Neutrophil adherence induced by lipopolysaccharide in vitro. Role of plasma component interaction with lipopolysaccharide.

Abstract

Endotoxemia results in neutrophil localization within a number of microcirculatory beds, reflecting in part an adhesive interaction between neutrophils and the vascular endothelial cell. In previous studies, endotoxin or lipopolysaccharide (LPS) treatment of rabbits resulted in neutrophil sequestration at LPS concentrations well below those effective at increasing neutrophil adherence in vitro. We hypothesized that LPS-induced neutrophil adherence involved a plasma component. In the absence of plasma, high concentrations of LPS (10 $tg/ml) were required to increase human neutrophil adherence to endothelial cells in vitro. With the inclusion of as little as 1% plasma or serum, however, the LPS dose-response curve was markedly shifted, resulting in increments in adherence at 10 ng/ml, and the time course of enhanced adherence was accelerated. Pretreatment studies suggested that the effect of LPS was on the neutrophil rather than the endothelial cell. Immunoprecipitation of 0111:B4 LPS paralleled the loss of functional activity, suggesting that LPS was an integral part of the active complex, rather than altering a plasma component to make it active. The incubation of plasma with LPS decreased the apparent molecular mass of LPS from 500-1,000 kD to -100 kD. The disaggregated 0111:B4 LPS eluted in the range of albumin and was able to increase adherence in the absence of additional plasma. Plasma depleted of lipoproteins or heat treated retained activity, suggesting that the interaction of LPS with HDL or complement did not account for the observed findings. An LPSbinding protein isolated from rabbit serum enhanced the adherence-inducing effects of both 0111:B4 and Re595 LPS. Furthermore, the activity of rabbit serum was abolished after incubation with an antibody directed against this LPS-binding protein (LBP). An antibody directed against CD14, the putative receptor of the LPS-LBP complex, prevented the adhesive response to LPS. These data suggest that LPS is disaggregated by an LBP in serum and plasma to form an active LPS-plasma component complex. This putative complex then interacts with CD14 on the neutrophil so as to induce an adhesive state. (J.

Lipopolysaccharide-Binding Protein Critically Regulates Lipopolysaccharide-Induced IFN-β Signaling Pathway in Human Monocytes

“…The results showed a sufficient effect of 1% AB serum in our experiment system (data not shown), and using human serum allowed us to use neutralizing anti-human LBP mAb and anti-human CD14 mAb. In addition, 1% human AB serum, which we used in our experiments, has been reported to be capable of maximally inducing adhesion of neutrophils upon stimulation with LPS (41).…”

Common and distinct intracellular signaling pathways in human neutrophils utilized by platelet activating factor and FMLP.

“…Neutrophil adherence after stimulation with PAF or FMLP was measured as previously described (27). Actin assembly was determined in triplicate by NBD-phallacidin staining and analyzed by a Coulter EPICs C cytofluorograph (28).…”

Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock

“…The CD14-mediated activation of peritoneal macrophages by heat-killed Staphylococcus aureus bacteria, LTA, cell wall PGN, or mycobacterial lipoproteins is not enhanced by LBP (345,357,478,524). In the presence of LBP, LPS induced an enhanced intracellular killing and secretion of TNF-␣ and NO by murine macrophages (68), increased adherence of human PMN to endothelial cells (590), LBP and CD14 release by HepG2 human hepatoma cells (383), and release of tissue factor by THP-1 cells in vitro (500), whereas the addition of anti-LBP or anti-CD14 antibodies abrogated the effect of LBP (154,500,590). Application of anti-LBP antibodies together with LPS protected D-galactosamine-sensitized mice from death (155, 156, 307a).…”