Aggregates Are the Biologically Active Units of Endotoxin

Abstract

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta-and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity. Bacterial lipopolysaccharide (LPS)1 is one of the most potent activators of the immune system in mammals. During cell growth or as a result of the action of antibacterial host factors or antimicrobial peptides, LPS is released from the outer leaflet of the cell wall of Gram-negative bacteria. Manifold interactions of LPS with host factors have been described, such as the activation of the complement system, activation of immune cells, and interaction with a variety of serum proteins, to name only a few. The most prominent activity of LPS is its immunostimulatory potency leading to the complex clinical syndrome of Gram-negative sepsis when the initial host response to an infection becomes dysregulated. The clinical manifestation of sepsis is characterized by fever, hypotension, respiratory and renal failure, and intravascular disseminated coagulation (1). These effects are not the result of LPS toxicity but are rather a consequence of cell activation by LPS and a subsequent dysregulation of the inflammatory host response. The biological activity of LPS is harbored in the lipid anchor of the molecule, termed lipid A or "the endotoxic principle" of LPS (2).A variety of investigations in the structural prerequisites ...

Influence of CD14 on Ligand Interactions between Lipopolysaccharide and Its Receptor Complex

“…For example, it may be that the differences we observed are due to different degrees of aggregation and/or differing physical states of the micellar aggregates that are formed by the various forms of LPS used in these studies (42)(43)(44). Studies by Mueller et al (45) suggest that aggregates, rather than monomers, are the active units of endotoxin, and that the physical states of these aggregates are a function of their hydrophobicity. In contrast, several other studies have suggested that LPS monomers, rather than LPS aggregates, are important for function (46 -48).…”

Supramolecular structure of enterobacterial wild-type lipopolysaccharides (LPS), fractions thereof, and their neutralization by Pep19-2.5

“…Unfortunately, the exact chemical structure of the lipid A moieties of the two LPS fractions was not be analyzed in detail, it seems only that fraction 4 may be slightly underacylated as compared to complete hexaacyl lipid A. Such change of the acyl chain moiety, however, would not be observable in the bioactivity test, because in this even an only 50 % hexaacylated lipid A with significant presence of pentaand tetraacylated species can nearly not be differentiated from a 100 % hexaacylated form (34). More important, however, is the finding (Figs.…”

The Preparation of a Lipidic Endotoxin Affects Its Biological Activities

“…Previously, Mueller et al demonstrated that the aggregate is an active unit of endotoxin; Endotoxin aggregates induced the secretion of TNF-a from human mononuclear cells, but the monomers did not. 15) Their observations suggest that the formation of aggregates is essential for recognition by endotoxin receptors, including TLR4. Our observations were consistent with theirs in that endotoxin aggregates, such as aq-OL, aq-lipid A, or-OL and or-lipid A, induced the secretion of TNF-a from mouse macrophages; however, the reduced ability of or-OL and or-lipid A to induce the secretion of IL1b from mouse macrophages could not be explained by their observations.…”