Glycobiology Advance Access originally published online on June 22, 2005
Glycobiology 2005 15(11):1125-1135; doi:10.1093/glycob/cwi097
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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
2 Department of Molecular Biology, The Scripps Research Institute, San Diego, CA 92037; and 3 Division of Cell Biology and Immunology, The Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK
1 To whom correspondence should be addressed; e-mail: jpaulson{at}scripps.edu
Received on May 11, 2005; revised on June 17, 2005; accepted on June 19, 2005
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Abstract |
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Mouse sialic acid-binding immunoglobulin-like lectin F (Siglec-F) is an eosinophil surface receptor, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, implicating it as a regulator of cell signaling as documented for other siglecs. Here, we show that the sialoside sequence 6'-sulfo-sLeX (Neu5Ac
2–3[6-SO4] Galß1–4[Fuc1–3]GlcNAc) is a preferred ligand for Siglec-F. In glycan array analysis of 172 glycans, recombinant Siglec-F-Fc chimeras bound with the highest avidity to 6'-sulfo-sLeX. Secondary analysis showed that related structures, sialyl-Lewis X (sLeX) and 6-sulfo-sLeX containing 6-GlcNAc-SO4 showed much lower binding avidity, indicating significant contribution of 6-Gal-SO4 on Siglec-F binding to 6'-sulfo-sLex. The lectin activity of Siglec-F on mouse eosinophils was "masked" by endogenous cis ligands and could be unmasked by treatment with sialidase. Unmasked Siglec-F mediated mouse eosinophil binding and adhesion to multivalent 6'-sulfo-sLeX structure, and these interactions were inhibited by anti-Siglec-F monoclonal antibody (mAb). Although there is no clear-cut human ortholog of Siglec-F, Siglec-8 is encoded by a paralogous gene that is expressed selectively by human eosinophils and has recently been found to recognize 6'-sulfo-sLeX. These observations suggest that mouse Siglec-F and human Siglec-8 have undergone functional convergence during evolution and implicate a role for the interaction of these siglecs with their preferred 6'-sulfo-sLeX ligand in eosinophil biology. Key words: 6'-sulfo-sialyl-Lewis X / eosinophils / functionally convergent paralog / Siglec-8 / Siglec-F
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Sialic acid-binding immunoglobulin-like lectins (siglecs) are a structurally and evolutionarily related family of cell surface lectins (Crocker, 2002
). Siglecs can be divided into two categories: the CD33-related siglecs whose composition varies amongst mammals and a second group that includes CD22 (Siglec-2), sialoadhesin (Siglec-1), and MAG (Siglec-4). So far, eleven human and eight mouse siglecs have been identified. Most siglecs are expressed by cells of the immune system and contain two or more tyrosine-based motifs in their cytoplasmic tail, suggesting a role for siglecs in immune regulation (Crocker and Varki, 2001).
Although all siglecs bind to sialic acid-containing glycans, they exhibit different sialic acid linkage specificity. For example, CD22 shows high specificity for 2–6-linked sialic acids (Blixt et al., 2003), whereas Siglec-7 prefers NeuAc2–8NeuAc linkages (Yamaji et al., 2002). Ligand recognition by siglecs is complicated by the fact that these receptors interact with their ligands containing sialic acids both in cis and in trans (Crocker and Varki, 2001). In this regard, interactions with cis ligands are a common feature of siglecs, resulting in masking of lectin activity to exogenous sialoside probes, unless unmasked by prior treatment with sialidase or mild periodate treatment (Razi and Varki, 1998; Crocker and Varki, 2001; Collins et al., 2002; Crocker, 2002; Nakamura et al., 2002).
Mouse Siglec-F (Siglec-F) is a typical CD33-related siglec that consists of four Ig-like domains, a transmembrane domain, and cytoplasmic tail, which contains a putative immunoreceptor tyrosine-based inhibitory motif (ITIM) in addition to a C-terminal tyrosine-based motif, suggesting a role for Siglec-F as an inhibitory receptor (Angata et al., 2001). It was first proposed as a likely ortholog of human Siglec-5 based on phylogenetic analysis, gene structure, and gene maps (Angata et al., 2001; Aizawa et al., 2003). However, subsequent analyses indicated that Siglec-F is a hybrid gene that arose in the common rodent ancestor through gene conversion (Angata et al., 2004). Furthermore, Siglec-F is expressed predominantly on mouse eosinophils (Zhang et al., 2004), whereas Siglec-5 is not expressed on human eosinophils, but is instead expressed on human neutrophils and monocytes (Cornish et al., 1998). In contrast, Siglec-8 is a Siglec-F paralog that is also expressed selectively on human eosinophils, raising the possibility that these two molecules have undergone convergent evolution (Zhang et al., 2004).
Eosinophils are bone marrow-derived leukocytes that circulate through the blood stream and transmigrate from vascular endothelium into various tissues and to sites of allergic inflammation. The interaction of eosinophils with the vascular endothelium has been shown to be mediated by eosinophil cell surface receptors, including L-selectin (Sriramarao et al., 1994; Kitayama et al., 1997; Teixeira and Hellewell, 1998) and integrins (VLA-4, Mac-1) (Dobrina et al., 1991; Sriramarao et al., 1994; Kitayama et al., 1997; Teixeira and Hellewell, 1998; Jia et al., 1999; Ulfman et al., 1999). L-selectin is also a sialic acid specific lectin of the C-type lectin family, which mediates the tethering and rolling of lymphocytes along high endothelial venules (HEVs) in peripheral lymph nodes (Kansas, 1996; Shailubhai et al., 1997; van Zante and Rosen, 2003). L-selectin exhibits preferential specificity for sulfated-sLeX (NeuAc2–3Galß1–4[Fuc1–3]GlcNAc) as a receptor. It is generally accepted that sulfation at C-6 of GlcNAc in the context of 6-sulfo-sLeX enhances L-selectin binding relative to sLeX (Scudder et al., 1994; Mitsuoka et al., 1998; Galustian et al., 1999), but the contribution of Gal-6-SO4 is controversial (Sanders et al., 1996; Tsuboi et al., 1996; Mitsuoka et al., 1998; Galustian et al., 1999).
Bochner et al. recently showed that Siglec-8 preferentially recognizes 6'-sulfo-sLex as a glycan ligand (Guo et al., 2004; Bochner et al., 2005). Here, we show that mouse Siglec-F also preferentially recognizes 6'-sulfo-sLeX as a glycan ligand. The lectin activity of Siglec-F was constitutively masked on mouse eosinophils and could be unmasked by removing cis sialic acids. Unmasked eosinophils showed Siglec-F-dependent binding and adhesion to 6'-sulfo-sLeX structure, suggesting a role for Siglec-F as an eosinophil adhesion receptor. Based on their predominant expression on eosinophils and unique specificity for 6'-sulfo-sLeX, we propose that mouse Siglec-F and human Siglec-8 are functionally convergent paralogs.
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Results |
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Glycan array screening reveals 6'-sulfo-sLeX as the preferred ligand for mouse Siglec-F and human Siglec-8
Potential ligands of recombinant Siglec-F-Fc and Siglec-8-Fc chimeras were screened using the glycan array developed by the Consortium for Functional Glycomics (Guo et al., 2004
; Bochner et al., 2005), which contained 172 synthetic glycans including 50 sialosides (Figure 1). Two different Siglec-F-Fc chimera constructs containing two or four N-terminal Ig-like domains were pre-complexed with Alexa 488-labeled goat anti-human IgG and were incubated with biotinylated glycosides immobilized on streptavidin-coated microtiter plates. As shown in Figure 1, both chimeras bound to 6'-sulfo-sLeX (Neu5Ac2–3[6-SO4]Galß1–4[Fuc1–3]GlcNAc). Results shown in the top panel with the two domain chimera show two prominent peaks that are nonspecific as indicated by the error bars larger than the signal itself. The Siglec-F-Fc chimera containing four Ig-like domains, which gave a better signal-to-noise ratio (middle panel), was also observed to bind two other Neu5Ac2–3Gal containing sialosides, 3'-SiaDi-LN (Neu5Ac2–3Galß1–4GlcNAcß1–4Galß1–4GlcNAc) and GM2 (Neu5Ac2–3(GalNAcß1–4)Galß1–4Glc). By comparison, Siglec-8-Fc chimera bound strongly and selectively to 6'-sulfo-sLeX, as also shown recently by Bochner et al. (2005). Thus, Siglec-F and Siglec-8, the predominant siglecs on mouse and human eosinophils, respectively, bound most strongly to the same glycan ligand, 6'-sulfo-sLeX in this glycan array screen.
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Analysis of sialoside specificity of Siglec-F
To understand the structural requirement of 6'-sulfo-sLeX that confers increased affinity to Siglec-F, we further verified the glycan array results by reverse assay using various sialylated oligosaccharides coupled to polyacrylamide backbones (sialoside-PAA probes) and immobilized Siglec-F-Fc chimera. As seen in Figure 2, strong binding of the 6'-sulfo-sLeX was also observed in this assay. Several sialosides containing the ligand NeuAc2–3Galß1–4Glc(NAc) also bound to a lesser extent as observed previously by Angata et al. (2001). The substitution of N-acetylneuraminic acid (Neu5Ac) with N-glycolylneuraminic acid (Neu5Gc) or 2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) decreased avidity, showing no binding over background. Addition of Fuc1–3 and 6-sulfate to the GlcNAc of the NeuAc2–3Gal 1–4GlcNAc sequence (sLeX and 6-sulfo-sLex) showed no increase in binding. Thus, the 6-sulfate addition to the Gal in 6'-sulfo-sLex appears to be primarily responsible for the preferred binding of this glycan (Figure 2). In summary, the results of this assay support the glycan array results that 6'-sulfo-sLeX is the preferred structure for Siglec-F-Fc chimera.
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Comparison of the binding of sialoside-PAA probes to Siglec-F and Siglec-8 expressed on CHO cells
To assess the specificity of ligand binding to cell surface-expressed Siglec-F, we next examined the binding of sialoside-PAA probes to Chinese hamster ovary (CHO) cells stably expressing Siglec-F (SigF-CHO) (Figure 3). CHO cells with or without sialidase pretreatment were incubated with sialoside-PAA probes, and bound probes were detected by flow cytometry with phycoerythrin (PE)-conjugated streptavidin. No binding of any probes was observed to untreated SigF-CHO or mock-transfected control cells (Mock-CHO). The 6'-sulfo-sLeX-PAA probe bound strongly to sialidase-treated SigF-CHO cells, but not to the control cells. sLeX and 6-sulfo-sLeX showed lower binding than 6'-sulfo-sLeX, consistent with the results obtained in Figure 2.
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The binding of sialoside-PAA probes to CHO cells stably expressing Siglec-8 (Sig8-CHO) was also examined. Remarkably, 6'-sulfo-sLeX bound to Sig8-CHO cells even without sialidase pretreatment, and its binding was enhanced after sialidase treatment of the cells. Interestingly, sLeX showed no binding to Sig8-CHO cells, although 6-sulfo-sLeX bound to sialidase treated but not native cells. Taken together, the results suggest that Siglec-8 binds with sufficiently high affinity to 6'-sulfo-sLex that it competes with cis ligands on CHO cells, whereas the lower avidity 6-sulfo-sLeX only binds after cells are treated with sialidase to destroy cis ligands.
We conclude here that both cell surface Siglec-F and Siglec-8 bind 6'-sulfo-sLeX-based probe as a preferred glycan ligand, but their specificities are distinguishable through their differential recognition of other sialoside probes and masking by cis ligands on CHO cells.
Comparison of Siglec-F expression on eosinophils of wild-type and IL-5 transgenic mice
As shown in Figure 4, eosinophils were gated by the properties of side scatter (SSC) and forward scatter (FSC). Eosinophils were present as 3% of both blood and bone marrow leukocytes from C57BL/6 wild-type mice (WT). Most blood eosinophils expressed a chemokine receptor CCR3, a mouse mature eosinophil-specific marker (Grimaldi et al., 1999), whereas bone marrow contained both immature (CCR3low)and mature (CCR3high) eosinophils (Figure 4A). Siglec-F was selectively expressed on eosinophils as previously reported (Zhang et al., 2004). The expression of L-selectin, a major sulfo-sLeX receptor on lymphocytes, was low on both blood and bone marrow eosinophils (Figure 4A).
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IL-5-overexpressing transgenic mice (NJ. 1638) displayed a marked increase of total white blood cells due to an increase in eosinophils resulting in a compositional shift to 52–73% eosinophils in blood, spleen, and bone marrow as previously reported (Lee et al., 1997) (Figure 4B). Lymph nodes contained a lesser amount of eosinophils (20%). Gated eosinophils from WT and IL-5 transgenic mice were Fc
RI negative, indicating that basophils are not contaminated. It is notable that eosinophils from IL-5 transgenic mice expressed significantly higher level of Siglec-F than WT mice. In contrast, L-selectin surface expression was very low on eosinophils from IL-5 transgenic mice. At present, the reason for this differential expression of these glycan-binding proteins on eosinophils of IL-5 transgenic mice is not clear.
The expression of Siglec-F on mouse bone marrow-derived mast cells (mBMMC) was also examined (Takai et al., 1994) but none was detected, nor was Siglec-F mRNA detected by PCR (data not shown).
6'-sulfo-sLeX-PAA probe binds exclusively to Siglec-F on mouse eosinophils
We next examined binding of sialoside-PAA probes to splenic eosinophils from IL-5 transgenic mice (Figure 5A). No binding of any probes was observed with untreated eosinophils. Following sialidase pretreatment, significant binding of 6'-sulfo-sLeX to eosinophils was observed, indicating that Siglec-F is masked by endogenous cis ligands on mouse eosinophils. sLeX and 6-sulfo-sLeX showed much weaker binding to sialidase-treated eosinophils, consistent with the results obtained with SigF-CHO cells (Figure 3). It is notable that the binding of all the probes was weaker than to the Siglec-F-expressing CHO cells, likely due to the higher expressing of Siglec-F on these cells. Binding of the probes was completely abolished by anti-Siglec-F monoclonal antibody (mAb), but not by the anti-L-selectin antibody MEL-14 (data not shown), indicating that Siglec-F is the sole lectin that mediates eosinophil binding to the 6'-sulfo-sLeX structure.
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Binding of 6'-sulfo-sLeX-PAA probe to sialidase-treated bone marrow eosinophils from wild-type mice was lower than to eosinophils from IL-5 transgenic mice, presumably because of lower surface expression of Siglec-F on wild-type eosinophils (Figure 5B). These results indicate that 6'-sulfo-sLeX-PAA probe specifically bind to Siglec-F on mouse eosinophils.
Siglec-F mediates adhesion of mouse eosinophils to 6'-sulfo-sLeX
We examined binding of eosinophils to microtiter plates coated with the 6'-sulfo-sLeX-PAA probe (Figure 6). Biotinylated 6'-sulfo-sLeX- or spacer-PAA was immobilized on streptavidin-coated wells and then incubated with mouse eosinophils with or without sialidase pretreatment. After washing, bound cells were lysed and their ß-glucuronidase activity was measured. Sialidase-treated eosinophils bound to wells coated with 6'-sulfo-sLeX-PAA probe, but not with spacer-PAA probe. The binding was abolished by preincubation of eosinophils with anti-Siglec-F mAb on ice for 15 min. Untreated eosinophils showed no binding to wells coated with 6'-sulfo-sLeX-PAA probe. These results indicate that unmasked Siglec-F is able to mediate eosinophil adhesion to 6'-sulfo-sLeX.
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Discussion |
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Human and mouse eosinophils have each been demonstrated to express two siglecs. Human eosinophils express Siglec-8 and Siglec-10, whereas mouse eosinophils express Siglec-F and have been shown to express mRNA of Siglec-G, the murine ortholog of Siglec-10 (Aizawa et al., 2003
). Although the precise functions of these siglecs are not known, siglecs in general are believed to play important roles as negative regulators of cell signaling through ITIM-like regulatory motifs in their cytoplasmic domains, as exemplified by the well-documented role of CD22 in B cell receptor signaling (Nitschke et al., 1997; Sato et al., 1998; Nitschke and Tsubata, 2004). Indications that siglecs may play similar roles in eosinophil biology are suggested by a report by Nutku et al. demonstrating that anti-Siglec-8 antibodies induce eosinophil apoptosis (Nutku et al., 2003). Phylogenetic analysis of the human and mouse siglecs has demonstrated that Siglec-G is the mouse ortholog of human Siglec-10 (Aizawa et al., 2003). In contrast, Siglec-F and Siglec-8 arose independently via gene duplication events during evolution of the rodent and primate lineages and are, therefore, paralogs rather than orthologs (Fitch, 2000; Angata et al., 2001, 2004). Despite this, Siglec-F and Siglec-8 share a unique cellular distribution, being expressed predominantly on eosinophils in their respective species (Floyd et al., 2000, Zhang et al., 2004). In this report we show, in addition, that Siglec-F and Siglec-8 remarkably share a unique specificity for the sialoside sequence 6'-sulfo-sLeX. The fact that they are both expressed preferentially on eosinophils and bind the same sialoside ligand provides compelling evidence that they have undergone convergent evolution to mediate parallel functions on human and mouse eosinophils.
It was important to document that the 6'-sulfo-sLeX probe was specifically binding Siglec-F rather than L-selectin, because it has been proposed to bind this structure (Tsuboi et al., 1996). Binding and adhesion of wild type and IL-5 transgenic eosinophils to 6'-sulfo-sLeX-PAA probe were inhibited by blocking anti-Siglec-F mAb, indicating that these interactions are Siglec-F dependent. Using enzyme-linked immunosorbant assay (ELISA), we also observed that L-selectin-Fc chimera binds to sLeX- and 6-sulfo-sLeX-PAA probes, but not to 6'-sulfo-sLeX-PAA probe (data not shown). Therefore, it appears that Siglec-F is the dominant receptor recognizing 6'-sulfo-sLeX-containing glycans on mouse eosinophils.
Siglec-F binding to sialoside-PAA probes was masked by cis ligands when expressed on eosinophils or CHO cells, a phenomenon first demonstrated for CD22 on B cells (Razi and Varki, 1998; Collins et al., 2002), and subsequently for other siglecs (Barnes et al., 1999; Nicoll et al., 1999, 2003; Angata and Brinkman-Van der Linden, 2002; Crocker, 2002; Yamaji et al., 2002). Razi and Varki have suggested that a small portion of human CD22 becomes unmasked during activation of B cells (Razi and Varki, 1998, 1999), although unmasking of mouse CD22 on B cells and Siglec-7 on human NK cells following activation has not been observed so far (Collins et al., 2002; Nicoll et al., 2003). Using 6'-sulfo-sLeX-PAA as a probe, we also failed to detect unmasked Siglec-F following activation of eosinophils from IL-5 transgenic mice with phorbol-12-myristate-13-acetate (PMA) (data not shown). It should be recognized that although Siglec-F is masked from binding sialoside-PAA probes, this may not prevent interaction of Siglec-F with ligands presented in trans by other cells, as documented by Collins et al. (2004) for CD22.
Surprisingly, binding of the 6'-sulfo-sLeX-PAA probe to Siglec-8 on CHO cells was not masked by cis ligands (Figure 3). This is likely due to differences in the detailed specificity of Siglec-F and Siglec-8. As shown in Figure 3B, Siglec-F also bound with less avidity to the sLex-PAA and 6-sulfo-sLex probes, whereas Siglec-8 bound only weakly to the latter probe. Because the fucose substitution on sLex binding does not seem to be important for Siglec-F binding (Figure 2), masking of Siglec-F is likely due to the NeuAc2–3Gal sequence known to be abundantly expressed on CHO cell glycoproteins (Takeuchi et al., 1988). The lack of masking of Siglec-8 could, therefore, be due to its lower avidity for endogenous sialoside ligands on CHO cells and/or higher avidity for the 6'-sulfo-sLeX-PAA probe.
Based on the specificity of Siglec-F, glycoproteins or glycolipids containing 6'-sulfo-sLeX could be candidate ligands. To date, only a few glycoproteins have been reported to carry this structure. One is GlyCAM-1 (Hemmerich and Rosen, 1994; Hemmerich et al., 1995). GlyCAM-1 is one of several HEV-expressed L-selectin counter-receptors and was reported to contain O-linked sugar chains containing two different sulfation modifications of sialyl-Lewis X (sLeX), Gal-6-sulfate and GlcNAc-6-sulfate (Hemmerich et al., 1995). Mucins secreted by the human colonic cancer cell line CL. 16E are also reported to contain 6'-sulfo-sLex structure (Capon et al., 1992). A candidate sulfotransferase responsible for the sulfation of sLex is keratan sulfate Gal-6-sulfotransferase (KSGal6ST) cloned from human fetal brain (Fukuta et al., 1997). This enzyme can transfer sulfate to the position 6 of Gal of Neu5Ac2–3Galß1–4GlcNAc (Habuchi et al., 1997; Torii et al., 2000). Because this enzyme is widely expressed, other glycoproteins containing 6'-sulfo-sLex are likely to be identified in the future. In addition to Siglec-8 and Siglec-F, langerin of C-type lectin family expressed on Langerhans cells was reported to bind to 6'-sulfo-sLeX as preferred glycan ligand (Galustian et al., 2004). Thus, sulfation on glycans seems to be important not only for selectins, but also for other type of lectins.
Eosinophils accumulate at the sites of allergic inflammation and promote the pathogenesis of allergic diseases by releasing cytotoxic mediators (Duez et al., 2004). However, it is reported that eosinophils can also act as antigen-presenting cells by interacting with T cells and promoting Th2 polarization (Del Pozo et al., 1992; Shi et al., 2000; Duez et al., 2004). It has been shown that eosinophils can process inhaled antigens and then migrate to lymph nodes to effectively present these antigens to CD4+ T cells after allergen challenge (Shi et al., 2000). But the mechanism of eosinophil migration to lymph nodes has not been investigated. Because one of HEV-expressed glycoprotein GlyCAM-1 expresses 6'-sulfo-sLeX structure, it is tempting to speculate that endogenous Siglec-F mediates eosinophil recruitment to lymph node during allergic inflammation.
Asthma is a chronic inflammatory disorder of the airways in which many inflammatory cells including eosinophils play a role. The expression of sulfated sialyl Lewis X glycans was reported to increase in endothelium of bronchial asthma, but not that of other chronic inflammatory diseases (Toppila et al., 2000). Adhesion molecules expressed on eosinophils such as Siglec-F might mediate eosinophil adhesion and infiltration into the airway. Because mouse Siglec-F shares similar properties to human Siglec-8, Siglec-F would be a good mouse model to study the function of siglecs in allergic disease such as asthma.
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Materials and methods |
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Reagents
Purified rat anti-Siglec-F (clone E50-2440), PE-conjugated anti-Siglec-F (clone E50-2440), anti-CD62L (clone MEL-14) mAbs were purchased from BD Biosciences Pharmingen (San Diego, CA). Affinity-purified sheep anti-Siglec-8 IgG was prepared as described (Nicoll et al., 2003
). Fluorescein-conjugated anti-mouse CCR3 (clone 83101) was purchased from R & D systems (Minneapolis, MN). sLeX-, 6-sulfo-sLeX-, and 6'-sulfo-sLeX-polyacrylamide (PAA) were purchased from Glycotech (Gaithersburg, MD). All other glycans were obtained from the Consortium for Functional Glycomics. Anti-CD45 and anti-CD90 antibody-conjugated magnetic beads were purchased from Miltenyi Biotech (Auburn, CA). CHO cell line stably expressing Siglec-F-Fc chimera containing two N-terminal Ig-like domains was a generous gift from Dr Ajit Varki at the University of California, San Diego, California.
Mice
The IL-5 transgenic mouse, line NJ.1638, was the generous gift of Dr James J. Lee at Mayo Clinic Scottsdale, Arizona. C57B1/6 mice were obtained from the Scripps Research Institute. Protocols were conducted in accordance with the National Institutes of Health and the Scripps Research Institute.
Preparation of Siglec-F-Fc chimera
A CHO cell line stably expressing a Siglec-F-Fc protein, comprising the two N-terminal Ig domains of Siglec-F fused to the Fc region of human IgG, was grown in ultraculture serum free medium (Cambrex, Rockland, MA). Fusion protein secreted into medium was purified by affinity chromatography on Protein A-agarose (Invitrogen, Carlsbad, CA). A similar Siglec-F-Fc protein containing the N-terminal four Ig domains of Siglec-F was prepared using the pDEF vector as described (Zhang et al., 2004), with a full-length Siglec-F cDNA as template kindly provided by A. Varki. Siglec-8-Fc containing all three extracellular Ig domains was prepared by transfecting CHO cells with Siglec-8-Fc cDNA cloned into the pIGplus vector (Bebbington et al., 1992; Floyd et al., 2000). Stably expressing CHO cell clones were expanded in X-VIVO-10 serum free medium (Biowhittaker, Walkersville, MD) and the concentrations of Fc-proteins determined by ELISA.
Glycan array
The sugar-binding specificities of Siglec-F and Siglec-8 were analyzed using a glycan array (Glycan Array v2.3) by the Consortium for Functional Glycomics as previously described (Bochner et al., 2005). Briefly, streptavidin-coated microtiter plates (Reacti-Bind NeutrAvidin Coated High Binding Capacity Black 384-Well Plates, Pierce, Rockford, IL) were coated with various biotinylated-glycosides (30 pmol/well) in phosphate-buffered saline pH 7.4, overnight at 4°C. After washing, wells were incubated with Siglec-F-Fc or Siglec-8-Fc chimeras precomplexed with Alexa 488-conjugated goat anti-human IgG (Molecular Probes, Eugene, OR) at room temperature for 1 h and measured at excitation 485/emission 535. The glycosides used in this study are listed in the Consortium for Functional Glycomics website (http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh2.shtml).
Standard ELISA
Sialic acid-binding specificity of Siglec-F-Fc chimera containing two N-terminal Ig-like domains was analyzed by a standard ELISA assay as previously described (Collins et al., 2002). Microtiter wells (Corning Costar, Park Acton, MA) were coated with Protein A (1 µg/well, Sigma) in 50 mM Na bicarbonate pH 9.5, overnight at 4°C. Wells were blocked with ELISA buffer (20 mM HEPES, 125 mM NaCl, 10 mg/mL BSA, pH 7.5) and incubated with Siglec-F-Fc chimera (2 µg/well) pretreated with 10 mU V. cholerae sialidase (Roche Molecular Biochemicals, Indianapolis, IN), for 1h at 37°C. After washing wells with ELISA buffer, biotin-conjugated sialoside-PAA probe was added and allowed to bind for 1 h at 37°C. After washing, alkaline phosphatase-conjugated streptavidin was added and incubated for 1 h at 37°C. After the final wash, p-nitrophenyl phosphate substrate (Sigma, St. Louis, MO) was added and absorbance at 405 nm was measured after incubation for 3 h at 37°C.
Flow cytometry
Sialic acid-binding specificity of Siglec-F was analyzed by flow cytometry as previously described (Collins et al., 2002). Cells (1 x 105) suspended in FACS buffer (10 mM phosphate-buffered saline pH 7.0 containing 10 mg/mL BSA) were incubated with 1 µg of sialoside-PAA probe for 30 min on ice. After washing twice with FACS buffer, cells were incubated with 1 µg of streptavidin-PE (Jackson ImmunoResearch Laboratories, West grove, PA) for 30 min on ice. For sialidase pretreatment, cells in FACS buffer were incubated with 25 mU of Arthrobacter ureafaciens sialidase (Roche Molecular Biochemicals) for 30 min at 37°C. Cells were also stained with anti-mouse Siglec-F-PE and anti-mouse CCR3-FITC. Flow cytometry data was acquired on a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) and analyzed using the CellQuest software.
Construction of CHO cell lines stably expressing Siglec-F and Siglec-8
Total RNA from mouse bone marrow and human granulocytes was prepared for use in cDNA cloning. The full-length cDNA fragments of Siglec-F (AF293371
[GenBank]
) and Siglec-8 long variant (AF287892
[GenBank]
) were amplified by PCR, cloned into pcDNA5/FRT/V5-His vector (Invitrogen), and transfected into a Flp-In-CHO cell line (Invitrogen) by Lipofectamine 2000 (Invitrogen). The CHO cells stably expressing Siglec-F or Siglec-8 were selected with 0.5 mg/mL hygromycin B (Roche Molecular Biochemicals).
Preparation of single cell suspensions
Single cell suspensions from spleen, peripheral lymph node, mesenteric lymph node, and bone marrow were obtained from mice aged 8–12 weeks. To obtain single cell suspensions, tissues were ground between two frosted glass slides and passed through a cotton-plugged Pasteur pipette. Erythrocytes were lysed with 150 mM ammonium chloride buffer (pH 7.2) containing 10 mM potassium carbonate and 0.1 mM EDTA.
Adhesion assay
Eosinophil adhesion was monitored in triplicate by measuring the content of eosinophil lysosomal enzyme ß-glucuronidase of adherent cells (Triggiani et al., 2003). Mouse eosinophils were isolated from splenocytes of IL-5 transgenic mice by negative selection using anti-CD90 (Thy1.2)- and anti-CD45R-microbeads to remove B cells and T cells (Shen et al., 2003). Purity was 95% as determined by flow cytometry. Biotinylated spacer-PAA or 6'-sulfo-sLeX-PAA were immobilized on streptavidin-coated wells of 96-well plates for 1 h at 37°C. Mouse eosinophils (1 x 106) with or without anti-Siglec-F mAb (1 µg) pretreatment were added to the wells and then incubated for 30 min at 4°C. After gentle washing, the bound eosinophils were lysed with 0.1 M sodium acetate buffer pH 4.5 for 30 min at room temperature. Extracted cell lysates were added to p-nitrophenyl ß-D-glucuronide solution (5 mg/mL) (EMD Biosciences, San Diego, CA) in 0.1 M sodium acetate buffer and incubated at 37°C for 3 h. The reaction was stopped by adding an equal amount of 0.4 M glycine buffer (pH 10.5). Absorbance at 405 nm was measured, and the percentage of adhesion was calculated as a percentage of the ß-glucuronidase extracted from adherent cells according to the formula: % adhesion = (ß-glucuronidase in adherent fraction/total ß-glucuronidase added to the well). Data are represented as the percentage of adherence.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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The authors thank Drs. Brian E. Collins, Per Bengtson, Karin Norgard-Sumnicht, Tyrone Bowes, Jiquan Zhang, and Joanna Warren for technical assistance and advice; Ms. Anna Tran-Crie for help in preparing this manuscript; Dr. James J. Lee for IL-5 transgenic mice, line NJ. 1638; Dr. Ajit Varki for a CHO cell line stably expressing Siglec-F-Fc chimera and the full-length cDNA encoding Siglec-F. We thank the Consortium for Functional Glycomics (GM62116) for sialosides and PAA-probes and for glycan array analysis conducted by Dr. Rick Alvarez of the glycan array Core. This work was supported by grants GM60938 and AI050143 from the National Institutes of Health, by a Wellcome Trust Senior Fellowship awarded to P.R.C., and by a JSPS Postdoctoral Fellowship for Research Abroad awarded to H.T.
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CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbant assay; FSC, forward scatter; HEVs, high endothelial venules; ITIM, immunoreceptor tyrosine-based inhibitory motif; KDN, 2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid; mAb, monoclonal antibody; PAA, polyacrylamide; PE-conjugated, phycoerythrin-conjugated; Siglecs, sialic acid-binding immunoglobulin-like lectins; sLeX, sialyl-Lewis X; SSC, side scatter; WT, wild-type C57BL/6 mice
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