Sherry A. Hudson

Activation of Human Leukocytes Reduces Surface P-Selectin Glycoprotein Ligand-1 (PSGL-1, CD162) and Adhesion to P-Selectin In Vitro

P-selectin glycoprotein ligand-1 (PSGL-1), the primary ligand for P-selectin, is constitutively expressed on the surface of circulating leukocytes. The objective of this study was to examine the effect of leukocyte activation on PSGL-1 expression and PSGL-1-mediated leukocyte adhesion to P-selectin. PSGL-1 expression was examined via indirect immunofluorescence and flow cytometry before and after leukocyte stimulation with platelet activating factor (PAF) and PMA. Human neutrophils, monocytes, and eosinophils were all demonstrated to have significant surface expression of PSGL-1 at baseline, which decreased within minutes of exposure to PAF or PMA. PSGL-1 was detected in the supernatants of PAF-activated neutrophils by immunoprecipitation. Along with the expression data, this suggests removal of PSGL-1 from the cell surface. Soluble PSGL-1 was also detected in human bronchoalveolar lavage fluids. Down-regulation of PSGL-1 was inhibited by EDTA. However, inhibitors of L-selectin shedding and other sheddase inhibitors did not affect PSGL-1 release, suggesting that PSGL-1 may be shed by an as yet unidentified sheddase or removed by some other mechanism. Functionally, PSGL-1 down-regulation was associated with decreased neutrophil adhesion to immobilized P-selectin under both static and flow conditions, with the most profound effects seen under flow conditions. Together, these data indicate that PSGL-1 can be removed from the surface of activated leukocytes, and that this decrease in PSGL-1 expression has profound effects on leukocyte binding to P-selectin, especially under conditions of flow.

Siglec‐F antibody administration to mice selectively reduces blood and tissue eosinophils

Background:  Sialic acid‐binding immunoglobulin‐like lectins (Siglecs) are a family of receptors that bind sialic acid and mostly contain immunoreceptor tyrosine‐based inhibitory motifs, suggesting that these molecules possess inhibitory functions. We have recently identified Siglec‐8 as an eosinophil‐prominent Siglec, and cross‐linking of Siglec‐8 on human eosinophils induces apoptosis. In this article, we address the in vivo consequences of Siglec engagement. We and others have identified mouse Siglec‐F as the closest functional paralog of human Siglec‐8, based on shared ligand‐binding and expression pattern. We therefore hypothesized that Siglec‐F engagement would affect levels and viability of eosinophils in vivo.

E-selectin receptors on human leukocytes

Selectins on activated vascular endothelium mediate inflammation by binding to complementary carbohydrates on circulating neutrophils. The human neutrophil receptor for E-selectin has not been established. We report here that sialylated glycosphingolipids with 5 N-acetyllactosamine (LacNAc, Galbeta1-4GlcNAcbeta1-3) repeats and 2 to 3 fucose residues are major functional E-selectin receptors on human neutrophils. Glycolipids were extracted from 10(10) normal peripheral blood human neutrophils. Individual glycolipid species were resolved by chromatography, adsorbed as model membrane monolayers and selectin-mediated cell tethering and rolling under fluid shear was quantified as a function of glycolipid density. E-selectin-expressing cells tethered and rolled on selected glycolipids, whereas P-selectin-expressing cells failed to interact. Quantitatively minor terminally sialylated glycosphingolipids with 5 to 6 LacNAc repeats and 2 to 3 fucose residues were highly potent E-selectin receptors, constituting more than 60% of the E-selectin-binding activity in the extract. These glycolipids are expressed on human blood neutrophils at densities exceeding those required to support E-selectin-mediated tethering and rolling. Blocking glycosphingolipid biosynthesis in cultured human neutrophils diminished E-selectin, but not P-selectin, adhesion. The data support the conclusion that on human neutrophils the glycosphingolipid NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-3[Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3](2)[Galbeta1-4GlcNAcbeta1-3](2)Galbeta1-4GlcbetaCer (and closely related structures) are functional E-selectin receptors.

Eosinophil-selective binding and proapoptotic effect in vitro of a synthetic siglec-8 ligand, polymeric 6′-sulfated sialyl lewis X

The lectin Siglec-8 (sialic acid-binding, immunoglobulin-like lectin), which is selectively expressed on eosinophil surfaces and regulates eosinophil survival, preferentially binds to the glycan 6′-sulfo-sialyl Lewis X (6′-sulfo-sLex). Antibody engagement of Siglec-8 on eosinophils causes their apoptosis, suggesting that engagement of Siglec-8 with its natural glycan ligands in vivo may control allergic inflammation. We report that a soluble synthetic polymer displaying 6′-sulfo-sLex glycan selectively binds to human eosinophils and human embryonic kidney 293 cells expressing Siglec-8. Binding was inhibited by anti-Siglec-8 antibody. In whole blood, eosinophils were the only leukocyte subtype to detectably bind polymeric 6′-sulfo-sLex. Interleukin-5-primed eosinophils underwent apoptosis when incubated with either anti-Siglec-8 monoclonal antibody or polymeric 6′-sulfo-sLex, although the glycan polymer was less effective. These data demonstrate that a soluble, multivalent glycan selectively binds to human eosinophils and induces their apoptosis in vitro and provide proof-of-concept that such a reagent could be used to selectively target eosinophils.

CCR3-Active Chemokines Promote Rapid Detachment of Eosinophils from VCAM-1 In Vitro

Selective eosinophil recruitment is the result of orchestrated events involving cell adhesion molecules, chemokines, and their receptors. The mechanisms by which chemokines regulate eosinophil adhesion and migration via integrins are not fully understood. In our study, we examined the effect of CCR3-active chemokines on eosinophil adhesion to VCAM-1 and BSA under both static and flow conditions. When eotaxin-2 or other CCR3-active chemokines were added to adherent eosinophils, it induced rapid and sustained eosinophil detachment from VCAM-1 in a concentration-dependent manner. Adhesion was detectably reduced within 3 min and was further reduced at 10–60 min. Simultaneously, eotaxin-2 enhanced eosinophil adhesion to BSA. Preincubation of eosinophils with the CCR3-blocking mAb 7B11 completely prevented chemokine-induced changes in adhesion to VCAM-1 and BSA. Using a different protocol, pretreatment of eosinophils with chemokines for 0–30 min before their use in adhesion assays resulted in inhibition of VCAM-1 adhesion and enhancement of BSA adhesion. By flow cytometry, expression of α4 integrins and a β1 integrin activation epitope on eosinophils was decreased by eotaxin-2. In a flow-based adhesion assay, eotaxin-2 reduced eosinophil accumulation and the strength of attachment to VCAM-1. These results show that eotaxin-2 rapidly reduced α4 integrin function while increasing β2 integrin function. These findings suggest that chemokines facilitate migration of eosinophils by shifting usage away from β1 integrins toward β2 integrins.

IL-33 enhances Siglec-8 mediated apoptosis of human eosinophils

IL-33 activates eosinophils directly via the ST2 receptor. Like IL-5, IL-33 induces eosinophilia and eosinophilic airway inflammation in mouse models and primes human eosinophil responses. Previously, we reported that IL-5 priming enhances Siglec-8 mediated mitochondrial and reactive oxygen species (ROS)-dependent eosinophilic apoptosis and eliminates caspase dependence of this cell death process. Whether IL-33, like IL-5, augments pro-apoptotic pathways involving receptors such as Siglec-8 and in a similar manner has not been explored. Annexin-V labeling was performed to detect apoptosis in human eosinophils pre-incubated with or without a range of concentrations of IL-33 and/or IL-5 in the presence or absence of Siglec-8 monoclonal antibody (mAb) 2C4 and inhibitors of caspases. Tetramethyl-rhodamine staining was used as a marker of mitochondrial membrane potential loss and injury. ROS production was determined by measuring the superoxide dismutase-inhibitable reduction of cytochrome c. Cleavage of poly(ADP-ribose) polymerase (PARP) was assessed using Western blotting. Eosinophils cultured alone or with mAb 2C4 underwent low levels of apoptosis at 24h. 2C4-induced eosinophil apoptosis was markedly and equally enhanced after culture for 24h with either IL-33 or IL-5, although IL-5 was more potent. Effects on apoptosis with IL-33 and IL-5 were synergistic. In contrast, percentages of cells exhibiting reduced mitochondrial membrane potential were greater with IL-33 than IL-5 and effects of these cytokines were also synergistic. Antimycin, an inhibitor of mitochondrial electron transport, almost completely inhibited 2C4-induced apoptosis with either IL-33 or IL-5. Surprisingly, 2C4-induced eosinophil ROS production was significantly enhanced with IL-5 but not IL-33. Siglec-8-mediated apoptosis in the presence of IL-33 was more sensitive in magnitude than IL-5 to inhibition by the pan-caspase inhibitor Z-VAD-FMK, yet both cytokine conditions were associated with PARP cleavage. These data demonstrate that IL-33 is as effective but less potent than IL-5 in enhancing Siglec-8-mediated eosinophil apoptosis, and can synergize with IL-5. Eosinophils primed by IL-33 and/or IL-5 in vivo would be expected to display enhanced susceptibility to undergoing Siglec-8-induced apoptosis.

Endogenous airway mucins carry glycans that bind Siglec-F and induce eosinophil apoptosis

BACKGROUND Sialic acid-binding, immunoglobulin-like lectin (Siglec) F is a glycan-binding protein selectively expressed on mouse eosinophils. Its engagement induces apoptosis, suggesting a pathway for ameliorating eosinophilia in the setting of asthma and other eosinophil-associated diseases. Siglec-F recognizes sialylated sulfated glycans in glycan-binding assays, but the identities of endogenous sialoside ligands and their glycoprotein carriers in vivo are unknown. OBJECTIVES To use mouse lung-derived materials to isolate, biochemically identify, and biologically characterize naturally occurring endogenous glycan ligands for Siglec-F. METHODS Lungs from normal and mucin-deficient mice, as well as mouse tracheal epithelial cells, were investigated in vitro and in vivo for the expression of Siglec-F ligands. Western blotting and cytochemistry used Siglec-F-Fc as a probe for directed purification, followed by liquid chromatography-tandem mass spectrometry of recognized glycoproteins. Purified components were tested in mouse eosinophil-binding assays and flow cytometry-based cell death assays. RESULTS We detected mouse lung glycoproteins that bound to Siglec-F; binding was sialic acid dependent. Proteomic analysis of Siglec-F binding material identified Muc5b and Muc4. Cross-affinity enrichment and histochemical analysis of lungs from mucin-deficient mice assigned and validated the identity of Muc5b as one glycoprotein ligand for Siglec-F. Purified mucin preparations carried sialylated and sulfated glycans, bound to eosinophils and induced their death in vitro. Mice conditionally deficient in Muc5b displayed exaggerated eosinophilic inflammation in response to intratracheal installation of IL-13. CONCLUSIONS These data identify a previously unrecognized endogenous anti-inflammatory property of airway mucins by which their glycans can control lung eosinophilia through engagement of Siglec-F.

Developmental, Malignancy-Related, and Cross-Species Analysis of Eosinophil, Mast Cell, and Basophil Siglec-8 Expression

ObjectiveThe aim of this study is to determine when during hematopoiesis Siglec-8 gets expressed, whether it is expressed on hematologic malignancies, and if there are other non-human species that express Siglec-8.MethodsSiglec-8 mRNA and cell surface expression was monitored during in vitro maturation of human eosinophils and mast cells. Flow cytometry was performed on human blood and bone marrow samples, and on blood samples from dogs, baboons, and rhesus and cynomolgus monkeys.ResultsSiglec-8 is a late maturation marker. It is detectable on eosinophils and basophils from subjects with chronic eosinophilic leukemia, chronic myelogenous leukemia, and on malignant and non-malignant bone marrow mast cells, as well as the HMC-1.2 cell line. None of the Siglec-8 monoclonal antibodies tested recognized leukocytes from dogs, baboons, and rhesus and cynomolgus monkeys.ConclusionsSiglec-8-based therapies should not target immature human leukocytes but should recognize mature and malignant eosinophils, mast cells, and basophils. So far, there is no suitable species for preclinical testing of Siglec-8 monoclonal antibodies.

Polymorphisms in the sialic acid-binding immunoglobulin-like lectin-8 (Siglec-8) gene are associated with susceptibility to asthma

Sialic acid-binding immunoglobulin-like lectin-8 (Siglec-8) promotes the apoptosis of eosinophils and inhibits FcɛRI-dependent mediator release from mast cells. We investigated the genetic association between sequence variants in Siglec-8 and diagnosis of asthma, total levels of serum IgE (tIgE), and diagnosis of eosinophilic esophagitis (EE) in diverse populations. The effect of sequence variants on Siglec-8 glycan ligand-binding activity was also examined. Significant association with asthma was observed for SNP rs36498 (odds ratios (OR), 0.69, P=8.8 × 10−5) among African Americans and for SNP rs10409962 (Ser/Pro) in the Japanese population (OR, 0.69, P=0.019). Supporting this finding, we observed association between SNP rs36498 and current asthma among Brazilian families (P=0.013). Significant association with tIgE was observed for SNP rs6509541 among African Americans (P=0.016), and replicated among the Brazilian families (P=0.02). In contrast, no association was observed with EE in Caucasians. By using a synthetic polymer decorated with 6′-sulfo-sLex, a known Siglec-8 glycan ligand, we did not find any differences between the ligand-binding activity of HEK293 cells stably transfected with the rs10409962 risk allele or the WT allele. However, our association results suggest that the Siglec8 gene may be a susceptibility locus for asthma.

Stimulation of Human Endothelium with IL-3 Induces Selective Basophil Accumulation In Vitro

Basophils have been shown to accumulate in allergic airways and other extravascular sites. Mechanisms responsible for the selective recruitment of basophils from the blood into tissue sites remain poorly characterized. In this study, we characterized human basophil rolling and adhesion on HUVECs under physiological shear flow conditions. Interestingly, treatment of endothelial cells with the basophil-specific cytokine IL-3 (0.01–10 ng/ml) promoted basophil and eosinophil, but not neutrophil, rolling and exclusively promoted basophil adhesion. Preincubation of HUVECs with an IL-3R-blocking Ab (CD123) before the addition of IL-3 inhibited basophil rolling and adhesion, implicating IL-3R activation on endothelial cells. Incubation of basophils with neuraminidase completely abolished both rolling and adhesion, indicating the involvement of sialylated structures in the process. Abs to the β1 integrins, CD49d and CD49e, as well as to P-selectin and P-selectin glycoprotein ligand 1, inhibited basophil rolling and adhesion. Furthermore, blocking chemokine receptors expressed by basophils, such as CCR2, CCR3, and CCR7, demonstrated that CCR7 was involved in the observed recruitment of basophils. These data provide novel insights into how IL-3, acting directly on endothelium, can cause basophils to preferentially interact with blood vessels under physiological flow conditions and be selectively recruited to sites of inflammation.