Role of plasma, lipopolysaccharide-binding protein, and CD14 in response of mouse peritoneal exudate macrophages to endotoxin

Abstract

Plasma lipopolysaccharide (LPS)-binding protein (LBP) and membrane CD14 function to enhance the responses of monocytes to low concentrations of endotoxin. Surprisingly, recent reports have suggested that LBP or CD14 may be dispensable for macrophage responses to low concentrations of LPS or may even exert an inhibitory effect in the case of LBP. We therefore investigated whether LBP and CD14 participated in the response of mouse peritoneal exudate macrophages (PEM) to LPS stimulation. In the presence of a low amount of plasma (<1%) or of recombinant mouse or human LBP, PEM were found to respond to low concentrations of LPS (<5 to 10 ng/ml) in an LBP- and CD14-dependent manner. However, tumor necrosis factor production (not interleukin-6 production) by LPS-stimulated PEM was reduced when cells were stimulated in the presence of higher concentrations of plasma or serum (5 or 10%). Yet, the inhibitory effect of plasma or serum was not mediated by LBP. Taken together with previous results obtained with LBP and CD14 knockout mice in models of experimental endotoxemia, the present data confirm a critical part for LBP and CD14 in innate immune responses of both blood monocytes and tissue macrophages to endotoxins.

FIG. 1

TNF and IL-6 production by PEM stimulated with LPS in the presence of low doses of plasma. PEM (50,000 cells/well) were stimulated for 6 h with LPS in the presence of RPMI medium with no plasma (squares), RPMI medium containing 0.2% human plasma (circles, panel A), or 0.2% autologous mouse plasma (triangles, panel B). Data are the mean ± SD of five different experiments run in duplicates. ∗, P < 0.05 by ANOVA.

FIG. 2

Effect of neutralization of mouse plasma LBP on the cytokine response of PEM to LPS. PEM (50,000 cells/well) were stimulated for 6 h with LPS in the presence of RPMI medium containing 1% autologous mouse plasma (A) or RPMI medium with no plasma (B) and in the presence of nonneutralizing anti-LBP MAb (clone M306-5) (squares) or neutralizing anti-LBP MAb (clone M 330-19) (circles). Data are the mean ± SD of five different experiments run in duplicates. ∗, P < 0.05 by ANOVA.

FIG. 3

Effect of heterozygous LBP^+/− mice and LBP^−/− plasma on the cytokine response by LPS-stimulated PEM. PEM (50,000 cells/well) were stimulated for 6 h with LPS in the presence of RPMI medium containing either 1% LBP^+/− plasma (squares) or 1% LBP^−/− plasma (circles). Data are the mean ± SD of five different experiments run in duplicates. ∗, P < 0.05 by ANOVA.

FIG. 4

Effect of recombinant mouse LBP on TNF production by PEM stimulated with LPS. PEM (50,000 cells/well) were stimulated for 6 h with 1 ng of LPS/ml in the presence of increasing concentrations of purified rmLBP (squares) or of SF9 supernatant containing known concentrations of LBP (circles). TNF was not detected in cultures containing control SF9 supernatant or RPMI medium (data not shown). Data are the mean ± SD of four different experiments run in duplicates.

FIG. 5

Effect of anti-CD14 MAb on cytokine production by PEM stimulated with LPS. PEM (50,000 cells/well) were pretreated with 10 μg of the anti-CD14 MAb/ml (circles) or with an irrelevant MAb (squares) and were stimulated for 6 h with LPS in the presence of RPMI medium containing 1% OF1 autologous mouse plasma (A) or RPMI medium with no plasma (B). Data are the mean ± SD of five different experiments run in duplicates. ∗, P < 0.05 by ANOVA.

FIG. 6

TNF and IL-6 production by PEM stimulated with LPS in the presence of high doses of plasma. PEM (50,000 cells/well) were stimulated for 6 h with LPS in the presence of RPMI medium with no plasma (squares), RPMI medium containing 5% human plasma (circles, panel A) or RPMI medium containing 5% autologous mouse plasma (triangles, panel B). Data are the mean ± SD of five different experiments run in duplicates. ∗, P < 0.05 by ANOVA.