Circulating neutrophils initiate locomotion on the luminal surface of endothelial cells after the rolling leukocyte firmly arrests at a site of inflammation. The adhesive mechanisms are partially understood, and generic models have evolved in which a cascade of adhesive and signaling events leads from tethering under shear to migration into extravascular spaces.1 2 The cells pass through stages supported by both distinct and overlapping sets of adhesion molecules that are clearly more than simple tethers. Some of these molecules have been shown to trigger signaling pathways involved in subsequent effector functions and successful translocation of neutrophils from the vessel lumen to the extravascular tissue. Each stage follows events that apparently alter the neutrophil's reaction to new surfaces and stimuli.

Members of the selectin family of adhesion molecules (L-, E- and P-selectin)3 exhibit binding characteristics that are sufficiently rapid to catch flowing leukocytes and sustain a dynamic interaction between leukocytes and endothelial cells that is evident as cell rolling. This rolling phenomenon in experimental models in vitro has been resolved by high-speed videomicroscopy as a series of discrete pauses whose duration (in msec at physiologic shear rates) and step distance are affected by a number of biophysical factors, including site density of the selectin or the ligand.4 These steps apparently represent receptor-ligand dissociation events, and it has been estimated that as few as two adhesive bonds per step between leukocyte and substrate are sufficient to support rolling.5At this stage, neutrophils are spherical in shape, and the dominant anatomical display of the adhesion molecules that tether under shear and initiate and sustain rolling (eg, L-selectin and the principal ligand for P-selectin, PSGL-1, CD162)6 is at the tips of microvillus-like projections from the neutrophil surface.7-9 

For reasons that are yet poorly defined, rolling cells may arrest on the endothelial surface. This arrest is clearly dependent on β2 (CD18) integrins,10 and both CD11b/CD18 (Mac-1) and CD11a/CD18 (LFA-1)11 may serve this function. Intravital microscopic observations have shown that anti-CD18 antibodies prevent firm adhesion in vivo without altering leukocyte rolling. Blocking selectin functions also inhibits firm adhesion at the physiologic shear rates of post capillary venules.12 The apparent requirement for selectin-dependent rolling may have at least three components. The prolonged contact duration between leukocytes and endothelial surfaces is permissive for integrin bond formation. It has been estimated that contact durations of more than 25 msec are needed for CD18 integrin bonds to form,13 much longer than the contacts of free-flowing cells. In addition, there is evidence that L-selectin cross-linking can signal14 a number of functional responses in neutrophils (eg, upregulation of CD11b/CD18).15 Also, rolling provides enhanced opportunity for contact with surface bound chemokines (eg, IL-8). Evidence for possible synergy between L-selectin and chemokine signaling of CD18 integrin upregulation has been published.16 

Stationary neutrophils adherent to the luminal surface of endothelium frequently change shape and assume the characteristic bipolar configuration of motile cells.17 This event presumably results from contact with surface-bound chemokines or chemotactic factors.18 Observed in vitro, this event usually occurs within 1 to 2 minutes of contact with cytokine-activated endothelial cells and appears much the same as isolated neutrophils exposed to exogenous chemotactic stimulation19 (ie, initial ruffling followed by formation of lamellipodium and uropod). Transendothelial migration usually follows and is complete within 1 to 2 minutes. This stage of migration is relatively poorly understood with regard to the specific mechanisms that allow leukocytes to pass through the endothelial monolayer. Antibodies that inhibit LFA-1 (CD11a/CD18) adhesion are particularly effective in blocking transmigration,20 and mice with targeted deletion of CD11a exhibit marked reductions in neutrophil emigration at sites of inflammation.21,22 In contrast, antibodies that block Mac-1 (CD11b/CD18) adhesion are marginally effective,20 and mice deficient in CD11b exhibit no deficit in neutrophil emigration at inflammatory sites.23,24 In addition, there is recent evidence that neutrophils express α9β1,25 an integrin that binds VCAM-1 (CD106) and some extracellular matrix molecules. Blocking antibody to α9 is reported to inhibit transendothelial migration,25 though some uncertainty remains regarding the contributions of β1 integrins to the process of neutrophil transendothelial migration.26 There is essentially nothing known about the adhesive events that occur as the neutrophil penetrates the endothelial monolayer. There may be important tissue or stimulus-specific factors that determine these events since there is evidence that neutrophils may pass through interendothelial clefts in restricted regions27 or transcellularly with specific stimuli.28 

The shape change that follows chemotactic stimulation of neutrophils is accompanied by a number of potentially important changes in surface molecules. L-selectin is shed, and the mechanisms of this shedding have been partially defined.29 CD43 and CD44 are partially shed from the surface. PSGL-1,7 FcRII, and Mac-1 (CD11b/CD18)30 are translocated to the uropod, while chemotactic receptors31 (eg, formyl peptide receptors), CR4 (CD11c/CD18),32 and urokinase plasminogen activator receptor (uPAR)30 are translocated to the forward regions of the cell. Some of these changes occur within the time frame of transendothelial migration, and others are delayed. For example, the shedding of L-selectin is pronounced within 2 to 3 minutes. The translocation of Mac-1 to the uropod, and the movement of formyl peptide receptors CR4 and uPAR to the front follows the kinetics of the initial shape change.

In the current issue of Blood, Seveau et al provide new evidence that CD43 is directly related to neutrophil polarity and motility. They demonstrate that chemotactic stimulation leading to neutrophil motility is accompanied by translocation of CD43 to the uropod with kinetics that follows shape change, and they define a cytoskeletal link of CD43 through moesin. It is reasonable to assume that this redistribution of CD43 may occur with the initial shape change event of firmly adherent neutrophils on the endothelial surface. If this is true, the leading edge of the neutrophil would likely begin transendothelial migration leukocyte/endothelial membrane contacts without CD43. If CD43 is antiadhesive as has been proposed, its absence may facilitate adhesion at the point of transmigration. However, one important published observation raises concern with this model. Woodman et al33 found that transendothelial migration, as observed by intravital microscopy, was markedly depressed in mice with targeted deletion of CD43, even though adhesion was significantly increased. Thus, it appears that the absence of CD43 is not sufficient. An alternative possibility is that CD43 is necessary for the initial shape change and migration. Seveau et al demonstrate that cross-linking CD43 induces shape change and motility, and others have shown that cross-linking CD43 stimulates neutrophil adhesion.34 In this model, the signaling function of CD4335 may be more important than relief from its antiadhesive effects that attend shedding or redistribution. It remains to be seen what mechanisms could possibly trigger a signaling event through CD43 at the endothelial surface.

With transendothelial migration, neutrophils encounter extracellular matrix, a context where β1 integrins are important. Additional cellular changes occur with this transmigration that appear to be relevant to neutrophil functions in this context. A large portion of cell surface L-selectin is shed,36 and CD43 surface levels are significantly reduced.37 There are increases in surface Mac-1 (CD11b/CD18) and α4β1. Kubes et al have shown that, following transendothelial migration, neutrophils exhibit significant increases in α4β1-dependent adhesion.38 The contribution this integrin makes to cell locomotion in the extravascular space is uncertain, but neutrophil adhesion to parenchymal cells is significantly augmented with consequent cell injury.39 In addition to these integrins, Shang et al have reported that α9β1 contributes to neutrophil migration through monolayers of fibroblasts in vitro.26 

Mac-1 (CD11b/CD18), an integrin that binds to a variety of ligands, has the potential to support cell locomotion. During migration on protein-coated glass surfaces, Francis et al40 examined the surface distribution of Mac-1 on neutrophils responding to fMLP stimulation. Using sequential two-color labeling with antibodies to CD11b, they found that the initial label was translocated to the uropod and retraction fibers. Once this distribution pattern was evident, addition of a second labeled anti-CD11b antibody revealed binding to the body of the cells. When neutrophils were fixed and permeabilized prior to the second label, the second label was localized to a granular compartment near the lamellipodia. They concluded that Mac-1, which is contained in the secondary granule compartment of the neutrophil, is delivered to the lamellipodium and cell body and then translocated to the uropod and retraction fibers with cell locomotion. In similar experiments, Hughes et al41 examined the distribution of albumin-coated latex beads (ACLB) on the surface of fMLP-stimulated neutrophils in suspension. Binding of these beads is completely inhibited by anti-CD11b antibodies, indicating that the beads reflect Mac-1–dependent adhesion sites. Following exposure of neutrophils to the beads and a single concentration of fMLP, surface-bound beads translocated to the uropod. If these cells with uropod-bound beads were then exposed to a step increase in the concentration of fMLP coincident with exposure to additional ACLB, the newly bound beads were consistently (within 20 to 30 seconds of stimulation) on the lamellipodia of polarized cells. The newly bound beads then translocated to the uropod. Cell locomotion on albumin-coated planar surfaces followed the same set of events, consistent with the interpretation that Mac-1–dependent adhesion sites on the lamellipodia could be upregulated by increases in chemotactic stimulation. These results suggest that an internal pool of Mac-1 in secondary granules42 could be mobilized and respond to increases in chemotactic stimulation.

In the current issue of Blood, Pierini et al demonstrate an important new observation regarding a role for α5β1. In contrast to studies with Mac-1, where there is a substantial storage pool in the secondary granules, α5β1 recycles from uropod to lamellipodium through an endocytic recycling compartment. This integrin is then displayed on the cell surface in the advancing regions of motile cells and subsequently translocated to the uropod. The recycling compartment retains its localization just behind the leading lamellipodium as the neutrophil migrates. Such a mechanism would contribute to neutrophil locomotion through connective tissue in the extravascular space, a compartment where α5β1 has been shown to be important for migration.43 These observations were evident with neutrophils migrating on fibronectin-coated surfaces (a ligand for this integrin). These and previous studies from these investigators regarding αvβ3-dependent interactions of neutrophils migrating on vitronectin-coated surfaces44raise our understanding of the multiplicity of mechanisms for locomotion that neutrophils can use as they encounter different substrates on their trek from blood into tissue.

Reprints:Wayne Smith, Section of Leukocyte Biology, Children's Nutrition Research Center, 1100 Bates, Room 6014, Houston, TX 77030.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Issekutz
AC
Adhesion molecules mediating neutrophil migration to arthritis in vivo and across endothelium and connective tissue barriers in vitro.
Inflamm Res.
47(suppl 3)
1998
S123
2
Repo
H
Harlan
JM
Mechanisms and consequences of phagocyte adhesion to endothelium.
Ann Med.
31
1999
156
3
Vestweber
D
Blanks
JE
Mechanisms that regulate the function of the selectins and their ligands.
Physiol Rev.
79
1999
181
4
Smith
MJ
Berg
EL
Lawrence
MB
A direct comparison of selectin-mediated transient, adhesive events using high temporal resolution [In Process Citation].
Biophys J.
77
1999
3371
5
Alon
R
Chen
S
Puri
KD
Finger
EB
Springer
TA
The kinetics of L-selectin tethers and the mechanics of selectin-mediated rolling.
J Cell Biol.
138
1997
1169
6
Yang
J
Furie
BC
Furie
B
The biology of P-selectin glycoprotein ligand-1: its role as a selectin counterreceptor in leukocyte-endothelial and leukocyte-platelet interaction.
Thromb Haemost.
81
1999
1
7
Bruehl
RE
Moore
KL
Lorant
DE
et al
Leukocyte activation induces surface redistribution of P-selectin glycoprotein ligand-1.
J Leukoc Biol.
61
1997
489
8
Bruehl
RE
Springer
TA
Bainton
DF
Quantitation of L-selectin distribution on human leukocyte microvilli by immunogold labeling and electron microscopy.
J Histochem Cytochem.
44
1996
835
9
von Andrian
UH
Hasslen
SR
Nelson
RD
Erlandsen
SL
Butcher
EC
A central role for microvillous receptor presentation in leukocyte adhesion under flow.
Cell.
82
1995
989
10
Bienvenu
K
Granger
DN
Molecular determinants of shear rate-dependent leukocyte adhesion in postcapillary venules.
Am J Physiol.
264
1993
H1504
11
Gopalan PK, Burns AR, Simon SI, Sparks S, McIntire LV, Smith CW. Preferential sites for stationary adhesion of neutrophils to cytokine-stimulated HUVEC under flow conditions. J Leukoc Biol. In press.
12
von Andrian
UH
Hansell
P
Chambers
JD
et al
L-selectin function is required for beta-2 integrin-mediated neutrophil adhesion at physiologic shear rates in vivo.
Am J Physiol.
263
1992
H1034
13
Taylor
AD
Neelamegham
S
Hellums
JD
Smith
CW
Simon
SI
Molecular dynamics of the transition from L-selectin- to β2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear.
Biophys J.
71
1996
3488
14
Simon
SI
Cherapanov
V
Nadra
I
et al
Signaling functions of L-selectin in neutrophils: alterations in the cytoskeleton and colocalization with CD18.
J Immunol.
163
1999
2891
15
Gopalan
PK
Smith
CW
Lu
H
Berg
EL
McIntire
LV
Simon
SI
Neutrophil CD18-dependent arrest on ICAM-1 in shear flow can be activated through L-selectin.
J Immunol.
158
1997
367
16
Tsang
YTM
Neelamegham
S
Hu
Y
et al
Synergy between L-selectin signaling and chemotactic activation during neutrophil adhesion and transmigration.
J Immunol.
159
1997
4566
17
Smith
CW
Marlin
SD
Rothlein
R
Toman
C
Anderson
DC
Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro.
J Clin Invest.
83
1989
2008
18
Rot
A
Neutrophil attractant/activation protein-1 (interleukin-8) induces in vitro neutrophil migration by haptotactic mechanism.
Eur J Immunol.
23
1993
303
19
Zigmond
SH
Levitsky
HI
Kreel
BJ
Cell polarity: an examination of its behavioral expression and its consequences for polymorphonuclear leukocyte chemotaxis.
J Cell Biol.
89
1981
585
20
Furie
MB
Tancinco
MCA
Smith
CW
Monoclonal antibodies to leukocyte integrins CD11a/CD18 and CD11b/CD18 or intercellular adhesion molecule-1 (ICAM-1) inhibit chemoattractant-stimulated neutrophil transendothelial migration in vitro.
Blood.
78
1991
2089
21
Schmits
R
Kundig
TM
Baker
DM
et al
LFA-1-deficient mice show normal CTL responses to virus but fail to reject immunogenic tumor.
J Exp Med.
183
1996
1415
22
Ding
Z-M
Babensee
JE
Simon
SI
et al
Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.
J Immunol.
163
1999
5029
23
Lu
H
Smith
CW
Perrard
J
et al
LFA-1 is sufficient in mediating neutrophil emigration in Mac-1 deficient mice.
J Clin Invest.
99
1997
1340
24
Coxon
A
Rieu
P
Barkalow
FJ
et al
A novel role for the beta 2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation.
Immunity.
5
1996
653
25
Taooka
Y
Chen
J
Yednock
T
Sheppard
D
The integrin α9β1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1.
J Cell Biol.
145
1999
413
26
Shang
T
Yednock
T
Issekutz
AC
α9β1 integrin is expressed on human neutrophils and contributes to neutrophil migration through human lung and synovial fibroblast barriers.
J Leukoc Biol.
66
1999
809
27
Burns
AR
Walker
DC
Brown
ES
et al
Neutrophil transendothelial migration is independent of tight junctions and occurs preferentially at tricellular corners.
J Immunol.
159
1997
2893
28
Feng
D
Nagy
JA
Pyne
K
Dvorak
HF
Dvorak
AM
Neutrophils emigrate from venules by a transendothelial cell pathway in response to FMLP.
J Exp Med.
187
1998
903
29
Feehan
C
Darlak
K
Kahn
J
Walcheck
B
Spatola
AF
Kishimoto
TK
Shedding of the lymphocyte L-selectin adhesion molecule is inhibited by a hydroxamic acid-based protease inhibitor.
J Biol Chem.
271
1996
7019
30
Kindzelskii
AL
Laska
ZO
Todd
RF
III
Petty
HR
Urokinase-type plasminogen activator receptor reversibly dissociates from complement receptor type 3 (αMβ2, CD11b/CD18) during neutrophil polarization.
J Immunol.
156
1996
297
31
Sullivan
SJ
Daukas
G
Zigmond
SH
Asymmetric distribution of the chemotactic peptide receptor on polymorphonuclear leukocytes.
J Cell Biol.
99
1984
1461
32
Kindzelskii
AL
Eszes
MM
Todd
RF
Petty
HR
Proximity oscillations of complement type 4 (alphaX beta2) and urokinase receptors on migrating neutrophils.
Biophys J.
73
1997
1777
33
Woodman
RC
Johnston
B
Hickey
MJ
et al
The functional paradox of CD43 in leukocyte recruitment: a study using CD43-deficient mice.
J Exp Med.
188
1998
2181
34
Kuijpers
TW
Hoogerwerf
M
Kuijpers
KC
Schwartz
BR
Harlan
JM
Cross-linking of sialophorin (CD43) induces neutrophil aggregation in a CD18-dependent and a CD18-independent way.
J Immunol.
149
1992
998
35
Anzai
N
Gotoh
A
Shibayama
H
Broxmeyer
HE
Modulation of integrin function in hematopoietic progenitor cells by CD43 engagement: possible involvement of protein tyrosine kinase and phospholipase C-gamma.
Blood.
93
1999
3317
36
Kishimoto
TK
Jutila
MA
Berg
EL
Butcher
EC
Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors.
Science.
245
1989
1238
37
Lopez
S
Halbwachs-Mecarelli
L
Ravaud
P
Bessou
G
Dougados
M
Porteu
F
Neutrophil expression of tumour necrosis factor receptors (TNF-R) and of activation markers (CD11b, CD43, CD63) in rheumatoid arthritis.
Clin Exp Immunol.
101
1995
25
38
Kubes
P
Niu
X
Smith
CW
Kehrli
ME
Jr
Reinhardt
PH
Woodman
RC
A novel β1-dependent adhesion pathway on neutrophils: a mechanism invoked by dihydrocytochalasin B or endothelial transmigration.
FASEB J.
9
1995
1103
39
Poon
BY
Ward
CA
Giles
WR
Kubes
P
Emigrated neutrophils regulate ventricular contractility via alpha-4 integrin.
Circ Res.
84
1999
1245
40
Francis
JW
Todd
RF
III
Boxer
LA
Petty
HR
Sequential expression of cell surface C3bi receptors during neutrophil locomotion.
J Cell Physiol.
140
1989
519
41
Hughes
BJ
Hollers
JC
Crockett-Torabi
E
Smith
CW
Recruitment of CD11b/CD18 to the neutrophil surface and adherence-dependent cell locomotion.
J Clin Invest.
90
1992
1687
42
Jones
DH
Schmalstieg
FC
Hawkins
HK
et al
Characterization of a new mobilizable Mac-1 (CD11b/CD18) pool that co-localizes with gelatinase in human neutrophils.
Leukocyte Adhesion Molecules: Structure, Function, and Regulation.
Springer
TA
Anderson
DC
Rosenthal
AS
Rothlein
R
1989
106
NY Springer-Verlag
New York
43
Werr
J
Xie
X
Hedqvist
P
Ruoslahti
E
Lindbom
L
Beta1 integrins are critically involved in neutrophil locomotion in extravascular tissue in vivo.
J Exp Med.
187
1998
2091
44
Lawson
MA
Maxfield
FR
Ca(2+)- and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils.
Nature.
377
1995
75
Sign in via your Institution