In view of the usefulness of mouse models for functional manipulations, it was imperative to identify a suitable mouse homolog of Siglec-8. In mice, there is no Siglec-8 ortholog, but Siglec-F has been found to be the closest functional paralog. Siglec-F is a 569-aa, CD33-related siglec that contains four Ig-like domains (Siglec-8 contains three) and, like Siglec-8, it contains both ITIM and ITIM-like motifs in its cytoplasmic tail. Using sequence homology comparisons, it was initially proposed that Siglec-F was the likely ortholog of human Siglec-5, but the homology was limited to the extracellular domains of both receptors (47). Furthermore, initial studies revealed that Siglec-F was predominantly expressed in bone marrow cells of the myelomonocytic lineage, and it was not expressed on mature neutrophils and monocytes (47), which have been shown to express Siglec-5 (52), further suggesting that Siglec-F may not be the true ortholog of Siglec-5.

Later efforts to fully characterize the expression pattern of Siglec-F and determine its functional counterpart in humans revealed that Siglec-F shared 38% similarity with human Siglec-8 (53). Using monoclonal antibodies to Siglec-F, it was found that Siglec-F, like Siglec-8, was predominantly expressed on the surface of mature eosinophils and on bone marrow eosinophils (54). However, Siglec-F is not expressed on mouse mast cells and surprisingly is instead expressed on mouse alveolar macrophages and subpopulations of intestinal epithelial cells (55–57) (Figure 3). Although they are structurally different and are expressed on different cell types, Siglec-F, like Siglec-8, has a binding preference for α2-3-linked sialic acids and recognizes 6′-sulfo-sLe^x (58). While subsequent studies have reproduced these findings, access to additional glycan structures for screening has allowed the identification of several multi-antennary structures that are recognized by Siglec-F but not Siglec-8 (38) (Figure 1). Indeed, this may explain why mouse lung ligands are recognized by Siglec-F but not by Siglec-8 (see below) (59). Based on these reports, it was concluded that Siglec-F and Siglec-8 are functionally convergent paralogs rather than orthologs.

Subsequent studies to examine the biological roles of Siglec-F in vivo revealed that its expression is upregulated following allergen challenge in a mouse lung allergy model and the congenital deficiency via genetic deletion of the Siglec-F gene led to enhanced eosinophil numbers in the bone marrow, peripheral blood, and lungs during allergic inflammation but not at baseline. Furthermore, Siglec-F-null mice had diminished eosinophil death, suggesting a role for Siglec-F in mediating eosinophil apoptosis (60). Indeed, administration of anti-Siglec-F antibody reduced peripheral blood and tissue eosinophil numbers in wild-type mice, IL-5 transgenic mice, and in mouse models of hypereosinophilic syndrome and eosinophilic esophagitis, which was attributed to induction of eosinophil apoptosis. Additionally, the effect of the anti-Siglec-F antibody was specific to eosinophils and had no effect on other cells, not even Siglec-F-expressing alveolar macrophages (61–64). Despite our advances in understanding the role of Siglec-F in eosinophil survival in vivo and in vitro, little is known about the signaling mechanism of this receptor. A report by Mao et al. showed that Siglec-F engagement on mouse eosinophils led to caspase cleavage; however, unlike Siglec-8, there was no detectable ROS production, and Siglec-F function did not involve the activation of SFKs or SHP-1 (65). Therefore, further studies are needed to fully characterize the signaling pathways for Siglec-F.

Although both Siglec-F and Siglec-8 preferentially recognize the glycan 6′-sulfo-sLe^x, the identity of their natural ligands is still under investigation. Initial studies to identify endogenous tissue ligands in mice using Siglec-F-Fc chimeras and immunohistochemistry showed that Siglec-F ligands were constitutively expressed on airway epithelial cells and their expression was dependent on ST3Gal-III, a sialyltransferase that can add α2,3 terminal sialic acids to glycans (66). Expression of these ligands was increased upon induction of allergic airway inflammation (67, 68). Glycoproteomic analysis of material derived from mouse tracheal epithelial cells revealed that Siglec-F-Fc bound to glycans displayed on Muc5b and Muc4, but not Muc5ac. Mouse lungs deficient in Muc5b had reduced Siglec-F-Fc binding, and mice conditionally deficient in Muc5b showed enhanced eosinophilic inflammation in response to airway instillation of IL-13, further validating that Muc5b carries glycan ligands for Siglec-F and suggesting that only subsets of airway mucins display the glycan structures necessary for Siglec-F binding (38). Although less is known about Siglec-8 tissue ligands, a recent study showed that Siglec-8 ligands were expressed selectively on serous cells, a subpopulation of submucosal gland cells in the inferior turbinate, and inflammation that occurs in chronic sinusitis led to increased expression of Siglec-8 tissue ligands in the upper airways (69). Additional studies show that mouse airways do not express Siglec-8 ligands and Siglec-8-Fc binding in human tracheal sections is restricted to serous cells in submucosal glands and cartilage (59) (Figure 4). The exact identity of these ligands is still under investigation, but given the fact that the galactose 6-O-sulfotransferase CHST-1 is dispensable for generating Siglec-F ligands in the mouse, it appears that the 6′ sulfation needed for Siglec-8 binding is not required for Siglec-F binding (70).

Beyond the physiological role of Siglec-8 in inducing cell death of eosinophils, Siglec-8 represents a promising target through which to deliver therapeutic payloads into eosinophils and other Siglec-8-expressing cells. Several studies have shown that siglecs are endocytic receptors and, once engaged, can carry their ligand—and presumably any associated cargo—into the cell (71–74). This strategy has been employed in the development of cancer therapeutics by targeting preferentially upregulated receptors such as the receptors for transferrin or folate or through antibodies targeting slightly more selectively expressed antigens, such as CD33 (Siglec-3) in acute myelogenous leukemia (75–77). An antibody-targeting CD22 (Siglec-2) is also under investigation to treat diseases involving B cells, such as B-cell acute lymphoblastic leukemia or systemic lupus erythematosus (78, 79). Due to the restricted expression pattern of Siglec-8, targeting of eosinophils through Siglec-8 offers promise. However, the capability to exploit Siglec-8 in this manner depends on whether Siglec-8 is endocytosed and is present and accessible on the surface of eosinophils in various circumstances. Siglec-F endocytosis has been studied in mouse eosinophils. Siglec-F is internalized in response to antibody ligation via a clathrin- and lipid raft-independent pathway that relies on ARF6 but not dynamin-1 (72). New data indicate that Siglec-8 is indeed internalized in response to antibody or synthetic ligand engagement on peripheral blood eosinophils and that this pathway can be exploited to deliver a toxin (the ribosome-inhibiting protein saporin) to eosinophils to induce cell death under conditions in which Siglec-8 engagement alone would be insufficient (i.e., in the absence of IL-5 priming) (80). Despite some similarities, including the lysosomal localization of the internalized siglec, the pathway utilized by Siglec-F internalization appears to be distinct from that of Siglec-8. The pathway of internalization can have profound effects on receptor function, leading to distinct signaling mechanisms and downstream functions or alterations in receptor turnover. For example, endocytosis of SR-A via a lipid raft/caveolae-dependent pathway is required for macrophage apoptosis in a ligand-dependent manner, whereas clathrin-mediated SR-A endocytosis is expendable for this effect (81). While there is abundant evidence linking endocytosis to the organization of signaling events (82), it remains to be determined whether the endocytosis of Siglec-8 affects its function.

Siglec-8 may also achieve part of its function by internalizing other surface proteins. Upon antigen stimulation, the B cell receptor (BCR) engages clathrin in lipid raft domains and thus is internalized via a mixed pathway (83, 84). While the siglec CD22 is initially excluded from lipid rafts, it colocalizes with the BCR and promotes its internalization when unmasked (85, 86). This downregulation of the BCR is thought to be one mechanism underlying the inhibitory function of CD22. Of note, the IL-5 receptor, which is critically important to the activation and survival of eosinophils, has been found to be internalized via distinct clathrin- and lipid raft-dependent pathways and is targeted for proteolytic degradation through the lipid raft-mediated endocytic pathway (87). It is an intriguing possibility that the endocytosis and trafficking of Siglec-8 and the IL-5 receptor may be linked in a way that influences the function of each receptor.

While Siglec-8 has garnered much attention as a cell-surface marker of eosinophils, there are a number of other glycans and glycan-binding proteins present on eosinophils that regulate their survival, trafficking, and adhesion and that may be useful markers of eosinophilic inflammation. In both mice and humans, eosinophils express siglec family members other than Siglec-F and Siglec-8, respectively.

Protein–glycan interactions are exceptionally important in the processes of cell adhesion and trafficking. In the initial steps of leukocyte extravasation, the cell must tether to and roll along the endothelium, which requires the interaction between selectins and their glycan ligands. Eosinophils depend to differing degrees on P-, E-, and L-selectin interactions in this process. Relative to neutrophils, eosinophils bind less well to E-selectin and bind to a greater extent to P-selectin through cell-surface glycan ligands (99), presumably due to increased levels of P-selectin glycoprotein ligand (PSGL)-1 (100). This same study found that L-selectin on the surface of eosinophils was important in tethering of eosinophils to human umbilical vein endothelial cells (HUVECs) but only due to establishing inter-eosinophil interactions rather than binding to the endothelial cells (99). However, others have found that diminished L-selectin expression or the use of blocking antibodies to L-selectin reduce eosinophil rolling and adhesion on HUVECs or on rabbit mesenteric venule endothelial cells under conditions of flow (101, 102). As demonstrated by the study by Sriramarao et al., human selectins are capable of interacting with ligands expressed in other species. Indeed, the selectin-binding sites in the best characterized P-selectin ligand, PSGL-1, are evolutionarily well conserved (103). However, distinct patterns of expression render cross-species comparisons more difficult. For example, P-selectin expression in mice but not in humans is induced by TNF-α or LPS, and cytokine regulation of human and primate P- and E-selectins is more selective than in mice (104, 105). However, mouse strains in which selectins have been knocked out have permitted elegant studies of their importance in mouse eosinophil migration. Using these mouse strains, several studies have shown that P-selectin is critical in eosinophil recruitment to the lung and peritoneum (106–108).

P-selectin glycoprotein ligand-1 is the best characterized P-selectin ligand on eosinophils, and its expression has been shown to be increased in patients with allergic asthma relative to healthy controls. This increase results in enhanced binding to P-selectin and IL-4-treated HUVECs (109). PSGL-1 contributes to eosinophil, but not neutrophil, adhesion to IL-13-activated HUVECs under conditions of physiological flow (110). Paradoxically, PSGL-1 expression is reduced on activated eosinophils and is shed from leukocytes (111). Although there have been reports that PSGL-1 can act as a ligand for E-selectin as well (112, 113), no changes in E-selectin binding were observed with eosinophils from allergic asthma patients in this study. There appear to be other significant ligands for P-selectin on eosinophils, however. In patients with atopic dermatitis, eosinophils are capable of binding substantially more soluble P-selectin than eosinophils from healthy donors but do not display more PSGL-1 on their surface (114).

Although the selectins bind to related sialylated glycans, these glycans can be present on a variety of different proteins or lipids that may be cell type- or tissue-specific. Eosinophils, for example, display far less sialyl Lewis^X antigen, a selectin ligand carbohydrate structure, than neutrophils, but a greater proportion is in the form of sialyl dimeric Lewis^X and is sensitive to endo-β-galactosidase treatment (115). Interestingly, neither the display of sialyl Lewis^X on eosinophils nor adhesion to immobilized E-selectin is protease-sensitive, indicating that these carbohydrate ligands may not be present on cell-surface proteins (115). Consistent with this, glycosphingolipids extracted from leukocytes were found to interact with E-selectin, and their biosynthesis was required for E-selectin-dependent, but not P-selectin-dependent, neutrophil adhesion (116). P-selectin ligands present on eosinophils clearly differ from those of E-selectin in that binding to immobilized P-selectin is protease-sensitive and endo-β-galactosidase-resistant (117). Indeed, although the expression of sialyl Lewis^X on eosinophils is not changed by cellular activation with platelet-activating factor (PAF), P-selectin binding is reduced following PAF activation and L-selectin is shed following transendothelial migration (117, 118).

Due to their critical role in eosinophil trafficking to peripheral tissues, selectins represent a potentially useful therapeutic target for diseases of eosinophilic inflammation. In fact, selectin antagonists interfere with eosinophil (and neutrophil) adhesion (119), and a pan-selectin antagonist glycomimetic agent is in clinical trials to modulate selectin-based adhesion in acute sickle cell crisis (120). Although lack of cell specificity is a concern, a more selective P-selectin antagonist or one that interacts with a eosinophil-selective P-selectin ligand would likely be effective in preventing further eosinophilic inflammation in the tissues with fewer potential complications.

The galectin family of proteins, previously known as S-type lectins, has a binding preference, generally, for β-galactosides, although there appear to be exceptions to this. Most of the members of the family are secreted but can cross-link cell-surface receptors due to the presence of more than one carbohydrate recognition domain (CRD) or through multimerization of a monomer containing one CRD (galectin-3).

The galectin family member most commonly associated with eosinophils is galectin-10, also known as Charcot–Leyden crystal (CLC) protein. Galectin-10 makes up about 10% of the total protein content of human eosinophils (121), and CLC deposition in tissues has long been considered a marker of eosinophilic (or basophilic) inflammation (122). The protein localizes to both the cytosol and a subset of core-less granules (123). The CLC protein was initially believed to function as a lysophospholipase within the eosinophil (121); however, this enzymatic activity has since been ascribed not the CLC protein but to another enzyme that can associate to a degree with it (124). Due to sequence identity, structural homology, and genomic structure, CLC protein became known also as galectin-10 (125–127). Unlike other members of the galectin family, however, galectin-10 appears not to bind β-galactosides to any substantial degree but instead appears to bind to mannose-containing carbohydrate moieties (128). The natural ligand or ligands of galectin-10 and the functional significance of its ability to bind to carbohydrates remain undetermined. Despite the lack of functional data, galectin-10 mRNA and protein levels remain useful biomarkers for eosinophilic airway inflammation, active eosinophilic esophagitis, aspirin-exacerbated respiratory disease, CRTH2 activation, and celiac disease (129–133).

On the eosinophil cell surface, galectin–ligand interactions have been found to be important in eosinophil recruitment, activation, and survival. The granulocyte-specific and heavily glycosylated protein CD66b (also known as CEACAM8) is expressed on eosinophils and is upregulated in response to cellular activation (134, 135). Sialylated glycans on CD66b interact with E-selectin, and this interaction has been shown to be important for neutrophil adherence to activated endothelium (136). Glycans on CD66b also interact with galectin-3, and engagement of CD66b using either soluble galectin-3 or antibody induced ROS production and degranulation (137). Cross-linking of CD66b also caused the eosinophils to become more adherent, perhaps through the clustering of the integrin subunit CD11b.

Several other galectin interactions may be important in eosinophil adhesion and chemoattraction, including those with galectins-1, -3, and -9 (138–140). However, the role of galectins in eosinophil recruitment is covered in greater detail in a review in this volume by Sriramarao et al. and will not be discussed further here. It should be noted, however, that galectins play other important roles in eosinophil biology and are biomarkers of disease activity. High concentrations of galectin-1, for example, can induce eosinophil cell death (140), and levels of galectin-3 before treatment in patients with severe asthma predict treatment responses to omalizumab (141).

While many of the cell-surface and intracellular eosinophil proteins have been identified and extensively described, the glycans that coat the various cell-associated proteins and likely play important roles in numerous biological pathways remain largely shrouded in mystery. As part of an effort to characterize these glycans, the glycome of human eosinophils has been analyzed in cell lysates and compared to those of basophils and mast cells to elucidate the identities of these glycans, their relative abundances, and key differences between these cell types (142). Although mast cells possess substantial amounts of terminally sialylated epitopes on their various glycoproteins, eosinophils and basophils have far more part-processed terminal N-acetylglucosamine (GlcNAc)-containing structures. For example, the most abundant N-glycan by far in both eosinophils and basophils is a bi-antennary structure with two terminal non-extended GlcNAc sugars, which is far less abundant in mast cells. While the functional relevance of these patterns is unclear, it is unlikely that these modifications to the cell surface are random. In addition, it is uncertain how cytokine priming and other signals that may be present under inflammatory conditions may affect glycan synthesis and processing. However, such changes may well affect processes such as adhesion, activation, cell–cell interaction, and even survival.

There remains a clinical need to effectively and selectively treat diseases of eosinophilic inflammation. Due to their roles in recruitment, adhesion, activation, and survival, glycan–glycan-binding protein interactions are beginning to garner attention as therapeutic prospects. Siglec-8 and its ligands offer a cell-selective pathway to induce cell death in primed eosinophils and deliver therapeutic payloads into the cell. Antagonists of selectin interactions may help limit eosinophilic inflammation, but significant hurdles remain for achieving a safe, cell-selective effect. Monomeric β-galactosides or glycomimetics may also be clinically useful in antagonizing eosinophil lectin interactions that are involved in cell adhesion and activation. While targets and biomarkers have been identified, further studies are necessary to elucidate the functions of glycan-binding proteins on eosinophils, such as those of other members of the siglec family; to identify their natural glycan ligands and how they are modulated; and to determine the functional significance of the glycans displayed on the eosinophil cell surface.

JO completed the sections on Siglec-8 endocytosis, selectins, galectins, and other members of the siglec family. DC completed all other sections regarding Siglec-8 as well as Siglec-F. BB organized the effort and wrote the introduction. All authors contributed to revisions of the manuscript.

BB has current or recent consulting or scientific advisory board arrangements with or has received honoraria from, Sanofi-Aventis, TEVA, AstraZeneca and Allakos, and owns stock in Allakos and Glycomimetics. He receives publication-related royalty payments from Elsevier and UpToDate™ and is a co-inventor on existing Siglec-8-related patents and thus may be entitled to a share of royalties received by Johns Hopkins University on the potential sales of such products. BB is also a co-founder of Allakos, which makes him subject to certain restrictions under University policy. The terms of this arrangement are being managed by the Johns Hopkins University and Northwestern University in accordance with their conflict of interest policies. The authors have no additional competing financial interests.