Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.

The addition of sulfate to glycan structures can regulate their ability to serve as ligands for glycan-binding proteins. Although sulfate groups present on the monosaccharides glucosamine, uronate, N-acetylglucosamine and N-acetylgalactosamine are recognized by defined receptors that mediate important functions, the functional significance of galactose-6-O-sulfate (Gal6S) is not known. However, in vitro studies using synthetic glycans and sulfotransferase overexpression implicate Gal6S as a binding determinant for the lymphocyte homing receptor, L-selectin. Only two sulfotransferases have been shown to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase-1 (C6ST-1). In the present study, we use mice deficient in KSGal6ST and C6ST-1 to test whether Gal6S contributes to ligand recognition by L-selectin in vivo. First, we establish that KSGal6ST is selectively expressed in high endothelial venules (HEVs) in lymph nodes and Peyer's patches. We also determine by mass spectrometry that KSGal6ST generates Gal6S on several classes of O-glycans in peripheral lymph nodes. Furthermore, KSGal6ST, but not C6ST-1, is required for the generation of the Gal6S-containing glycan, 6,6'-disulfo-3'sLN (Siaα2→3[6S]Galβ1→4[6S]GlcNAc) or a closely related structure in lymph node HEVs. Nevertheless, L-selectin-dependent short-term homing of lymphocytes is normal in KSGal6ST-deficient mice, indicating that the Gal6S-containing structures we detected do not contribute to L-selectin ligand recognition in this setting. These results refine our understanding of the biological ligands for L-selectin and introduce a mouse model for investigating the functions of Gal6S in other contexts.

Sialyl 6-sulfo Lewis X (6-sulfo sLeX) and its derivative sialyl 6-sulfo N-acetyllactosamine (LacNAc) are sialylated and sulfated glycans of sialomucins found in the high endothelial venules (HEVs) of secondary lymphoid organs. A component of 6-sulfo sLeX present in the core 1-extended O-linked glycans detected by the MECA-79 antibody was previously shown to exist in the lymphoid aggregate vasculature and bronchial mucosa of allergic and asthmatic lungs. The components of 6-sulfo sLeX in pulmonary tissues under physiological conditions remain to be analyzed. The CL40 antibody recognizes 6-sulfo sLeX and sialyl 6-sulfo LacNAc in O-linked and N-linked glycans, with absolute requirements for both GlcNAc-6-sulfation and sialylation. Immunostaining of normal mouse lungs with CL40 was performed and analyzed. The contribution of GlcNAc-6-O-sulfotransferases (GlcNAc6STs) to the synthesis of the CL40 epitope in the lungs was also elucidated. Here, we show that the expression of the CL40 epitope was specifically detected in the mesothelin-positive mesothelium of the pulmonary pleura. Moreover, GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5), but not GlcNAc6ST1 (encoded by Chst2) or GlcNAc6ST4 (encoded by Chst7), are required for the synthesis of CL40-positive glycans in the lung mesothelium. Furthermore, neither GlcNAc6ST2 nor GlcNAc6ST3 is sufficient for in vivo expression of the CL40 epitope in the lung mesothelium, as demonstrated by GlcNAc6ST1/3/4 triple-knock-out and GlcNAc6ST1/2/4 triple-knock-out mice. These results indicate that CL40-positive sialylated and sulfated glycans are abundant in the pleural mesothelium and are synthesized complementarily by GlcNAc6ST2 and GlcNAc6ST3, under physiological conditions in mice.