Endotoxin: physical requirements for cell activation

Lipopolysaccharide (LPS) is the eminent lipid component of the outer leaflet of the outer membrane of Gram-negative bacteria and the major initiator of innate immune response to bacterial infection. Below the critical micellar concentration (CMC), LPS is exclusively present as a monomer. Above this concentration, aggregates are formed. Increasing the concentration beyond the CMC leads to an increase in aggregate concentration, whereas the concentration of monomers remains constant or even decreases. The question how LPS activates immune cells and whether the aggregate or the monomer is the biologically active unit has been and still is controversial. To prepare clearly defined monomeric solutions, we utilized a dialysis set-up consisting of a donor and an acceptor chamber, separated by a dialysis diaphragm with a cut-off of 5 kDa, thus allowing only monomers to pass. Human mononuclear cells (MNCs) were then stimulated with equal concentrations of aggregates and monomers, respectively, of deep rough mutant LPS from Escherichia coli strain F515 (Re LPS) and TNF-alpha release was determined. In contrast to earlier and very recent work of others, we started with a preparation of aggregate-suspensions and pure monomer-solutions and show that monomers are significantly less active than aggregates in the absence and presence of serum proteins at identical concentrations. In our model, we propose that LPS aggregates are detected by membrane-associated LBP and intercalated into the cell membrane to bring LPS into close proximity to signaling proteins in the membrane, thus finally leading to cell activation. To support this model, we present data showing that LBP is indeed present in or at the cell membrane of human macrophages.