ROLE OF ACYL RESIDUES IN POLYCLONAL MURINE B-CELL ACTIVATION BY ACYLPOLY(1,3)GALACTOSIDES FROM KLEBSIELLA-PNEUMONIAE

Several components of Klebsiella pneumoniae including a membrane prote oglycan (Kp-MPG) were reponed to activate macrophages and to induce T- independent polyclonal activation of mouse B cells. Chemically defined derivatives of Kp-MPG were prepared and characterized, enabling us to approach the molecular substructures involved in the binding to lymph ocytes and the activation of B cells. Five derivatives were characteri zed: (i) an acylpoly(1,3)galactoside containing ester-linked fatty aci ds (EFA-APG) which was obtained by mild alkaline hydrolysis, (ii) a po lymer of EFA-APG (APG pol1), (iii) a preparation obtained by drastic a lkaline hydrolysis and delipidation which removed the esterified fatty acids (APG), (iv) a polymer of the latter compound (APG pol2), and (v ) an APG preparation submitted to mild acid hydrolysis which removed a ll fatty acids but left the galactose chain of APG (GC-APG) intact. Th e derivatives were studied for their capacity to bind to and to activa te mouse splenocytes. Binding was investigated on BALB/c and C3H/HeJ s plenocytes by indirect immunofluorescence using biotinylated F(ab')2 o f anti-Kp-MPG antibodies and the streptavidin-phycoerythrin amplificat ion system in flow cytometry and by competition of unlabeled APG with biotinylated APG. Activation was studied by measuring (i) [H-3]thymidi ne incorporation into spleen cells from BALB/c, C3H/HeJ, nude (nu+/nu) mouse strains, and purified B cells of BALB/c; (ii) immunoglobulin s ecretion in culture supernatants; and (iii) blastogenesis. The results demonstrate a specific uptake of EFA-APG and APG by T cells as well a s by B cells and exclude a contribution of the polygalactose part of t he APG molecule (GC-APG) to the binding to spleen lymphocytes. Unlike LPS from the same strain of K. pneumoniae, APG poll stimulated B cell activation in the LPS-resistant C3H/HeJ strain as well as in BALB/c mi ce. The compounds did not activate T cells and were T-independent B ce ll activators, stimulating nu+/nu+ spleen cells and inducing primarily IgM and IgG3 synthesis. Polymers were more potent activators than mon omers and removal of ester-linked fatty acids completely abrogated B c ell-activating properties. The monomer APG antagonized B cell activati on by Kp-MPG, LPS from K. pneumoniae, and APG pol1. The data indicate that within the EFA-APG molecule, distinct substructures are required for binding and for triggering B cell response.