Most leukocytes can roll along the walls of venules at low shear stress (1 dyn/cm 2 ), but neutrophils have the ability to roll at 10-fold higher shear stress in microvessels in vivo 1 , 2 . The mechanisms involved in this shear-resistant rolling are known to involve cell flattening 3 and pulling of long membrane tethers at the rear 4 – 6 . Here, we show that these long tethers do not retract as postulated 6 , 7 , but instead persist and appear as ‘slings’ at the front of rolling cells. We demonstrate slings in a model of acute inflammation in vivo and on P-selectin in vitro , where P-selectin-glycoprotein-ligand-1 (PSGL-1) is presented as discrete sticky patches while LFA-1 is expressed over the entire length on slings. As neutrophils roll forward, slings wrap around the rolling cells and undergo a step-wise peeling from the P-selectin substrate enabled by the failure of PSGL-1 patches under hydrodynamic forces. The ‘step-wise peeling of slings’ is distinct from the ‘pulling of tethers’ reported previously 4 – 6 , 8 . Each sling effectively lays out a cell-autonomous adhesive substrate in front of neutrophils rolling at high shear stress during inflammation.

B cells play critical roles in the pathogenesis of lupus. To examine the influence of B cells on disease pathogenesis in a murine lupus model, New Zealand Black and New Zealand White F1 hybrid (NZB/W) mice were generated that were deficient for CD19 (CD19−/− NZB/W mice), a B cell-specific cell surface molecule that is essential for optimal B cell signal transduction. The emergence of anti-nuclear antibodies was significantly delayed in CD19−/− NZB/W mice in comparison with wild type NZB/W mice. However, the pathologic manifestations of nephritis appeared significantly earlier and survival was significantly reduced in CD19−/− NZB/W mice in comparison with wild type mice. These results demonstrate both disease promoting and protective roles for B cells in lupus pathogenesis. Recent studies have identified a potent regulatory B cell subset (B10 cells) within the rare CD1dhiCD5+ B cell subset of the spleen that regulates acute inflammation and autoimmunity through the production of IL-10. In wild type NZB/W mice, the CD1dhiCD5+B220+ B cell subset that includes B10 cells was increased by 2.5-fold during the disease course, while CD19−/− NZB/W mice lacked this CD1dhiCD5+ regulatory B cell subset. However, the transfer of splenic CD1dhiCD5+ B cells from wild type NZB/W mice into CD19−/− NZB/W recipients significantly prolonged their survival. Furthermore, regulatory T cells were significantly decreased in CD19−/− NZB/W mice, but the transfer of wild type CD1dhiCD5+ B cells induced Treg cell expansion in CD19−/− NZB/W mice. These results demonstrate an important protective role for regulatory B10 cells in this systemic autoimmune disease.

Human blood neutrophils rolling on E-or P-selectin reduced their rolling velocity when intercellular adhesion molecule (ICAM)-1 was available. Similar to mouse neutrophils, this was dependent on P-selectin glycoprotein ligand 1 (PSGL1), ␣ L ␤ 2 integrin, the Src family tyrosine kinase FGR and spleen tyrosine kinase SYK. Blocking phospholipase C or p38 MAP kinase attenuated, but did not abolish the velocity reduction. To test expression of integrin activation epitopes, we adapted an immobilized reporter assay and developed a new homogeneous microfluidics-based reporter antibody binding assay. Rolling on E-or P-selectin induced the extension reporter epitopes KIM127 and NKI-L16, but not the high affinity reporter epitope monoclonal antibody (mAb) 24. This enabled rolling neutrophils to bind to immobilized extension reporter, but not activation reporter antibodies and allowed binding of soluble KIM127 during rolling. We conclude that human neutrophil rolling on E-or P-selectin induces the extended ␣ L ␤ 2 integrin conformation through signaling triggered by PSGL-1 engagement. (Blood. 2010;116(4):617-624) Introduction E-selectin or P-selectin binding to human neutrophils has long been known to induce activation as demonstrated by phosphorylation of p38 MAP kinase. [1][2][3] In Ficoll-isolated human neutrophils, this process leads to arrest from rolling, polarization, and firm adhesion to endothelial monolayers under flow. [4][5][6][7] The proximal signal transduction pathway leading from E-selectin binding to integrin-dependent adhesion is unknown. Whether neutrophil integrins assume the extended or high affinity conformation is also unknown.Mouse neutrophils in whole blood reduce their rolling velocity on E-selectin when intercellular adhesion molecule 1 (ICAM1) is also available. 8 This requires P-selectin glycoprotein ligand 1 (PSGL1), 8,9 a cell surface expressed O-glycan that is a ligand for all 3 selectins. 10 Slow rolling probably involves extension of the integrin LFA-1, which requires the Src family tyrosine kinase Fgr, the immunoreceptor tyrosine-based activation motif (ITAM)-containing adapter molecules 11 and Syk. 8 Binding of isolated human neutrophils to E-selectin increases intracellular calcium levels, 7 which is blocked by phospholipase C inhibition. Indeed, PLC␥2 was recently shown to be activated downstream of Syk after integrin engagement. 12 We found little evidence for LFA-1 activation during mouse neutrophil rolling on P-selectin, but McEver et al reported that rolling on P-selectin can trigger LFA-1 activation. 9 Like other integrins, 13,14 LFA-1 undergoes dramatic conformational changes when activated. 15 Fluorescence resonance energy transfer (FRET) studies show that the cytoplasmic and transmembrane domains of the ␣ L and ␤ 2 subunits of LFA-1 move apart, 16 forcing the extracellular domain of LFA-1 into the extended conformation. 15 Conformational unbending of ␣ 4 ␤ 1 integrin was also shown more directly using FRET between a fluorophore on ␣ 4 subunit and an acceptor in the lipid bila...

The integrin CD11b/CD18 (also known as Mac-1), which is a heterodimer of the αM (CD11b) and β2 (CD18) subunits, is critical for leukocyte adhesion and migration and for immune functions. Blocking integrin-mediated leukocyte adhesion, although beneficial in experimental models, has had limited success in treating inflammatory diseases in humans. Here, we used an alternative strategy of inhibiting leukocyte recruitment by activating CD11b/CD18 with small-molecule agonists, which we term leukadherins. These compounds increased the extent of CD11b/CD18-dependent cell adhesion of transfected cells and of primary human and mouse neutrophils, which resulted in decreased chemotaxis and transendothelial migration. Leukadherins also decreased leukocyte recruitment and reduced arterial narrowing after injury in rats. Moreover, compared to a known integrin antagonist, leukadherins better preserved kidney function in a mouse model of experimental nephritis. Leukadherins inhibited leukocyte recruitment by increasing leukocyte adhesion to the inflamed endothelium, which was reversed with a blocking antibody. Thus, we propose that pharmacological activation of CD11b/CD18 offers an alternative therapeutic approach for inflammatory diseases.