Abstract
Introduction: Mechanical force acting on selectin bonds facilitates neutrophil (PMN) rolling and transduction of signals that subsequently activate integrins that support cell arrest under hydrodynamic shear flow. E-selectin upregulated on inflamed endothelium has the unique capacity to bind multiple sialyl Lewisx (sLex) presenting ligands on circulating leukocytes to mediate slow rolling and activation of the β2 integrin- lymphocyte function-associated antigen 1 (LFA-1), which in turn binds ICAM-1 during PMN arrest at sites of inflammation. It was demonstrated in a mouse model that PSGL-1 is the primary E-selectin ligand (ESL) that facilitates cell rolling. L-selectin/PSGL-1 clustering at the site of adhesive contact provides an outside-in signal that in turn activates LFA-1. A fundamental difference between mouse and human PMN with respect to ESL recognition is the biosynthesis of glycosylated ligands. Human L-selectin (CD62L) expresses N- and O- linked sLex that is recognized by the lectin domain of E-selectin, whereas mouse L-selectin is not decorated with sLex. We report that CD62L binding and clustering by E-selectin is necessary and sufficient to transduce signals that activate LFA-1, even in the absence of PSGL-1 engagement. Our study aims to elucidate how sLexexpressed on CD62L is preferentially bound by E-selectin and how tension induced clustering at sites of adhesive contact transduces integrin mediated PMN arrest. Recognition of CD62L can be selectively blocked by rivipansel (GMI-1070), a small molecule glycomimetic pan-selectin antagonist.
Materials and Methods: PMN are perfused into a microfluidic device at 2 dynes/cm2 over a glass substrate coated with ICAM-1/E-selectin to mimic the inflamed vasculature and allow for quantification of rolling to arrest. Total internal reflection fluorescence (TIRF) microscopy employing quantitative dynamic footprinting (qDF) enables imaging of a membrane dye in conjunction with fluorescent antibodies to record cell adhesion and ligation of receptors by a molecular substrate. Rivipansel along with antibodies are used to block CD62L and PSGL-1 receptors. Fluorescent antibodies are used to image localization of receptors at the substrate. Immunoprecipitation of PMN lysates by E-selectin in presence or absence of antibody and rivipansel were followed up by Western blot analysis to identify their relative capacity to block recognition of CD62L versusPSGL-1.
Results and Discussion: Isolated human PMN rolling to arrest was recorded on a substrate of recombinant human E-selectin/ICAM-1. Rivipansel inhibited PMN rolling at IC50 ~6.5µM, whereas antagonism of β2-integrin activation resulted in tenfold more potent activity with an IC50 ~0.5 μM. Blocking PSGL-1 with antibody did not alter this inhibition, indicating that it is not required for CD62L mediated outside-in signaling of β2-integrin dependent rolling to arrest. Western blots of PMN lysates immunoprecipitated against E- versus P-selectin revealed that rivipansel at 6.5 µM inhibits E-selectin recognition of sLex on CD62L by ~70%, while PSGL-1 binding was unaltered at this concentration. qDF imaging of the distribution of CD62L/PSGL-1 during human PMN rolling on E-selectin/ICAM-1 revealed that PSGL-1 was not necessary for tethering interactions of CD62L on E-selectin, nor signaling of integrin activation and arrest.
Conclusions: E-selectin recognition of sLex expressed on CD62L is necessary and sufficient to generate outside-in signaling and activation of LFA-1 dependent arrest on ICAM-1. PSGL-1 contributes to PMN capture and rolling in shear flow, but is not requisite for conversion to arrest. Rivipansel binds tightly to the lectin domain on E-selectin and displaces sLex presented by CD62L, thereby preventing subsequent signal transduction associated with integrin activation and stable bond formation to ICAM-1. Blocking activation and arrest of leukocytes by rivipansel may play a central role in its reported clinical benefit in treatment of vaso-occlusive crisis in sickle cell patients.
Disclosures
Magnani: GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Spreading of human neutrophils on an ICAM-1-immobilized substrate under shear flow
