SIGLEC8

Sialic acid-binding Ig-like lectin 8 is a is_associated_with::protein that in humans is encoded by the SIGLEC8 is_associated_with::gene. This gene is located on chromosome 19q13.4, about 330 kb downstream of the is_associated_with::SIGLEC9 gene. Within the siglec family of transmembrane proteins, Siglec-8 belongs to the is_associated_with::CD33-related siglec subfamily, a subfamily that has undergone rapid evolution.

Initial characterization
Siglec-8 was first identified by is_associated_with::CD33 homology screening of ESTs from a cDNA library generated from a patient diagnosed with idiopathic is_associated_with::hypereosinophilic syndrome and was originally termed SAF-2 (sialoadhesin family 2). At the tissue level, Siglec-8 mRNA was found to be most highly expressed in lung, PBMCs, spleen, and kidney.

Expression
Siglec-8 is expressed by human is_associated_with::eosinophils, is_associated_with::mast cells, and, to a lesser extent, is_associated_with::basophils. It has thus garnered attention as a molecule that is uniquely expressed by immune effector cells involved in is_associated_with::asthma and is_associated_with::allergy. In both eosinophils and mast cells, Siglec-8 is expressed late in development. Siglec-8 transcript and protein are detectable at day 12 during the in vitro differentiation of eosinophils from cord blood precursors, whereas the transcription factor is_associated_with::GATA-1 peaks at day 2 and the secondary granule protein MBP-1 peaks at day 4 in this differentiation system. In mast cells generated from is_associated_with::CD34+ precursors, Siglec-8 expression peaks at 4 weeks of differentiation, in parallel with FcεRIα surface expression.

Consistent with the concept that Siglec-8 is a late differentiation marker, Siglec-8 has not been detected on the surface of relatively undifferentiated eosinophilic cell lines, such as EoL-1, AML14, AML14.3D10, or K562, the basophilic leukemia cell line KU812, nor on cells such as is_associated_with::HL60 or EoL-3 that have been differentiated towards an eosinophil-like lineage. Only low levels are detected on the human mast cell sub-line HMC-1.1; however, the HMC-1.2 cell line, which bears a second KIT mutation (D816V, in addition to the V560G mutation found in both HMC-1.1 and HMC-1.2 cells) that may induce further differentiation, expresses Siglec-8 at the cell surface. However, based on a small sampling of patients, all eosinophils from patients with is_associated_with::chronic eosinophilic leukemia (CEL), is_associated_with::hypereosinophilic syndrome, or is_associated_with::chronic myeloid leukemia (CML), all basophils from patients with CEL or CML, and all bone marrow mast cells from patients with indolent systemic is_associated_with::mastocytosis or is_associated_with::aplastic anemia express Siglec-8, providing a potential target for these cells in the context of these hematologic malignancies.

In addition, baboon eosinophils as well as is_associated_with::monocytes, a subset of is_associated_with::lymphocytes, and is_associated_with::neutrophils express on their cell surface a protein or proteins that are recognized by polyclonal human Siglec-8-specific antibody, consistent with genetic analyses indicating the existence of a Siglec-8 is_associated_with::ortholog in this species. However, the 2C4, 2E2, and 7C9 monoclonal antibodies against human Siglec-8 were not found to bind to targets on baboon cells, indicating that these particular is_associated_with::epitopes are not conserved.

Structure
Two splice variants of Siglec-8 exist. The initially characterized form contains 431 amino acid residues in total, 47 of which comprise an uncharacteristically short cytoplasmic tail compared to most CD33-associated siglecs. Subsequently, a longer form of Siglec-8, initially termed Siglec-8L, that contains 499 amino acid residues was identified. This longer form of Siglec-8 shares the same extracellular region but includes a longer cytoplasmic tail with two tyrosine-based motifs (an is_associated_with::immunoreceptor tyrosine-based inhibitory motif [ITIM] and an immunoreceptor tyrosine-based switch motif [ITSM]). Both forms of Siglec-8 are found in eosinophils and contain a V-set domain with is_associated_with::lectin activity and two C2-type Ig repeat domains in the extracellular region. Given that the longer version is felt to be the normal version, the term Siglec-8 is best used to refer to the 499 amino acid version, while the 431 amino acid version is best referred to as the “short form” of Siglec-8.

Ligand binding
Potential glycan ligands for Siglec-8 have been screened by glycan array. The glycan NeuAcα2–3(6-O-sulfo)Galβ1–4[Fucα1–3]GlcNAc, also known as 6′-sulfo-sialyl Lewis X, binds with high affinity to both Siglec-8 and to a mouse siglec, Siglec-F, which appears to have acquired a similar but not identical function and pattern of expression to human Siglec-8 through convergent evolution (the two siglecs are not orthologous). Rescreening on a more expanded glycan array reconfirmed this finding, but also identified a second closely related ligand in which the is_associated_with::fucose is absent (NeuAcα2–3(6-O-sulfo)Galβ1–4GlcNAc, or 6′-sulfated sialyl N-acetyl-D-lactosamine. These interactions are quite specific; no binding could be detected between these siglecs and unsulfated sialyl Lewis X or sialyl Lewis X sulfated at carbon 6 of GlcNAc (6-sulfo-sialyl Lewis X) rather than carbon 6 of galactose as in 6′-sulfo-sialyl Lewis X. Similarly, no other siglecs bind effectively to these Siglec-8 ligands, as demonstrated by selective binding to eosinophils in human blood of a polymer decorated with 6′-sulfo-sialyl Lewis X. The natural ligand or ligands for Siglec-8 have not yet been positively identified, but ongoing studies have determined that there are is_associated_with::sialidase-sensitive glycoprotein ligands for Siglec-F in mouse airways that require the activity of the α2,3 sialyltransferase 3 (ST3Gal-III) enzyme for their generation.

Eosinophils
Consistent with the role of most siglecs and the presence of the intracellular ITIM, Siglec-8 has been found to function as an inhibitory immunoregulatory receptor. Ligation of Siglec-8 induces apoptosis in eosinophils, and, surprisingly, the normally pro-survival cytokines interleukin (IL)-5 and is_associated_with::GM-CSF have been found to potentiate this apoptotic effect. IL-33, which activates and maintains eosinophils, also exerts a similar potentiating effect on Siglec-8-induced apoptosis. Inhibitor studies demonstrate that apoptosis induced by crosslinking Siglec-8 through the use of an anti-Siglec-8 mAb and a secondary antibody is mediated sequentially through is_associated_with::reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and is_associated_with::caspase activation. In the presence of IL-5, the loss of mitochondrial membrane integrity is accelerated and the secondary crosslinking antibody is no longer necessary to induce apoptosis. IL-5 stimulation also appears to alter the mode of cell death of eosinophils induced by Siglec-8 ligation in that cell death becomes a caspase-independent process. Costimulation of the IL-5 receptor and Siglec-8 leads to a type of cell death resembling regulated necrosis that is promoted by is_associated_with::MEK1/ERK signaling. Because inhibition of MEK1 does not alter ROS generation but the ROS inhibitor diphenyleneiodonium inhibits ERK1/2 phosphorylation and cell death, the production of ROS appears to be upstream of MEK1/ERK signaling in this pathway. Cell death induced by Siglec-8 in the presence of IL-33, in contrast, is mediated primarily by a caspase-dependent pathway, and IL-33 is capable of synergizing with IL-5 in potentiating cell death induced by Siglec-8 ligation.

Mast cells and basophils
While Siglec-8 ligation does not cause mast cell apoptosis, it inhibits FcεRIα-mediated Ca2+ flux and release of is_associated_with::prostaglandin D2 and is_associated_with::histamine. However, the release of IL-8 is not prevented by Siglec-8 ligation in mast cells. In experiments using the rat basophilic leukemia cell line RBL-2H3 stably transfected with Siglec-8, the inhibitory effect of Siglec-8 ligation on FcεRIα-mediated degranulation and Ca2+ flux was found to be dependent on the intact ITIM. There are no published data regarding the function of Siglec-8 on basophils.

CD33-related siglec subfamily
Due to its high level of sequence homology with CD33 (Siglec-3), Siglec-8 is grouped within the CD33-related siglec subfamily. This family is composed of a rapidly evolving group of siglecs that share 50–99% sequence identity. Most members of the subfamily also possess conserved cytoplasmic ITIM and ITIM-like sequences.

Mouse Siglec-F
While SIGLEC8 and mouse Siglecf  do not appear to derive from the same ancestral gene (they are paralogous, not orthologous), they share a binding preference for 6′-sulfo-sialyl Lewis X and 6′-sulfated sialyl N-acetyl-D-lactosamine, similar but distinct patterns of cellular expression, and similar inhibitory functions. For example, Siglec-F is expressed by eosinophils, like Siglec-8, but is also expressed by alveolar macrophages and has not been detected on mouse mast cells or basophils. This functional convergence of Siglec-8 and Siglec-F has permitted in vivo studies to be performed in mouse models of eosinophil-mediated disorders that may provide information about the human system. In a chicken is_associated_with::ovalbumin (OVA) model of allergic airway inflammation, the Siglec-F knockout mouse exhibits increased lung eosinophilia, enhanced inflammation, delayed resolution, and exacerbated peribronchial fibrosis. Antibody ligation of Siglec-F has also been shown to inhibit eosinophil-mediated intestinal inflammation and airway remodeling in OVA challenge models. The ST3Gal-III enzyme is necessary for the generation of the natural Siglec-F ligand, which remains unknown but is induced by IL-4 and IL-13 in the airway. Loss of this enzyme leads to enhanced allergic eosinophilic airway inflammation. Despite evidence that Siglec-F binds specifically to 6′-sulfo-sialyl Lewis X and 6′-sulfated sialyl N-acetyl-D-lactosamine, in which galactose is sulfated at carbon 6, mice deficient in the two known galactose 6-O-sulfotransferases, keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1), express equivalent levels of Siglec-F ligand. These models may shed some light on the regulation of human eosinophil biology by Siglec-8 and the production of natural Siglec-8 ligands in humans. Also like Siglec-8, Siglec-F ligation leads to the apoptosis of eosinophils. However, Siglec-F–induced eosinophil apoptosis is mediated by a mechanism distinct from that employed by Siglec-8, hindering direct comparisons between the mouse and human systems. Siglec-F-induced apoptosis is mediated by caspase activation in mouse eosinophils and does not involve ROS, in contrast to the mechanism reported in Siglec-8–induced apoptosis of human eosinophils. This apoptotic mechanism also does not involve is_associated_with::Src family kinases, SHP-1, or NADPH.