E-selectin extends from the plasma membrane of inflamed endothelium and serves to capture leukocytes from flowing blood via long-lived catch-bonds that support slow leukocyte rolling under shear stress. Its ligands are glycosylated with the tetrasaccharide sialyl Lewis (sLe), which contributes to bond affinity and specificity. E-selectin-mediated rolling transmits signals into neutrophils that trigger activation of high-affinity β-integrins necessary for transition to shear-resistant adhesion and transendothelial migration. Rivipansel is a glycomimetic drug that inhibits E-selectin-mediated vaso-occlusion induced by integrin-dependent sickle-red blood cell-leukocyte adhesion. How Rivipansel antagonizes ligand recognition by E-selectin and blocks outside-in signaling of integrin-mediated neutrophil arrest while maintaining rolling immune-surveillance is unknown. Here, we demonstrate that sLe expressed on human L-selectin is preferentially bound by E-selectin and, on ligation, initiates secretion of MRP8/14 that binds TLR4 to elicit the extension of β-integrin to an intermediate affinity state. Neutrophil rolling over E-selectin at precise shear stress transmits tension and catch-bond formation with L-selectin via sLe, resulting in focal clusters that deliver a distinct signal to upshift β-integrins to a high-affinity state. Rivipansel effectively blocked formation of selectin catch-bonds, revealing a novel mechanotransduction circuit that rapidly converts extended β-integrins to high-affinity shear-resistant bond clusters with intracellular adhesion molecule 1 on inflamed endothelium.
Application of mechanical force to bonds between selectins and their ligands is a requirement for these adhesion receptors to optimally perform functions that include leukocyte tethering and activation of stable adhesion. Although all three selectins are reported to signal from the outside-in subsequent to ligand binding, E-selectin is unique in its capacity to bind multiple sialyl Lewisx presenting ligands and mediate slow rolling on the order of a micron per second. A diverse set of ligands are recognized by E-selectin in the mouse, including ESL-1, CD44, and PSGL-1 which are critical in transition from slow rolling to arrest and for efficient transendothelial migration. The molecular recognition process is different in humans as L-selectin is a major ligand, which along with glycolipids constitute more than half of the E-selectin receptors on human polymorphonuclear neutrophils (PMN). In addition, E-selectin is most efficient at raising the affinity and avidity of CD18 integrins that supports PMN deceleration and trafficking to sites of acute inflammation. The mechanism is only partially understood but known to involve a rise in cytosolic calcium and tyrosine phosphorylation that activates p38 MAP kinase and Syk kinase, both of which transduce signals from clustered E-selectin ligands. In this review we highlight the molecular recognition and mechanical requirements of this process to reveal how E-selectin confers selectivity and efficiency of signaling for extravasation at sites of inflammation and the mechanism of action of a new glycomimetic antagonist targeted to the lectin domain that has shown efficacy in blocking neutrophil activation and adhesion on inflamed endothelium.
Anaplasma phagocytophilum is an obligate intracellular bacterium that has evolved mechanisms to hijack polymorphonuclear neutrophil (PMN) receptors and signaling pathways to bind, infect, and multiply within the host cell. E-selectin is upregulated during inflammation and is a requisite endothelial receptor that supports PMN capture, rolling, and activation of integrin-mediated arrest. Ligands expressed by PMN that mediate binding to endothelium via E-selectin include sialyl Lewis x (sLe(x))-expressing ligands such as P-selectin glycoprotein ligand-1 (PSGL-1) and other glycolipids and glycoproteins. As A. phagocytophilum is capable of binding to sLe(x)-expressing ligands expressed on PMN, we hypothesized that acute bacterial adhesion to PMN would subsequently attenuate PMN recruitment during inflammation. We assessed the dynamics of PMN recruitment and migration under shear flow in the presence of a wild-type strain of A. phagocytophilum and compared it with a strain of bacteria that binds to PMN independent of PSGL-1. Acute bacterial engagement with PMN resulted in transient PMN arrest and minimal PMN polarization. Although the wild-type pathogen also signaled activation of beta2 integrins and elicited a mild intracellular calcium flux, downstream signals including PMN transmigration and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were inhibited. The mutant strain bound less well to PMN and failed to activate beta2 integrins and induce a calcium flux but did result in decreased PMN arrest and polarization that may have been partially mediated by a suppression of p38 MAPK activation. This model suggests that A. phagocytophilum binding to PMN under shear flow during recruitment to inflamed endothelium interferes with normal tethering via E-selectin and navigational signaling of transendothelial migration.
851FN2 Introduction: E-selectin expression by endothelium plays dual roles in inflammation by supporting slow rolling and subsequently eliciting integrin activation and arrest of leukocytes. This process may be important for immune surveillance. In a previous mouse model of sickle cell disease, E-selectin mediated outside-in signaling results in upregulated leukocyte Mac-1 and increased red cell capture, exacerbating vaso-occlusion. This process was attenuated by infusion of GMI-1070, a novel synthetic small molecule pan-selectin antagonist. GMI-1070 is now in Phase II clinical trial to determine efficacy in treatment of vaso-occlusive crisis (VOC) of sickle cell disease (SCD). Here, we studied its dose dependent effects in SCD subjects not in VOC on neutrophil activation and its effects on E-selectin mediated β2 integrin activation and rolling and arrest in shear flow. Methods: Samples were obtained from 4 SCD subjects not in VOC enrolled in a Phase I study of the effects of GMI-1070, a pan-selectin antagonist that preferentially inhibits E-selectin. An intravenous (IV) loading dose of 20 mg/kg was followed 10 hours later by a dose of 10 mg/kg. Samples were drawn before the loading dose, then 4 and 8 hours after the initial infusion. Polymorphonuclear Neutrophil (PMN) activation was analyzed in whole blood and isolated cell assays. Monoclonal antibodies and fluorescent-activated cell sorting (FACS) were used to assay expression of CD11b/CD18 (Mac-1), CD62L (L-selectin), and the high affinity active conformation of CD18 (327C) as markers of neutrophil activation. E-selectin mediated activation of CD18 was achieved in isolated PMN by incubating with E-selectin-IgG and goat antibody F(ab')2 fragment to crosslink E-selectin-IgG PMN activation was assessed by surface expression of high affinity CD18 by the mAb 327C and FACS in the presence of various concentrations of GMI-1070. PMN rolling and arrest on an inflammatory substrate was quantified using a lab on a chip assay. Whole blood or isolated PMN were perfused through a microfluidic flow chamber at a shear stress of 2 dynes/cm2. The flow chamber had immobilized E-selectin and ICAM-1 to support PMN rolling and adhesion. Video recordings of PMN interacting with this substrate were taken to quantify the number of rolling versus arrested cells and to measure rolling velocity in the presence of GMI-1070. Results: An inverse relationship between the serum concentration of GMI-1070 and either activated CD18 or upregulated CD11b was observed. In the flow chamber assays, 6 of 7 samples showed GMI-1070 diminished PMN arrest that also correlated with diminished integrin activation. Incubation of PMN with GMI-1070 blocked CD18 activation in response to E-selectin-IgG cross-linking, with an IC50 of 0.5 mM. PMN rolling and arrest was measured following shearing of isolated PMN on E-selectin and ICAM-1 using a lab on a chip assay. The mean rolling velocity of 2 mm/sec was increased to 6 mm/sec at a GMI-1070 concentration of 20 mM, with an IC50 of 5.5 mM for the increase in rolling velocity. In contrast, an IC50 = 0.8 mM was required to antagonize the fraction converting from rolling to arrest under shear flow. Summary and Conclusions: There was a dose dependent inhibitory effect on PMN activation after IV administration of GMI-1070 as measured in ex vivo whole blood samples in a small cohort of SCD subjects at steady state. A systematic study of the activity of GMI-1070 on isolated PMN revealed two distinct patterns of inhibition. At low concentrations (IC50 = 0.5mM), GMI-1070 effectively blocked the capacity of E-selectin to activate CD18, in response to cross-linked ligands, and mediate arrest. Only at higher concentrations (IC50 = 5.5mM) did we observe a significant alteration in the capacity of GMI-1070 to increase the rolling velocity and frequency of capture on a substrate of E-selectin under fluid shear stress. This differential capacity to alter function reveals that slow rolling, which is mediated by recognition of a number of sialylated ligands on the surface of PMN can be distinguished from the processes associated with the transition to arrest. Thus, treatment with GMI-1070 at doses that efficiently block vascular occlusion may spare some PMN rolling and immune surveillance function. Disclosures: Simon: GlycoMimetics Inc.: Research Funding. Chase:GlycoMimetics Inc.: Research Funding. Thackray:GlycoMimetics, Inc.: Employment, Equity Ownership. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership.
262 Adhesion molecules are critically involved in the pathophysiology of sickle cell disease (SCD). A growing body of evidence from animal models and humans supports the role of selectin-mediated cell adhesion in the pathophysiology of vaso-occlusion. Leukocyte adhesion has been demonstrated to reduce microvascular blood flow in a sickle cell mouse model, and there is evidence to suggest leukocyte adhesion in humans contributes to vaso-occlusion (Stuart et al, Lancet 2004; Turhan et al, PNAS 2002). GMI-1070 is a pan-selectin inhibitor that targets E-, P-, and L-selectins and has previously been shown to restore blood flow and improve survival in a mouse model of vaso-occlusion (Chang et al, Blood 2010). Also, it is potent inhibitor of adhesion of human neutrophils to immobilized E-selectin and ICAM-1 under flow conditions in vitro. As part of a Phase 1/2 study of GMI-1070 in patients with SCD we determined the effects of the drug on biomarkers of adhesion in vivo and ex vivo. Methods: An open-label phase 1/2 study of the safety, PK, and activity of GMI-1070 was performed, enrolling adults with SCD at steady state. Here we are reporting data on GMI-1070 anti-adhesion activity. GMI-1070 was administered in two IV doses given on the same day: 20 mg/kg as a loading dose, followed 10 hours later by 10 mg/kg. Serial WBC count with differential (all 15 subjects), computer-assisted intravital microscopy (CAIM) (4 subjects), and ex vivo activity of GMI-1070 in plasma (4 subjects) were measured. CAIM is a non-invasive technique for quantitative measurement of microvascular blood flow in vessels of the bulbar conjunctiva. In this study it was used to measure RBC velocity before and after dosing (at 30 minutes, 2, 4, 8, and 24 hours) with GMI-1070. Ex vivo evaluation of plasma GMI-1070 activity in a cell adhesion assay was also performed, with samples taken at 0, 8, 24, and 48 hours after first infusion. Results: Adults were enrolled at three centers: 13 with HbSS, 2 with HbSB0thal. All were African-American, and 9 were male. All subjects received both doses of study drug. The t1/2 was 7.7 hours. Mean baseline WBC was 10 K/mm3, and baseline absolute neutrophil count (ANC) was 5.4 K/mm3. The mean WBC was 10.4, 11.6, 10.2, and 8.7 K/mm3 at 8, 24, 48 hours and 7 days, respectively. The ANC mean was 5.5, 7.5, 5.6, and 4.2 K/mm3 at the same time points. ANC % change from baseline was significant at 24 and 48 hours (p=0.001, 0.025 mixed effects model) (Figure). There were no significant changes in absolute monocyte or lymphocyte counts. There was no correlation with any clinical adverse events. In one subject, the WBC rose from 10.4 to a peak of 28, with no clinical symptoms or significant changes in other lab values. CAIM (n=4) evaluating microvascular blood flow in the bulbar conjunctiva (measured in screen pixels/sec), showed mean RBC velocity at baseline was 335 (SD 70) pixels/sec, with mean values at 30 min., 2, 4, and 8 hours of 368 (59), 345 (63), 341 (59), and 338 (83) pixels/sec respectively, and returned to baseline of 317 (82) pixels/sec at 24 h. These differences did not reach statistical significance (mixed effects model). In ex vivo evaluation of neutrophil adhesion to matrix proteins (as quantified by # of bound neutrophils per 50× field) from samples 0, 4, 8, 24, and 48 hours after the first infusion revealed reduction in adhesions at 4 and 8 hours; mean adhered neutrophils were 33 (17), 20 (11), 18 (9), 47 (49), and 42 (15) respectively. However, these differences did not reach statistical significance. In conclusion, GMI-1070 infusion resulted in neutrophilia, a trend towards increased RBC velocity in the vessels of the bulbar conjunctiva immediately after infusion, and reduced leukoctye adhesion in an ex vivo assay despite neutrophilia. All of these findings are consistent with an anti-adhesive effect on leukocyte adhesion in vivo and suggest that the findings in sickle mouse studies can be translated into SCD patients. This study supports further evaluation of GMI-1070 for the treatment of vaso-occlusive episodes in SCD.Figure:Observed (mean/SE) absolute WBC and ANC over timeFigure:. Observed (mean/SE) absolute WBC and ANC over time Disclosures: Wun: GlycoMimetics: Clinical Trial Sponsorship, Consultancy; Eli Lilly: Clinical Trial Sponsorship, Consultancy. Off Label Use: This drug (GMI-1070) has not been approved for any clinical indication. De Castro:GlycoMimetics: clinical trial sponsorship. Styles:GlycoMimetics: Clinical Trial Sponsorship, Consultancy. Cheung:GlycoMimetics: clinical trial sponsorship. Chase:GlycoMimetics: clinical trial sponsorship. Simon:GlycoMimetics: Research Funding. Magnani:GlycoMimetics: Employment, Equity Ownership. Thackray:GlycoMimetics: Employment, Equity Ownership.
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