title: Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages.
abstract: The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF-alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170-kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal-lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate.
authors.affiliation: Institute of Parasitology of McGill University, Ste. Anne-de-Bellevue, PQ Canada.
authors.forename: R
authors.initials: R
authors.lastname: Séguin
authors.forename: B J
authors.initials: BJ
authors.lastname: Mann
authors.forename: K
authors.initials: K
authors.lastname: Keller
authors.forename: K
authors.initials: K
authors.lastname: Chadee
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D001854:Bone Marrow Cells; D016466:CHO Cells; D002448:Cell Adhesion; D002478:Cells, Cultured; D006224:Cricetinae; D004748:Entamoeba histolytica / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D037102:Lectins; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D015800:Protozoan Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000939:Epitopes; D037102:Lectins; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D014409:Tumor Necrosis Factor-alpha; C052452:galactose inhibitable adherence protein, Entamoeba histolytica