Activity in the Limulus amebocyte lysate assay and induction of tumor necrosis factor-α by diverse Helicobacter pylori lipopolysaccharide preparations

Abstract

Different chemically characterized H. pylori LPS preparations, such as smooth (S)- and rough (R)-form LPS, a completely dephosphorylated R-LPS, and three lipid A chemotypes, from the S- and R- form LPS (S- and R-lipid A) as well as a dephosphorylated derivative of S-lipid A, respectively, were evaluated for expression of potency in a quantitative chromogenic Limulus amebocyte (CLAL) lysate assay and for release of tumor necrosis factor-α (TNF-α) from activated human mononuclear cells. As far as the CLAL activity is concerned, no statistically significant differences could be observed between S- and R-LPS. Dephosphorylation of both R-LPS and S-lipid A caused a significant decrease of CLAL activity. In general terms, all the lipid A chemotypes were significantly less effective than the native LPS molecule and, in particular, R-lipid A expressed the lowest Limulus activity of all preparations. With regard to TNF-α release, R-LPS was the most potent inducer of this cytokine, even though its dephosphorylation reduced activity. In conclusion, the results show that phosphate groups influence both CLAL activity and, to a lesser extent, TNF-α release, and that the core oligosaccharide synergically cooperates with lipid A for the production of this cytokine, being, however, not essential for the expression of CLAL activity. Furthermore, preliminary structural data show that H. pylori D-glucosamine disaccharide backbone, besides being underphosphorylated at position 4', is also characterized by a reduced number of acyloxyacyl residues in comparison with enterobacterial lipid A. These findings, besides providing useful information on the structure-bioactivity relationships within H. pylori LPS, further support the evidence that this non-invasive, slow bacterium possesses the ability to modulate the local cellular immune response via LPS and related inflammatory cytokines.