Discovery of new genes and proteins directly supporting leukocyte adhesion is waning, whereas there is heightened interest in the cell mechanics and receptor dynamics that lead from transient tethering via selectins to affinity shifts and adhesion strengthening through integrins. New optical tools enable real-time imaging of leukocyte rolling and arrest in parallel plate flow channels (PPFCs), and detection of single-molecule force spectroscopy provides an inner view of the intercellular adhesive contact region. Leukocyte recruitment during acute inflammation is triggered by ligation of G protein-coupled chemotactic receptors (GPCRs) and clustering of selectins. This, in turn, activates beta(2)-integrin (CD18), which facilitates cell capture and arrest in shear flow. This review provides a conceptual model for the molecular events supporting leukocyte recruitment.

Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewisx ligands and activation of β2-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 β2-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 Lewisx 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 β2-integrin to high-avidity clusters and elicited efficient neutrophil capture of β2-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 β2-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.

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency that manifests as increased susceptibility to many pathogens. Although the spectrum of infections suffered by WAS patients is consistent with defects in neutrophil (PMN) function, the consequences of WAS protein (WASp) deficiency on this innate immune cell have been unclear. We report that deficiency of WASp in both human and murine PMNs resulted in profound defects in clustering of beta2 integrins, leading to defective adhesion and transendothelial migration under conditions of physiologic shear flow. Wild-type PMNs redistributed clustered beta2 integrins to the uropod of the cell during active migration, whereas WASp-deficient cells remain unpolarized. The WASp-deficient PMNs also showed reduced integrin-dependent activation of degranulation and respiratory burst. PMNs from a WAS patient manifested similar defects in integrin clustering and signaling. These results suggest that impaired beta2 integrin function in WASp-deficient PMNs may contribute substantially to the clinical immunodeficiency suffered by WAS patients.

Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate ␤ 2 integrin (CD18), deemed a critical step in facilitating cell capture and arrest under the shear force of blood flow. A conformational switch in the I domain allosteric site (IDAS) and in CD18 regulates LFA-1 affinity for endothelial ligands including intercellular adhesion molecule 1 (ICAM-1). We examined the dynamics of CD18 activation in terms of the efficiency of neutrophil capture of ICAM-1, and we correlated this with the membrane topography of 327C, an antibody that recognizes the active conformation of CD18 I-like domain. Adhesion increased in direct proportion to chemotactic stimulus rising 7-fold over a log range of interleukin-8 (IL-8). A threshold dose of ϳ75 pM IL-8, corresponding to ligation of only ϳ10 -100 receptors, was sufficient to activate ϳ20,000 CD18 and a rapid boost in the capture efficiency on ICAM-1. This was accompanied by a rapid redistribution of active LFA-1, but not Mac-1, into membrane patches, a necessary component for optimum adhesion efficiency. Shear-resistant arrest on a monolayer of ICAM-1 was reversed within minutes of chemotactic stimulation correlating with a shift from high to low affinity CD18 and dispersal of patches of active CD18. Mobility of active CD18 into high avidity patches was dependent on phosphatidylinositol 3-kinase activity and not F-actin polymerization. The data reveal that the number of chemotactic receptors bound and the topography and lifetime of high affinity LFA-1 tightly regulate the efficiency of neutrophil capture on ICAM-1.Neutrophils are among the first cells to respond to acute inflammation through a multistep process of specific bond formation with adhesion molecules up-regulated on the surface of activated endothelium (1, 2). Endothelial and leukocyte selectins function to capture leukocytes from the circulation by rapid bond formation. However, selectins form transient bonds that last a second or less in shear flow and only mediate cell capture and rolling on endothelium (3, 4). Unlike selectins, ␤ 2 integrins (CD18) do not constitutively recognize ligand but require cellular activation to form stable shear-resistant bonds with endothelial ligands including ICAM-1.

Polymorphonuclear leukocyte (PMN) recruitment to vascular endothelium during acute inflammation involves cooperation between selectins, G-proteins, and ␤ 2 -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 ␤ 2 -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. IntroductionNeutrophils are recruited at vascular sites of acute inflammation by the sequential binding of selectins, CXC chemokines, and ␤ 2 -integrins that function cooperatively to elicit rolling, arrest, and transmigration. From observations of neutrophil recruitment in the murine microcirculation and on endothelial monolayers grown in tissue culture, a number of rules of engagement have emerged. First, Mac-1 (␣ M ␤ 2 ) and LFA-1 (␣ L ␤ 2 ) are necessary and sufficient for neutrophil arrest and transmigration, with each subunit providing distinct adhesive contributions throughout the process from rolling to transmigration. 1-3 Second, polymorphonuclear leukocyte (PMN) rolling on a monolayer of cells coexpressing E-selectin and intercellular adhesion molecule-1 (ICAM-1) is sufficient to induce selectin ligand clustering (PSGL-1 and L-selectin) and to signal a shift in LFA-1 and Mac-1 from low to high affinity to bind ICAM-1. This process is synergistic with chemokine signaling on rolling PMN to amplify the efficiency of arrest. 4,5 LFA-1 appears to function early in this process in that it participates in tethering to ICAM-1 as it shifts from low to intermediate and high affinity. 6,7 How changes in conformation of the heterodimer result in changes in affinity to interact with ICAM-1 and mediate rolling, arrest, and outside-in signaling is only partially defined. 8 Structural studies of LFA-1 reveal that extension and activation of ICAM-1 binding involves an inserted or I-domain on the ␣-subunit and an I-like domain on the ␤-subunit that exerts a pull on the C-terminal ␣ 7 -helix of the ␣-subunit, leading to the open shape of the heterodimer and high-affinity ligand binding. 9,[10][11][12] This conformational shift to high affinity can be stabilized by binding of Mg 2ϩ or Mn 2ϩ or by inside-out signaling by chemokine receptors. 9,13,14 Once activated by a chemokine such as IL-8 or SDF-1, extension and opening of the LFA-1 heterodimer initiate rapid arrest on ICAM-1, as do activated I-domain mutants. 6,[15][16][17][18] Counteracting a shift to high affinity, small molecule allosteric anti-inflammatory inhibitors of LFA-1 function by effectively stabilizing the low-affinity state and antagonize binding to ICAM-1 and leukocyte adhesion. 19,20 XVA143 is one such ...

Cross-linking of L-selectin on leukocytes signals phosphorylation of mitogen-activated protein kinases (MAPKs) leading to activation of CD18 function and enhanced transmigration on inflamed endothelium. We examined how alterations in the topography of L-selectin correlate with the dynamics of CD18 activation and phosphorylation of MAPK. Simultaneous ligation of humanized antibodies DREG55 and DREG200 provided a strategy for regulating the extent of cross-linking. Triggering of CD11b/CD18 upregulation and adhesion required clustering of L-selectin to microvillus-sized patches of approximately 0.2 microm(2). Immunofluorescence revealed that L-selectin was colocalized with high-affinity CD18. Anti-L-selectin-coated protein A microspheres indicated that a single site of contact to a 5.5-microm bead, or multiple contacts to 0.94- or 0.3-microm beads, elicited maximum neutrophil activation. Adhesion signaled via L-selectin coincided with the kinetics of MAPK phosphorylation and was inhibited by blocking p38 or p42/44 activity. These data demonstrate the capacity of L-selectin to transduce signals effecting rapid ( approximately 1 s) neutrophil adhesion that is regulated by the size and frequency of receptor clustering.

selectin (CD62L) amplifies neutrophil capture within the microvasculature at sites of inflammation. Activation by G protein-coupled stimuli or through ligation of L-selectin promotes clustering of Lselectin and serves to increase its adhesiveness, signaling, and colocalization with ␤2-integrins. Currently, little is known about the molecular process regulating the lateral mobility of L-selectin. On neutrophil stimulation, a progressive change takes place in the organization of its plasma membrane, resulting in membrane domains that are characteristically enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins and exclude the transmembrane protein CD45. Clustering of L-selectin, facilitated by E-selectin engagement or antibody cross-linking, resulted in its colocalization with GPI-anchored CD55, but not with CD45 or CD11c. Disrupting microfilaments in neutrophils or removing a conserved cationic motif in the cytoplasmic domain of L-selectin increased its mobility and membrane domain localization in the plasma membrane. In addition, the conserved element was critical for L-selectin-dependent tethering under shear flow. Our data indicate that L-selectin's lateral mobility is regulated by interactions with the actin cytoskeleton that in turn fortifies leukocyte tethering. We hypothesize that both membrane mobility and stabilization augment L-selectin's effector functions and are regulated by dynamic associations with membrane domains and the actin cytoskeleton. membrane domains; adhesion; leukocyte; inflammation NEUTROPHIL RECRUITMENT from the bloodstream to sites of inflammation requires overcoming tremendous shear forces to initially tether to the vascular wall. Participation by the selectin adhesion protein family (E-, L-, and P-selectin) is critical for this process. The selectins are type 1 integral membrane proteins that contain a C-type lectin domain and recognize specific glycan moieties. L-selectin (CD62L) is expressed constitutively by leukocytes of myeloid and lymphoid origin; E-and P-selectin (CD62E and CD62P) are expressed by activated endothelial cells and by activated platelets (P-selectin). Neutrophils attach and roll along the vascular endothelium via the selectins, and then, if properly stimulated, integrins on the surface of neutrophils become activated and participate in adhesion strengthening and transmigration (18,40,41,78).Neutrophils that have accumulated within the microvasculature capture free-flowing leukocytes (13,69). This process of indirect leukocyte tethering accelerates neutrophil accumulation and is facilitated by L-selectin (2,4,74,85). Consistent with its role in amplifying the extent and rate of neutrophil accumulation, L-selectin is tightly regulated, which involves rapid and transient increases in its binding activity (26,70). C-type lectins achieve high-affinity binding through the presentation of multiple carbohydrate recognition domains in a single polypeptide or by clustering (reviewed in Ref. 86). L-selectin, which contains a single carbohydrate recognition domai...

Abstract:Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also docu mented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.Key words: nitroreductase, TNT, RDX, Enterobacter cloacae, Morganella morganii.Résumé : Différentes bactéries entériques produisent une nitroréductase de type I insensible à l'oxygène capable de ré duire les groupements nitro de divers composés nitroaromatiques dans des conditions anaérobies. La réduction enzyma tique des nitramines en anaérobiose est aussi connue chez les entérobactéries. La présente étude indique que la réduction des nitramines par les entérobactéries était catalysée par une nitroréductase de type I non sensible à l'oxygène plutôt que par une enzyme de type II. La souche B2 de Morganella morganii reconnue pour son activité hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroréductase, et la souche 96-3 d'Enterobacter cloacae reconnue pour son activité 2,4,6 trinitrotoluène (TNT) nitroréductase, ont toutes deux servi à démontrer que les deux explosifs TNT et RDX étaient réduits par une nitroréductase de type I. Les RDX et TNT nitroréductases de M. morganii et d'E. cloacae ont été observées lors d'essais avec des bactéries entières. La nitroréductase de type I, purifiée de l'E. cloacae, oxydait le NADPH en présence de TNT ou de RDX comme substrat. L'induction de l'expression du gène de la nitroréductase de type I de l'E. cloacae chez une souche d'Escherichia coli porteuse d'un plasmide a causé une augmentation spon tanée de l'activité des TNT et RDX nitroréductases. Par contre aucune activité TNT ou RDX nitroréductase n'a été ob servée chez des mutants de M. morganii résistants au nitrofurazone. La présente étude permet de conclure que la nitroréductase de type I ret...