Force as a facilitator of integrin conformational changes during leukocyte arrest on blood vessels and antigen-presenting cells.
ABSTRACT Integrins comprise a large family of cell-cell and cell-matrix adhesion receptors that rapidly modulate their adhesiveness. The arrest of leukocyte integrins on target vascular beds involves instantaneous conformational switches generating shear-resistant adhesions. Structural data suggest that these integrins are maintained in low-affinity conformations and must rapidly undergo conformational switches transduced via cytoplasmic changes ("inside-out" signaling) and simultaneous ligand-induced rearrangements ("outside-in"). This bidirectional activation is accelerated by signals from endothelial chemoattractants (chemokines). Recent studies predict that shear forces in the piconewton (pN) range per integrin can facilitate these biochemical switches. After extravasation, antigen recognition involves smaller internal forces from cytoskeletal motors and actin polymers forming the immune synapse. In this review, we address how forces facilitate allosteric integrin activation by biochemical signals. Evidence suggests that preformed cytoskeletal anchorage rather than free integrin mobility is key for force-enhanced integrin activation by chemokines and TCR signals.
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ABSTRACT: T. gondii is a highly successful global pathogen that is remarkable in its ability to infect nearly any nucleated cell in any warm-blooded animal. Infection with T. gondii typically occurs through the ingestion of contaminated food or water, but the parasite then breaches the intestinal epithelial barrier and spreads from the lamina propria to a large variety of other organs in the body. A key feature of T. gondii pathogenesis is the parasite's ability to cross formidable biological barriers in the infected host and enter tissues such as the brain, eye, and placenta. The dissemination of T. gondii into these organs underlies the severe disease that accompanies human toxoplasmosis. In this review we will focus on seminal studies as well as exciting recent findings that have shaped our current understanding of the cellular and molecular mechanisms by which T. gondii journeys throughout the host and enters the vital organs to cause disease.This article is protected by copyright. All rights reserved.Parasite Immunology 11/2014; 37(3). · 1.85 Impact Factor
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ABSTRACT: The investigation of nano-mechanical properties and nano-architectures of CD8 + T cells activated in vitro will help further interpret the immune response/recognition of these cells at nanoscale resolution. In this work, the local biophysical properties of human peripheral blood CD8 + T cells were obtained, revealing the adhesion force of membrane was increased after CD8 + T cells were stimulated with phorbol dibutyrate plus ionomycin, though the measured local stiffness for resting and activated cells was similar. The 3-D distribu-tion pattern and grayscale map together revealed that membrane receptors (CD8 and activation marker CD69) distributed non-uniformly on the membrane and were shown as micro-, nano-scale clusters or domains. This work describes a relatively simple approach for ex-ploiting the distribution pattern of receptor molecules on a single T cell surface.
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ABSTRACT: Leukocyte migration through activated venular walls is a fundamental immune response that is prerequisite to the entry of effector cells such as neutrophils, monocytes, and effector T cells to sites of infection, injury, and stress within the interstitium. Stimulation of leukocytes is instrumental in this process with enhanced temporally controlled leukocyte adhesiveness and shape-changes promoting leukocyte attachment to the inner wall of blood vessels under hydrodynamic forces. This initiates polarized motility of leukocytes within and through venular walls and transient barrier disruption facilitated sequentially by stimulated vascular cells, i.e., endothelial cells and their associated pericytes. Perivascular cells such as macrophages and mast cells that act as tissue inflammatory sentinels can also directly and indirectly regulate the exit of leukocytes from the vascular lumen. In this review, we discuss current knowledge and open questions regarding the mechanisms involved in the interactions of different effector leukocytes with peripheral vessels in extralymphoid organs. Copyright © 2014 Elsevier Inc. All rights reserved.Immunity 11/2014; 41(5):694-707. · 19.75 Impact Factor
Force as a Facilitator of Integrin Conformational
Changes during Leukocyte Arrest on Blood
Vessels and Antigen-Presenting Cells
Ronen Alon1,* and Michael L. Dustin2
1Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel
2Program in Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine and Department of Pathology, New York
University School of Medicine, New York, NY 10016, USA
Integrins comprise a large family of cell-cell and cell-matrix adhesion receptors that rapidly modulate
their adhesiveness. The arrest of leukocyte integrins on target vascular beds involves instantaneous
conformational switches generating shear-resistant adhesions. Structural data suggest that these
integrins are maintained in low-affinity conformations and must rapidly undergo conformational
switches transduced via cytoplasmic changes (‘‘inside-out’’ signaling) and simultaneous ligand-
induced rearrangements (‘‘outside-in’’). This bidirectional activation is accelerated by signals from
endothelialchemoattractants (chemokines). Recent studies predict that shear forces in the piconew-
ton(pN) rangeperintegrincanfacilitatethesebiochemicalswitches. Afterextravasation,antigenrec-
ognition involves smaller internal forces from cytoskeletal motors and actin polymers forming the
immune synapse. In this review, we address how forces facilitate allosteric integrin activation by bio-
chemical signals. Evidence suggests that preformed cytoskeletal anchorage rather than free integrin
mobility is key for force-enhanced integrin activation by chemokines and TCR signals.
Leukocytes circulating in the blood are recruited to lym-
phoid organs and peripheral sites of injury, infection, and
inflammation by a series of sequential but overlapping
steps mediated by members of two major adhesion
receptor families, selectins and integrins (Butcher, 1991).
Multiple molecular options at each step provide large
sue selectivity in leukocyte-endothelial cell recognition. In
order to appreciate the contributions of integrins to leuko-
cyte rolling and arrest on blood vessels, we will first intro-
duce concepts initially elucidated from the selectin family.
Selectins Are Structurally Adapted to Mediate
the Capture and Rolling of Circulating
Leukocytes on Target Endothelial Sites
Selectins are the main receptors that mediate the initial
capture of circulating leukocytes to ligands expressed
on the endothelium (McEver, 2002). Leukocyte capture
is followed by rolling adhesions, which are maintained
on a timescale of seconds to a few minutes, depending
prise a three-member family (L-, P-, and E-selectins) shar-
inghighlyconservedN-terminal C-typelectindomains fol-
lowed by regulatory epidermal growth factor (EGF)-like
domains and short consensus repeats. All selectins rec-
ognize sialyl Lewis X and related carbohydrate ligands,
presented mainly by sialomucin-like surface molecules
(McEver, 2002; Rosen, 2004). The leukocyte selectin,
L-selectin, is expressed on most circulating leukocytes
and is the key receptor that initiates leukocyte capture
events in secondary lymphoid tissues and at peripheral
sites of injury and inflammation (Rosen, 2004). P- and
E-selectins are inducibly expressed in both acutely and
chronically stimulated endothelial beds and are critical
mediators of slow rolling adhesions (Ley et al., 1995;
McEver, 2002). Selectin-mediated adhesions are charac-
terized by fast on and off rates and exceptional resistance
to disruptive shear forces exerted on the leukocyte at the
vessel wall (Alon et al., 1995). Selectins and their ligands
are localized to microvilli-like projections, favorable sites
for leukocyte-endothelial collisions (von Andrian et al.,
1995). Selectin bonds undergo conformational changes
that decrease their off rate under low tensile forces, giving
them properties of ‘‘catch bonds’’ (Thomas et al., 2002;
Marshall et al., 2003; Sarangapani et al., 2004; Zhu
et al., 2005), and their lectin-EGF interdomain hinge
may critically regulate this key mechanical property (Lou
et al., 2006; Phan et al., 2006). The interdomain hinge
may also control the rotational freedom of the selectin lec-
is a consistent but mild increase in the off rate of selectin
bonds, a behavior characteristic of ‘‘slip bonds’’ (Dembo
et al., 1988). The mechanical stability of selectin interac-
tions is also conferred by selectin or ligand dimerization,
which disperses high shear forces applied on the leuko-
cyte-endothelial contact over multiple bonds (Chen and
Evidence suggests that, in order to resist shear-derived
detaching forces, both selectins and their ligands also
need to be properly anchored to the cytoskeleton (Kansas
et al., 1993; Setiadi et al., 1998; Dwir et al., 2001; Ivetic
Immunity 26, January 2007 ª2007 Elsevier Inc.
et al., 2002; Snapp et al., 2002). Thus, conformations of
selectin-ligand pairs that are stabilized by force (Phan
et al., 2006) may require anchorage of selectins and
ligands within their respective cell membranes. In agree-
ment with this possibility, bonds mediated by tailless
L-selectin failtostabilize adhesiveinteractions evenunder
low-force conditions and break within milliseconds (Dwir
et al., 2001; Dwir et al., 2003). Microvillar localization
of L-selectin, which enhances the selectin availability on
the leukocyte surface (von Andrian et al., 1995), may
bond (Fritz et al., 1998) since microvilli are highly elastic
projections (Shao et al., 1998). To disperse forces along
the microvilli axis, ligand-occupied selectins must be
properly anchored to the leukocyte microvilli (Figure 1).
Indeed, chemical stiffening of microvilli destabilizes
selectin-mediated adhesions under shear flow (Yago
et al., 2002).
Leukocyte Integrin Activation: Structural Basis
The arrest of tethered or rolling leukocytes on target endo-
thelium is nearly exclusively mediated by members of the
integrin superfamily and their endothelial immunoglobulin
superfamily (IgSF) ligands (Hynes, 2002). Integrins consti-
tute a family of over 20 heterodimers whose ligand-
binding activity can be rapidly regulated by conforma-
tional changes, clustering, and redistribution from surface
and intracellular pools (Carman and Springer, 2003;
Dustin et al., 2004). The most relevant integrins for leuko-
myeloid-specific integrin Mac-1 (aMb2), as well as the two
a4integrins, a4b1(VLA-4) and a4b7(Hynes, 2002). With the
exception of T cell and B cell blasts (Vajkoczy et al., 2001)
and subsets of innate immune cells that express highly
adhesive integrins, circulating leukocytes maintain their
integrins in largely inactive states. They must therefore
undergo in situ modulation to develop high-affinity and
shear-resistant adhesiveness for their specific endothelial
ligands. Both in vitro and in vivo data suggest that integrin
adhesiveness can be increased during subsecond con-
tacts with stimulatory endothelial signals (Alon et al.,
regulation of LFA-1, the most thoroughly investigated leu-
kocyte integrin, have been provided by studies of its main
Figure 1. Integrin Activation on Selectin-Occupied Leukocytes Rolling on Vessel Walls Presenting Chemoattractants
Adhesive cascades generated by leukocytes recruited to different endothelial sites and their timescales. Leukocyte microvilli are preferential sites of
collision and serve as elastic projections that reduce the forces applied to given selectin or integrin bonds. Selectin-mediated adhesions bring leu-
kocytes into proximity with clusters of chemoattractants (chemokines) and integrin ligands. Initial activation of integrins by the chemoattractant re-
ceptor GPCR occurs within a subsecond period, possibly within singular microvillar contacts. For simplicity, selectin and integrin bonds are shown to
form on two separate microvilli, but at high densities of integrin ligands and activating chemokines, a given microvillus may simultaneously occupy
receptors into contact with their endothelial ligands and facilitate adhesion strengthening after the initial arrest.
Immunity 26, January 2007 ª2007 Elsevier Inc.
ligand-binding domains and detailed structural analysis of
related integrins, including the platelet integrin aIIbb3, the
vitronectin receptor avb3, the b2 integrins Mac-1 and
axb2, and the fibronectin receptor a5b1 (Adair et al.,
2005; Shimaoka et al., 2003; Takagi et al., 2001; Xiong
et al., 2001, 2002; Nishida et al., 2006). These studies
took advantage of X-ray-based structural analysis, NMR,
or ultrastructural imaging by negative stain electron mi-
croscopy. Use of monoclonal antibodies that recognize
neoepitopes induced by conformational switches on na-
tive and mutated integrins has also provided key insights
into integrin rearrangements and activation states (Be-
glova et al., 2002; Xie et al., 2004). The a and b subunit
of all integrin heterodimers fold to form an extracellular
headpiece connectedto themembranebytwo‘‘legs,’’fol-
lowed by a short transmembrane and cytoplasmic tail
(Figure 2). The headpiece of all integrins is formed by
a b-propeller domain of the a subunit, which closely inter-
acts with a dinucleotide fold domain within the b subunit,
termed the b I domain (also known as the I-like domain).
The b I domain is linked to the hybrid domain, part of the
two b subunit domains controls the b I domain conforma-
tion and the ligand-binding affinity of the headpiece (Fig-
ure 2). The integrin legs are formed by multiple globular
domains that incorporate two knee-like joints referred to
as a ‘‘genu.’’
Inactive integrins are compact and bent (Xiong et al.,
2001; Takagi et al., 2002), with their genu folded and the
headpiece only 5 nm from the membrane (Figure 2, top).
Separation of the a and b subunit legs, a critical step in
inside-out integrin activation, destabilizes their interface
with the headpiece, converting the bent structure to an
overall extended conformation and relieving constraints
on headpiece activation (Figure 2, top). This separation
is driven by unclasping intersubunit associations between
the transmembrane and cytoplasmic integrin domains.
When the genu is straightened through this switchblade-
like unbending, the integrin headpiece can project 20–
25 nm above the membrane, becoming readily available
for surface-presented ligand (Beglova et al., 2002; Salas
et al., 2004; Zhang et al., 2005; Nishida et al., 2006)
NMR and X-ray structures of the EGF-like domains
C-terminal to the b subunit hybrid domain suggest that
these domains, and in particular the EGF-like domain 2
(Figure 2, middle right ribbon diagram), are key in translat-
ing cytoplasmic tail rearrangements into separation of the
a and b subunit legs and straightening of the integrin ecto-
domain (Beglova et al., 2002; Xiong et al., 2001).
Affinity states of all integrins are also tightly controlled
by local rearrangements of their headpiece. Notably,
these rearrangements can be initiated both by extrinsic li-
gands andby binding of specific adaptors to the cytoplas-
mic domain clasp (Arnaout et al., 2002; Carman and
Springer, 2003). The conformation of a critical ligand-
binding site termed the metal-ion-dependent adhesion
site (MIDAS)dictates theaffinityof ligandbinding tothein-
tegrin headpiece. The metal ion bound at this site is coor-
dinated by a key carboxylate residue shared by all integrin
ligands (Hynes, 2002), and the coordination of this metal
dictates the overall conformation of the MIDAS loops
and the headpiece affinity to ligand. Whereas the ligand-
binding sites of most integrins are composed of residues
from both their a subunit b-propeller and b I domains (Fig-
ure 2, lower row), the ligand-binding site of LFA-1, as well
as of other b2integrins and subsets of b1and b7integrins,
is located in an a I domain, inserted atop the a subunit
b-propeller domain (Shimaoka et al., 2003) (Figure 2, mid-
dle row). The b I and the a I domains share a similar fold
and MIDAS motifs. Lateral movements of loops that
form the MIDAS upon ligand binding result in allosteric
ure 2, middle and bottom rows). Activation and opening of
the cytoplasmic domains (Takagi et al., 2001) that exert
a swing-out of the b subunit hybrid domain (Figure 2,
inside-out). This critical conformational switch can pull
ilar to a bell rope. Notably, in a I domain-containing integ-
rins, the b I domain does not directly participate in ligand
downward through an intramolecular bond between an
invariant glutamate on the a subunit and the b I MIDAS
(Figure 2, middle row). This in turn exerts a second down-
ward pull on the seventh a helix of the b I domain (Shi-
maoka et al., 2003).
To summarize, changes in conformation initiated at the
cytoplasmic clasp and transmitted to the headpiece
MIDAS domain (or domains) via the leg domains are re-
ferred to as inside-out activation (Dustin and Springer,
1989). When ligand binding to the headpiece domain in-
duces changes in the headpiece as well as in the orienta-
tion ofthelegdomains andthe cytoplasmicregions, thisis
referred to as outside-in activation. Consistent with this
model, LFA-1 tail unclasping, a marker of integrin activa-
tion, can also be induced by integrin occupancy with its
ligand, ICAM-1 (Kim et al., 2003). Intersubunit interactions
including the cytoplasmic clasp, transmembrane do-
mains, and leg interactions will tend to maintain the integ-
rin in its low-affinity closed state. Although high-affinity in-
tegrin states can be artificially induced by chemically
locking extended and open integrin conformations or by
freezing these states with monoclonal antibodies prior to
ligand binding (Carman and Springer, 2003), physiological
activation of integrins appears to involve simultaneous
bidirectional activation induced by both inside-out and
outside-in rearrangements (Figure 2).
VLA-4 and a4b7
In contrast to the great advances in our understanding of
b2and b3integrin affinity modulation, there is still limited
tional activation by inside-out and outside-in signals.
Although there is no current evidence for overall extension
of these two integrins, ligand binding by three non-b2
integrins, VLA-5, aIIbb3, and avb3, induces a canonical
swing-out of their b subunit hybrid domain (Chen et al.,
2004). Since a4integrins on both resting lymphocytes
Immunity 26, January 2007 ª2007 Elsevier Inc.
and monocytes can spontaneously interact with their re-
spective endothelial ligands when present at high density
(Berlin et al., 1995), it is possible that these integrins exist
in overall extended conformations with high accessibility
to their endothelial ligands. Accessibility of a4integrins
may also result from their relatively high occupancy on mi-
crovilli (Berlin et al., 1995). Notably, chemokines that po-
tently stimulate T cell VLA-4 adhesiveness to VCAM-1 un-
der shear flow fail to trigger activation epitopes on VLA-4
that report high affinity to ligands (Grabovsky et al., 2000)
but do trigger LFA-1 extension and activation epitopes
(Shamri et al., 2005). a4integrin adhesiveness under shear
forces can be therefore augmented by conformational
switches in their cytoplasmic domains that do not alter
Figure 2. Proposed Role for External (Shear) Forces Applied on Ligand-Integrin Complexes in a and b I Domain Activation
Integrin activation is triggered by a switchblade-like extension of its bent form that increases headpiece accessibility to surface-bound ligand (top
row). The middle row depicts bidirectional headpiece activation of the a I domain-containing integrin LFA-1 as a prototype. A partial opening of
the bIdomainis drivenbyaswing-out of thebsubunit hybrid domain(purple) throughinside-outactivationsignals. Theextrinsic ligand thenoccupies
and further activates (outside-in) the a I domain. In addition, the b I domain must be occupied with an intrinsic ligand to enable maximal stabilization.
Bottom row: Bidirectional activation of an integrin lacking an a I domain. When loaded with low forces (F, blue arrows, middle and bottom rows), the
various ligand-occupied I domains are predicted to undergo these activation events within less than a microsecond (Puklin-Faucher et al., 2006),
whereas in the absence of force, these events take seconds. Bottom right: Integrin anchorage is required for the integrin to load low forces
(<30 pN) and undergo instantaneous activation by surface-bound ligand (t1, millisecond time range). Subsequent dispersion of the applied forces
across the much softer microvillus may take place via microvillus extension (t2, subsecond range). At t > 1 s, the ligand-occupied integrin can break
apart fromthecytoskeleton(notshown),allowing alongmembranecylindertoextendbeforefinalbonddissociation(Shaoetal.,1998)(Heinrichetal.,
2005). (Modified from Carman and Springer, 2003; Zhang et al., 2005).
Immunity 26, January 2007 ª2007 Elsevier Inc.
integrin affinity to ligand in shear-free conditions (Alon
et al., 2005). Recent findings also indicate that VLA-4
can undergo direct conformational activation by shear
stress generated by circular stirring (Zwartz et al., 2004).
Taken together, externally applied shear force is an addi-
tional factor in VLA-4 activation by ligand overlooked by li-
gand-binding assays conducted in shear-free conditions.
Force Exerted on Leukocyte Integrins at Vessel
Walls: A Barrier or a Positive Regulator?
Low forces (<30 pN) appear to stabilize extended
headpiece conformations of selectins associated with
strengthened selectin-ligand bonds (Marshall et al.,
2003; Sarangapani et al., 2004; Phan et al., 2006). Since
full a I domain integrin activation requires sequential
pull-down steps with a simultaneous swing-out of the
b subunit hybrid domain, these rearrangements may
also benefit from low forces applied on the ligand-head-
piece interface (Figure 2). Even though rupture analysis
of single integrin-ligand bonds using atomic force micros-
copy has not detected net stabilization of these bonds by
low forces (Zhang et al., 2002) as was recently found for
selectin bonds (Zhu et al., 2005), there are accumulating
data suggesting that low shear forces exerted on cells ex-
pressing an isolated LFA-1-derived a I domain augment I
domain adhesiveness to ICAM-1 by stabilizing the open
conformation of this domain (Salas et al., 2002) (Astrof
ness to its ligand MadCAM-1 above a critical threshold of
shear stress (de Chateau et al., 2001). Optimal integrin ac-
tivation by chemokines in T cells is also greatly facilitated
by externally applied forces (E. Woolf, A. Sagiv, Z. Shul-
man, V. Grabovsky, R. Pasvolsky, S. Feigelson, M. Sixt,
and R.A., unpublished data). Notably, recent computer
simulation of integrin dynamics also predicts that the
ligand-bound avb3 transitions into a high-affinity open
headpiece state within nanoseconds if force is applied
to the ligand-headpiece complex (Puklin-Faucher et al.,
2006). In contrast, ligand-driven head rearrangements of
integrins may take seconds to complete in the absence
of force (Bednar et al., 1997). Thus, low forces may theo-
retically accelerate ligand-driven outside-in activation of
the integrin headpiece by up to nine orders of magnitude.
To load forces, whether externally applied by shear
stress or internally generated by filamentous actin, integ-
rin-ligand complexes must be properly anchored within
the plasma membrane. The role of preexisting cytoskele-
tal integrin links in integrin adhesiveness of leukocytes
under shear flow has only begun to unfold. One line of
evidence that leukocyte integrin anchorage is essential
for mechanical strengthening of integrin bonds is based
on recent findings indicating that strong a4 integrin
anchorage to the cytoskeleton is key for subsecond stabi-
not for VLA-4 adhesiveness under shear-free conditions
(Alon et al., 2005). Although preformed integrin anchorage
was considered to be antiadhesive due to its negative
effects on lateral integrin mobility to contact sites (Kucik
et al., 1996), for initial mechanical activation of integrin-
ligand bonds at leukocyte-vessel contacts, the opposite
may be true. A recent study using single-molecule track-
ing on total, closed (low-affinity), and extended forms of
LFA-1 identified a large fraction of open and extended
LFA-1 conformations as being preanchored to the cyto-
skeleton (Cairo et al., 2006). This analysis revealed that
the population of anchored and extended LFA-1 confor-
mations increases upon inside-out activation by either
phorbol esters or T cell receptor (TCR) ligation, two key in-
side-out modalities of LFA-1 activation (Cairo et al., 2006).
Ligand-engaged LFA-1 molecules showed the greatest
degree of cytoskeletal association, consistent withligand-
induced outside-in conformational changes that favor
integrin cytoskeletal associations (Shamri et al., 2005). In
contrast, closed low-affinity LFA-1 conformations were
found to be poorly anchored to the cytoskeleton and ap-
peared to further decrease their cytoskeletal associations
upon activation. These low-affinity unanchored LFA-1
species may therefore serve as a reserve pool that can
diffuse to sites of adhesion, initiated by the anchored
extended LFA-1 subsets. The properly anchored and
extended integrin appears to be ideally suited to translate
ligand recognition into high-affinity and shear-resistant
binding necessary for the rapid arrest of the rolling leuko-
cyte (Shamri et al., 2005). Released and unclasped integ-
rins with high affinity to ligand may, on the other hand, fail
to generate the mechanically stable ligand complexes
necessary for initial arrests under shear flow. These integ-
rins are still likely to be recruited by ligand-bound integrins
and contribute to postarrest adhesion strengthening.
Increasing evidence suggests that subsets of circulat-
ing leukocytes express a fraction of their integrins in pre-
formed intermediate-affinity states. These integrins may
contain partially closed I domains that temporarily arrest
derson et al., 2001; Salas et al., 2004). In lymphocytes
homing to Peyer’s patch HEV, a4b7-MadCAM-1 inter-
actions decelerate rapid L-selectin-mediated rolling prior
to GPCR activation (Bargatze et al., 1995); in eosinophils,
VLA-4-VCAM-1 interactions slow down eosinophil rolling
on inflamed venules (Sriramarao et al., 1994); and in neu-
trophils, both LFA-1 and Mac-1 have been reported to re-
tard selectin-mediated rolling (Dunne et al., 2003). These
integrin states may be preanchored to the cytoskeleton
and stabilized by membrane effectors such as tetraspa-
nins (Feigelson et al., 2003) and surface receptors such
as CD44 (Nandi et al., 2004).
Chemoattractants May Stimulate Abrupt Integrin
Adhesiveness by Accelerating Bidirectional
Conformational Changes of Anchored Integrins
In situ activation of integrins on leukocytes rolling on
endothelial targets is rapidly triggered by the binding of
specialized chemoattractants, or chemotactic cytokines
(chemokines), to cognate G protein-coupled receptors
(GPCRs) (Bargatze and Butcher, 1993; Campbell et al.,
1998). A recent study suggests that subsets of GPCRs,
when occupied by endothelial-presented ligands but not
Immunity 26, January 2007 ª2007 Elsevier Inc.
by soluble ligands, can trigger, within subseconds, both
inside-out and outside-in activation of LFA-1 in T lympho-
cytes (Shamri et al., 2005). A critical step in this LFA-1 ac-
tivation is the instantaneous stabilization of the extended
integrin state through a chemokine signal, which must
be immediately coupled to an ICAM-1-induced activation
integrin ligand (Figure 1) and an intact actin cytoskeleton
appear to be also required for chemoattractant-triggered
activation in multiple types of leukocyte and integrin sys-
tems (Alon et al., 2003).
Numerous downstream effectors have been so far sug-
gested to mediate this integrin activation step (Kinashi,
2005). However, many integrin-associated adhesive pro-
cesses have been assessed in the absence of shear
forces or have involved late adhesive and spreading steps
downstream of the initial bidirectional integrin activation
described above (Alon et al., 2003). So far, only a few reg-
ulators, mainly GTPases, have been implicated in rapid
integrin activation at leukocyte-endothelial contacts. The
small GTPase RhoA was originally shown to be involved
in rapid integrin activation by CXCL8 in neutrophils (Lau-
danna et al., 1996) as well as LFA-1 activation by the
CCL21 and CXCL12 chemokines in lymphocytes (Giagulli
et al., 2004). The downstream target of chemokine-
activated RhoA in rapid integrin activation is still unclear.
A second GTPase, Rap1, has emerged as another key
regulator of early integrin activation by chemokine signals
and shear stress signals (Katagiri et al., 2004; Shimonaka
et al., 2003). The role of Rap1 in integrin triggering was
genetic defect, LAD III, in which a deficiency in chemo-
kine-triggered integrin activation correlates with impair-
ment of Rap1 activation (Kinashi et al., 2004).
Akey potential adaptor capable of translating these and
anchored integrins is talin (Shamri et al., 2005). Talin is
a large and extended homodimer that links integrins to
the actin cytoskeleton (Critchley, 2000). Talin has an
amino-terminal FERM head domain that, when exposed,
binds an NPXY/F tail motif shared by all major b-integrin
subunits. Rap1 has been recently shown to link inside-
out signals to talin activation of integrins (Han et al.,
2006). Talin head can bind b subunits of multiple integrins
and unclasp their tails, driving integrin extension or rein-
forcing integrin activation by extracellular ligand (Tado-
koro et al., 2003). As talin exists in multiple conformational
a manner that restricts their tail unclasping (Sampath
et al., 1998), whereas, when properly activated by inter-
action with the phosphoinositide PI(4,5)P2 or by phos-
phorylation, talin can productively trigger conformational
integrin activation (Kim et al., 2003; Tadokoro et al.,
2003). Talin may also crosslink correctly spaced ligand-
occupied integrins and thereby further strengthen adhe-
sions (Jiang et al., 2003). Notably, proteolytic talin cleav-
age and release of the head domain, although reported
to induce integrin activation, may release the activated
integrin from the actin cytoskeleton, a counterproductive
outcome for optimal stabilization of the integrin bond
under tensile forces. Intact talin is therefore most suited
to both anchor and unclasp the integrin cytoplasmic
domains upon chemoattractant activation of leukocytes
under shear stress conditions.
In conclusion, multiple features are required for an in-
tegrin to successfully generate firm adhesion at endothe-
lial contacts. Selectin- and integrin-mediated rolling may
slow down freely flowing leukocytes and thereby allow
them to survey the endothelial target for proper arrays of
gands (Figure 1). Chemoattractant signals may either in-
integrin unclasping. Preformed binding of extended integ-
rins to the cortical cytoskeleton (directly or via transmem-
brane associations with other anchored proteins or with
cytoskeleton-tethered lipids) will allow anchored integrins
to load low forces and undergo rapid (subsecond) ligand-
induced activation (Figure 2, outside-in). Integrins pre-
in these rapid steps (Figure 1). Integrins may also need to
adhesion (Cambi et al., 2006), and endothelial integrin li-
gands are more adhesive in their dimeric states (Miller
et al., 1995), which rapidly drive integrin microclustering
(Kim et al., 2004). Positive feedback loops between che-
moattractant-driven integrin unclasping, ligand-induced
headpiece activation, and dimerization of integrin-ligand
complexes may also be possible. Once nascent adhesion
isgenerated, pools ofrecruited high-mobilityintegrinscan
further enhance leukocyte adhesion through multivalent
interactions (Giagulli et al., 2004; Kim et al., 2004).
Earliest LFA-1 Activation Events Triggered in the
Immune Synapse: A Role for Intracellular Forces?
LFA-1 is used for arrest not only on the blood-vessel wall
but within the T cell zones of secondary lymphoid tissues
(Dustin, 2004). In the steady state, T cells undergo rapid
amoeboid locomotion in a variety of tissue contexts
(Geissmann et al., 2005; Miller et al., 2003). This motility
is thought to be critical in the search for rare antigen-
expressing cells. Once T lymphocytes locate cells with
antigenic MHC-peptide complexes, they decelerate from
>10 mm/min to <2 mm/min in order to form stable interac-
tions with antigen-presenting cells (APCs) (Shakhar et al.,
2005). Such interactions include both priming of naive T
cells and activation of effector cells, both of which can in-
volve rapid arrest (Mempel et al., 2006). While slow by
comparison to the blood-flow-induced velocities at the
vessel wall, the amoeboid motion of T cells within tissue
is rapid, reaching speeds of up to 30 mm/min. Unlike fibro-
blasts, which contract collagen gels by generating high
levels of forcethrough focal adhesion-like structures, cells
undergoing amoeboid locomotion do not contract colla-
gen gels and do not dramatically remodel their environ-
ment (Wolf et al., 2003).
apse, and LFA-1 is the best-studied molecule involved in
this process (Grakoui et al., 1999; Monks et al., 1998).
Immunity 26, January 2007 ª2007 Elsevier Inc.
Ligand-induced LFA-1 activation may take place more
readily when LFA-1 is subjected to internally applied
forces during immunological synapse formation (Varma
et al., 2006). The observation that supported planar bila-
yers, in which proteins have free lateral mobility, support
immunological synapse formation and full T cell activation
would seem to indicate that cytoskeletal anchorage of
molecules in the APC may not be important. However, un-
like biological membranes from which nonanchored pro-
teins pull long elastic membrane tethers when subjected
to ?10 pN forces, the planar bilayer does not allow pulling
of such tethers and instead is rigid in the vertical dimen-
sion due to trapping at the glass surface. Thus, molecules
in the bilayer have the physical signature of cytoskeletally
anchored proteins when pulled vertically. The earliest
LFA-1 activation events underlying the initiation of firm T
et al., 2005; Dustin et al., 1997; Negulescu et al., 1996) but
involve weak inside-out conformational switches prior to
ligand binding (R. Pasvolsky and R.A., unpublished
data). These events may therefore involve the local trig-
gering of anchored LFA-1 subsets, which are prone to
load internally generated forces via Ca2+-stimulated myo-
sin II activation and contraction (Figure 3).
The immunological synapse formed on round APCs or
on planar substrates is a radially symmetric contact inter-
intermediate ring, and a core containing accumulated
TCRs and protein-sorting and secretory compartments
(Grakoui et al., 1999; Monks et al., 1998; Stinchcombe
et al., 2001). Kupfer and colleagues (Monks et al., 1998)
defined the central TCR/sorting/secretory region as the
central supramolecular activation cluster (cSMAC). The
Figure 3. A Proposed Role for Internal, Cytoskeleton-Driven Forces in the Interactions of TCR and Integrin Clusters
in the Immunological Synapse
Schematic representation of a region in the pSMAC of the immunological synapse. Integrins on lymphocytes appear to be extensively preclustered
(Cambi et al., 2006).These clusters may be inactive on resting lymphocytes (1);following TCRmicrocluster activation, inside-out signaling (2) induces
cytoskeletal association of LFA-1 and LFA-1 extension (3), rendering the integrin competent for ICAM-1 binding. Within seconds, myosin II-mediated
contraction or retrograde actin flow exerts low forces on the integrin-ligand complexes to induce rapid and full outside-in activation in a ligand- and
anchorage-dependent manner (4). The LFA-1 and ICAM-1clusters may also work against eachother across thesynapseto maintain tension and fully
arrest the T cell as it locomotes over the APC.
Immunity 26, January 2007 ª2007 Elsevier Inc.
(Figure 3). The outer F-actin ring is also enriched in CD45
and has been defined as the distal SMAC (dSMAC). The
dSMAC undergoes cycles of actin polymerization-depen-
move around the periphery of the synapse in circular
waves (Dobereiner et al., 2005). This periodic extension/
contraction activity, together with intermediate filaments
and microtubules, may allow diverse cell types to sense
the physical properties of the substrate, whether soft or
rigid (Giannone and Sheetz, 2006). Peak forces evolve
during the contraction phase, and integrins engaged dur-
ing the actin-extension phase would be subjected to ver-
tical and lateral forces. The magnitude of these forces
would depend upon the rigidity of the substrate and the
cytoskeletal anchorage of ICAM-1 and stimulatory mole-
cules on the APC surface. LFA-1-ICAM-1 complexes
that areformedin thedSMAC coupleto the retrograde ac-
tin flow and translocate to the pSMAC, where they accu-
mulate and generate forces that are significant at the level
of single receptor-ligand pairs. Therefore, while there is
little translocation of cells during immunological synapse
through LFA-1-ICAM-1 complexes. Force-dependent
conformational changes in LFA-1 may therefore play an
important role in transduction of costimulatory signals in
T cells during the process of T cell activation. The ability
of the cell to exert forces on molecules held by the APC
may also be important in evaluating the affinity of antigen
receptors (Fleire et al., 2006).
Leukocyte integrins are versatile adhesion molecules
known to play important functions in both extravasation
and tissue interactions. Conformational flexibility of integ-
rins has been appreciated traditionally through studies
with conformation-sensitive antibodies and more recently
from structural studies and molecular dynamic simula-
tions. Force is an accelerator of integrin-mediated lym-
phocyte adhesion in the context of arrest from flow and
immunological synapse formation. This new awareness
of force as a critical signal in rapid integrin activation by
its own ligand may change the model for LFA-1 activation
by inside-out signals. When external (shear-based) or in-
ternal (cytoskeleton-based) forces on individual bonds
are negligible during leukocyte motility, freely mobile
LFA-1 may contribute to adhesion by clustering and inter-
acting with ligand and only then undergoing various cyto-
skeletal associations. However, in the presence of exter-
nal shear forces, cytoskeletal anchorage of LFA-1 and
other integrins can be essential even prior to ligand bind-
ing. We propose that in the immunological synapse,
forces internally applied by the cytoskeleton to nascent
LFA-1-ICAM-1 interactions could also facilitate LFA-1 ac-
tivation. Thus, we can no longer study how GPCRs and
TCRs transmit inside-out signals to integrins without
also fully dissecting how cytoskeletal assemblies of these
integrins promote ligand-induced force-facilitated integrin
activation. Future studies will need to resolve how differ-
ent cytoskeletal assemblies are regulated in distinct types
of immune cells to translate external and internal forces
into rapid generation of integrin adhesions in endothelial
and immunological synapses.
We wish to thank Drs. Sara Feigelson and Shelley Schwarzbaum for
comments on the manuscript and Chana Vega for help with figures.
R.A. is the Linda Jacobs Chair in Immune and Stem Cell Research
gram for Migration and Inflammation, and the Minerva Foundation.
M.L.D. is supported by NIH grants AI044931 and EY016586 (Nano-
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