Mechanical shedding of L-selectin from the neutrophil surface during rolling on sialyl Lewis x under flow

The interaction of L-selectin expressed on leukocytes with endothelial cells leads to capture and rolling and is critical for the recruitment of leukocytes into sites of inflammation. It is known that leukocyte activation by chemoattractants, the change of osmotic pressure in cell media, or cross-linking of L-selectin all result in rapid shedding of L-selectin. Here we present a novel mechanism for surface cleavage of L-selectin on neutrophils during rolling on a sialyl Lewis x-coated surface that involves mechanical force. Flow cytometry and rolling of neutrophils labeled with Qdot(R)-L-selectin antibodies in an in vitro flow chamber showed that the mechanical shedding of L-selectin occurs during rolling and depends on the amount of shear applied. In addition, the mechanical L-selectin shedding causes an increase in cell rolling velocity with rolling duration, suggesting a gradual loss of L-selectin and is mediated by p38 mitogen-activated protein kinase activation. Thus, these data show that mechanical force induces the cleavage of L-selectin from the neutrophil surface during rolling and therefore decreases the adhesion of cells to a ligand-presenting surface in flow.     

A potential role for annexin 1 as a physiologic mediator of glucocorticoid-induced L-selectin shedding from myeloid cells

Glucocorticoids can dampen inflammatory responses by inhibiting neutrophil recruitment to tissue sites. The detailed mechanism by which glucocorticoids exert this affect on neutrophils is unknown. L-selectin is a leukocyte cell surface receptor that is implicated in several steps of neutrophil recruitment. Recently, several studies have shown that systemic treatment of animals and humans with glucocorticoids induces decreased L-selectin expression on neutrophils, suggesting one mechanism by which inflammation may be negatively regulated. However, when neutrophils are treated in vitro with glucocorticoids, no effect on L-selectin expression is observed. Thus, the existence of an additional mediator is plausible. In this study, we investigate whether annexin 1 (ANX1), a recognized second messenger of glucocorticoids, could be such a mediator. We show that ANX1 induces a dose- and time-dependent decrease in L-selectin expression on both peripheral blood neutrophils and monocytes but has no effect on lymphocytes. The loss of L-selectin from neutrophils is due to shedding that is mediated by a cell surface metalloprotease ("sheddase"). Using cell shape and a beta(2) integrin activation epitope, we show that the ANX1-induced shedding of L-selectin appears to occur without overt cell activation. These data may provide the basis for further understanding of mechanisms involved in the down-regulation of inflammatory responses.     

Neutrophil adhesion is impaired in the mesentery but not in the liver sinusoids of portal hypertensive rats

Altered leukocyte/cytokine response to inflammation has been observed in human and experimental portal hypertension. The aim of this study was to characterize leukocyte adhesion in portal hypertensive (PPVL) rats stimulated with endotoxin. Leukocyte rolling, adhesion, and migration assessed by intravital microscopy were impaired in mesenteric venules after lipopolysaccharide administration (150 microg/kg) in PPVL vs. sham-operated rats. Analysis of leukocyte L-selectin expression and soluble L-selectin showed that this defective adhesion was related to increased L-selectin shedding. In vitro experiments using isolated leukocytes treated with phorbol 12-myristate 13-acetate showed that monocytes and neutrophils but not lymphocytes were hyperreactive to cell activation, as measured by CD11b overexpression and increased L-selectin shedding in PPVL rats. However, neutrophil emigration in liver sinusoids and in the lung 3 h after endotoxin injection were similar in both groups of animals. Thus the alterations in leukocyte activation and adhesion molecule expression observed in this study may contribute to a better understanding of the higher susceptibility and severity of bacterial infections in cirrhotic patients with portal hypertension.     

Shear-induced resistance to neutrophil activation via the formyl peptide receptor

The application of fluid shear stress on leukocytes is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. The formyl peptide receptor (FPR) on neutrophils, which binds to formyl-methionyl-leucyl-phenylalanine (fMLP) and plays a role in neutrophil chemotaxis, has been implicated as a fluid shear stress sensor that controls pseudopod formation. The role of shear forces on earlier indicators of neutrophil activation, such as L-selectin shedding and α(M)β(2) integrin activation, remains unclear. Here, human neutrophils exposed to uniform shear stress (0.1-4.0 dyn/cm(2)) in a cone-and-plate viscometer for 1-120 min showed a significant reduction in both α(M)β(2) integrin activation and L-selectin shedding after stimulation with 0.5 nM of fMLP. Neutrophil resistance to activation was directly linked to fluid shear stress, as the response increased in a shear stress force- and time-dependent manner. Significant shear-induced loss of FPR surface expression on neutrophils was observed, and high-resolution confocal microscopy revealed FPR internalized within neutrophils. These results suggest that physiological shear forces alter neutrophil activation via FPR by reducing L-selectin shedding and α(M)β(2) integrin activation in the presence of soluble ligand.     

Tangential Tether Extraction and Spontaneous Tether Retraction of Human Neutrophils

Membrane tethers are extracted when neutrophils roll on the endothelium to initiate their transendothelial migration. Tether extraction from both neutrophils and endothelial cells stabilizes neutrophil rolling, so it has been studied extensively and the force-velocity relationship for tether extraction is of great interest. Due to limitations of the techniques used in previous studies, this relationship has been obtained only from tethers perpendicular to the cell surface. Here, with the microcantilever technique, where latex beads affixed on silicon cantilevers were used as the force transducer, we extracted tethers either perpendicular or tangential to the neutrophil surface. We found that the force-velocity relationship was not sensitive to tether pulling direction. Little movement of the tether-cell junction was observed during tangential tether extraction, and no coalescence was observed during multiple tether extraction. Following adhesion rupture, spontaneous tether retraction was visualized by membrane staining, which revealed two phases: one was fast and exponential, whereas the other was slow and linear. Both phases can be reproduced with a mechanical model. These results show for the first time, to our knowledge, how neutrophil tethers shorten upon instantaneous force removal, and furthermore, they illustrate how membrane tethers contribute to neutrophil rolling stability during the inflammatory response.     

Mechanical anchoring strength of L-selectin, beta2 integrins, and CD45 to neutrophil cytoskeleton and membrane.

The strength of anchoring of transmembrane receptors to cytoskeleton and membrane is important in cell adhesion and cell migration. With micropipette suction, we applied pulling forces to human neutrophils adhering to latex beads that were coated with antibodies to CD62L (L-selectin), CD18 (beta2 integrins), or CD45. In each case, the adhesion frequency between the neutrophil and bead was low, and our Monte Carlo simulation indicates that only a single bond was probably involved in every adhesion event. When the adhesion between the neutrophil and bead was ruptured, it was very likely that receptors were extracted from neutrophil surfaces. We found that it took 1-2 s to extract an L-selectin at a force range of 25-45 pN, 1-4 s to extract a beta2 integrin at a force range of 60-130 pN, and 1-11 s to extract a CD45 at a force range of 35-85 pN. Our results strongly support the conclusion that, during neutrophil rolling, L-selectin is unbound from its ligand when the adhesion between neutrophils and endothelium is ruptured.     

Sulfhydryl regulation of L-selectin shedding: phenylarsine oxide promotes activation-independent L-selectin shedding from leukocytes

The L-selectin adhesion molecule mediates leukocyte recruitment to inflammatory sites and lymphocyte trafficking through the peripheral lymph nodes. In response to leukocyte activation, L-selectin is proteolytically released from the cell surface, disabling leukocytes from the subsequent L-selectin-dependent interactions. We have found that L-selectin shedding is sensitive to sulfhydryl chemistry; it is promoted by thiol-oxidizing or -blocking reagents and inhibited by reducing reagents. Phenylarsine oxide (PAO), a trivalent arsenical that interacts with vicinal dithiols, is most potent in inducing rapid shedding of L-selectin from isolated neutrophils, eosinophils, and lymphocytes as well as from neutrophils in whole blood. PAO does not cause cell activation, nor does it interfere with integrin function or alter the expression of several other cell surface molecules at the low concentrations that induce L-selectin shedding. PAO is not required to enter the cell to induce L-selectin shedding. TAPI-2 ((N-(D,L-[2-(hydroxyaminocarbonyl)-methyl]-4-methylpentanoyl)-L-3-(tert-butyl)-alanyl-l -alanine, 2-aminoethyl amide), which has previously been shown to inhibit the activation-dependent L-selectin shedding, is also capable of inhibiting PAO-induced L-selectin shedding. We hypothesize that PAO-induced L-selectin shedding involves a regulatory molecule, such as protein disulfide isomerase (PDI), an enzyme that plays a role in the formation and rearrangement of disulfide bonds, contains PAO-binding, vicinal dithiol-active sites, and is expressed on the neutrophil surface. Cell surface expression of PDI, L-selectin shedding induced by PDI-blocking Abs and by bacitracin, a known inhibitor of PDI activity, and direct binding of PDI to PAO, provide supporting evidence for this hypothesis.     

P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells

Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L- selectin/IgM heavy chain (mu) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34+ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and leukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P- and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/mu chimera to inhibit L-selectin/mu binding to leukocytes and by the complete inhibition of both selectin interactions with myeloid cells treated with mocarhagin, a cobra venom metalloproteinase that cleaves the amino terminus of PSGL-1 at Tyr-51. Finally, the abrogation of L- and P-selectin binding to myeloid cells treated with a polyclonal antibody, raised against a peptide corresponding to the amino acid residues 42-56 of PSGL-1, indicated that L- and P-selectin interact with a domain located at the amino- terminal end of PSGL-1. The ability of the anti-PSGL-1 mAb PL-1 to inhibit L- and P-selectin binding to KG-1 cells further supported that possibility. Thus, apart from being involved in neutrophil rolling on P- and E-selectin, PSGL-1 also plays a critical role in mediating neutrophil attachment to adherent neutrophils. Interaction between L- selectin and PSGL-1 may be of major importance for increasing leukocyte recruitment at inflammatory sites.     

Painful stimulation suppresses joint inflammation by inducing shedding of L-selectin from neutrophils.

Although the inflammatory response is essential for protecting tissues from injury and infection, unrestrained inflammation can cause chronic inflammatory diseases such as arthritis, colitis and asthma. Physiological mechanisms that downregulate inflammation are poorly understood. Potent control might be achieved by regulating early stages in the inflammatory response, such as accumulation of neutrophils at the site of injury, where these cells release chemical mediators that promote inflammatory processes including plasma extravasation, bacteriocide and proteolysis. To access an inflammatory site, neutrophils must first adhere to the vascular endothelium in a process mediated in part by the leukocyte adhesion molecule L-selectin. This adhesion is prevented when L-selectin is shed from the neutrophil membrane. Although shedding of L-selectin is recognized as a potentially important mechanism for regulating neutrophils, its physiological function has not been demonstrated. Shedding of L-selectin may mediate endogenous downregulation of inflammation by limiting neutrophil accumulation at inflammatory sites. Here we show that activation of nociceptive neurons induces shedding of L-selectin from circulating neutrophils in vivo and that this shedding suppresses an ongoing inflammatory response by inhibiting neutrophil accumulation. These findings indicate a previously unknown mechanism for endogenous feedback control of inflammation. Failure of this mechanism could contribute to the etiology of chronic inflammatory disease.     

Rolling dynamics of a neutrophil with redistributed L-selectin

The most common white blood cell is the neutrophil, which slowly rolls along the walls of blood vessels due to the coordinated formation and breakage of chemical selectin-carbohydrate bonds. We show that L-selectin receptors are rapidly redistributed to form a cap at one end of the cell membrane during rolling via selectins or chemotactic stimulation. This topography significantly alters the adhesive dynamics as demonstrated by computer simulations of neutrophils rolling on a carbohydrate selectin-ligand substrate under flow. It was found that neutrophils with a redistributed L-selectin cap roll on sialyl Lewis-x with a quasi-periodic motion, as characterized by relatively low velocity intervals interspersed with regular jumps in the rolling velocity. On average, neutrophils with redistributed L-selectin rolled at a lower velocity when compared with cells having a uniform L-selectin distribution of equal average density. We speculate on the possible biological implications that these differences in adhesion dynamics will have during the inflammatory response.     

Inhibition of Na+/H+ exchanger enhances low pH-induced L-selectin shedding and beta2-integrin surface expression in human neutrophils

Ischemia-reperfusion injury is a common pathological occurrence causing tissue damage in heart attack and stroke. Entrapment of neutrophils in the vasculature during ischemic events has been implicated in this process. In this study, we examine the effects that lactacidosis and consequent reductions in intracellular pH (pH(i)) have on surface expression of adhesion molecules on neutrophils. When human neutrophils were exposed to pH 6 lactate, there was a marked decrease in surface L-selectin (CD62L) levels, and the decrease was significantly enhanced by inclusion of Na(+)/H(+) exchanger (NHE) inhibitor 5-(N,N-hexamethylene)amiloride (HMA). Similar effects were observed when pH(i) was reduced while maintaining normal extracellular pH, by using an NH(4)Cl prepulse followed by washes and incubation in pH 7.4 buffer containing NHE inhibitors [HMA, cariporide, or 5-(N,N-dimethyl)amiloride (DMA)]. The amount of L-selectin shedding induced by different concentrations of NH(4)Cl in the prepulse correlated with the level of intracellular acidification with an apparent pK of 6.3. In contrast, beta(2)-integrin (CD11b and CD18) was only slightly upregulated in the low-pH(i) condition and was enhanced by NHE inhibition to a much lesser extent. L-selectin shedding was prevented by treating human neutrophils with inhibitors of extracellular metalloproteases (RO-31-9790 and KD-IX-73-4) or with inhibitors of intracellular signaling via p38 MAP kinase (SB-203580 and SB-239063), implying a transmembrane effect of pH(i). Taken together, these data suggest that the ability of NHE inhibitors such as HMA to reduce ischemia-reperfusion injury may be related to the nearly complete removal of L-selectin from the neutrophil surface.     

Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding

A disintegrin and metalloproteinase domain (ADAM) proteins are a family of transmembrane glycoproteins with heterogeneous expression profiles and proteolytic, cell-adhesion, -fusion, and -signaling properties. One of its members, ADAM-8, is expressed by several cell types including neurons, osteoclasts, and leukocytes and, although it has been implicated in osteoclastogenesis and neurodegenerative processes, little is known about its role in immune cells. In this study, we show that ADAM-8 is constitutively present both on the cell surface and in intracellular granules of human neutrophils. Upon in vitro neutrophil activation, ADAM-8 was mobilized from the granules to the plasma membrane, where it was released through a metalloproteinase-dependent shedding mechanism. Adhesion of resting neutrophils to human endothelial cells also led to up-regulation of ADAM-8 surface expression. Neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis expressed higher amounts of ADAM-8 than neutrophils isolated from peripheral blood and the concentration of soluble ADAM-8 in synovial fluid directly correlated with the degree of joint inflammation. Remarkably, the presence of ADAM-8 both on the cell surface and in suspension increased the ectodomain shedding of membrane-bound L-selectin in mammalian cells. All these data support a potential relevant role for ADAM-8 in the function of neutrophils during inflammatory response.     

Shear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation of high-avidity beta2-integrin on neutrophils

Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewis(x) ligands and activation of beta(2)-integrins that support neutrophil arrest by binding ICAM-1. We have previously reported that neutrophils rolling on E-selectin are sufficient for signaling cell arrest through beta(2)-integrin binding of ICAM-1 in a process dependent upon ligation of L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1). Unresolved are the spatial and temporal events that occur as E-selectin binds to human neutrophils and dynamically signals the transition from neutrophil rolling to arrest. Here we show that binding of E-selectin to sialyl Lewis(x) on L-selectin and PSGL-1 drives their colocalization into membrane caps at the trailing edge of neutrophils rolling on HUVECs and on an L-cell monolayer coexpressing E-selectin and ICAM-1. Likewise, binding of recombinant E-selectin to PMNs in suspension also elicited coclustering of L-selectin and PSGL-1 that was signaled via mitogen-activated protein kinase. Binding of recombinant E-selectin signaled activation of beta(2)-integrin to high-avidity clusters and elicited efficient neutrophil capture of beta(2)-integrin ligands in shear flow. Inhibition of p38 and p42/44 mitogen-activated protein kinase blocked the cocapping of L-selectin and PSGL-1 and the subsequent clustering of high-affinity beta(2)-integrin. Taken together, the data suggest that E-selectin is unique among selectins in its capacity for clustering sialylated ligands and transducing signals leading to neutrophil arrest in shear flow.     

Human neutrophil surface protrusion under a point load: location independence and viscoelasticity

Mechanical properties of neutrophils have been recognized as key contributors to stabilizing neutrophil rolling on the endothelium during the inflammatory response. In particular, accumulating evidence suggests that surface protrusion and tether extraction from neutrophils facilitate stable rolling by relieving the disruptive forces on adhesive bonds. Using a customized optical trap setup, we applied piconewton-level pulling forces on targeted receptors that were located either on the microvillus tip (CD162) or intermicrovillus surface of neutrophils (CD18 and CD44). Under a constant force-loading rate, there always occurred an initial tent-like surface protrusion that was terminated either by rupture of the adhesion or by a "yield" or "crossover" to tether extraction. The corresponding protrusional stiffness of neutrophils was found to be between 0.06 and 0.11 pN/nm, depending on the force-loading rate and the cytoskeletal integrity, but not on the force location, the medium osmolality, nor the temperature increase from 22 degrees C to 37 degrees C. More importantly, we found that neutrophil surface protrusion was accompanied by force relaxation and hysteresis. In addition, the crossover force did not change much in the range of force-loading rates studied, and the protrusional stiffness of lymphocytes was similar to that of neutrophils. These results show that neutrophil surface protrusion is essentially viscoelastic, with a protrusional stiffness that stems primarily from the actin cortex, and the crossover force is independent of the receptor-cytoskeleton interaction.     

Lactoferrin inhibits the binding of lipopolysaccharides to L-selectin and subsequent production of reactive oxygen species by neutrophils

The activation of leukocytes by lipopolysaccharides (LPS), resulting in the oxidative burst, contributes to the pathogenesis of septic shock. The binding of LPS to L-selectin, which was reported as a serum-independent LPS receptor on neutrophils, induces the production of oxygen free radicals. Human lactoferrin (hLf), an anti-inflammatory glycoprotein released from neutrophil granules during infection, binds to LPS. In this study, we investigated the capacity of hLf to inhibit the L-selectin-mediated activation of neutrophils. Our experiments revealed that hLf prevents the binding of LPS to L-selectin in a concentration-dependent manner. Inhibition was maximum (87.7+/-0.5%) at a concentration of 50 microg/ml of hLf. Furthermore, hLf inhibited up to 55.4+/-0.5% of the intracellular hydrogen peroxide production induced by LPS in neutrophils. These findings suggest that the anti-inflammatory properties of hLf are due, at least in part, to their ability to prevent the binding of LPS to neutrophil L-selectin.     

Rho signaling inhibition mitigates lung injury via targeting neutrophil recruitment and selectin-AKT signaling

Neutrophils, the early responders of the immune system, eliminate intruders, but their over-activation can also instigate tissue damage leading to various autoimmune and inflammatory disease conditions. As approaches causing neutropenia are associated with immunodeficiency, targeting aberrant neutrophil infiltration offers an attractive strategy in neutrophil-centered diseases including acute lung injury. Rho GTPase family proteins Rho, Rac and Cdc42 play important role as regulators of chemotaxis in diverse systems. Rho inhibitors protected against lung injuries, while genetic Rho-deficiency exhibited neutrophil hyperactivity and exacerbated lung injury. These differential outcomes might be due to distinct effects on different cell types or activation/ inhibition of specific signaling pathways responsible for neutrophil polarity, migration and functions. In this study, we explored neutrophil centric effects of Rho signaling mitigation. Consistent with previous reports, Rho signaling inhibitor Y-27632 provided protection against acute lung injury, but without regulating LPS mediated systemic increase of neutrophils in the circulation. Interestingly, the adoptive transfer approach identified a specific defect in neutrophil migration capacity after Rho signaling mitigation. These defects were associated with loss of polarity and altered actin dynamics identified using time-lapse in vitro studies. Further analysis revealed a rescue of stimulation-dependent L-selectin shedding on neutrophils with Rho signaling inhibitor. Surprisingly, functional blocking of L-selectin (CD62L) led to defective recruitment of neutrophils into inflamed lungs. Further, single-cell level analyses identified MAPK signaling as downstream mechanism of Rho signaling and L-selectin mediated effects. p-AKT levels were diminished in detergent resistance membrane-associated signalosome upon Rho signaling inhibition and blockade of selectin. Moreover, inhibition of AKT signaling as well as selectin blocking led to defects in neutrophil polarity. Together, this study identified Rho-dependent distinct L-selectin and AKT signaling mediated regulation of neutrophil recruitment to inflamed lung tissue.     

Structure-function relationship and role of tumor necrosis factor-alpha-converting enzyme in the down-regulation of L-selectin by non-steroidal anti-inflammatory drugs

It has been recently described that some non-steroidal anti-inflammatory drugs (NSAIDs) are able to induce the shedding of L-selectin in neutrophils, an adhesion molecule that plays an essential role in the inflammatory response. We have found that, according to this capability, NSAIDs could be grouped into three categories. A high releaser group (flufenamic, meclofenamic, and mefenamic acids, diclofenac and aceclofenac), a group of moderate releasers (aspirin, indomethacin, nimesulide, flurbiprofen, and ketoprofen), and a non-releaser group (phenylbutazone and the oxicams, piroxicam and meloxicam). Only NSAIDs from the high releaser group shared diphenylamine in their chemical structure. The amine group of this chemical agent proved to be essential for the anti-L-selectin activity of diphenylamine-based NSAIDs. The presence of a carboxylic acid group in the diphenylamine (N-phenylanthranilic acid) highly increased its ability to reduce the L-selectin surface expression in neutrophils. Diphenylamine and N-phenylanthranilic acid neither affected COX activity in platelets nor modified the activation state of neutrophils. Diphenylamine-related compounds, which include the diphenylamine-based NSAIDs caused a variable reduction in the neutrophil intracellular ATP concentration, which correlated with the differential ability of such compounds to trigger L-selectin shedding (r = 0.97, p < 0.01). Diphenylamine-related compounds failed to down-regulate L-selectin in a tumor necrosis factor-alpha-converting enzyme (TACE)-deficient murine monocytic cell line. Our data indicate that diphenylamine seems to be the structural core of NSAIDs accounting for their down-regulatory activity of L-selectin leukocyte expression. Diphenylamine and its related compounds exert this action on L-selectin through a prostaglandin-independent, TACE-dependent mechanism that seems to be linked to the capability of these agents to uncouple the mitochondrial oxidative phosphorylation.     

Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis

Leukocyte infiltration into the liver is paramount to the development of liver injury in hepatitis. Hepatitis occurring after the administration of Con A in mice is felt to be a T lymphocyte-mediated disease. In this study, we report that neutrophils are the key initiators of lymphocyte recruitment and liver injury caused by Con A. The objectives of this study were to investigate the involvement of neutrophils in Con A-induced hepatitis in vivo via intravital microscopy. After Con A administration, we observed a significant increase in leukocyte rolling flux, a decrease in rolling velocity, and an increase in leukocyte adhesion to the hepatic microvasculature. Fluorescence microscopy identified that within 4 h of Con A administration only a minority of the recruited leukocytes were T lymphocytes. Furthermore, immunohistochemistry showed a significant increase in neutrophils recruited to the liver post-Con A treatment in association with liver cell damage, as reflected by elevated serum alanine aminotransferase levels. Using flow cytometry, we observed that Con A could bind directly to neutrophils, which resulted in a shedding of L-selectin, an increase in beta(2)-integrin expression, and the production of reactive oxidants. Following neutrophil depletion, a significant inhibition of Con A-induced CD4+ T lymphocyte recruitment to the liver resulted and complete reduction in hepatic injury, as assessed by serum alanine aminotransferase levels. In summary, the present data support the concept that neutrophils play an important and previously unrecognized role in governing Con A-induced CD4+ T cell recruitment to the liver and the subsequent development of hepatitis.     

Actin polymerization induces shedding of FcgammaRIIIb (CD16) from human neutrophils

FcgammaRIIIb (CD16) is a glycosyl phosphatidylinositol (GPI)-anchored low-affinity IgG receptor, exclusively expressed on human neutrophils. FcgammaRIIIb associates with complement receptor 3 (CR3, Mac-1, CD11b/CD18), which may indirectly link FcgammaRIIIb to the actin cytoskeleton. Upon neutrophil activation, apoptosis, or chemotaxis, FcgammaRIIIb is shed from the cell surface. In all of these events, actin rearrangements play an important role. To establish a role for the actin cytoskeleton in the control of FcgammaRIIIb shedding, we treated human neutrophils with jasplakinolide, an actin-polymerizing peptide. We show that enhanced actin polymerization induces time- and dose-dependent shedding of FcgammaRIIIb. This effect was not restricted to FcgammaRIIIb, because the cell surface expression of CD43, CD44, and L-selectin was also downregulated after induction of actin polymerization. This actin-dependent pathway is staurosporine sensitive but does not appear to involve activation of PKC or CR3. These data show that the actin cytoskeleton can regulate protein ectodomain shedding from human neutrophils.     

Dynamics of adhesion molecule domains on neutrophil membranes: surfing the dynamic cell topography

Lateral organization and mobility of adhesion molecules play a significant role in determining the avidity with which cells can bind to target cells or surfaces. Recently, we have shown that the lateral mobility of the principal adhesion molecules on neutrophils is lower for rolling associated adhesion molecules (RAAMs: L-selectin and PSGL-1) than for β₂ integrins (LFA-1 and Mac-1). Here we report that all four adhesion molecules exhibit distinct punctate distributions that are mobile on the cell surface. Using uniform illumination image correlation microscopy, we measure the lateral mobility of these topologically distinct domains. For all four molecules, we find that diffusion coefficients calculated from domain mobility agree with measurements we made previously using fluorescence recovery after photobleaching. This agreement indicates that the transport of receptors on the surface of the resting neutrophil is dominated by the lateral movement of domains rather than individual molecules. The diffusion of pre-assembled integrin domains to zones of neutrophil/endothelial contact may provide a mechanism to facilitate high avidity adhesion during the earliest stages of firm arrest.