Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2459-2461
FOCUS ON HEMATOLOGY
Introduction: functional polarity of motile neutrophils
C. Wayne Smith
From the Section of Leukocyte Biology, Children's Nutrition
Research Center, 1100 Bates, Room 6014, Houston, TX 77030.
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Article |
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.5
At 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
2 (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 
91,25 an integrin that binds
VCAM-1 (CD106) and some extracellular matrix molecules. Blocking
antibody to 9 is reported to inhibit transendothelial
migration,25 though some uncertainty remains regarding the
contributions of 1 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 1 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 41. Kubes
et al have shown that, following transendothelial migration,
neutrophils exhibit significant increases in
41-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 91 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
51. In contrast to studies with Mac-1,
where there is a substantial storage pool in the secondary granules,
51 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 51 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 v3-dependent interactions of
neutrophils migrating on vitronectin-coated surfaces44
raise 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.
| |
Footnotes |
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.
| |
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