Introduction: functional polarity of motile neutrophils

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 α_vβ₃-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.