Distribution and kinetics of lipoprotein-bound lipoteichoic acid

Abstract

Lipoteichoic acid (LTA), a major cell wall component of gram-positive bacteria, is an amphipathic anionic glycolipid with structural similarities to lipopolysaccharide (LPS) from gram-negative bacteria. LTA has been implicated as one of the primary immunostimulatory components that may trigger the systemic inflammatory response syndrome. Plasma lipoproteins have been shown to sequester LPS, which results in attenuation of the host response to infection, but little is known about the LTA binding characteristics of plasma lipid particles. In this study, we have examined the LTA binding capacities and association kinetics of the major lipoprotein classes under simulated physiological conditions in human whole blood (ex vivo) by using biologically active, fluorescently labeled LTA and high-performance gel permeation chromatography. The average distribution of an LTA preparation from Staphylococcus aureus in whole blood from 10 human volunteers revealed that >95% of the LTA was associated with total plasma lipoproteins in the following proportions: high-density lipoprotein (HDL), 68% +/- 10%; low-density lipoprotein (LDL), 28% +/- 8%; and very low density lipoprotein (VLDL), 4% +/- 5%. The saturation capacity of lipoproteins for LTA was in excess of 150 micro g/ml. The LTA distribution was temperature dependent, with an optimal binding between 22 and 37 degrees C. The binding of LTA by lipoproteins was essentially complete within 10 min and was followed by a subsequent redistribution from HDL and VLDL to LDL. We conclude that HDL has the highest binding capacity for LTA and propose that the loading and redistribution of LTA among plasma lipoproteins is a specific process that closely resembles that previously described for LPS (J. H. M. Levels, P. R. Abraham, A. van den Ende, and S. J. H. van Deventer, Infect. Immun. 68:2821-2828, 2001).

FIG. 1.

Comparison of TNF-α-inducing capacities of labeled and unlabeled LTA in whole blood. Whole-blood stimulations with LTA and TNF-α measurements were done as described in Materials and Methods. Shown are the means of TNF-α values from five volunteers, with the error bars indicating the SD.

FIG. 2.

Chromatographic profiles of the distribution of LTA and cholesterol among the main lipoprotein classes. Fluorescence and cholesterol content of the separated lipoprotein classes were determined by HPGC by using Superose 6 HR as described in Materials and Methods. The LTA chromatogram was corrected for inherent fluorescence of plasma components.

FIG. 3.

Relative distribution of LTA and cholesterol among the main lipoprotein classes. LTA and cholesterol contents of the separated lipoprotein classes were determined by HPGC by using Superose 6 HR as described in Materials and Methods. The bars represent the means of values from 10 volunteers, and the error bars indicate the SD.

FIG. 4.

LTA saturation curves of the major lipoprotein classes. Binding capacity was determined as described in Materials and Methods. The LTA chromatogram was corrected for inherent fluorescence of plasma components. Nonlinear regression was used to generate the curves (R = 0.98).

FIG. 5.

The effect of temperature on the LTA distribution among plasma lipoprotein classes. Plasma samples containing added labeled LTA at 25 μg/ml were incubated for 1 h at the indicated temperatures and analyzed in duplicate by HPGC as described in Materials and Methods. A nonlinear regression data fit was used to generate the curves.

FIG. 6.

LTA binding kinetics of plasma lipoproteins. Plasma samples containing labeled LTA at 40 μg/ml were incubated for up to 2 h at 37°C. Aliquots were extracted at the time points indicated in the figure and analyzed by HPGC as described in Materials and Methods. Peak areas were corrected for inherent background fluorescence of plasma components at the used excitation and emission wavelengths. Nonlinear regression was used for the generation of the curves. The data represent the means of results from duplicate experiments with samples from two healthy individuals. Bars, standard errors of the means.