An analogue of lipid A and LPS from Rhodobacter sphaeroides inhibits neutrophil responses to LPS by blocking receptor recognition of LPS and by depleting LPS-binding protein in plasma

Abstract

When incubated with lipopolysaccharide (LPS) in the presence of plasma, neutrophils become primed for enhanced release of superoxide in response to triggering by formyl-Met-Leu-Phe (fMLP). The effect of LPS on phagocytes is inhibited by a synthetic lipid A precursor, LA-14-PP (lipid IVa) or by LPS from Rhodobacter sphaeroides (Rs). We studied the mechanisms by which LA-14-PP or Rs-LPS inhibited LPS-induced responses. When neutrophils were exposed to LA-14-PP or Rs-LPS for 3 min and then to Escherichia coli-LPS, the antagonists inhibited priming for superoxide release, and also blocked up-regulation of CD11b and adherence. This inhibition was dependent on plasma, was not overcome by higher amounts of E. coli-LPS or plasma, and was not observed at 0 degrees C, suggesting that E. coli-LPS was not able to interact with its receptor or other cellular recognition molecule in neutrophils that had been exposed to the antagonists. The alternative possibility that LA-14-PP or Rs-LPS depleted a plasma cofactor, resulting in inhibition of priming, was investigated by using LPS from Porphyromonas gingivalis (Pg) and Bordetella pertussis (Bp). These LPS primed neutrophils in a plasma-dependent and CD14-dependent manner, but were not blocked by LA-14-PP or Rs-LPS. When sub-optimal concentrations of plasma were exposed to LA-14-PP or Rs-LPS, and then mixed with Pg-LPS or Bp-LPS, followed by incubation with neutrophils, priming and up-regulation of CD11b were inhibited, and this inhibition was overcome by increasing the concentration of plasma. Binding of LPS-binding protein (LBP) in plasma to immobilized E. coli-LPS was inhibited by pre-incubation of plasma with LA-14-PP or Rs-LPS. Together with the result that treatment of plasma with anti-LBP antibody abolished the cofactor activity of plasma, these results indicated that LA-14-PP and Rs-LPS depleted LBP from plasma, resulting in inability of LPS to act on neutrophils. Thus LA-14-PP and Rs-LPS inhibited the action of LPS on neutrophils by at least two mechanisms, blocking of LPS receptor recognition and depletion of the cofactor LBP.

Neutrophil migration during endotoxemia

“…Probably the most often cited example is augmentation of superoxide production elicited by phorbol esters or fMLP [150][151][152][153]. Exposure of human PMNs to LPS causes the membrane localization of cytochrome b 558 , a subunit of the superoxide-generating enzyme, NADPHoxidase [154].…”

Neutrophils responded to immobilized lipopolysaccharide in the absence of lipopolysaccharide-binding protein

“…Although neutrophils responded to LPS in immobilized form and in solution with similar sensitivity, the requirement for cofactor was different, comparing the various forms of immobilized LPS and LPS in solution. Treatment of immobilized LPS in the form of LPS/plasma with anti-LBP antibody caused inhibition of priming, as has been shown with priming by LPS in solution [24]. But the antibody did not affect priming by immobilized LPS in other forms, namely plasma/LPS, fibrinogen/ LPS, and LPS/fibrinogen.…”

“…We showed that immobilized LPS acted on neutrophils, causing priming for enhanced release of O 2 Ϫ in response to fMLP, up-regulation of CD11b/CD18 and latent alkaline phosphatase, and enhanced adherence, all of which have been observed with LPS in solution [17,24]. The immobilized LPS was estimated to be 20% of the LPS used for coating.…”

“…After the washing, peroxidase activity was determined using 3, 3', 5, 5'-tetramethylbenzidine as substrate. The reaction was stopped with 1 N H 2 SO 4 , and the absorbance at 450 nm was measured as described [24]. The ELISA assay showed that about 20% of the added LPS was firmly immobilized on the polystyrene surface, and that this 20% relationship between the amount of LPS added and the amount firmly immobilized was the same for LPS concentrations up to 400 ng/mL.…”

“…LPS also causes up-regulation of CD11b/CD18 and latent alkaline phosphatase and down-regulation of L-selectin [17,21,22]. The responses of neutrophils to LPS have been shown to be CD14-dependent, to require plasma or LBP, and to be sensitive to the LPS antagonist LA-14-PP that appears to block unidentified LPS-signaling molecules [17,21,[23][24][25][26][27].…”