Mice deficient in the St3gal3 gene product α2,3 sialyltransferase (ST3Gal-III) exhibit enhanced allergic eosinophilic airway inflammation

Abstract

Background: Sialic acid-binding immunoglobulin-like lectin (Siglec)-F is a proapoptotic receptor on mouse eosinophils, but little is known about its natural tissue ligand.

Objective: We previously reported that the St3gal3 gene product α2,3 sialyltransferase (ST3Gal-III) is required for constitutive Siglec-F lung ligand synthesis. We therefore hypothesized that attenuation of ST3Gal-III will decrease Siglec-F ligand levels and enhance allergic eosinophilic airway inflammation.

Methods: C57BL/6 wild-type mice and St3gal3 heterozygous or homozygous deficient (St3gal3(+/-) and St3gal3(-/-)) mice were used. Eosinophilic airway inflammation was induced through sensitization to ovalbumin (OVA) and repeated airway OVA challenge. Siglec-F human IgG1 fusion protein (Siglec-F-Fc) was used to detect Siglec-F ligands. Lung tissue and bronchoalveolar lavage fluid (BALF) were analyzed for inflammation, as well as various cytokines and chemokines. Serum was analyzed for allergen-specific immunoglobulin levels.

Results: Western blotting with Siglec-F-Fc detected approximately 500-kDa and approximately 200-kDa candidate Siglec-F ligands that were less abundant in St3gal3(+/-) lung extracts and nearly absent in St3gal3(-/-) lung extracts. After OVA sensitization and challenge, Siglec-F ligands were increased in wild-type mouse lungs but less so in St3gal3 mutants, whereas peribronchial and BALF eosinophil numbers were greater in the mutants, with the following rank order: St3gal3(-/-) ≥ St3gal3(+/-) > wild-type mice. Levels of various cytokines and chemokines in BALF were not significantly different among these 3 types of mice, although OVA-specific serum IgG1 levels were increased in St3gal3(-/-) mice.

Conclusions: After OVA sensitization and challenge, St3gal3(+/-) and St3gal3(-/-) mice have more intense allergic eosinophilic airway inflammation and less sialylated Siglec-F ligands in their airways. One possible explanation for these findings is that levels of sialylated airway ligands for Siglec-F might be diminished in mice with attenuated levels of ST3Gal-III, resulting in a reduction in a natural proapoptotic pathway for controlling airway eosinophilia.

Keywords: 6’-su-sLacNAc; 6′-Sulfated sialyl Lewis X; 6′-Sulfated sialyl N-acetyl-D-lactosamine; 6′-su-sLe(x); 6′-sulfated sialyl Lewis X; 6′-sulfated sialyl N-acetyl-D-lactosamine; BALF; Bronchoalveolar lavage fluid; Eosinophils; MBP; Major basic protein; OVA; Ovalbumin; ST3Gal-III; Sialic acid–binding immunoglobulin-like lectin; Siglec; Siglec-8 human IgG(1) Fc chimera; Siglec-8-Fc; Siglec-F; Siglec-F human IgG(1) Fc chimera; Siglec-F-Fc; St3gal3; St3gal3 gene product α2,3 sialyltransferase; WT; Wild-type; apoptosis; asthma; glycan ligands; lung.

Fig 1

A, Glycan array screening with Version 4.2 of the Consortium for Functional Glycomics glycan array (http://www.functionalglycomics.org) reveals 2 shared ligands for Siglec-F-Fc (1 μg/mL) and Siglec-8-Fc (200 μg/mL) among 465 glycans tested, namely 6′-su-sLe^x and 6′-su-sLacNAc. Values represent means ± SDs of replicates from a single assay. Structures are displayed with nomenclature used in the Essentials of Glycobiology textbook, second edition (http://www.ncbi.nlm.nih.gov/books/NBK1908/). Diamonds, N-acetylneuraminic acid (sialic acid); circles, galactose; squares, N-acetylglucosamine; triangles, fucose. B, Chemical structures of the 2 shared ligands, 6′-su-sLe^x and 6′-su-sLacNAc, that differ only with respect to the presence or absence, respectively, of the fucose residue.

Fig 2

St3gal3^+/− and St3gal3^−/− mice demonstrate decreased Siglec-F ligand expression in the lung and airway. A, Constitutive ligand characterization in WT, St3gal3^+/−, and St3gal3^−/− mouse whole-lung paren-chymal extracts with or without sialidase pretreatment probed by means of Western blotting with Siglec-F-Fc and for β-actin as a positive control. For each lane, 150 μg of lung extract protein was loaded. Data are representative of 3 experiments with similar results. B, Western blotting of mouse whole-lung parenchymal extracts in control and OVA-sensitized and challenged mice probed with Siglec-F-Fc or β-actin. Data are representative of 3 experiments with similar results. C, Representative detection and localization of Siglec-F ligand by using Siglec-F-Fc in immunohistochemistry from lung tissues of normal WT, St3gal3^+/−, and St3gal3^−/− mice in tracheal epithelium (panels a-f) and submucosal tracheal glands (panels g-l). All samples were obtained 24 hours after final challenge with PBS or OVA. Data are representative of 3 experiments with similar results.

Fig 3

Attenuation of St3gal3 is associated with enhanced OVA-induced allergic eosinophilic airway inflammation. A, BALF cytology of each indicated cell type using samples obtained 24 hours after PBS or OVA final challenge from WT, St3gal3^+/−, and St3gal3^−/− mice (n = 8-12). B, Representative lung tissue sections from WT (panels a and b), St3gal3^+/− (panels c and d), and St3gal3^−/− (panels e and f) mice obtained 24 hours after PBS (panels a, c, and e) or OVA (panels b, d, and f) final challenge analyzed by using immunohistochemistry after being labeled with anti-MBP mAb. Representative photomicrographs are provided for each group (magnification ×200). C, Peribronchial eosinophils (MBP⁺ cells) in lung tissue sections (like those in Fig 3, B) were enumerated. Values represent means ± SDs (n = 5-7). For Fig 3, A and C, horizontal bars indicate direct statistical comparisons between OVA-challenged WT, St3gal3^+/−, or St3gal3^−/− mice. NS, Not significant.

Fig 4

Effect of attenuation of St3gal3 on OVA-induced serologic markers associatedwith allergic sensitization. Total IgE (A), OVA-specific IgE (B), OVA-specific IgG₁ (C), and OVA-specific IgG_2a (D) levels in serum obtained 24 hours after PBS or OVA final challenge (n = 9-13). *P < .05, **P < .01, and ***P < .001 relative to the WT-PBS control mice. Horizontal bars indicate direct statistical comparisons between OVA-challenged WT, St3gal3^+/−, or St3gal3^−/− mice. NS, Not significant.