Adhesive dynamics simulations of the mechanical shedding of L-selectin from the neutrophil surface

Here we accurately recreate the mechanical shedding of L-selectin and its effect on the rolling behavior of neutrophils in vitro using the adhesive dynamics simulation by incorporating the shear-dependent shedding of L-selectin. We have previously shown that constitutively expressed L-selectin is cleaved from the neutrophil surface during rolling on a sialyl Lewis x-coated planar surface at physiological shear rates without the addition of exogenous stimuli. Utilizing a Bell-like model to describe a shedding rate which presumably increases exponentially with force, we were able to reconstruct the characteristics of L-selectin-mediated neutrophil rolling observed in the experiments. First, the rolling velocity was found to increase during rolling due to the mechanical shedding of L-selectin. When most of the L-selectin concentrated on the tips of deformable microvilli was cleaved by force exerted on the L-selectin bonds, the cell detached from the reactive plane to join the free stream as observed in the experiments. In summary, we show through detailed computational modeling that the force-dependent shedding of L-selectin can explain the rolling behavior of neutrophils mediated by L-selectin in vitro.     

Molecular mechanisms of L-selectin-induced co-localization in rafts and shedding [corrected

Leukocyte recruitment to lymph nodes or inflammatory sites is regulated by adhesion and activation. L-selectin (CD62L) is expressed on leukocytes and mediates tethering and rolling of leukocytes on endothelial cells. Upon stimulation L-selectin is down-regulated by proteolytic cleavage but the molecular mechanisms regulating this shedding step are poorly defined. To study intracellular mechanisms, we induced shedding of L-selectin by cross-linking with an immobilized L-selectin antibody (Dreg56) in Jurkat cells. The loss of surface expression was quantitated by flow cytometry and the increase of soluble L-selectin was determined by Western blot analysis. We find that Jurkat and p56(lck)-deficient JCaM1.6 cells released L-selectin to similar extent (18+/-4% and 17+/-3%, respectively) and revealed comparable inhibition with the src-tyrosine kinase inhibitor PP2. Glutathione (GSH), an inhibitor of the neutral sphingomyelinase, PD98059, a MAP-kinase (MAP-K) inhibitor and metalloprotease inhibitors (MPI) (TAPI, Ro 31-9790, and BB-3103) reduced significantly L-selectin-induced shedding by 60-80%. In Jurkat cells, L-selectin was present in Triton X-100 insoluble membrane rafts and was constitutively tyr-phosphorylated. Dreg56 cross-linking enhanced phosphorylation and recruitment of L-selectin into rafts which was significantly decreased by pretreatment of cells with PD98059. We conclude, that the metalloproteinase-mediated cleavage of L-selectin from cell surface is triggered by intracellular signaling pathways that are independent of p56(lck) tyrosine kinase activity, but require other tyrosine kinases and the neutral sphingomyelinase. The cleavage of L-selectin might involve membrane rafts as signaling platform.     

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.     

Regulation of membrane metalloproteolytic cleavage of L-selectin (CD62l) by the epidermal growth factor domain

The adhesion molecule L-selectin is cleaved rapidly from the surface of activated leukocytes by tumor necrosis factor-alpha converting enzyme, a cell surface metalloprotease, and also undergoes slower constitutive shedding in unactivated cells. The structural features that render it susceptible to shedding are poorly understood. We therefore analyzed the shedding of a series of mutant and chimeric L-selectin molecules. Although murine L-selectin is cleaved at a specific location in the juxtamembrane region 11 amino acids distal to the cell membrane, this cleavage has little sequence specificity. However, proline substitution at the P2' or P3' position or deletion of the epidermal growth factor (EGF) domain completely blocks the rapid phorbol ester-induced cleavage, but does not affect the slower basal proteolytic shedding. Insertion of the 15-residue membrane-proximal region (MPR) of L-selectin into the heterologous protein B7.2 results in a molecule that undergoes constitutive proteolytic turnover. In contrast, insertion of both the EGF domain and the MPR confers susceptibility to both slow constitutive shedding and the rapid proteolytic cleavage induced by phorbol 12-myristate 13-acetate. These results demonstrate that constitutive and induced L-selectin cleavage are separable processes and that the rapid phorbol ester-induced shedding requires the presence of the EGF domain, a sequence that is remote from the cleavage site.     

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.     

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.     

Ectodomain shedding of TGF-alpha and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades

A variety of transmembrane proteins, such as transforming growth factor-alpha (TGF-alpha), tumor necrosis factor-alpha (TNF-alpha) and L-selectin, undergo shedding, i.e. cleavage of the ectodomain, resulting in release of a soluble protein. Although the physiological relevance of ectodomain shedding is well recognized, little is known about the signaling mechanisms activating this process. We show that growth factor activation of cell surface tyrosine kinase receptors induces ectodomain cleavage of transmembrane TGF-alpha through activation of the Erk MAP kinase signaling cascade without the need for new protein synthesis. In addition, expression of constitutively activated MEK1 or its downstream target Erk2 MAP kinase was sufficient to stimulate TGF-alpha shedding. The basal cleavage level in the absence of exogenous growth factor stimulation was due to p38 MAP kinase signaling. Accordingly, a constitutively activated MKK6, a p38 activator, activated TGF-alpha shedding in the absence of exogenous stimuli. In addition to TGF-alpha shedding, these mechanisms also mediate L-selectin and TNF-alpha cleavage. Thus, L-selectin shedding by neutrophils, induced by N-formylmethionyl-leucyl-phenylalanine, was strongly inhibited by inhibitors of Erk MAP kinase or p38 MAP kinase signaling. Our results indicate that activation of Erk and p38 signaling pathways may represent a general physiological mechanism to induce shedding of a variety of transmembrane proteins.     

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.     

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.     

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.     

L-selectin shedding is independent of its subsurface structures and topographic distribution

L-selectin (CD62L), a lectin-like adhesion molecule, mediates lymphocyte homing and leukocyte accumulation at sites of inflammation. Its transmembrane (TM) and intracellular (IC) domains confer clustering of L-selectin on microvilli of resting leukocytes, which is important for L-selectin function. Following activation of protein kinase C (PKC) or calmodulin inhibition, the wild-type (WT) protein is rapidly cleaved in its membrane-proximal ectodomain. To examine whether L-selectin topography or TM/IC domains are involved in this shedding process, we used stable transfectants expressing WT L-selectin (on microvilli) or chimeric molecules consisting of the L-selectin ectodomain linked to the TM/IC domains of CD44 (excluded from microvilli) or CD31 (randomly distributed). PKC activation by PMA altered the cells' surface morphology, but did not induce a redistribution of L-selectin ectodomains. All cell lines shed ectodomains upon PMA activation in a dose-dependent fashion and with similar kinetics. Calmodulin inhibition by trifluoperazine induced shedding in both WT and chimera transfectants. At high trifluoperazine concentrations, shedding of WT L-selectin was significantly more pronounced than that of chimeric molecules. Regardless of the activating stimulus, shedding was blocked by a hydroxamate-based metalloprotease inhibitor, suggesting that ectodomain down-regulation occurred through proteolytic cleavage by identical protease(s). These results show that the recognition site(s) for PKC-induced L-selectin shedding is exclusively contained within the ectodomain; the nature of subsurface structures and surface topography are irrelevant. Shedding induced by calmodulin inhibition has two components: one requires the L-selectin TM/IC domain, and the other is independent of it.     

Mutagenesis of the ezrin-radixin-moesin binding domain of L-selectin tail affects shedding, microvillar positioning, and leukocyte tethering

L-selectin is a cell adhesion molecule that mediates the initial capture (tethering) and subsequent rolling of leukocytes along ligands expressed on endothelial cells. We have previously identified ezrin and moesin as novel binding partners of the 17-amino acid L-selectin tail, but the biological role of this interaction is not known. Here we identify two basic amino acid residues within the L-selectin tail that are required for binding to ezrin-radixinmoesin (ERM) proteins: arginine 357 and lysine 362. L-selectin mutants defective for ERM binding show reduced localization to microvilli and decreased phorbol 12-myristate 13-acetate-induced shedding of the L-selectin ectodomain. Cells expressing these L-selectin mutants have reduced tethering to the L-selectin ligand P-selectin glycoprotein ligand-1, but rolling velocity on P-selectin glycoprotein ligand-1 is not affected. These results suggest that ERM proteins are required for microvillar positioning of L-selectin and that this is important both for leukocyte tethering and L-selectin shedding.     

Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions

Interaction of leukocytes in flow with adherent leukocytes may contribute to their accumulation at sites of inflammation. Using L- selectin immobilized in a flow chamber, a model system that mimics presentation of L-selectin by adherent leukocytes, we characterize ligands for L-selectin on leukocytes and show that they mediate tethering and rolling in shear flow. We demonstrate the presence of L- selectin ligands on granulocytes, monocytes, and myeloid and lymphoid cell lines, and not on peripheral blood T lymphocytes. These ligands are calcium dependent, sensitive to protease and neuraminidase, and structurally distinct from previously described ligands for L-selectin on high endothelial venules (HEV). Differential sensitivity to O-sialo- glycoprotease provides evidence for ligand activity on both mucin-like and nonmucin-like structures. Transfection with fucosyltransferase induces expression of functional L-selectin ligands on both a lymphoid cell line and a nonhematopoietic cell line. L-selectin presented on adherent cells is also capable of supporting tethering and rolling interactions in physiologic shear flow. L-selectin ligands on leukocytes may be important in promoting leukocyte-leukocyte and subsequent leukocyte endothelial interactions in vivo, thereby enhancing leukocyte localization at sites of inflammation.     

Neutrophil activation in smokers

Smoking and elevated leukocyte counts are risk factors for cardiovascular disease. Experimental studies suggest that leukocyte activation may be a requirement for certain cardiovascular complications. Clinical studies have demonstrated activated leukocytes in the peripheral blood of stroke victims. Accordingly, neutrophil activation in unseparated whole blood of smokers as well as naive neutrophils of non-smokers exposed to plasma of smokers was investigated. Both spontaneous Superoxide formation as determined by nitroblue tetrazolium reduction, as well as pseudopod formation, are significantly elevated in autologous neutrophils of smokers. The surface expression of CD 18 and L-selectin on autologous circulating neutrophils of smokers is not significantly different from non-smoker controls. In contrast, incubation of naive neutrophils with smoker plasma leads to significantly higher levels of Superoxide formation, pseudopod formation, and L-selectin shedding, compared with non-smoker plasma, suggesting that the plasma of smokers contains a transferable factor which causes leukocyte activation. The results indicate that analysis of blood samples from large peripheral veins may not accurately reflect leukocyte activation in the circulation since activated leukocytes have a higher probability to be trapped in the microcirculation.     

c-Abl is involved in the F-actin assembly triggered by L-selectin crosslinking

L-selectin is a cell adhesion molecule mediating the initial capture and subsequent rolling of leukocytes along the endothelial cells expressing L-selectin ligands. In addition to its action in adhesion, an intracellular signaling role for L-selectin has been recognized. Its cytoplasmic domain is involved in signal transduction following antibody crosslinking and in the regulation of receptor binding activity in response to intracellular signals. In this work, we demonstrated that L-selectin crosslinking led to F-actin polymerization and redistribution in human neutrophils. Using immuno-fluorescence microscopy, we observed that F-actin redistribution spatiotemporally related to the polarization of L-selectin. STI571, a specific inhibitor for cytoplasmic tyrosine kinase c-Abl, can inhibit F-actin polymerization and c-Abl redistribution in the activated neutrophils. Furthermore, we determined that c-Abl redistributed to the region where L-selectin polarized and associated with L-selectin in the activated neutrophils. The association between L-selectin and c-Abl was reduced by cytochalasin B. These results suggested that c-Abl was involved in the F-actin alteration triggered by L-selectin crosslinking in human neutrophils.     

L-选择素在白细胞活化中的作用

白细胞沿着血管内皮滚动、稳定黏附,最终到达炎症部位是一个复杂的、多步骤的过程,该过程需要众多分子协同完成。选择素家族分子对于白细胞沿着血管内皮的滚动起重要作用。L-选择素是选择素家族的一员,组成性的表达在白细胞微绒毛顶端,在白细胞沿血管内皮起始黏附过程中起主要作用。除具有黏附作用外,L-选择素还作为信号分子在黏附事件中发挥作用。该文结合作者的研究工作,综述了L-选择素在白细胞活化过程中的功能。     

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.     

The cytoplasmic tail of L-selectin interacts with members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent binding of Moesin but not Ezrin.

L-selectin regulates the recruitment of naive lymphocytes from the bloodstream to secondary lymphoid organs, mediating their initial capture and subsequent rolling along high endothelial cell surface-expressed ligands in peripheral lymph nodes. In vivo, distribution of L-selectin and cell surface levels determine the tethering efficiency and rolling velocity of leukocytes, respectively. Treatment of naive lymphocytes with phorbol myristate acetate (PMA) induces rapid ectodomain proteolytic down-regulation (shedding) of surface L-selectin via a protein kinase C (PKC)-dependent pathway. In an attempt to isolate proteins that are involved in regulating L-selectin expression, an affinity column was constructed using the 17-amino acid cytoplasmic tail of L-selectin. Affinity purification of extracts from lymphocytes, pre-treated with or without PMA, allowed identification of proteins that interact with the affinity column under one condition but not the other. By using this approach, members of the Ezrin-Radixin-Moesin family of proteins were found to interact specifically with the cytoplasmic tail of L-selectin. Moesin from PMA-stimulated lymphocytes, but not from unstimulated lymphocytes, bound to L-selectin tail. In contrast, ezrin from unstimulated or PMA-stimulated lymphocytes associated with L-selectin tail with equal affinity. Furthermore, the PKC inhibitor Ro 31-8220 significantly reduced the interaction of moesin, but not ezrin, with L-selectin. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of L-selectin identified arginine 357 as a critical residue for both ezrin and moesin interaction. Finally, BIAcore affinity analysis confirmed that N-terminal moesin interacts specifically with L-selectin cytoplasmic tail, with relatively high affinity (K(d) approximately 40 nm). Based on these findings, although moesin and ezrin bind to a similar region of the cytoplasmic tail of L-selectin, moesin binding is dependent on PKC activation, which suggests that ezrin and moesin are regulated differently in lymphocytes.     

Activation of human leukocytes reduces surface P-selectin glycoprotein ligand-1 (PSGL-1, CD162) and adhesion to P-selectin in vitro

P-selectin glycoprotein ligand-1 (PSGL-1), the primary ligand for P-selectin, is constitutively expressed on the surface of circulating leukocytes. The objective of this study was to examine the effect of leukocyte activation on PSGL-1 expression and PSGL-1-mediated leukocyte adhesion to P-selectin. PSGL-1 expression was examined via indirect immunofluorescence and flow cytometry before and after leukocyte stimulation with platelet activating factor (PAF) and PMA. Human neutrophils, monocytes, and eosinophils were all demonstrated to have significant surface expression of PSGL-1 at baseline, which decreased within minutes of exposure to PAF or PMA. PSGL-1 was detected in the supernatants of PAF-activated neutrophils by immunoprecipitation. Along with the expression data, this suggests removal of PSGL-1 from the cell surface. Soluble PSGL-1 was also detected in human bronchoalveolar lavage fluids. Down-regulation of PSGL-1 was inhibited by EDTA. However, inhibitors of L-selectin shedding and other sheddase inhibitors did not affect PSGL-1 release, suggesting that PSGL-1 may be shed by an as yet unidentified sheddase or removed by some other mechanism. Functionally, PSGL-1 down-regulation was associated with decreased neutrophil adhesion to immobilized P-selectin under both static and flow conditions, with the most profound effects seen under flow conditions. Together, these data indicate that PSGL-1 can be removed from the surface of activated leukocytes, and that this decrease in PSGL-1 expression has profound effects on leukocyte binding to P-selectin, especially under conditions of flow.