Differential induction of tumor necrosis factor by bacteria expressing rough and smooth lipopolysaccharide phenotypes

Abstract

The lipid A portion of the lipopolysaccharide (LPS) molecule of gram-negative bacteria has the ability to turn on the production of tumor necrosis factor (TNF) in macrophage cells. The question addressed in this paper was whether the presence of the polysaccharide moiety on the LPS molecule had any bearing on this ability. The question was asked (i) by using isolated LPS from a series of Salmonella mutants having progressively less polysaccharide attached to the lipid A portion of the molecule and (ii) by using whole bacteria expressing alternatively the smooth or rough LPS phenotype. Isolated LPS and bacteria were examined for their abilities to induce bioactive TNF in the mouse macrophage cell line RAW 264.7. The results indicated that the presence of long- or short-chain polysaccharide moieties had no bearing on the ability of the isolated LPS molecule to induce TNF. However, the presence of long-chain polysaccharides attached to the lipid moiety on the intact smooth bacterium was associated with a decreased ability to induce TNF. To test whether the bacteria were inducing TNF by a cell (bacterium)-to-cell (macrophage) contact mechanism or through a releasable product, the bacteria were removed from direct contact with the macrophage cells by using a Transwell filter insert. Under these conditions the rough bacteria continued to induce TNF, while the smooth bacteria were no longer capable of doing so. When filtrates from the bacteria were examined in the Limulus amebocyte lysate assay, the results showed that the rough bacteria were releasing approximately a log order more Limulus amebocyte lysate activity than the smooth bacteria. The results of this study suggest that rough bacteria may be superior inducers of TNF compared with their smooth counterparts because of a greater propensity to shed their LPS.