Neutrophil Rolling Research Articles

Nitric oxide inhibits neutrophil migration by a mechanism dependent on ICAM-1: Role of soluble guanylate cyclase

In the present study, we addressed the role of intercellular adhesion molecule type 1 (ICAM-1/CD54) in neutrophil migration to inflammatory site and whether the inhibitory effect of nitric oxide (NO) upon the neutrophil rolling, adhesion and migration involves down-modulation of ICAM-1 expression through a cyclic GMP (cGMP) dependent mechanism. It was observed that neutrophil migration induced by intraperitoneal administration of endotoxin (LPS), carrageenan (Cg) or N-formyl peptide (fMLP) in ICAM-1 deficient (ICAM-1-/-) is similar to that observed in wild type (WT) mice. The treatment of mice with NO synthase (NOS) inhibitors, NG-nitro-l-arginine, aminoguanidine or with a soluble guanylate cyclase (sGC) inhibitor, ODQ enhanced LPS- or Cg-induced neutrophil migration, rolling and adhesion on venular endothelium. These parameters induced by LPS were also enhanced by 1400 W, a specific iNOS inhibitor, treatment. On the other hand, the treatment of the mice with S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, reduced these parameters induced by LPS or Cg by a mechanism sensitive to ODQ pretreatment. The NOS inhibitors did not enhance LPS-, Cg- or fMLP-induced migration and adhesion in ICAM-1-/- mice. Moreover, genetic (iNOS-/- mice) or pharmacological inhibition of NOS or of sGC enhanced LPS-induced ICAM-1 expression on mesenteric microcirculation vessels of WT mice. By contrast, SNAP reduced the ICAM-1 expression by a mechanism dependent on cGMP. In conclusion, the results suggest that although during inflammation, ICAM-1 does not contribute to neutrophil migration, it is necessary for the down-modulatory effect of inflammation-released NO on the adhesion and transmigration of neutrophils. Moreover, these NO effects are mediated via cGMP.

Induction of LFA‐1‐Dependent Neutrophil Rolling on ICAM‐1 by Engagement of E‐Selectin

To study rolling of mouse neutrophils on E-selectin and ICAM-1 in an ex vivo flow chamber system. The authors developed a small autoperfused flow chamber (20 x 200-microm cross section) that allows direct visualization of cells with and without fluorescent labeling and does not require recirculation of blood. Neutrophils rolled on E-selectin alone, but were unable to interact with immobilized ICAM-1. When ICAM-1 was co-immobilized with E-selectin, the number of cells that rolled was doubled, but no significant firm adhesion was observed. This phenomenon was specific for E-selectin, and no enhancement of rolling was observed when P-selectin was immobilized with ICAM-1. The increased neutrophil rolling seen on E-selectin and ICAM-1 substrates required beta2 integrins. Treating mice with antibodies to the beta2 integrins LFA-1 and Mac-1 showed that LFA-1 was primarily responsible for mediating rolling on ICAM-1 in this model. Increased rolling on E-selectin and ICAM-1 was significantly reduced following administration of a specific p38 mitogen-activated protein kinase (MAPK) inhibitor. The data show that neutrophil rolling on E-selectin leads to partial activation of LFA-1, enabling LFA-1-dependent rolling on ICAM-1. This mechanism is likely to amplify and accelerate neutrophil recruitment in inflammation.

Dynamic shifts in LFA-1 affinity regulate neutrophil rolling, arrest, and transmigration on inflamed endothelium

Polymorphonuclear leukocyte (PMN) recruitment to vascular endothelium during acute inflammation involves cooperation between selectins, G-proteins, and beta2-integrins. LFA-1 (CD11a/CD18) affinity correlates with specific adhesion functions because a shift from low to intermediate affinity supports rolling on ICAM-1, whereas high affinity is associated with shear-resistant leukocyte arrest. We imaged PMN adhesion on cytokine-inflamed endothelium in a parallel-plate flow chamber to define the dynamics of beta2-integrin function during recruitment and transmigration. After arrest on inflamed endothelium, high-affinity LFA-1 aligned along the uropod-pseudopod major axis, which was essential for efficient neutrophil polarization and subsequent transmigration. An allosteric small molecule inhibitor targeted to the I-domain stabilized LFA-1 in an intermediate-affinity conformation, which supported neutrophil rolling but inhibited cell polarization and abrogated transmigration. We conclude that a shift in LFA-1 from intermediate to high affinity during the transition from rolling to arrest provides the contact-mediated signaling and guidance necessary for PMN transmigration on inflamed endothelium.

Putting the brakes on neutrophils

Comment on Hayhoe et al, page [2123][1] The glucocorticoid-induced annexin 1 system controls neutrophil trafficking. Hayhoe and colleagues report the receptors and anti-inflammatory mechanisms for annexin 1 and its N-terminal peptide Ac2-26 that limit neutrophil rolling, adhesion, and diapedesis.

Neutrophil Recruitment by Fetal Porcine Endothelial Cells: Implications in Scarless Fetal Wound Healing

Fetal dermal wounds heal scarlessly and with a minimal inflammatory response. When a robust inflammatory response is induced at the site of fetal dermal wounds by the application of cytokines, healing results in fibrosis. To test the hypothesis that the reduced inflammatory response in fetal wounds is due to impaired fetal leukocyte-endothelial interactions, the contributions of fetal endothelial cells to the inflammatory response in the fetus were investigated. Endothelial cells isolated from blood vessels of adult and mid-gestational fetal pigs were cultured in media until confluent monolayers were established. Adult porcine neutrophils were isolated and resuspended at a concentration of 1 million cells/mL. Interactions between neutrophils and endothelial cells were observed under static and flow conditions. Endothelial monolayers were exposed to neutrophils with and without prior stimulation of the endothelial cells with tumor necrosis factor alpha (TNF-alpha) for 4 h. The neutrophil-endothelial interactions were observed and analyzed for neutrophil adhesion, rolling velocity, and transmigration. Endothelial P-selectin mRNA expression was determined by real-time polymerase chain reaction (PCR). A novel in vitro model of fetal inflammation is described. Both adult and fetal endothelial cells demonstrated a dose-dependent increase in neutrophil adhesion and transmigration with increasing doses of TNF-alpha. The fetal response was significantly lower than the adult. As expected, rolling velocity was lower at higher cytokine concentrations and had an inverse correlation with P-selectin mRNA expression. Fetal endothelial cells are less permissive to adhesion and transmigration of neutrophils than adult endothelial cells. This may contribute to the paucity of inflammation seen in the fetal response to dermal injury.

The Principal Eosinophil Peroxidase Oxidant, HOSCN, Is a Uniquely Potent Phagocyte Oxidant Inducer of E-Selectin, ICAM-1 and VCAM-1 Expression on Endothelial Cells and Promotes Leukocyte-Endothelial Cell Adhesion. A Potential Mechanism Regulating Leukocyte Trafficking in Eosinophilic Inflammatory States.

We have shown that SCN- is the principal substrate for EPO in vivo and that its product, HOSCN, a weak sulfhydryl-reactive oxidant, is a singularly effective oxidant inducer of tissue factor activity in human umbilical vein endothelial cells (HUVEC) through activation of the NF-κB transcription factor. Because their genes contain upstream NF-κB binding sites, we hypothesized that HOSCN would induce expression of the cell adhesion molecules E-selectin, ICAM-1 and VCAM-1 and thereby enhance leukocyte-endothelium adhesion. HUVEC monolayers were exposed to various phagocyte-derived oxidants or 10 μg/ml LPS in M199 medium with 10% FCS and assayed by western blotting and flow cytometry. We find that 150 μM HOSCN induces VCAM-1 and ICAM-1 expression starting at 2 h that peaks stably at up to 10-fold from 4–12 h while upregulation of E-selectin is first detectable by 2h, peaks up to 10-fold at 4 hours, then rapidly diminishes to baseline. This induction is dose-dependent and does not occur in the presence of the other phagocyte oxidants HOCl, HOBr and H2O2. HOSCN inducton of adhesion molecule expression is transcriptionally mediated as documented by semi-quantitative RT-PCR analysis. Moreover, HOSCN, but not HOCl, HOBr or H2O2 strongly activates the NF-κB p65/p50 heterodimer assayed by EMSA and cytoplasmic IκB-α degradation assayed by western blotting. To test the functional significance and specificity of these findings we performed neutrophil/HUVEC static adhesion assays with blocking monoclonal antibodies. 150 μM HOSCN induces a 6-fold increase in neutrophil adhesion that is totally blocked by 10 μg/ml of andrographolide, a specific inhibitor of the NF-κB pathway. Adhesion peaks at 4 h then wanes, consistent with the kinetics of E-selectin expression. Neutrophil/HUVEC adhesion was decreased 30–40% by blocking antibodies to either E-selectin or ICAM-1 but not VCAM-1. In an in vivo model of neutrophil rolling and adhesion, mice were intraperitoneally injected with reagent HOSCN (10 nmol/g) or buffer control 2 h before the cremaster muscle was externalized and leukocyte/endothelial interactions in post-capillary venules were quantitated by intravital microscope using a CCD video camera. Peritoneal cavities were lavaged and leukocytes enumerated and identified. HOSCN treatment induces a 4.6-fold (n = 8, p = 0.006) increase in total endothelium-adherent leukocytes but no significant change in leukocyte rolling flux. HOSCN also stimulates a 20-fold increase in peritoneal neutrophils. These findings show that HOSCN is a uniquely potent oxidant inducer of HUVEC E-selectin, ICAM-1 and VCAM-1 expression and promotes neutrophil-HUVEC adhesion. We propose that HOSCN generated by eosinophils adhering to and infiltrating endothelium may stimulate the recruitment and activation of neutrophils, eosinophils and other leukocytes to sites of eosinophilic inflammation and thereby participate in the pathogenesis of allergic diseases and the hypereosinophilic syndrome. The recent unexpected finding that neutrophil myeloperoxidase generates equimolar amounts of HOSCN and HOCl as its principal physiologic products raises the possiblility that neutrophils also utilize HOSCN as an inflammatory amplification mechanism.

Synthesis of α(1,3) Fucosyltransferases IV- and VII-Dependent Eosinophil Selectin Ligand and Recruitment to the Skin

Selectins mediate the initial adhesion of leukocytes to endothelial cells in many contexts of inflammation-dependent leukocyte recruitment. The glycans that contribute to P- and E-selectin counterreceptor activity arise through glycosylation reactions in which the terminal steps are catalyzed by alpha(1,3) fucosyltransferases (FTs). We examined how selectin ligand activities are controlled in eosinophils by characterizing FT expression profiles and regulatory mechanisms in eosinophils isolated from human blood. We found that FT-IV and FT-VII mRNAs were up-regulated by transforming growth factor-beta1, but the FT-IV transcript consistently predominated in eosinophils. To further define the physiological role of FT-IV and FT-VII in expression of eosinophil selectin ligand, we characterized models of dermal eosinophilia in FT-IV- and/or FT-VII-deficient mice in vivo. FT-IV deficiency yielded a significant decrease in eosinophil recruitment to the skin. Likewise, deficiency of FT-VII also yielded a decrease in eosinophil recruitment. Eosinophil recruitment that remained in the absence of FT-VII was further inhibited by blocking P- or E-selectin and was essentially absent in mice deficient in both enzymes. These observations indicate that FT-IV and FT-VII are both important contributors to selectin-dependent eosinophil recruitment to the skin and may represent therapeutic targets for treating diseases in which eosinophil recruitment contributes to pathophysiology.

Leukocyte Function-associated Antigen 1-mediated Adhesion Stability Is Dynamically Regulated through Affinity and Valency during Bond Formation with Intercellular Adhesion Molecule-1

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the beta(2) integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.

Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses

Here we have studied the involvement of endothelial heparan sulfate in inflammation by inactivating the enzyme N-acetyl glucosamine N-deacetylase-N-sulfotransferase-1 in endothelial cells and leukocytes, which is required for the addition of sulfate to the heparin sulfate chains. Mutant mice developed normally but showed impaired neutrophil infiltration in various inflammation models. These effects were due to changes in heparan sulfate specifically in endothelial cells. Decreased neutrophil infiltration was partially due to altered rolling velocity correlated with weaker binding of L-selectin to endothelial cells. Chemokine transcytosis across endothelial cells and presentation on the cell surface were also reduced, resulting in decreased neutrophil firm adhesion and migration. Thus, endothelial heparan sulfate has three functions in inflammation: by acting as a ligand for L-selectin during neutrophil rolling; in chemokine transcytosis; and by binding and presenting chemokines at the lumenal surface of the endothelium.

Protection against Renal Ischemia Reperfusion Injury by CD44 Disruption

Inflammation contributes to renal ischemia reperfusion (I/R) injury, potentially causing renal dysfunction. The inflammatory infiltrate mainly consists of neutrophils, which are deleterious for the renal tissue. Because CD44 is expressed by neutrophils and is rapidly upregulated by capillary endothelial cells after I/R injury, it was hypothesized that CD44 might play an important role in the development of I/R injury. This study showed that rapid CD44 upregulation on renal capillary endothelial cells mediates neutrophil recruitment to the postischemic tissue. Hence, CD44 deficiency led to decreased influx of neutrophils regardless of comparable levels in chemotactic factors expressed in the kidney. The reduced influx of neutrophils was associated with preserved renal function and morphology. Adoptive transfer experiments of labeled neutrophils revealed that endothelial CD44 rather than neutrophil CD44 mediates neutrophil migration. Activation of neutrophils increased cell-surface expression of hyaluronic acid (HA). Altogether, a novel mechanism in the recruitment of neutrophils that involves interaction of endothelial CD44 and neutrophil HA was found. Either blocking endothelial CD44 or removal of neutrophil HA decreased rolling and adhesion of neutrophils. Administration of anti-CD44 to mice reduced the influx of neutrophils into the postischemic tissue, associated with renal function preservation. Therefore, anti-CD44-based therapies may contribute to prevent or reduce renal I/R injury.

Controversies in Experimental Dermatology

Controversies in Experimental Dermatology

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.

P-Selectin Activates Leukocyte Integrin alphaMbeta2 by Recruiting Phosphatidylinositol-3 Kinase to P-Selectin Glycoprotein Ligand-1 Cytoplasmic Domain through Adaptor Protein Naf1.

The interaction of endothelial P-selectin (CD62P) with neutrophil PSGL-1 (P-selectin glycoprotein ligand-1, CD162) mediates neutrophil rolling, which acts in concert with cytokines and chemoattractants for integrin-mediated firm adhesion of neutrophils to vascular endothelial cells. Numerous studies have established that this sequence of events is critical to the extravasation of neutrophils during an inflammatory response. We have recently shown that cross-linking of PSGL-1 with P-selectin acts cooperatively with extracellular stimuli, such as cytokines and chemoattractants released from and displayed on the activated endothelial lining of the vessel walls, for optimal activation of beta2-integrins, an increase in their apparent affinity/avidity for cognate ligands, which in turn supports firm adhesion and transendothelial migration. To identify cellular proteins which interact with human PSGL-1 and trigger this cascade of events, we used the cytoplasmic domain of human PSGL-1 as the bait for screening of human leukocyte yeast two-hybrid library. During this genetic screening, we identified Naf-1 (Nef-associated factor 1; also called ABIN, A20 binding inhibitor of NF-kB, or VAN, virion-associated nuclear shuttle protein) as a binding partner to the cytoplasmic domain of PSGL-1. The specific interaction of Naf1 with PSGL-1 was confirmed by GST-fusion protein pull-down and co-immunoprecipitation experiments. As YPPM at 552–555 of Naf1a amino acid sequence is a known binding motif for p85 subunit of phosphatidylinositol-3 kinase (PI3K; YXXM in which phosphorylated Y is required for p85 binding), we performed co-immunoprecipitation experiments to show that Naf1a actually acted as an adaptor protein for PSGL-1 and p85 subunit of PI3K. In addition, we found that P-selectin activated the enzyme activity of PI3K in human neutrophils and PI3K specific inhibitors, wortmannin and LY294002, inhibited alphaMbeta2 activation and clustering induced by P-selectin. Our data thus delineate a novel PSGL-1 signal transduction pathway essential for transactivation of beta2-integrins.

Bovine respiratory syncytial virus infection influences the impact ofα4- andβ2-integrin-mediated adhesion of peripheral blood neutrophils

Neutrophil migration into the airways and pulmonary tissue is a common finding in bovine respiratory syncytial virus (BRSV) infections. Although neutrophil trans-endothelial migration in general depends on beta2-integrins, alternative integrins such as the alpha4-integrins have been implicated. In this study, rolling and firm adhesion of peripheral blood neutrophils isolated from healthy and BRSV-infected calves to tumour necrosis factor (TNF)-alpha activated pulmonary endothelium was investigated under flow conditions in vitro. For neutrophils obtained from healthy animals, inhibition of the beta2-integrin reduced firm adhesion to 63% and inhibition of alpha4-integrin to 73% compared with untreated controls. Inhibition of both integrins reduced firm adhesion to 25%. Rolling velocity, which is used as a parameter for integrin involvement in neutrophil rolling, increased 1.7-fold by blocking beta2-integrin and was significantly augmented to 2.5-fold by blocking both alpha4- and beta2-integrins. For neutrophils obtained from BRSV-infected animals, however, rolling velocities at 10 days after infection (p.i.) were not influenced by blocking adhesion of alpha4- and beta2-integrins, indicating that these integrins did not support neutrophil rolling. In addition, the inhibition of firm adhesion by blocking both alpha4- and beta2-integrins was reduced significantly 9 days post-infection, resulting in a residual 68% neutrophil binding at 9 days p.i. Non-blocked firm adherence was not reduced, indicating that binding was achieved by other mechanisms than through alpha4- and beta2-integrins. These results demonstrate an important function for alpha4- and beta2-integrins in rolling and firm adherence of bovine neutrophils, to TNF-alpha-activated endothelium and show the dynamic use of these integrins for adhesion and migration by neutrophils in the course of BRSV infection.

Membrane Tether Extraction from Human Umbilical Vein Endothelial Cells and Its Implication in Leukocyte Rolling

During the rolling of human neutrophils on the endothelium, tethers (cylindrical membrane tubes) are likely extracted from the neutrophil. Tether extraction reduces the force imposed on the adhesive bond between the neutrophil and endothelium, thereby facilitating the rolling. However, whether tethers can be extracted from the endothelium is still unknown. Here, with the micropipette-aspiration technique, we show that tethers can be extracted from either suspended or attached human umbilical vein endothelial cells. We also show that a linear relationship between the pulling force and tether growth velocity exists and this relationship does not depend on the receptor type (used to impose point forces), tumor necrosis factor- α stimulation, or cell attachment state. With linear regression, we determined that the threshold force was 50 pN and the effective viscosity was 0.50 pN·s/ μm. Therefore, tethers might be simultaneously extracted from the neutrophil and endothelial cell during the rolling and, more importantly, the endothelial cell might contribute much more to the total composite tether length than the neutrophil. Compared with tether extraction from the neutrophil alone, simultaneous tether extraction results in a larger increase in the lifetime of the adhesive bond, and thus further stabilizes the rolling of neutrophils under high physiological shear stresses.

Importance of platelets and fibrinogen in neutrophil-endothelial cell interactions in septic shock.

To examine the role of platelets, fibrin, and adhesion molecules in mediating neutrophil-endothelial cell interactions in septic shock. Controlled experiments using phase contrast microscopy to examine neutrophil, platelet, and endothelial cell interactions in flowing cell suspensions under simulated physiologic conditions. University research laboratory. Adult patients with septic shock and normal volunteers. Microslides were coated with human umbilical vein endothelial cells. Neutrophils were removed from control subjects and patients in septic shock and were perfused over endothelial cells at rates representing a range of physiologic shear stresses. In an attempt to examine the effects of fibrin deposition on neutrophil-endothelial cell interactions, neutrophils, with and without platelets, were suspended in plasma and serum was removed from patients in septic shock. In addition, blocking monoclonal antibodies against the platelet receptor P-selectin and neutrophil receptor CD11b/CD18, and a platelet glycoprotein IIb/IIIa inhibitor, were incubated with cells suspended in plasma. Phase contrast video microscopy was used to count the number of neutrophils/mm adherent to endothelial cells during cessation of flow. Neutrophil rolling velocity was calculated as the time required for neutrophils to move across a 1-mm field (mm/sec). Leukoaggregation was defined as the number of neutrophils in aggregates (three or more nuclei) across a 1-mm field. Normal neutrophils exposed to plasma from patients with septic shock demonstrated significant increases in aggregation and endothelial cell adherence with associated decreases in neutrophil rolling velocity. These changes were significantly enhanced in the presence of platelets and significantly attenuated in the presence of serum, which is fibrinogen depleted. Preincubation with antibodies to the surface receptors P-selectin, CD11b/CD18, and glycoprotein IIb/IIIa abrogated the changes in neutrophil aggregation, adhesion, and rolling velocity. These data suggest that platelets and fibrinogen play an important role in mediating neutrophil-endothelial cell adherence in septic shock.

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.

017 Lower Expression of P‐Selectin may Modulate the Fetal Inflammatory Response:

Fetal dermal wound healing is characterized by a minimal inflammatory response and absence of scar. In order to determine the factors contributing to the minimal inflammatory response in the fetus, the role of P‐selectin in the interaction and transmigration of leukocytes through vascular endothelium was investigated. Methods: Primary endothelial monolayers were established from adult porcine central veins and from umbilical veins of mid‐gestation porcine fetuses. The ability of the endothelial cells to capture and enable transmigration of adult leukocytes was studied under flow conditions of 4 dynes/sec, with and without prior stimulation of the endothelial cells with TNF‐α or IL‐1β. In addition, P‐selectin mRNA expression was determined by quantitative real time PCR. All data were analyzed by ANOVA. Results: In response to IL‐1β 10 ng/ml stimulation, adult endothelial cells manifested a 10 fold increase in P‐selectin mRNA expression while fetal endothelial cells mounted only a 3.5 fold increase. 100 ng/ml was required to mount a 10 fold increase in fetal P‐selectin expression while no further increase in adult P‐selectin expression was noted at this dose. Leukocyte capture, demonstrated by rolling of neutrophils over the endothelial surface, was more efficient on adult monolayers compared to the fetus. This is inversely related to the rolling velocity which was significantly slower in the adult (5.3 ± 0.6 vs 12.4 ± 1.0 μm/mm2). Ultimately, the number of neutrophils transmigrating across adult endothelial monolayers under flow conditions was significantly higher compared to the fetal monolayer (199 ± 18 vs 72 ± 9 cells/mm2). Conclusion: Lower P‐selectin expression on fetal endothelial cells may account, in part, for the minimal inflammatory response noted following fetal dermal injury.

Autoperfused mouse flow chamber reveals synergistic neutrophil accumulation through P-selectin and E-selectin.

To study rolling of mouse neutrophils on P- and E-selectins in whole blood and without cell isolation, we constructed an autoperfused flow chamber made from rectangular microslides (0.2x2 mm) perfused from a carotid artery catheter. A differential pressure transducer served to measure wall shear stress. Green fluorescent neutrophils rolled on P-selectin but not E-selectin coated at 50 ng/ml, with some rolling on E-selectin at 150 ng/ml. However, when P- and E-selectins were coimmobilized, the resulting number of rolling neutrophils was sixfold and fourfold higher than on P- or E-selectin alone. Velocity and flux analysis shows that P-selectin initiates neutrophil rolling, and a small amount of E-selectin, unable to capture many neutrophils, reduces the rolling velocity of all neutrophils by more than 90%. The unexpected synergism between E- and P-selectins explains why neutrophil recruitment is enhanced when both selectins are expressed.

Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow.

Flowing leukocytes roll on P-selectin that is mobilized from secretory granules to the surfaces of endothelial cells after stimulation with histamine or thrombin. Before it is internalized, P-selectin clusters in clathrin-coated pits, which enhances its ability to support leukocyte rolling. We found that thrombin and histamine induced comparable exocytosis of P-selectin on endothelial cells. However, compared with histamine, thrombin decreased the recruitment of P-selectin into clathrin-coated pits, slowed the internalization of P-selectin, and reduced the number and stability of neutrophils rolling on P-selectin. Significantly more RhoA was activated in thrombin- than in histamine-stimulated endothelial cells. Inhibitors of RhoA or its effector, Rho kinase, reversed thrombin's ability to inhibit the internalization and adhesive function of P-selectin in endothelial cells. Experiments with transfected cells confirmed that the inhibitory actions of thrombin and Rho kinase on P-selectin required its cytoplasmic domain. Thus, a signaling event affects both the function and clearance of a protein that enters the constitutive clathrin-mediated endocytic pathway.