Lactoferrin inhibits the endotoxin interaction with CD14 by competition with the lipopolysaccharide-binding protein

Abstract

Human lactoferrin (hLf), a glycoprotein released from neutrophil granules during inflammation, and the lipopolysaccharide (LPS)-binding protein (LBP), an acute-phase serum protein, are known to bind to the lipid A of LPS. The LPS-binding sites are located in the N-terminal regions of both proteins, at amino acid residues 28 to 34 of hLf and 91 to 108 of LBP. Both of these proteins modulate endotoxin activities, but they possess biologically antagonistic properties. In this study, we have investigated the competition between hLf and recombinant human LBP (rhLBP) for the binding of Escherichia coli 055:B5 LPS to the differentiated monocytic THP-1 cell line. Our studies revealed that hLf prevented the rhLBP-mediated binding of LPS to the CD14 receptor on cells. Maximal inhibition of LPS-cell interactions by hLf was raised when both hLf and rhLBP were simultaneously added to LPS or when hLf and LPS were mixed with cells 30 min prior to the incubation with rhLBP. However, when hLf was added 30 min after the interaction of rhLBP with LPS, the binding of the rhLPS-LBP complex to CD14 could not be reversed. These observations indicate that hLf competes with rhLBP for the LPS binding and therefore interferes with the interaction of LPS with CD14. Furthermore, experiments involving competitive binding of the rhLBP-LPS complex to cells with two recombinant mutated hLfs show that in addition to residues 28 to 34, another basic cluster which contains residues 1 to 5 of hLf competes for the binding to LPS. Basic sequences homologous to residues 28 to 34 of hLf were evidenced on LPS-binding proteins such as LBP, bactericidal/permeability-increasing protein, and Limulus anti-LPS factor.

FIG. 1

LPS binding to differentiated THP-1 cells in the presence of human serum. As described in Materials and Methods, cells were incubated with 1 μg of FITC-labeled E. coli 055:B5 LPS per ml in the presence of 0.4% BSA (bar 1), 10% human serum (bar 2), 10% human serum after preincubation of cells with isotype control IgG2a (bar 3), 10% human serum after previous incubation of cells with anti-CD14 monoclonal antibody (bar 4), 20 μg of hLf per ml (bar 5), or 10% human serum and 20 μg of hLf per ml added at the same time (bar 6). The mean fluorescence intensity was determined by flow cytometry. The results (means ± standard errors) were calculated from five separate experiments.

FIG. 2

Inhibition of LPS binding to differentiated THP-1 cells by hLf in the presence of human serum. Cells were incubated with 1 μg of FITC-labeled LPS per ml and 10% human serum in the presence of increasing concentrations of hLf, as described in Materials and Methods. The results are expressed as percentages of the total LPS bound to cells with 10% serum alone. Each point represents the mean ± standard error from four experiments.

FIG. 3

LPS binding to differentiated THP-1 cells in the presence of rhLBP. As described in Materials and Methods, cells were incubated with 1 μg of FITC-labeled LPS per ml in the presence of 0.4% BSA (bar 1), 1.5 μg of rhLBP per ml (bar 2), 1.5 μg of rhLBP per ml after preincubation of cells with anti-CD14 monoclonal antibody (bar 3), or 1.5 μg of rhLBP per ml and 20 μg of hLf per ml added at the same time (bar 4). The mean fluorescence intensity was determined. The results (means ± standard errors) were calculated from four separate experiments.

FIG. 4

Inhibition of LPS binding to differentiated THP-1 cells by hLf in the presence of rhLBP. The binding of FITC-labeled LPS (1 μg/ml) to cells was performed in the presence of 1.5 μg of rhLBP per ml and increasing concentrations of hLf, added simultaneously. The results are expressed as percentages of the total LPS bound to cells with rhLBP alone. Each point represents the mean ± standard error from four experiments.

FIG. 5

Time-dependent inhibition of rhLBP-mediated binding of LPS to differentiated THP-1 cells in the presence of hLf. The binding of FITC-labeled LPS was studied, as described in Materials and Methods, in the presence of rhLBP (1.5 μg/ml) and three concentrations of hLf added at various intervals: 5 μg/ml (filled bars), 10 μg/ml (hatched bars), and 20 μg/ml (stippled bars). hLf was added to LPS at the same time as rhLBP (A), 30 min before rhLBP (B), or 30 min after rhLBP (C). Results are percentages of the total LPS bound to cells in the presence of rhLBP alone. Data are expressed as the means ± standard errors from three replicates.

FIG. 6

Inhibition of LPS binding to differentiated THP-1 cells by rhLf and mutated rhLfs in the presence of rhLBP. Cells were incubated for 1 h at 4°C simultaneously with 1 μg of FITC-labeled LPS per ml, 1.5 μg of rhLBP per ml, and increasing concentrations of rhLf (•), EGS-rhLf (▴), or G4R-rhLf (○). Results are percentages of total LPS bound to cells in the presence of rhLBP alone. Each point represents the mean ± standard error from four experiments.

FIG. 7

Alignment of amino acid residues 24 to 36 of hLf, 87 to 99 of hLBP, 26 to 38 of hBPI, and 36 to 47 of LALF. Homologous residues are in boldface. The positions of sheets (β), helices (α), and loops (L) are indicated for hLf and LALF.