New scavenger receptor-like receptors for the binding of lipopolysaccharide to liver endothelial and Kupffer cells

Abstract

Lipopolysaccharide (LPS) is cleared from the blood mainly by the liver. The Kupffer cells are primarily responsible for this clearance; liver endothelial and parenchymal cells contribute to a lesser extent. Although several binding sites have been described, only CD14 is known to be involved in LPS signalling. Among the other LPS binding sites that have been identified are scavenger receptors. Scavenger receptor class A (SR-A) types I and II are expressed in the liver on endothelial cells and Kupffer cells, and a 95-kDa receptor, identified as macrosialin, is expressed on Kupffer cells. In this study, we examined the role of scavenger receptors in the binding of LPS by the liver in vivo and in vitro. Fucoidin, a scavenger receptor ligand, significantly reduced the clearance of 125I-LPS from the serum and decreased the liver uptake of 125I-LPS about 40%. Within the liver, the in vivo binding of 125I-LPS to Kupffer and liver endothelial cells was decreased 72 and 71%, respectively, while the binding of 125I-LPS to liver parenchymal cells increased 34% upon fucoidin preinjection. Poly(I) inhibited the binding of 125I-LPS to Kupffer and endothelial cells in vitro 73 and 78%, respectively, while poly(A) had no effect. LPS inhibited the binding of acetylated low-density lipoprotein (acLDL) to Kupffer and liver endothelial cells 40 and 55%, respectively, and the binding of oxidized LDL (oxLDL) to Kupffer and liver endothelial cells 65 and 61%, respectively. oxLDL and acLDL did not significantly inhibit the binding of LPS to these cells. We conclude that on both endothelial cells and Kupffer cells, LPS binds mainly to scavenger receptors, but SR-A and macrosialin contribute to a limited extent to the binding of LPS.

FIG. 1

Serum decay and liver uptake of ¹²⁵I-LPS. Rats were injected with ¹²⁵I-LPS without (•) or with (▴) a preinjection of fucoidin (15 mg/kg). At the indicated times, levels of radioactivity in serum (A) and liver (B) were determined. Data are means of two experiments ± variation.

FIG. 2

Cell distribution of ¹²⁵I-LPS. Rats were injected with ¹²⁵I-LPS without (open bars) or with (hatched bars) a preinjection of fucoidin (15 mg/kg). Fifteen minutes after ¹²⁵I-LPS injection, the liver was perfused at 8°C, cells were isolated, and binding of ¹²⁵I-LPS to the different liver cell types was determined. (A) Binding to endothelial cells (EC) and Kupffer cells (KC); (B) binding to liver tissue (L) and parenchymal cells (PC). Data are means of three experiments ± SD.

FIG. 3

Binding of ¹²⁵I-LPS to liver endothelial and Kupffer cells. Liver endothelial cells (A) and Kupffer cells (B) were isolated as described in Materials and Methods. Cells were incubated in DMEM–2% BSA (pH 7.4) for 2 h at 4°C with 5 μg of ¹²⁵I-LPS per ml and increasing amounts of unlabelled LPS. Cells were then washed, and radioactivity was counted. Data are means of three experiments ± SD.

FIG. 4

Effects of poly(I) and poly(A) on the binding of ¹²⁵I-LPS. Liver endothelial cells (A) and Kupffer cells (B) were isolated as described in Materials and Methods. Cells were incubated in DMEM–2% BSA (pH 7.4) for 2 h at 4°C with 5 μg of ¹²⁵I-LPS per ml and increasing amounts of poly(I) (•) or poly(A) (▴). Cells were then washed, and radioactivity was counted. Data are means of three experiments ± SD.

FIG. 5

Displacement of ¹²⁵I-oxLDL and ¹²⁵I-acLDL by LPS. Liver endothelial cells (A) and Kupffer cells (B) were isolated as described in Materials and Methods. Cells were incubated in DMEM–2% BSA (pH 7.4) for 2 h at 4°C with 5 μg of ¹²⁵I-acLDL (•) or ¹²⁵I-oxLDL (▴) per ml and increasing amounts of LPS. Cells were then washed, and radioactivity was counted. Data are means of three experiments ± SD.

FIG. 6

Effects of acLDL and oxLDL on the binding of ¹²⁵I-LPS. Liver endothelial cells (A) and Kupffer cells (B) were isolated as described in Materials and Methods. Cells were incubated in DMEM–2% BSA (pH 7.4) for 2 h at 4°C with 5 μg of ¹²⁵I-LPS per ml and increasing amounts of acLDL (•) or oxLDL (▴). Cells were then washed, and radioactivity was counted. Data are means of three experiments ± SD.

FIG. 7

Contribution of poly(I)-sensitive binding sites to the binding of LPS. Cells were isolated as described in Materials and Methods. Endothelial cells (A) and Kupffer cells (B) were incubated with increasing amounts of ¹²⁵I-LPS, without (•) or with (▴) an excess of poly(I) (200 μg/ml) for 2 h at 4°C.

FIG. 8

Effect of phospholipase C on the binding of ¹²⁵I-LPS to Kupffer cells. Isolated Kupffer cells were not treated (•) or were incubated with phospholipase C (3 U/ml) at 37°C for 30 min (▴) and subsequently incubated with 5 μg of ¹²⁵I-LPS per ml at 4°C for 2 h.