Dissociation between mitogenicity and immunogenicity of TNP- lipopolysaccharide, a T-independent antigen

Abstract

Two models have been proposed to explain triggering of B cells by so-called T-independent antigen. Feldmann and Basten (1) proposed that the interaction of multiple repeating determinants on polymeric antigens with specific Ig receptors on the B-cell surface is sufficient to provide the signals for division of these cells and differentiation to antibody-forming cells. In contrast, coutinho et al. (2, and see review, 3) have claimed that there is only one signal, a mitogenic signal, receptors acting merely as passive focusing devices to localize the antigen on specific cells where it delivers a mitogenic signal resulting in differentiation to an antibody-producing cell. This model rests primarily on the demonstration that at high concentration all T-independent antigens they have tested are mitogenic for B cells (4-6). Compatible with this hypothesis are the observations that hydrolysis of lipopolysaccharide (LPS) to remove the ester- linked fatty acids of the mitogenic lipid A component abrogates its mitogenic (7,8) activity as well as its ability, when substituted with the TNP hapten, to induce a T-independent anti- TNP response (9). However, alkali treatment of LPS, although not changing its antigenic component (8), may also modify the molecule physically or chemically which could account for loss of immunogenic properties (10). We therefore investigated other reagents which interact with LPS in a more chemically defined manner in an effort to clarify the relationship between the mitogenic and immunogenic properties of this molecule. Polymyxin B (PB) is one of a family of cyclic peptide antibiotics which are bactericidal for most gram-negative bacteria. It prevents the lethal endotoxic activity of LPS (11, 12) and changes the physical structure of LPS (13). We report here that low doses of PB added to cultures of mouse spleen cells inhibit the mitogenic activity of TNP-LPS, a T- independent antigen, and native LPS, but do not suppress the immune response to TNP-LPS. PB interacts with TNP-LPS and LPS causing a physical change in the molecule. In addition, polymyxin-treated LPS is no longer mitogenic. These results suggest a dissociation between the mitogenic and immunogenic properties of TNP-LPS.

Structural features of Salmonella typhimurium lipopolysaccharide required for activation of tissue factor in human mononuclear cells.

“…Similar dissociations of the properties of LPS have been demonstrated after interaction with the cyclic peptide antibiotic, polymyxin B, in vitro (37,(40)(41)(42)(43)(44)(45). Polymyxin B-treated LPS, though no longer mitogenic (37) nor anticomplementary (45), still retains its ability to generate an immune response when complexed with a hapten (37).…”

“…Polymyxin B is bactericidal for most Gram-negative bacteria, and it has been reported to prevent the lethal endotoxin activity of LPS (40)(41)(42)(43)(44) as well as to alter the physical structure of LPS (37,45 myxin B (Fig. 5).…”

“…Briefly, lipid A (2 mg) in water (1.0 ml) was solubilized by addition of triethylamine (10 ,ul) as reference standards (25). Polymyxin B-treated LPS was prepared as described by Jacobs and Morrison (37) and utilized in tissue culture after ovemight dialysis against pyrogen-free water or, polymyxin B itself (Aerosporin, Burroughs Wellcome Co., Research Triangle Park, N. C.) was added, uncomplexed, to LPS-stimulated cultures to achieve a final concentration of10,ug/ml. All materials were handled with pyrogen-free plastic pipettes (Falcon Plastics, Oxnard, Calif.) and serial log-dilutions of LPS (10 ,ug/ml to 10-5 ,g/ml) were made in pyrogen-free plastic tubes (Falcon Plastics), …”

B-cell activation by lipopolysaccharide. Distinct pathways for induction of mitosis and antibody production.

“…Our findings, summarized in Table III, indicate that the two responses represent separate pathways of B-cell activation by LPS, one representing direct interaction between LPS and B cells (mitosis), the other depending on macrophages or macrophage-produced factors (antigendependent production of antibody). A dissociation between the adjuvant activity of LPS and its mitogenic effect on B cells has also been suggested by Jacobs and Morrison (22) who reported that the cationic polypeptide antibiotic, polymyxin B, abrogates the mitogenic effect of LPS but enhances the immune response to TNP conjugated with LPS.…”

Characterization of lipopolysaccharide fractions and their interactions with cells and model membranes

“…From these results, we have made an assumption that the same molar amounts of lipid A of each LPS fraction should have the same effect on splenocyte mitogenesis. Mitogen assay was carried out as previously described (14). The result is shown in Fig.…”