strophic. In less granular cells, such as lymphocytes, even

fusion of all granular membrane would be insufficient to pro-

vide the additional membrane.

The third proposal is that the plasma membrane wrinkles

provide the membrane reservoir for expansion during extrava-

sation. The extent of this reservoir has been demonstrated by

applying suction to the neutrophil surface through a micropi-

pette. Under this physically applied force, the neutrophil

plasma membrane can undergo large expansions [79].

WRINKLES IN THE PLASMA MEMBRANE ASTHE EXPANSION RESERVOIR

A scanning electron micrograph (SEM) of a neutrophil or

macrophage shows immediately that the assumption that these

cells are spherical and that their surface area can be calculated

easily is incorrect. These cells have numerous surface wrinkles

and folds, which means that the actual surface area of these

cells is greatly in excess of that of a sphere of the same

diameter. A series of elegant biophysical studies about the

properties of the neutrophil plasma membrane has been per-formed using suction of the plasma membrane into micropi-

pettes [79]. Their findings show that a moderate amount of

suction can expand the membrane into the mouth of the mi-

cropipette by up to 5% of the membrane area, and further

expansion is possible by applying a greater force. These mea-

surements are consistent with there being a limited amount of

slack in the wrinkles. Additional force is required to un-

wrinkle the remainder of the membrane, as if the wrinkles are

held together by a molecular velcro-like mechanism [10].

Presumably, the molecular velcro is sufficiently strong to

hold the wrinkles in place against the osmotic pressure tending

Fig. 1. Cartoons showing the morphology change during flattening of a sphere of fixed volume against a flat surface. (a) The surface area was calculated as

(2x2h2), where x is the radius of the contact surface, and h is the height of the cell and is shown as a percentage of the initial area. (b) SEMs of the spherical,

nonadhered cell (showing multiple surface wrinkles) and the flattened morphology with loss of wrinkling. b, Reproduced with permission [2].

Dewitt and Hallett Leukocyte membrane expansion 1161


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to swell the cells. However, when neutrophils are activated to

expand their plasma membrane, the amount of available slack

membrane increases, and the force required to un-velcro the

wrinkles is reduced significantly [8]. As the velcro holding the

wrinkles in place has lost its grip under this condition, the

membrane in the wrinkles would now become available to

accommodate the change in shape as the cell flattens out (Fig.

1). The loss of surface wrinkles in neutrophils when they

flattened out is apparent in SEMs and can be seen in classic

textbooks (Fig. 1b). We are now in a position to define the

nature of the molecular velcro and suggest how its grip may be

loosened.

THE MOLECULAR VELCRO OFMEMBRANE WRINKLES

Meandering across the surfaces of myeloid cells, there are

linear ridges, projecting 0.8 m high and 0.1 m wide, with

lengths of 1015 m [11]. These dimensions make them

difficult to visualize by standard light microscopy, but they are

striking SEM structures (e.g., ref. [12]). Similarly on lympho-

cytes, projections from the surface, termed microvilli, can be

seen in transmission electron microscopy images (e.g., ref.

[13]). These wrinkles and microvilli are permanent (or at least

long-lived) structures, which influence the distribution of some

surface molecules. The distribution of two surface molecules,

which are important for leukocyte rolling and adhesion, L-selectin and integrin molecules, containing the 2-chain (re-

ferred to here simply as 2-integrins), is influenced profoundly

by the wrinkles, and selectins are located only on the wrinkles

and integrins in the valleys between wrinkles [12]. The lym-

phocyte microvilli have parallel actin filaments running within

their long axis [13] to which the cytoplasmic tail of selectins is

bound via a linker molecule, ezrin [14, 15]. In neutrophils,

there is a well-defined cortical network of polymerized actin,

but the molecules involved in maintaining the surface wrinkles

are not yet fully established but probably include talin. Two

general models, however, can be suggested (Fig. 2, a and b),

dependent on linkage to the cytoskeleton via ezrin or talin. In

both models, a wrinkled membrane could be formed by cross-

linking actin to membrane proteins (Fig. 2, a and b). The two

models are not, of course, mutually exclusive, but the velcro

may be attached to cytoskeleton outside the wrinkles (Fig. 2a)

or within the wrinkle (Fig. 2b). These models also depend on

the actin-membrane linkage via L-selectin and 2-integrin and

would explain their nonhomogenous distribution on the cell

surface, i.e., their exclusion or inclusion from the wrinkled

membrane Fig. 2, a and b).

Ca2-ACTIVATED RELEASE OFWRINKLED MEMBRANE

It has been established for over 20 years that a large rise incytosolic-free Ca2 accompanies macrophage and neutrophil

spreading [16 17]. It has also been shown that uncaging

cytosolic Ca2 or inositol 1,4,5-trisphosphate (IP3) to provide

controlled Ca2 elevation causes an acceleration in the rate of

flattening [19, 20]. This is similar to the relationship between

Ca2 and membrane expansion during pseudopod extension

around the particle during phagocytosis [21], where accelera-

Fig. 3. A stylized view of talin and ezrin showing the locations of the actin

binding and FERM domains at separate ends of the molecule with the calpain

cleavage site indicated. The FERM domain binds to the cytosolic tail of

2-integrin and selectin.

Fig. 2. Release of wrinkled membrane. The cartoon shows the wrinkles held in place (a) via 2-integrin/talin/actin linkage and (b) via selectin/ezrin/actin

linkage. In the lower cartoons (c and d), the talin and ezrin have been cleaved to release the tension holding the wrinkles in place.

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tion was dependent on -calpain activity [21, 22]. This is a

Ca2-activated protease [23, 24], which cleaves substrates in

vitro and within the cell. The flattening of lymphocytes during

adhesion via 2-integrin is dependent on the activity of calpain

[25, 26]. As a number of the substrates of calpain are cytoskel-

etal proteins involved in membrane linkage [27], this would

provide a mechanism for releasing the grip of the molecular

velcro. The calpain cleavage site lies between a 4.1/ezrin/

radixin/moesin (FERM) domain, binding to membrane-associ-

ated proteins, and an actin-binding domain, linking to thecytoskeleton (Fig. 3). Activation of calpain by Ca2 would

thus lead to the uncoupling of the link between the membrane

and the underlying actin cytoskeleton and permit additional

membrane to become available for cell flattening (Fig. 2, c and

d). However, for such a proposal to be viable, there must be

some selectivity in the activation of calpain, as activation of a

(relatively) nonspecific protease within the cytosol of a healthy

cell would be disastrous. Also in vitro, activation of-calpain

requires unusually high Ca2 concentrations, its dissociation

constant is 30 M [28], whereas physiological, cytosolic

Ca2 signals reach a maximum of1 M. In fact the selec-

tivity may arise from the existence of a subplasma membranemicrodomain of high Ca2. The Ca2 concentration near the

plasma membrane exceeds 50 M during the influx of Ca2

from the extracellular medium, although the concentration in

the bulk cytosol remains below 1 M [29, 30]. When uncaging

Ca2, it was necessary to elevate bulk, cytosolic-free Ca2 to

levels above the saturation point of the indicator, estimated to

be 50 M [19]. However, the more physiological signal

induced by uncaging IP3

(which induces release of Ca2 from

stores and then physiological Ca2 influx) induced neutrophil

flattening at physiological Ca2 concentrations in the cytosol

[20]. This again suggests that microdomains exist in the cell,

which experiences high Ca2

during physiological Ca2

influx.The Ca2 concentrations within individual wrinkles may reach

the highest level, as there is a larger localized surface area to

volume ratio. In addition, it has been reported that calpain

translocates to the plasma membrane [31], perhaps by virtue of

its C2-like domain. We have found that in the neutrophilic

NB4 cell line, elevation of Ca2 can cause profound and rapid

trapping of calpain at the plasma membrane [22]. Together,

these phenomena would limit calpain activity to the strategi-

cally required location and target subplasma membrane pro-

teins. It is, in fact, known that cleavage of talin, an important

calpain substrate, occurs during physiological Ca2 influx in

neutrophils [32], suggesting that when activated by Ca2

in-flux, calpain-mediated proteolysis has specificity. Furthermore,

a number of important calpain substrates, including ezrin,

Wiskott-Aldrich syndrome protein, and myosin X, undergo

-calpain-dependent cleavage in myeloid and lymphoid cells

[3335]. In nonimmune cells, calpains have also been impli-

cated in the local control membrane surface area leading to

membrane protrusions [36]. This proposed mechanism of mem-

brane expansion thus has a number of features, which make

this model attractive for explaining the nature of the membrane

reservoir and the way in which it is brought into play during

leukocyte extravasation

POTENTIAL THERAPEUTIC TARGET

In this short review, we have identified the need to understand

membrane expansion during adhesion and cell flattening of

myeloid and lymphoid cells onto the endothelium during ex-

travasation. We have also pointed to an attractive hypothesis as

to the nature of this reservoir of additional membrane. Al-

though more work will be required before such a proposal can

be fully accepted (or rejected), we hope this article will act as

a stimulus to such future work. This mechanism does, however,

open an exciting possibility for anti-inflammatory therapy. By

targeting inhibition of -calpain, it may be possible to slow

down or inhibit the influx of immune cells into inflamed sites.

As this approach targets a key step in the extravasation pro-

cess, it would be effective, regardless of the driving stimulus

and etiology. It is tantalizing that inhibition of calpain has been

shown to reduce inflammatory cell influx effectively in animal

models of experimentally induced inflammation and extrava-

sation. For example, inhibition of calpain reduces neutrophil

adhesion and improves myocardial dysfunction and inflamma-

tion induced by endotoxin in rats [37] and reduces neutrophil-

driven reperfusion injury in the intestine [38] and kidney [39].

Animal models of collagen-induced arthritis are potently in-hibited by calpain inhibition [40], and in an experimental

model of rheumatoid arthritis induced by antitype collagen

mAb, calpain inhibition using the calpain inhibitor, E64d,

produced a dramatic reduction in joint inflammation [41].

Although little mechanistic studies have been performed, it is

important to note that E64d is an ester derivation of E64, which

is de-esterified intracellularly to generate the calpain inhibi-

tory molecule. As it has no activity on extracellular proteases,

this strongly suggests that the important site of calpain activity

in this context is within cells rather than as an extracellularly

destructive protease. These studies obviously point to calpain

as an important target for anti-inflammatory therapy, one thatmay be the common Achilles heel of inflammatory disease.

ACKNOWLEDGMENTS

We thank Chris von Ruhland for the SEMs of neutrophils and

the Wellcome Trust (UK) for continued support. We apologize

to authors of many of the important publications in this field,

which we could not find space to cite or discuss in this short

review.

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leukocyte membrane "expansion": a central mechanism for leukocyte extravasation

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