Serum amyloid P component prevents high-density lipoprotein-mediated neutralization of lipopolysaccharide

Abstract

Lipopolysaccharide (LPS) is an amphipathic macromolecule that is highly aggregated in aqueous preparations. LPS-binding protein (LBP) catalyzes the transfer of single LPS molecules, segregated from an LPS aggregate, to high-density lipoproteins (HDL), which results in the neutralization of LPS. When fluorescein isothiocyanate-labeled LPS (FITC-LPS) is used, this transfer of LPS monomers to HDL can be measured as an increase in fluorescence due to dequenching of FITC-LPS. Recently, serum amyloid P component (SAP) was shown to neutralize LPS in vitro, although only in the presence of low concentrations of LBP. In this study, we show that SAP prevented HDL-mediated dequenching of FITC-LPS, even in the presence of high concentrations of LBP. Human bactericidal/permeability-increasing protein (BPI), a very potent LPS-binding and -neutralizing protein, also prevented HDL-mediated dequenching of FITC-LPS. Furthermore, SAP inhibited HDL-mediated neutralization of both rough and smooth LPS in a chemiluminescence assay quantifying the LPS-induced priming of neutrophils in human blood. SAP bound both isolated HDL and HDL in serum. Using HDL-coated magnetic beads prebound with SAP, we demonstrated that HDL-bound SAP prevented the binding of LPS to HDL. We suggest that SAP, by preventing LPS binding to HDL, plays a regulatory role, balancing the amount of LPS that, via HDL, is directed to the adrenal glands.

FIG. 1

SAP prevents the serum-induced dequenching of FITC-LPS. (A) Normal serum (2%) or SAP-depleted (SAP⁻) serum (2%), obtained from the same donor, replenished with increasing amounts of SAP was incubated with FITC-LPS (0.5 μg/ml) at 37°C under continuous agitation. (B) The contribution of SAP in the dequenching of FITC-LPS was also tested by preincubating normal serum with rabbit anti-human SAP antibodies (30 μg/ml) before the addition of FITC-LPS. Fluorescence, as a measure of serum-induced dequenching of FITC-LPS, was measured at set time periods on a fluorometer. Data represent the mean fluorescence ± SEM of three separate experiments performed in triplicate.

FIG. 2

SAP and BPI prevent the binding of FITC-LPS to HDL. HDL (30 μg/ml) was incubated with FITC-LPS (0.5 μg/ml) and LBP (1 μg/ml) in the presence of increasing amounts of SAP (A) or native BPI (B). Fluorescence, as a measure of HDL-induced dequenching of FITC-LPS, was measured at set time periods on a fluorometer. Data represent the mean fluorescence ± SEM of three separate experiments performed in duplicate.

FIG. 3

SAP prevents the HDL-mediated neutralization of LPS. HDL (30 μg/ml) was preincubated with ReLPS (A) and O111:B4 LPS (B) (1 ng/ml) in the presence of increasing amounts of SAP (0 to 10 μg/ml) for 90 min at 37°C at a volume of 20 μl. Then, 80 μl of undiluted heparinized human blood was added, and the mixture was incubated for 30 min at 37°C. The chemiluminescence response was measured for 10 min after automated injection of fMLP and luminol to 10-fold-diluted blood samples in a luminometer. The background (BG) represents the chemiluminescence response in the absence of LPS and HDL. Data represent the fold increase of the 10-min integral (area under the curve [AUC]) ± SEM of four separate experiments compared to the background.

FIG. 4

SAP binds to HDL and ApoAI. The binding of SAP was tested with isolated HDL (A), HDL in serum (B), and purified ApoAI (C). (A) For the binding of SAP to isolated HDL, HDL (3 μg/ml) was used to coat a microtiter plate overnight. After washing, increasing concentrations of SAP were tested for binding, as detected by a biotinylated anti-human SAP MAb and subsequent peroxidase-labeled streptavidin. (B) The binding of SAP to HDL in serum was tested by incubating serum in an anti-human SAP MAb-coated microtiter plate, followed by the detection of captured HDL with a polyclonal anti-human ApoAI antibody and a peroxidase-labeled goat anti-rabbit IgG. (C) The binding of SAP to ApoAI was tested by incubating increasing concentrations of SAP in an ApoAI-coated microtiter plate and detecting SAP binding as described above. Data represent the mean OD₄₅₀ ± SEM of two (A and C) and three (B) separate experiments.

FIG. 5

HDL-bound SAP prevents the binding of LPS to HDL. The effect of SAP on the binding of FITC-LPS to HDL-coated beads was studied to discriminate between HDL-bound SAP and fluid-phase SAP. The binding of FITC-LPS to HDL-coated beads preincubated with SAP (open bars) was compared to the binding of FITC-LPS to HDL-coated beads with no preincubation (solid bars). Briefly, the HDL-coated beads that were preincubated with SAP were washed to remove any unbound SAP. Then, the SAP-preincubated HDL-coated beads were incubated with FITC-LPS (50 ng/ml) and LBP (1 μg/ml) for 60 min at 37°C (∗, preincubation of HDL-coated beads with SAP [0.3 μg/ml] was not determined), while the non-SAP-preincubated HDL-coated beads were incubated with increasing concentrations of SAP, together with FITC-LPS and LBP, for 60 min at 37°C. The background (BG) represents the fluorescence of HDL-coated beads in the presence of LBP (1 μg/ml) only. LPS represents the fluorescence of the HDL-coated beads incubated with FITC-LPS and LBP. Data represent the fold increase of the fluorescence ± SEM of two separate experiments compared to the background.