Minimal penetration of lipopolysaccharide across the murine blood–brain barrier

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Abstract

LPS given peripherally or into the brain induces a neuroinflammatory response. How peripheral LPS induces its effects on brain is not clear, but one mechanism is that LPS crosses the blood–brain barrier (BBB). Alternatively, LPS acts outside the BBB by stimulating afferent nerves, acting at circumventricular organs, and altering BBB permeabilities and functions. Here, we labeled LPS with radioactive iodine (I-LPS) and coinjected it with radioactively labeled albumin (I-Alb) which acted as a vascular space marker. Measurable amounts of I-LPS associated with the BBB, most reversibly bound to brain endothelia. Brain endothelia also sequestered small amounts of I-LPS and about 0.025% of an intravenously injected dose of I-LPS crossed the BBB to enter the CNS. Disruption of the BBB with repeated injections of LPS did not enhance I-LPS uptake. Based on dose–response curves in the literature of the amounts of LPS needed to stimulate brain neuroimmune events, it is unlikely that enough peripherally administered LPS enters the CNS to invoke those events except possibly at the highest doses used and for the most sensitive brain functions. I-LPS injected into the lateral ventricle of the brain entered the circulation with the reabsorption of cerebrospinal fluid (bulk flow) as previously described. In conclusion, brain uptake of circulating I-LPS is so low that most effects of peripherally administered LPS are likely mediated through LPS receptors located outside the BBB.

Introduction

Lipopolysaccharide (LPS), or endotoxin, is the major component of the outer membrane leaflet of Gram-negative bacteria. The exact composition of LPS varies with bacterial species, but all consist of a hydrophilic polysaccharide domain covalently bound to a hydrophilic lipid component termed lipid A (Ulmer et al., 2002). Low levels of LPS are being discovered to circulate in the blood during an increasing number of conditions and diseases (Pussinen et al., 2004, Szeto et al., 2008, Brenchley et al., 2006, Zhang et al., 2009). These levels are thought to reflect bacterial translocation from the endogenous flora of the gastrointestinal tract, including the oral cavity. However, very high levels of LPS suggest infection, peritonitis, or sepsis.

The body has evolved to recognize LPS as a pathogen-associated molecular pattern, activating the innate immune system when significant levels of LPS are detected. The lipid A component with a molecular weight of less than 2000 Da is the main immunostimulatory component of LPS. The 55 kDa glycoprotein CD-14 and Toll-like receptor 4 are the major receptors binding LPS (Ulmer et al., 2002). Other proteins that can bind LPS (Ulmer et al., 2002) and that may participate in responses to LPS include Toll-like receptor 2, hsp90, TREM-1, decay accelerating factor (CD55), adhesion molecules of the β-integrin family, and the high-conductance calcium and voltage dependent potassium channel (MaxiK). Nod1 and Nod2 represent intracellular binding proteins capable of binding LPS, suggesting that sensing by the innate immune system may extend to intracellular infections (Girardin et al., 2001, Ulevitch et al., 2004).

Peripheral administration of LPS increases brain levels of interleukins, prostaglandins, nitric oxide, and other substances (Singh and Yiang, 2004, Larson and Dunn, 2001, Sugita et al., 2002). Injection of LPS directly into the CNS can induce a neuroimmune response similar to peripheral administration (Gottschall et al., 1992). In the study of Quan et al. (1994), central infusion of LPS first produced detectable IL-1 in the brain and later in the blood, whereas peripheral administration of LPS first produced detectable IL-1 in blood and later in the brain. With simultaneous administration, central LPS can oppose or even produce the opposite effect of peripheral LPS (Chen et al., 2000). These findings raise the question of how LPS on one side of the blood–brain barrier (BBB) is mediating changes on the other.

Peripheral LPS could induce CNS effects by crossing the BBB and directly activating cells within the CNS. This is how many authors have assumed that LPS works. Indeed, the CNS contains many cells that possess Toll-like 4 receptors (Chakravarty and Herkenham, 2005) or can directly respond to LPS, including brain endothelial cells (Reyes et al., 1999, Verma et al., 2006), microglia (Marzolo et al., 2000), and astrocytes (Chakravarty and Herkenham, 2005). However, work by Singh and Yiang suggests that LPS can associate with but does not cross the BBB (Singh and Yiang, 2004). LPS can act through indirect mechanisms (Watkins et al., 1995), such as vagal (Goehler et al., 1999) or other afferent nerve (Romeo et al., 2001) stimulation, release of substances from the periphery that can cross the BBB (Qin et al., 2007), enhancing interactions between the BBB and immune cells (Strosznajder et al., 1996), altering BBB permeability (Xaio et al., 2001), acting at circumventricular organs (Blatteis et al., 1983, Ulmer et al., 2002), or by inducing release of substances from the cells constituting the BBB (Quan et al., 2003, Verma et al., 2006). Here, we radioactively labeled LPS and examined with state-of-the-art methods the ability of LPS to cross the BBB.

Section snippets

Radioactive labeling and purification of LPS and albumin

LPS from Salmonella enterica (1 mg; Sigma Chemical Co., St. Louis, MO, Cat. No. L-6511) was radioactively labeled according to the method of Ulevitch (1978). LPS was incubated with a 1 ml solution of methyl 4-hydroxybenzimidate HCl (MHBI; 9.4 mg/ml of 50 mM borate buffer, pH 8.0) in a 37 °C water bath for 18 h. The MHBI–LPS mixture was added to a dialysis cassette (Slide-A-Lyzer; 10,000 Da cutoff and dialyzed 2× in 500 ml saline for 2 h at 4 °C and then overnight in 500 ml saline to remove unreacted MHBI.

Results

The relation between log(%Inj/ml) and time was statistically significant: r = 0.707, p < 0.001, n = 20 (Fig. 1, upper panel). The half-time clearance from blood was 259 min and the Vd was 1.50 ml. There was no relation between brain/serum ratios and exposure time indicating that there was no measurable transport over time across the BBB (Fig. 1, lower panel). The mean brain/serum ratio was 11.3 ± 0.26 μl/g (n = 20). To determine whether an early phase of uptake occurred for I-LPS, mice were studied at 1 and

Discussion

These studies examined the ability of LPS labeled with radioactive iodine to cross the blood–brain barrier. We examined the ability of I-LPS to cross in both the blood-to-brain direction and in the brain-to-blood direction. We determined the influences of circulating factors, saturable processes, enzymatic degradation, BBB disruption through activation of the innate immune system, and capillary adhesion on the permeability process. In general, we found that I-LPS reversibly adhered to and was

Acknowledgment

Supported by VA Merit Review, NS05047, and AG029839.

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