Haemophilus influenzae Lipopolysaccharide Disrupts Confluent Monolayers of Bovine Brain Endothelial Cells via a Serum-Dependent Cytotoxic Pathway

Abstract

An in vitro blood-brain barrier (BBB) model consisting of primary cultures of bovine brain microvascular endothelial cells was used to examine the effect of Haemophilus influenzae type b (Hib) on the BBB. Whole bacteria and purified lipopolysaccharide (LPS; greater than 10 ng/ml) caused marked cytotoxicity on the bovine brain endothelial cells. This effect could be completely blocked by polymyxin B. Similar cytotoxic effects were observed with a cultured bovine pulmonary endothelial cell line. Serum was essential for the LPS-mediated cytotoxic effect, and human, horse, bovine, or fetal calf serum all had similar effects. The serum factor was not a complement component. A monoclonal antibody against CD14, a receptor involved in mediating the effect of LPS in monocytes, completely blocked the cytotoxic effect in both brain and pulmonary endothelial cells. These results suggest that Hib LPS disrupts an in vitro BBB model via a serum- and CD14-dependent pathway and that LPS has cytotoxic effects on bovine endothelial cells without the involvement of monocytic cells, an effect that may be important in gram-negative meningitis and in endotoxic shock.

Concepts and Mechanisms: Crossing Host Barriers

“…In case of E. coli K1, cytotoxic necrotizing factor-1 (CNF-1) activates RhoA resulting in increased invasion of BMEC in vitro and increased penetration of the BBB in vivo (Khan et al , 2003. In contrast, b hemolysin (bh/c) of GBS (Doran et al 2003;Lembo et al 2010), pneumolysin of S. pneumonia (Zysk et al 2001) and HiB lipopolysaccharide (Patrick et al 1992) all cause apoptosis in brain endothelial cells, which lead to increased BBB permeability and bacterial penetration in vivo.…”

Molecular and cellular mechanisms for microbial entry into the CNS

“…In contrast, Lyme disease agent Borrelia burgdorferi (Szczepanski et al, 1990) and syphilis bacterium Treponema pallidum (Haake and Lovett, 1994;Thomas et al, 1988) appear to cross the endothelial barrier by travelling between the endothelial cells. Bacterial products and the CNS invasion BBB permeability can be modulated by bacterial components such as LPS (Patrick et al, 1992;Temesvari et al, 1993;Wispelwey et al, 1988), cell wall (Burroughs et al, 1992), and proteins (Spellerberg et al, 1995;Wispelwey et al, 1989). In a bacterial meningitis model, inoculation of animals with live H. In¯uenzae or bacterial LPS caused functional and ultrastructural changes of the BBB (Wispelwey et al, 1988(Wispelwey et al, , 1989.…”

“…Bacterial cell wall components such as LPS in E. coli and H. In¯uenzae (Patrick et al, 1992;Temesvari et al, 1993) and glycoproteins from S. pneumoniae (Spellerberg et al, 1995) have been known to affect BBB permeability. Release of these substances during bacteremia may facilitate bacterial entry into the CNS.…”

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

“…LPS ( 10 ng/ml) failed to increase endothelial cell LDH release over 6 h. LPS failed to increase 51Cr release after 1 or 2 h, but after 4 and 6 h, 51Cr release was minimally but significantly increased. LPS-induced EC cytotoxicity has been well described and is highly dependent on LPS concentration and exposure time, the presence of serum, as well as the species and possibly anatomical origin of the target EC (5,(30)(31)(32). Most of these earlier studies used higher LPS concentrations and longer LPS exposure times (4,5,30,31,33,34).…”