Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro.

Abstract

Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 011 1:B4 LPS on movement of '4C-BSA across bovine pulmonary artery EC monolayers. In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations 2 0.5 ng/ml, and LPS (10 ng/ml) exposures of 2 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 Ag/ml failed to increase 14C-BSA flux at 6 h. The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold. LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of . 2.5%. LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function. LPS-LBP had a more rapid onset than did LPS-sCD14. The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone. These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface. However, in the presence of serum both molecules are required. (J. Clin. Invest. 1994. 93:692-702.)

Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

“…To exclude the contribution of endotoxin or bacterial lipopolysaccharide (LPS), transendothelial 14 C-BSA flux was assayed after 6 h exposures to 30 g TSP/ml, TSP preincubated with polymixin B (PMB) immobilized onto agarose beads (Sigma), LPS derived from Escherichia coli: 0111:B4 (Sigma) 100 ng/ml, and LPS preincubated with PMB in the presence of 10% fetal bovine serum. Because LPS-induced barrier dysfunction is profoundly serum dependent, the TSP-induced changes were also studied under strict serum-starvation conditions as described previously (Goldblum et al, 1994).…”

“…To determine whether TSP-induced changes in endothelial barrier function could be explained by EC injury, TSP-exposed and medium control monolayers were studied for 51 Cr release as we have described previously (Goldblum et al, 1994). Briefly, ECs were labeled with [ 51 Cr]-sodium chromate (Amersham), and the labeled monolayers were incubated for 6 h with either TSP (30 g/ml) or medium alone.…”

“…Preincubation of TSP (30 g/ml) with Ab 133 (Schultz-Cherry and Murphy-Ullrich, 1993) reduced the TSP effect by Ͼ80%. Endotoxin, in the presence of serum accessory molecules, is known to influence endothelial barrier function (Goldblum et al, 1994). One TSP preparation analyzed for endotoxin activity by a chromogenic limulus amebocyte lysate assay (Associates of Cape Cod, Inc., Woods Hole, MA) was found to contain ϳ0.36 ng endotoxin/ g TSP.…”

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation

“…The systemic inflammatory response in sepsis can lead to rapid organ failure and death (1,5). Bacterial endotoxin (LPS) ranks highest among risk factors contributing to ALI in sepsis (8). Endotoxins are known to activate innate immune responses, resulting in the production of a vast spectrum of inflammatory cytokines (1,9).…”

CD44 Regulates Hepatocyte Growth Factor-mediated Vascular Integrity

“…LPS induces a delayed EC barrier-disrupting response by activating a receptor complex of TLR4, CD14, and MD2, with consequent Rho-dependent NF-B activation, cytokine production, and generation of low molecular weight HA degradation fragments (55)(56)(57)(58)(59)(60)(61). In particular, RhoA is important in LPS-mediated regulation of interleukin-8 production (62).…”