Chitin particles induce size-dependent but carbohydrate-independent innate eosinophilia.

Murine Mϕ that phagocytose CMP develop into M1; this response depends on the size and the chemical composition of the particles. In contrast, recent studies concluded that chitin particles induce M2 and eosinophil migration, promoting acquired Th2 immune responses against chitin-containing microbes or allergens. This study examined whether these apparently inconsistent responses to chitin could be induced by variation in the size and chemical composition of the chitin particles. We compared the responses of Mϕ with CMP, LCB, and Sephadex G-100 beads (>40 µm). Beads were given i.p. to WT mice and to mice deficient in a CRTH2, a receptor for the eosinophil chemoattractant PGD(2). In contrast to the M1 activation induced by CMP, i.p. administration of LCB or Sephadex beads induced within 24 h a CRTH2-dependent peritoneal eosinophilia, as well as CRTH2-independent activation of peritoneal Mϕ that expressed Arg I, an M2 phenotype. LCB-induced Mϕ exhibited elevated Arg I and a surface MR, reduced surface TLR2 levels, and no change in the levels of CHI3L1 or IL-10 production. Our results indicate that the effects of chitin in vivo are highly dependent on particle size and that large, nonphagocytosable beads, independent of their chemical composition, induce innate eosinophilia and activate Mϕ expressing several M2, but not M1, phenotypes.

Molecular design of N-linked tetravalent glycosides bearing N-acetylglucosamine, N,N'-diacetylchitobiose and N-acetyllactosamine: Analysis of cross-linking activities with WGA and ECA lectins.

Two types of nonspacer- and spacer-N-linked tetravalent glycosides bearing N-acetylglucosamine (GlcNAc), N,N'-diacetylchitobiose [(GlcNAc)(2)] and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. The interactions of wheat germ (Triticum vulgaris) agglutinin (WGA) and coral tree (Erythrina cristagalli) agglutinin (ECA) with a series of tetravalent glycosides and related compounds were studied using a hemagglutination inhibition assay, a precipitation assay, double-diffusion test, and an optical biosensor based on surface plasmon resonance (SPR). The tetravalent glycosides were found to be capable of binding and precipitating the lectins as tetravalent ligands. Strong interactions with WGA, due to a combination of multivalency effects and spacer effects, were observed for tetravalent glycosides bearing flexible tandem GlcNAc. The chelate effect leads to large rate enhancement for the tetravalent system with favorable orientation of ligands. Our simple strategy produced multivalent glycosides with strong cross-linking activity for lectin as a specific coagulant.

Efficient synthesis of spacer-N-linked double-headed glycosides carrying N-acetylglucosamine and N,N'-diacetylchitobiose and their cross-linking activities with wheat germ agglutinin.

We describe here an efficient synthetic route to spacer-N-linked double-headed glycosides via a simple two-step procedure. N-Acetylglucosamine (GlcNAc) and N,N'-diacetylchitobiose [(GlcNAc)(2)] were treated with ammonia and the resulting N-beta-glycosylamines were coupled to a series of dicarboxylic acids. Condensation with each dicarboxylic acid proceeded stereoselectively to give the corresponding beta-N-linked double-headed glycoside without the need for any protection/deprotection steps. Interaction of the resulting N-linked double-headed glycosides with wheat germ agglutinin (WGA) were then investigated using a precipitation assay and an optical biosensor based on surface plasmon resonance (SPR). Spacer-N-linked double-headed glycosides bearing GlcNAc and (GlcNAc)(2) were found to be capable of binding and precipitating WGA as divalent ligands. However, the length of the spacer groups between the two terminal sugar residues was found to greatly influence the cross-linking activities with the lectin.

Enzymatic synthesis of spacer-linked divalent glycosides carrying N-acetylglucosamine and N-acetyllactosamine: analysis of cross-linking activities with WGA.

Divalent glycosides carrying N-acetyl-d-glucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. First, hexan-1,6-diyl bis-(2-acetamido-2-deoxy-beta-d-glucopyranoside) (GlcNAc-Hx-GlcNAc) and 3,6-dioxaoct-1,8-diyl bis-(2-acetamido-2-deoxy-beta-d-glucopyranoside) (GlcNAc-Doo-GlcNAc) were enzymatically synthesized by transglycosylation of an N,N'N'',N'''-tetraacetylchitotetraose [(GlcNAc)(4)] donor with a primary diol acceptor, utilizing a chitinolytic enzyme from Amycolatopsis orientalis. The resulting divalent glycosides were further converted to the respective hexan-1,6-diyl bis-[beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside] (LacNAc-Hx-LacNAc) and 6-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-hexyl beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside (LacNAc-Hx-GlcNAc), and respective 3,6-dioxaoct-1,8-diyl bis-[beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside] (LacNAc-Doo-LacNAc) and 8-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-3,6-dioxaoctyl beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside (LacNAc-Doo-GlcNAc) by galactosyltransferase. The interaction of wheat germ agglutinin (WGA) with a series of divalent glycosides and related compounds were studied using a biosensor based on surface plasmon resonance (SPR) and by precipitation analysis. Our results demonstrated that divalent glycosides carrying GlcNAc on both sides and GlcNAc and LacNAc on each side are capable of precipitating WGA as divalent ligands, but that the corresponding monovalent controls and divalent glycosides carrying LacNAc on both sides are unable to precipitate the lectin and bind as univalent ligands.