Efflux transport of serum amyloid P component at the blood-brain barrier

Abstract

Serum amyloid P component (SAP), a member of the innate immune system, does not penetrate the brain in physiological conditions; however, SAP is a stabilizing component of the amyloid plaques in neurodegenerative diseases. We investigated the cerebrovascular transport of human SAP in animal experiments and in culture blood-brain barrier (BBB) models. After intravenous injection, no SAP could be detected by immunohistochemistry or ELISA in healthy rat brains. Salmonella typhimurium lipopolysaccharide injection increased BBB permeability for SAP and the number of cerebral vessels labeled with fluorescein isothiocyanate (FITC)-SAP in mice. Furthermore, when SAP was injected to the rat hippocampus, a time-dependent decrease in brain concentration was seen demonstrating a rapid SAP efflux transport in vivo. A temperature-dependent bidirectional transport of FITC-SAP was observed in rat brain endothelial monolayers. The permeability coefficient for FITC-SAP was significantly higher in abluminal to luminal (brain to blood) than in the opposite direction. The luminal release of FITC-SAP from loaded endothelial cells was also significantly higher than the abluminal one. Our data indicate the presence of BBB efflux transport mechanisms protecting the brain from SAP penetration. Damaged BBB integrity due to pathological insults may increase brain SAP concentration contributing to development of neurodegenerative diseases.

Keywords: blood–brain barrier; brain efflux transport; brain endothelial cells; lipopolysaccharide; neurodegenerative diseases; neuroimmunology; permeability; serum amyloid P component.

Fig. 1.

Immunohistochemistry of SAP on brain cortex sections in control (A), LPS- (B), SAP- (C), and LPS + SAP- (D) treated animals. Extravasation of human SAP can be seen around the brain vessels only in LPS-treated mice (D). Detection of i.v. injected FITC–SAP in brain tissue of mice by confocal microscopy (E). LPS treatment increased the FITC–SAP leakage to brain parenchyma in the frontal cortex (F). Bar = 50 μm

Fig. 2.

Time-dependent decrease of SAP content in rat brain after microinjection of SAP to the left hippocampus. SAP content of the left brain hemisphere decreased rapidly after SAP exposition and reached the base level of unexposed right hemisphere in three days (n = 5). Statistics: ANOVA followed by Newman–Keuls multiple comparison test. Significant changes: ^***P < 0.001, ^**P < 0.01, and ^*P < 0.05 compared to the values measured in the same time in the right hemisphere; in the samples from the left hemisphere: ^aP < 0.001 compared to the value measured at 0.02 day (i.e., 30 min), ^bP < 0.05 compared to the value measured at Day 1, ^cP < 0.05 compared to the value measured at Day 3, and ^dP < 0.05 compared to the value measured at Day 5. No time-dependent significant difference was found in the samples from the right hemispheres

Fig. 3.

Transendothelial permeability for FITC–SAP in rat brain endothelial cell monolayers at 37 °C and 4 °C from the luminal to abluminal and abluminal to luminal directions (A). All values presented are means ± S.E.M. (n = 5). Statistically significant differences (P < 0.001) are indicated between values measured. Fluorescent microscopy images show FITC–SAP uptake (green) to primary RBE cells in vitro at 37 °C (B). Cell nuclei are stained by bis-benzimide (blue), intercellular junctions are delineated by ZO-1 immunostaining (red). No labeled protein uptake to the cells can be observed at 4 °C (C). Bar = 10 μm

Fig. 4.

Measurement of FITC–SAP release into the luminal and abluminal compartments after loading RBE cells 25 μM FITC–SAP for 1 h. All values presented are means ± S.E.M. (n = 5). Statistically significant differences are indicated between values measured (^*P < 0.01, ^**P < 0.001)