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.

SUMMARY Cellular senescence suppresses cancer by arresting the proliferation of cells at risk for malignant transformation. Recently, senescent cells were shown to secrete numerous cytokines, growth factors and proteases that can alter the tissue microenvironment and may promote age-related pathology. To identify small molecules that suppress the senescence-associated secretory phenotype (SASP), we developed a screening protocol using normal human fibroblasts and a library of compounds that are approved for human use. Among the promising library constituents was the glucocorticoid corticosterone. Both corticosterone and the related glucocorticoid cortisol decreased the production and secretion of selected SASP components, including several pro-inflammatory cytokines. Importantly, the glucocorticoids suppressed the SASP without reverting the tumor suppressive growth arrest, and were efficacious whether cells were induced to senesce by ionizing radiation or strong mitogenic signals delivered by oncogenic RAS or MAP kinase kinase 6 overexpression. Suppression of the prototypical SASP component IL-6 required the glucocorticoid receptor, which, in the presence of ligand, inhibited IL-1α signaling and NF-κB transactivation activity. Accordingly, co-treatments combining glucocorticoids with the glucocorticoid antagonist RU-486 or recombinant IL-1α efficiently reestablished NF-κB transcriptional activity and IL-6 secretion. Our findings demonstrate feasibility of screening for compounds that inhibit the effects of senescent cells. They further show that glucocorticoids inhibit selected components of the SASP, and suggest that corticosterone and cortisol, two FDA-approved drugs, might exert their effects in part by suppressing senescence-associated inflammation.

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 ...

Radiation force produced by low-amplitude ultrasound at clinically relevant frequencies remotely translates freely flowing microbubble ultrasound contrast agents over distances up to centimeters from the luminal space to the vessel wall in order to enhance ligand-receptor contact in targeting applications. The question arises as to how the microbubble shell might be designed at the molecular level to fully take advantage of such physical forces in targeted adhesion for molecular imaging and controlled therapeutic release. Herein, we report on a novel surface architecture in which the tethered ligand is buried in a polymeric overbrush. Our results, with biotin-avidin as the model ligand-receptor pair, show that the overbrush conceals the ligand, thereby reducing immune cell binding and increasing circulation persistence. Targeted adhesion is achieved through application of ultrasound radiation force to instantly reveal the ligand within a well-defined focal zone and simultaneously bind the ligand and receptor. Our data illustrate how the adhesive properties of the contrast agent surface can be reversibly changed, from stealth to sticky, through the physical effects of ultrasound. This technique can be combined with any ligand-receptor pair to optimize targeted adhesion for ultrasonic molecular imaging.