Originally published online as doi:10.1189/jlb.1106710 on March 14, 2007
Published online before print March 14, 2007
(Journal of Leukocyte Biology. 2007;81:1160-1164.)
© 2007
by Society for Leukocyte Biology
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
1 Correspondence: Neutrophil Signalling Group, School of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK. E-mail: hallettmb{at}cf.ac.uk
ABSTRACT
The infiltration of inflamed tissues by leukocytes is a key
event in the development and progression of inflammation. Although
individual cytokines, which coordinate extravasation, have become
the targets for therapy, a mechanism that is common to white
cell extravasation, regardless of the specific molecular mechanism
involved, would represent a more attractive therapeutic target.
Such a target may be represented by the events underlying the
spreading of leukocytes on the endothelium, which is a necessary
prelude to extravasation. This leukocyte "spreading" involves
an apparent increase in the cell surface area. The aim of this
review is to examine whether the mechanism underlying the apparent
expansion of plasma membrane surface area during leukocyte extravasation
could be an "Achilles’ heel," which is amenable to therapeutic
intervention. 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."
The molecular pathway for the unwrinkling of the leukocyte plasma
membrane may involve Ca
2+ activation of µ-calpain and
cleavage of cytoskeletal linkage molecules such as talin and
ezrin. This route could be common to all extravasation signals
and thus, represents a potential target for anti-inflammatory
therapy.
Key Words: adhesion • neutrophil • calcium
INTRODUCTION
The infiltration of inflamed tissues by leukocytes, especially
lymphocytes and neutrophils, is a key event in the development
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 extravasation, regardless
of the specific molecular mechanism involved, 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 transmigration 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 MEMBRANE EXPANSION DURING EXTRAVASATION
When neutrophils flatten out onto endothelial cells, the transformation
from the spherical to the flattened (spread) morphology 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 reservoirs) required? If additional
membrane is required, the mechanism 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, therefore, 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 water molecules 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 required 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 intracellular membranes, 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 granules 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 surface. However, this brings the second major problem for proposing 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 µm (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 neutrophils 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 content experimentally, 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 catastrophic. In less granular cells, such as lymphocytes, even fusion of all granular membrane would be insufficient to provide the additional membrane.
The third proposal is that the plasma membrane wrinkles provide the membrane reservoir for expansion during extravasation. The extent of this reservoir has been demonstrated by applying suction to the neutrophil surface through a micropipette. Under this physically applied force, the neutrophil plasma membrane can undergo large expansions [7
8
9
].
WRINKLES IN THE PLASMA MEMBRANE AS THE 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 performed using suction of the plasma
membrane into micropipettes [
7
8
9
]. Their findings show that
a moderate amount of suction can expand the membrane into the
mouth of the micropipette by up to
5% of the membrane area,
and further expansion is possible by applying a greater force.
These measurements are consistent with there being a limited
amount of "slack" in the wrinkles. Additional force is required
to "unwrinkle" 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
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 OF MEMBRANE WRINKLES
Meandering across the surfaces of myeloid cells, there are linear
ridges, projecting
0.8 µm high and 0.1 µm wide,
with lengths of 10–15 µm [
11
]. These dimensions
make them difficult to visualize by standard light microscopy,
but they are striking SEM structures (e.g., ref. [
12
]). Similarly
on lymphocytes, 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 ß2-chain (referred
to here simply as ß2-integrins), is influenced profoundly
by the wrinkles, and selectins are located only on the wrinkles
and integrins in the valleys between wrinkles [
12
]. The lymphocyte
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. 2a
and 2b
),
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. 2a
and 2b)
. 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 ß2-integrin
and would explain their nonhomogenous distribution on the cell
surface, i.e., their exclusion or inclusion from the wrinkled
membrane
Fig. 2a
and 2b
).
Ca2+-ACTIVATED RELEASE OF WRINKLED MEMBRANE
It has been established for over 20 years that a large rise
in cytosolic-free Ca
2+ accompanies macrophage and neutrophil
spreading [
16
17
]. It has also been shown that uncaging cytosolic
Ca
2+ or inositol 1,4,5-trisphosphate (IP
3) to provide controlled
Ca
2+ elevation causes an acceleration in the rate of flattening
[
19
,
20
]. This is similar to the relationship between Ca
2+ and membrane expansion during pseudopod extension around the
particle during phagocytosis [
21
], where acceleration was dependent
on µ-calpain activity [
21
,
22
]. This is a Ca
2+-activated
protease [
23
,
24
], which cleaves substrates in vitro and within
the cell. The flattening of lymphocytes during adhesion via
ß2-integrin is dependent on the activity of calpain
[
25
,
26
]. As a number of the substrates of calpain are cytoskeletal
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-associated proteins, and
an actin-binding domain, linking to the cytoskeleton (
Fig. 3
).
Activation of calpain by Ca
2+ 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. 2c
and 2d)
. 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 µ-calpain requires unusually high
Ca
2+ concentrations, its dissociation constant is
30 µM
[
28
], whereas physiological, cytosolic Ca
2+ signals reach a
maximum of
1 µM. In fact the selectivity may arise from
the existence of a subplasma membrane microdomain of high Ca
2+.
The Ca
2+ concentration near the plasma membrane exceeds 50 µM
during the influx of Ca
2+ from the extracellular medium, although
the concentration in the bulk cytosol remains below 1 µM
[
29
,
30
]. When uncaging Ca
2+, it was necessary to elevate
bulk, cytosolic-free Ca
2+ to levels above the saturation point
of the indicator, estimated to be
50 µM [
19
]. However,
the more physiological signal induced by uncaging IP
3 (which
induces release of Ca
2+ from stores and then physiological Ca
2+ influx) induced neutrophil flattening at physiological Ca
2+ concentrations in the cytosol [
20
]. This again suggests that
microdomains exist in the cell, which experiences high Ca
2+ during physiological Ca
2+ influx. The Ca
2+ 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 Ca
2+ can cause profound and rapid trapping of calpain at
the plasma membrane [
22
]. Together, these phenomena would limit
calpain activity to the strategically required location and
target subplasma membrane proteins. It is, in fact, known that
cleavage of talin, an important calpain substrate, occurs during
physiological Ca
2+ influx in neutrophils [
32
], suggesting that
when activated by Ca
2+ influx, calpain-mediated proteolysis
has specificity. Furthermore, a number of important calpain
substrates, including ezrin, Wiskott-Aldrich syndrome protein,
and myosin X, undergo µ-calpain-dependent cleavage in
myeloid and lymphoid cells [
33
34
35
]. In nonimmune cells,
calpains have also been implicated in the local control membrane
surface area leading to membrane protrusions [
36
]. This proposed
mechanism of membrane 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 extravasation.
We have also pointed to an attractive hypothesis as to the nature
of this reservoir of additional membrane. Although 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 µ-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 process, 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 extravasation. For
example, inhibition of calpain reduces neutrophil adhesion and
improves myocardial dysfunction and inflammation 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 inhibited 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 inhibitory 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 that may be the common Achilles’
heel of inflammatory disease.
ACKNOWLEDGEMENTS
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.
Received November 30, 2006;
revised February 23, 2007;
accepted February 26, 2007.
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(2x2+h2), 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
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ABSTRACT
INTRODUCTION