Immunological Responses of Mice to Native Protoplasmic Polysaccharide and Lipopolysaccharide

Abstract

There have been several recent hypotheses about the nature of the signals which cause activation of bursa-equivalent cells (B cells) during an immunological response (cf. 1). In one theory it was proposed that a single, antigen-specific signal induced B cells to produce antibodies (2). On the other hand, Coutinho et al. (3) suggested that activation of B cells was the result of one nonspecific signal which was mitogenic for B cells. In a third theory it was postulated that two signals were required to activate B cells to produce antibodies (4-8). These two signals were thought to be (a) the antigenic signal, or the binding of a specific immunodeterminant group to immunoglobulin-like receptors on the surface of a B cell and (b) a second, nonspecific signal. The second signal might have been some product of antigen-activated thymus-derived cells (T cells) in the case of T-cell dependent antigens (9-22) or, as postulated for T-cell independent antigens, the second signal could have been associated with the antigen itself (8,(23)(24)(25). With respect to lipopolysaccharide (LPS), 1 a T-cell independent antigen (26-28), the second signal was reported to reside in the Lipid A portion of the LPS molecule (8,(29)(30)(31)(32). W o r k in our l a b o r a t o r y has b e e n d i r e c t e d t o w a r d i n v e s t i g a t i n g the i m m u n o l o g i c a l r e s p o n s e s of m i c e i n j e c t e d w i t h L P S (33, 34) or a r e l a t e d m a t e r i a l , n a t i v e p r o t o p l a s m i c p o l y s a c c h a r i d e ( N P P ) (35). N P P , e x t r a c t e d from the p r o t o p l a s m of E s c h e r i c h i a coli (36, 37), was a s i m p l e p o l y s a c c h a r i d e w i t h an a v e r a g e mQ1 wt of 163,000 d a l t o n s (38). O n c e t h o u g h t to be a p r o t o p l a s m i c p r e c u r s o r to cell wall*This investigation was supported in part by U. S. Public Health Service Training Grant 5-T01-AI00385. A preliminary report of this work was presented, in part, at the Annual Meeting of the American Society for Microbiology, Chicago, Ill., May 17, 1974. ~Abbreviations used in this paper: LPS, lipopolysaccharide; NPP, native protoplasmic polysaccharide; RML mice, outbred mice from the Rocky Mountain Laboratory stock; nude mice, congenitally athymic nude mice; LPS-0113, LPS from E. coli 0113; LPS-0111, LPS from E. coil 0111; NPP-0113, NPP from E. coli 0113; ReGL-G30/C21, glycolipid from the Re mutant G30/C21 of S. typhimurium; PBS, phosphate-buffered saline; PFC, direct plaque-forming cells; HGG, human gamma globulin.

Antibody response of immunodeficient (xid) CBA/N mice to Escherichia coli 0113 lipopolysaccharide, a thymus-independent antigen.

“…Nevertheless, these findings confirm that the antibody produced after immunization with LPS 01 13 is directed mainly against the O-polysaccharide determinant of LPS 0113, rather than the lipid-A-associated protein or some other component not present in NPP. these findings, in addition to those of T a b l e III, strongly suggest that (a) the antibody response produced is directed against the polysaccharide moiety of LPS 0113 rather than lipid-A-associated protein, and that (b) priming with N P P can prepare C B A / N , as well as other strains of mice (20), to give a secondary or increased antibody response to LPS 0113. W h e n C B A / N mice were h y p e r i m m u n i z e d (i.e., given three weekly injections of LPS 0113) before i m m u n i z a t i o n with 10 ~g LPS 0113, the response produced was less (P < 0.05) than that for a primary response (Table IV).…”

“…The degree of inhibition noted with NPP appeared to be lower than that obtained with LPS 0113. However, this could be attributed to the fact that NPP has a lower molecular size and epitope density (19,20); thus, larger amounts of NPP might be required for complete inhibition. Nevertheless, these findings confirm that the antibody produced after immunization with LPS 01 13 is directed mainly against the O-polysaccharide determinant of LPS 0113, rather than the lipid-A-associated protein or some other component not present in NPP.…”

“…The preparation of LPS 0113 used in this work has been shown unequivocally to be a TI antigen (20,(23)(24)(25); because it was prepared by the phenol-water method, it is thus not likely to contain significant amounts, if any, of lipid-A-associated protein (16,17). LPS 0113 produced substantial mitogenic responses in spleen cells from CBA/N, BALB/cCum, and LPS-sensitive C3H/HeN-MTV + mice.…”

“…LPS 0113 was extracted from the cell walls of E. coil 0113 (Braude) by the phenol-water method (18). Details concerning its preparation, as well as its immunological properties, have been described (22); there is ample evidence to indicate that LPS 0113 is a TI antigen (20,(23)(24)(25). NPP was extracted with cold trichloroacetic acid (26) from the protoplasmic fraction of E. coli 0113 (Braude).…”

“…Also, to establish that the antibody response of xid mice is specific for the O-polysaccharide moiety of LPS, the native protoplasmic polysaccharide (NPP) of E. coli 0113 was used to prime mice for a secondary antibody response to LPS 0113, as well as to inhibit the detection of antibody-producing plaque-forming cells (PFC) found after immunization with LPS 0113. NPP is a simple polysaccharide product of aberrant bacterial metabolism; it is free of lipid A but, in mice, behaves as an antigen with specificity for LPS 0113 (19,20) Recently, we derived a series of hybridomas that make monoclonal antibodies specific for LPS 0113; these monoclonal antibodies share a cross-reactive idiotype (IdX) expressed on LPS 0113-specific antibodies of all strains of mice examined (21). The expression of this IdX, which is neither allotype-nor major histocompatibility complex-restricted, also was examined for CBA/N mice immunized with LPS 0113.…”