Anti-N-Methyl-D-Aspartic Acid Receptor 2 (Anti-NR2) Antibody in Neuropsychiatric Lupus Serum Damages the Blood-Brain Barrier and Enters the Brain

Abstract

BACKGROUND Brain microvessel endothelial cells constitute an important component in the blood-brain barrier. Cell-culture-based models of the blood-brain barrier (BBB) have been extensively applied in pharmacology, pathology and physiology. This study investigated effects of anti-N-methyl-D-aspartic acid receptor 2 (anti-NR2), N-methyl-D-aspartic acid (NMDA) receptor antibodies, NMDA receptor antagonists, and NMDA receptor agonists on brain microvessel endothelial cell models, and verified the effect of anti-NR2 antibody on the BBB as a receptor agonist. MATERIAL AND METHODS The primary brain microvessel endothelial cells were isolated and cultured, and an in vitro BBB model was established based on microvessel endothelial cells. Anti-NR2 antibody, glutamic acid, ifenprodil, and memantine were added in the BBB model to analyze changes in transepithelial electrical resistance (TEER) and to examine the permeability of the brain microvessel endothelial cell model. RESULTS The results showed that TEER values were significantly decreased by the addition of anti-NR2 antibody and glutamate, but were significantly increased by the addition of ifenprodil and memantine. TEER values showed no changes when treated by anti-NR2 antibody and ifenprodil, as well as anti-NR2 antibody and memantine. When dexamethasone was added, the TEER values increased by 23.8%, 39.4%, and 29.6% by treating with anti-NR2 antibody, positive cerebrospinal fluid, and positive serum, respectively. CONCLUSIONS Our findings show that anti-NR2 antibody in neuropsychiatric lupus serum can damage the BBB and enter the brain.

Figure 1

Isolation and identification of rat brain microvessel endothelial cells. (A) Isolated microvessel segments and/or single cells. (B) Short spindle and/or polygonal cells were observed around microvessel fragments. (C) Paving stone-like cells. (D) Monolayer growth endothelial cells. (E) Passaged rat brain microvessel endothelial cells. (F) Immunocytochemistry identified brain microvessel endothelial cells. Scale bars are illustrated in figures.

Figure 2

The dynamic changes in transepithelial electrical resistance in the brain microvessel endothelial cells. Results are averages of 3 independent experiments. Data are represented as mean ±SEM.

Figure 3

The effects of different additives on transepithelial electrical resistance in the brain microvessel endothelial cells. 1: normal serum (100 μl), 2: anti-NR2 antibody (100 μl, 10 μg/ml), 3: positive cerebrospinal fluid (CSF, 100 μl), 4: negative CSF (100 μl), 5: positive serum (100 μl), 6: negative serum (100 μl). After culture for 12 h, the TEER was measured and measured. Results are averages of 3 independent experiments. Data are represented as mean ± SEM.

Figure 4

The effects of NMDA receptor antagonists and agonists on transepithelial electrical resistance in the brain microvessel endothelial cells. 1: anti-NR2 antibody (100 μl, 10 μg/ml), 2: glutamate (5 mM), 3: ifenprodil (10 μg/ml), 4: memantine (10 μg/ml), 5: ifenprodil (10 μg/ml) + anti-NR2 antibody (100 μl, 10 μg/ml), 6: memantine (10 μg/ml) + anti-NR2 antibody (100 μl, 10 μg/ml). After culture for 12 h, the TEER was measured. Results are averages of 3 independent experiments. Data are represented as mean ±SEM.

Figure 5

The effects of dexamethasone on transepithelial electrical resistance in the brain microvessel endothelial cells. 1: anti-NR2 antibody (100 μl, 10 μg/ml), 2: positive cerebrospinal fluid (CSF, 100 μl), 3: positive serum (100 μl). The holes with increased permeability in the blood-brain barrier were added by dexamethasone (1 μg), and the TEER was measured after 12 h. Results are averages of 3 independent experiments. Data are represented as mean ±SEM.