Sialic acid-binding Ig-like lectin 8 is a protein that in humans is encoded by the SIGLEC8 gene. This gene is located on chromosome 19q13.4, about 330 kb downstream of the SIGLEC9 gene. Within the siglec family of transmembrane proteins, Siglec-8 belongs to the CD33-related siglec subfamily, a subfamily that has undergone rapid evolution.
Siglec-8 was first identified by CD33 homology screening of ESTs from a cDNA library generated from a patient diagnosed with idiopathic 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.
Siglec-8 is expressed by human eosinophils, mast cells, and, to a lesser extent, basophils. It has thus garnered attention as a molecule that is uniquely expressed by immune effector cells involved in asthma and 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 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 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 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 chronic eosinophilic leukemia (CEL), hypereosinophilic syndrome, or chronic myeloid leukemia (CML), all basophils from patients with CEL or CML, and all bone marrow mast cells from patients with indolent systemic mastocytosis or 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 monocytes, a subset of lymphocytes, and 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 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 epitopes are not conserved.
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 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 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.
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 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 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.
Signaling and function
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 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 reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and 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 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 prostaglandin D2 and 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.
Relationships with other siglecs
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.
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 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 Src family kinases, SHP-1, or NADPH.
- Floyd H, Ni J, Cornish AL, Zeng Z, Liu D, Carter KC, Steel J, Crocker PR (January 2000). "Siglec-8. A novel eosinophil-specific member of the immunoglobulin superfamily". The Journal of Biological Chemistry. 275 (2): 861–6. doi:10.1074/jbc.275.2.861. PMID 10625619.
- "Entrez Gene: SIGLEC8 sialic acid binding Ig-like lectin 8".
- Foussias G, Yousef GM, Diamandis EP (November 2000). "Molecular characterization of a Siglec8 variant containing cytoplasmic tyrosine-based motifs, and mapping of the Siglec8 gene". Biochemical and Biophysical Research Communications. 278 (3): 775–81. doi:10.1006/bbrc.2000.3866. PMID 11095983.
- Kikly KK, Bochner BS, Freeman SD, Tan KB, Gallagher KT, D'alessio KJ, Holmes SD, Abrahamson JA, Erickson-Miller CL, Murdock PR, Tachimoto H, Schleimer RP, White JR (June 2000). "Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells, and basophils". The Journal of Allergy and Clinical Immunology. 105 (6 Pt 1): 1093–100. doi:10.1067/mai.2000.107127. PMID 10856141.
- Angata T, Margulies EH, Green ED, Varki A (September 2004). "Large-scale sequencing of the CD33-related Siglec gene cluster in five mammalian species reveals rapid evolution by multiple mechanisms". Proceedings of the National Academy of Sciences of the United States of America. 101 (36): 13251–6. doi:10.1073/pnas.0404833101. PMC 516556. PMID 15331780.
- Padler-Karavani V, Hurtado-Ziola N, Chang YC, Sonnenburg JL, Ronaghy A, Yu H, Verhagen A, Nizet V, Chen X, Varki N, Varki A, Angata T (March 2014). "Rapid evolution of binding specificities and expression patterns of inhibitory CD33-related Siglecs in primates". FASEB Journal. 28 (3): 1280–93. doi:10.1096/fj.13-241497. PMC 3929681. PMID 24308974.
- Hudson SA, Herrmann H, Du J, Cox P, Haddad el-B, Butler B, Crocker PR, Ackerman SJ, Valent P, Bochner BS (December 2011). "Developmental, malignancy-related, and cross-species analysis of eosinophil, mast cell, and basophil siglec-8 expression". Journal of Clinical Immunology. 31 (6): 1045–53. doi:10.1007/s10875-011-9589-4. PMC 3329870. PMID 21938510.
- Ellis AK, Ackerman SJ, Crawford L, Du J, Bedi R, Denburg JA (June 2010). "Cord blood molecular biomarkers of eosinophilopoiesis: kinetic analysis of GATA-1, MBP1 and IL-5R alpha mRNA expression". Pediatric Allergy and Immunology. 21 (4 Pt 1): 640–8. doi:10.1111/j.1399-3038.2010.01003.x. PMID 20337967.
- Yokoi H, Myers A, Matsumoto K, Crocker PR, Saito H, Bochner BS (June 2006). "Alteration and acquisition of Siglecs during in vitro maturation of CD34+ progenitors into human mast cells". Allergy. 61 (6): 769–76. doi:10.1111/j.1398-9995.2006.01133.x. PMID 16677248.
- Aizawa H, Plitt J, Bochner BS (January 2002). "Human eosinophils express two Siglec-8 splice variants". The Journal of Allergy and Clinical Immunology. 109 (1): 176. doi:10.1067/mai.2002.120550. PMID 11799386.
- Bochner BS, Alvarez RA, Mehta P, Bovin NV, Blixt O, White JR, Schnaar RL (February 2005). "Glycan array screening reveals a candidate ligand for Siglec-8". The Journal of Biological Chemistry. 280 (6): 4307–12. doi:10.1074/jbc.M412378200. PMID 15563466.
- Tateno H, Crocker PR, Paulson JC (November 2005). "Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs that are selectively expressed on eosinophils and recognize 6'-sulfo-sialyl Lewis X as a preferred glycan ligand". Glycobiology. 15 (11): 1125–35. doi:10.1093/glycob/cwi097. PMID 15972893.
- Kiwamoto T, Brummet ME, Wu F, Motari MG, Smith DF, Schnaar RL, Zhu Z, Bochner BS (January 2014). "Mice deficient in the St3gal3 gene product α2,3 sialyltransferase (ST3Gal-III) exhibit enhanced allergic eosinophilic airway inflammation". The Journal of Allergy and Clinical Immunology. 133 (1): 240–7.e1-3. doi:10.1016/j.jaci.2013.05.018. PMC 3874253. PMID 23830412.
- Guo JP, Brummet ME, Myers AC, Na HJ, Rowland E, Schnaar RL, Zheng T, Zhu Z, Bochner BS (February 2011). "Characterization of expression of glycan ligands for Siglec-F in normal mouse lungs". American Journal of Respiratory Cell and Molecular Biology. 44 (2): 238–43. doi:10.1165/rcmb.2010-0007OC. PMC 3049235. PMID 20395633.
- Suzukawa M, Miller M, Rosenthal P, Cho JY, Doherty TA, Varki A, Broide D (June 2013). "Sialyltransferase ST3Gal-III regulates Siglec-F ligand formation and eosinophilic lung inflammation in mice". Journal of Immunology. 190 (12): 5939–48. doi:10.4049/jimmunol.1203455. PMC 3679360. PMID 23677475.
- Nutku E, Aizawa H, Hudson SA, Bochner BS (June 2003). "Ligation of Siglec-8: a selective mechanism for induction of human eosinophil apoptosis". Blood. 101 (12): 5014–20. doi:10.1182/blood-2002-10-3058. PMID 12609831.
- Cherry WB, Yoon J, Bartemes KR, Iijima K, Kita H (June 2008). "A novel IL-1 family cytokine, IL-33, potently activates human eosinophils". The Journal of Allergy and Clinical Immunology. 121 (6): 1484–90. doi:10.1016/j.jaci.2008.04.005. PMC 2821937. PMID 18539196.
- Na HJ, Hudson SA, Bochner BS (January 2012). "IL-33 enhances Siglec-8 mediated apoptosis of human eosinophils". Cytokine. 57 (1): 169–74. doi:10.1016/j.cyto.2011.10.007. PMC 3282301. PMID 22079334.
- Suzukawa M, Koketsu R, Iikura M, Nakae S, Matsumoto K, Nagase H, Saito H, Matsushima K, Ohta K, Yamamoto K, Yamaguchi M (November 2008). "Interleukin-33 enhances adhesion, CD11b expression and survival in human eosinophils". Laboratory Investigation; A Journal of Technical Methods and Pathology. 88 (11): 1245–53. doi:10.1038/labinvest.2008.82. PMID 18762778.
- Nutku E, Hudson SA, Bochner BS (October 2005). "Mechanism of Siglec-8-induced human eosinophil apoptosis: role of caspases and mitochondrial injury". Biochemical and Biophysical Research Communications. 336 (3): 918–24. doi:10.1016/j.bbrc.2005.08.202. PMID 16157303.
- Nutku-Bilir E, Hudson SA, Bochner BS (January 2008). "Interleukin-5 priming of human eosinophils alters siglec-8 mediated apoptosis pathways". American Journal of Respiratory Cell and Molecular Biology. 38 (1): 121–4. doi:10.1165/rcmb.2007-0154OC. PMC 2176128. PMID 17690326.
- Kano G, Almanan M, Bochner BS, Zimmermann N (August 2013). "Mechanism of Siglec-8-mediated cell death in IL-5-activated eosinophils: role for reactive oxygen species-enhanced MEK/ERK activation". The Journal of Allergy and Clinical Immunology. 132 (2): 437–45. doi:10.1016/j.jaci.2013.03.024. PMC 4042061. PMID 23684072.
- Yokoi H, Choi OH, Hubbard W, Lee HS, Canning BJ, Lee HH, Ryu SD, von Gunten S, Bickel CA, Hudson SA, Macglashan DW, Bochner BS (February 2008). "Inhibition of FcepsilonRI-dependent mediator release and calcium flux from human mast cells by sialic acid-binding immunoglobulin-like lectin 8 engagement". The Journal of Allergy and Clinical Immunology. 121 (2): 499–505.e1. doi:10.1016/j.jaci.2007.10.004. PMID 18036650.
- Crocker PR, Paulson JC, Varki A (April 2007). "Siglecs and their roles in the immune system". Nature Reviews. Immunology. 7 (4): 255–66. doi:10.1038/nri2056. PMID 17380156.
- Stevens WW, Kim TS, Pujanauski LM, Hao X, Braciale TJ (October 2007). "Detection and quantitation of eosinophils in the murine respiratory tract by flow cytometry". Journal of Immunological Methods. 327 (1–2): 63–74. doi:10.1016/j.jim.2007.07.011. PMC 2670191. PMID 17716680.
- Zhang M, Angata T, Cho JY, Miller M, Broide DH, Varki A (May 2007). "Defining the in vivo function of Siglec-F, a CD33-related Siglec expressed on mouse eosinophils". Blood. 109 (10): 4280–7. doi:10.1182/blood-2006-08-039255. PMC 1885492. PMID 17272508.
- Zimmermann N, McBride ML, Yamada Y, Hudson SA, Jones C, Cromie KD, Crocker PR, Rothenberg ME, Bochner BS (September 2008). "Siglec-F antibody administration to mice selectively reduces blood and tissue eosinophils". Allergy. 63 (9): 1156–63. doi:10.1111/j.1398-9995.2008.01709.x. PMC 2726770. PMID 18699932.
- Cho JY, Song DJ, Pham A, Rosenthal P, Miller M, Dayan S, Doherty TA, Varki A, Broide DH (Nov 2010). "Chronic OVA allergen challenged Siglec-F deficient mice have increased mucus, remodeling, and epithelial Siglec-F ligands which are up-regulated by IL-4 and IL-13". Respiratory Research. 11 (154): 154. doi:10.1186/1465-9921-11-154. PMC 2988013. PMID 21040544.
- Song DJ, Cho JY, Lee SY, Miller M, Rosenthal P, Soroosh P, Croft M, Zhang M, Varki A, Broide DH (October 2009). "Anti-Siglec-F antibody reduces allergen-induced eosinophilic inflammation and airway remodeling". Journal of Immunology. 183 (8): 5333–41. doi:10.4049/jimmunol.0801421. PMC 2788790. PMID 19783675.
- Song DJ, Cho JY, Miller M, Strangman W, Zhang M, Varki A, Broide DH (April 2009). "Anti-Siglec-F antibody inhibits oral egg allergen induced intestinal eosinophilic inflammation in a mouse model". Clinical Immunology. 131 (1): 157–69. doi:10.1016/j.clim.2008.11.009. PMC 2683248. PMID 19135419.
- Patnode ML, Cheng CW, Chou CC, Singer MS, Elin MS, Uchimura K, Crocker PR, Khoo KH, Rosen SD (September 2013). "Galactose 6-O-sulfotransferases are not required for the generation of Siglec-F ligands in leukocytes or lung tissue". The Journal of Biological Chemistry. 288 (37): 26533–45. doi:10.1074/jbc.M113.485409. PMC 3772201. PMID 23880769.
- Mao H, Kano G, Hudson SA, Brummet M, Zimmermann N, Zhu Z, Bochner BS (Jun 2013). "Mechanisms of Siglec-F-induced eosinophil apoptosis: a role for caspases but not for SHP-1, Src kinases, NADPH oxidase or reactive oxygen". PLoS One. 8 (6): e68143. doi:10.1371/journal.pone.0068143. PMC 3695997. PMID 23840825.
- Munday J, Kerr S, Ni J, Cornish AL, Zhang JQ, Nicoll G, Floyd H, Mattei MG, Moore P, Liu D, Crocker PR (April 2001). "Identification, characterization and leucocyte expression of Siglec-10, a novel human sialic acid-binding receptor". The Biochemical Journal. 355 (Pt 2): 489–97. doi:10.1042/0264-6021:3550489. PMC 1221762. PMID 11284738.
- Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T, Sugano S (January 2006). "Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes". Genome Research. 16 (1): 55–65. doi:10.1101/gr.4039406. PMC 1356129. PMID 16344560.