Involvement of the Galβ1-3GalNAcβ structure in the recognition of apoptotic bodies by THP-1 cells

Abstract

A specific apoptotic glycosylation pattern may play an assistant or even a causative role in phagocytosis of apoptotic bodies. To elucidate the role of macrophages in lectin-mediated phagocytosis, an experimental system was used, where monocyte-derived THP-1 cells engulf the apoptotic bodies from the melanoma cell line MELJUSO. A flow cytometry assay was performed to reveal lectin expression and quantify the phagocytosis of apoptotic bodies. Taking into account that siglecs, a mannose receptor and galectins expressed on macrophages could be involved in engulfment of apoptotic bodies we studied their potential expression on THP-1 cells by means of polyacrylamide glycoconjugates. A strong binding of the cells to siglec ligands (3′SiaLac, 6′SiaLac, [Neu5Acα2-8]2) and galectin ligands (LacNAc, GalNAcβ1-4GlcNAc, Galβ1-3GalNAcβ and asialoGM1) was observed. To reveal the corresponding targets on apoptotic bodies, the carbohydrate pattern of MELJUSO cells was analyzed. The apoptotic membrane was characterized by a high level of glycans terminated by galactose or sialic acid. To study lectin-mediated phagocytosis of apoptotic bodies by THP-1 cells, an inhibitory phagocytosis assay was performed. Binding of Galβ1-3Gal-NAc- or LacNAc-specific reagents (lectins and antibodies) to apoptotic bodies abolished their engulfment by the THP-1 cells whereas blocking of Neu5Acα2-6 or Neu5Acα2-3 sites by the corresponding lectins was not effective. Furthermore, Galβ1-3GalNAcβ-PAA or asialoGM1-PAA binding to the THP-1 cells decreased phagocytosis, whereas two other potent THP-1-binding probes, LacNAc-PAA and GalNAcβ1-4GlcNAc-PAA did not inhibit phagocytosis. Thus, Galβ1-3GalNAcβ-terminated chains represented on the apoptotic bodies but not the other tested galectin ligands appear to be a target for THP-1 cells.