Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, E-selectin and ICAM-1, induced by the CD14-lipopolysaccharide complex

Abstract

Lipopolysaccharides (LPS), either in the free form or complexed to CD14, a LPS receptor, are elicitors of the immune system. Lactoferrin (Lf), a LPS-chelating glycoprotein, protects animals against septic shock. Since optimal protection requires administration of Lf prior to lethal doses of LPS, we hypothesized that interactions between Lf and soluble CD14 (sCD14) exist. In a first step, human sCD14 and human Lf (hLf) were used to determine the kinetic binding parameters of hLf to free sCD14 in an optical biosensor. The results demonstrated that hLf bound specifically and with a high affinity (K(d) = 16+/-7 nM) to sCD14. Affinity chromatography studies showed that hLf interacted not only with free sCD14 but also, though with different binding properties, with sCD14 complexed to LPS or lipid A-2-keto-3-deoxyoctonic acid-heptose. In a second step, we have investigated whether the capacity of hLf to interact with sCD14 could modulate the expression of endothelial-leukocyte adhesion molecule 1 (E-selectin) or intercellular adhesion molecule 1 (ICAM-1) induced by the sCD14-LPS complex on human umbilical vein endothelial cells (HUVEC). Our experiments show that hLf significantly inhibited both E-selectin and ICAM-1 expressions at the surface of HUVEC. In conclusion, these observations suggest that the anti-inflammatory effects of hLf are due not only to the ability of the molecule to chelate LPS but also to its ability to interact with sCD14 and with the sCD14 complexed to LPS, thus modifying the activation of endothelial cells.

FIG. 1

Analysis of sCD14 binding to hLf in an optical biosensor. Biotinylated hLf was immobilized on a streptavidin-derivatized biotin surface as described in Materials and Methods. The binding of different concentrations of sCD14 (14 to 73 nM) to immobilized hLf was monitored in real time for about 300 s. Four independent sets of binding reactions were performed, of which one is presented. The inset shows that a plot of k_on against ligand concentration yields a straight line (r = 0.989), the slope of which corresponds to k_ass. The k_on of sCD14 for hLf at each concentration of sCD14 was determined using the FastFit software as described in Materials and Methods.

FIG. 2

Affinity chromatography of sCD14 to immobilized hLf in the absence of LPS (A) or in the presence of LPS (B), lipid A (C), or lipid A-KDO-heptose (D). sCD14 was preincubated 1 h with or without the different LPS moieties, and affinity chromatography was performed on hLf bound to Ultralink hydrazide gel as described in Materials and Methods. After 3 h of incubation, the gels were washed three times with 20 mM sodium phosphate, pH 7.4, buffers containing 0.5 M and 1 M NaCl, twice with a 0.2 M glycine–HCl, pH 2.3, buffer containing 0.5% (vol/vol) Triton X-100 (Gly/HCl), and once with 300 μl of SDS (10%, wt/vol). Identical volumes of the corresponding washing solutions were subjected to SDS-PAGE (7.5% polyacrylamide) and transferred to nitrocellulose. Immunostaining was performed with specific anti-CD14 polyclonal antibodies. Lanes labeled 1, 2, and 3 correspond to the successive washes with each buffer.

FIG. 3

Effect of hLf on the LPS-induced ICAM-1 expression on HUVEC. Various mixtures were preincubated for 30 min at room temperature before 24 h of incubation at 37°C with cells (per milliliter) as follows: 2 μg of sCD14 (sCD14); 50 μg of hLf alone (hLf) or with 2 μg of sCD14 (hLf + sCD14); 100 ng of LPS alone (LPS) or with 2 μg of sCD14 (sCD14 + LPS); 50 μg of hLf with 100 ng of LPS (hLf + LPS); or 2 μg of sCD14, 100 ng of LPS, and 50 μg of hLf (sCD14 + LPS + hLf). In some cases, the sCD14-LPS, LPS-hLf, and sCD14-hLf mixtures were preincubated for 30 min at room temperature prior to addition of hLf [(sCD14 + LPS) + hLf], sCD14 [(LPS + hLf) + sCD14], and LPS [(sCD14 + hLf) + LPS], respectively, and further incubation with cells. A control was performed with cells in the absence of hLf, sCD14, and LPS (none). The expression of ICAM-1 on HUVEC was estimated by enzyme immunoassay as described in Materials and Methods. Results are expressed as mean values of optical density at 490 nm (O. D. 490 nm) ± SE (error bars) from quadruplicates, after subtracting nonspecific binding of antibodies, and are representative of at least two separate experiments conducted with HUVEC isolated from human umbilical veins from different donors.

FIG. 4

Effect of hLf on the LPS-induced E-selectin expression on HUVEC. Various mixtures were preincubated for 30 min at room temperature before 5 h of incubation at 37°C with cells (per milliliter) as follows: 2 μg of sCD14 (sCD14); 50 μg of hLf alone (hLf) or with 2 μg of sCD14 (hLf + sCD14); 1 μg of LPS alone (LPS) or with 2 μg of sCD14 (sCD14 + LPS); 50 μg of hLf with 1 μg of LPS (hLf + LPS); or 2 μg of sCD14, 1 μg of LPS, and 50 μg of hLf (sCD14 + LPS + hLf). In some cases, the sCD14-LPS, LPS-hLf, and sCD14-hLf mixtures were preincubated for 30 min at room temperature prior to addition of hLf [(sCD14 + LPS) + hLf], sCD14 [(LPS + hLf) + sCD14], and LPS [(sCD14 + hLf) + LPS], respectively, and further incubation with cells. A control was performed with cells in the absence of hLf, sCD14, and LPS (none). The expression of E-selectin on HUVEC was estimated by enzyme immunoassay as described in Materials and Methods. Results are expressed as mean values of optical density at 490 nm (O. D. 490 nm) ± SE (error bars) from quadruplicates, after subtracting nonspecific binding of antibodies, and are representative of at least four separate experiments conducted with HUVEC isolated from human umbilical veins from different donors.

FIG. 5

N-terminal sequences of human sCD14 and hLf. The underlined amino acids in the sCD14 sequence represent the cell signaling site (22, 40), and the residues in boldface type are those implicated in the LPS binding (23, 48). The amino acids in boldface type in the hLf sequence are the two N-terminal basic stretches involved in the interactions of hLf with LPS (2, 13, 14, 47).