Mechanism Of Activation And Leukocyte Recruitment In Mesencephalic Endothelial Cells Of Central Nervous System

Part I CXCR2 is essential for cerebral endothelial activation and leukocyte recruitment during neuroinflammationBackground:Chemokines and chemokine receptors cooperate to promote immune cell recruitment to the central nervous system (CNS). In this study, we investigated the roles of CXCR2 and CXCL1 in leukocyte recruitment to the CNS using a murine model of neuroinflammation.Methods:Wild-type (WT), CXCL1-/-, and CXCR2-/-mice each received an intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS). Esterase staining and intravital microscopy were performed to examine neutrophil recruitment to the brain. To assess endothelial activation in these mice, the expression of adhesion molecules was measured viaquantitative real-time polymerase chain reaction (PCR) and Western blotting. To identify the cellular source of functional CXCR2, chimeric mice were generated by transferring bone marrow cells between the WT and CXCR2-/-mice.Results:Expression levels of the chemokines CXCL1, CXCL2, and CXCL5 were significantly increased in the brain following the i.c.v. injection of LPS. CXCR2 or CXCL1 deficiency blocked neutrophil infiltration and leukocyte recruitment in the cerebral micro vessels. In the CXCR2-/- and CXCL1-/- mice, the cerebral endothelial expression of adhesion molecules such as P-selectin and VCAM-1 was dramatically reduced. Furthermore, the bone marrow transfer experiments demonstrated that CXCR2 expression on CNS-residing cells is essential for cerebral endothelial activation and leukocyte recruitment. Compared with microglia, cultured astrocytes secreted a much higher level of CXCL1 in vitro. Astrocyte culture conditioned medium significantly increased the expression of VCAM-1 and ICAM-1 in cerebral endothelial cells in a CXCR2-dependent manner. Additionally, CXCR2 messenger RNA (mRNA) expression in cerebral endothelial cells but not in microglia or astrocytes was increased following tumor necrosis factor-a (TNF-a)stimulation. The intravenous injection of the CXCR2 antagonist SB225002 significantly inhibited endothelial activation and leukocyte recruitment to cerebral microvessels.Conclusions:CXCL1 secreted by astrocytes and endothelial CXCR2 play essential roles in cerebral endothelial activation and subsequent leukocyte recruitment during neuroinflammation.Part Ⅱ Complement component C3a plays a critical role in endothelial activation and leukocyte recruitment into the brainBackground:The complement system is becoming increasingly recognized as a key participant in many neurodegenerative diseases of the brain. Complement-deficient animals exhibit reduced neuroinflammation.Methods:In the present study, we administered intracerebroventricularly lipopolysaccharide (LPS) to mimic local infection of the brain and investigated the role of key complement component C3 in brain vasculature endothelial activation and leukocyte recruitment. The degree of neutrophil infiltration was determined by esterase staining. Leukocyte-endothelial interactions were measured using intravital microscopy. Cerebral endothelial activation was evaluated using real-time PCR and Western blotting.Results:Neutrophil infiltration into the brain cortex and hippocampus was significantly reduced in C3-/- mice and C3aR-/-mice but not in C6-/- mice. We detected markedly attenuated leukocyte-endothelial interactions in the brain microvasculature of C3-/-mice. Accordingly, in response to LPS administration, the brain microvasculature in these mice had decreased expression of P-selectin, E-selectin, intercellular cell adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). Depletion of C3 from the circulation also caused reduction in VCAM-1 and E-selectin expression and leukocyte recruitment, suggesting that C3 in the circulation contributed to brain endothelial activation. Furthermore, C3-/- mice exhibited decreased leukocyte recruitment into the brain upon tumor necrosisfactor-a (TNF-a) stimulation. C3a activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB(NF-κB) and induced the upregulation of VCAM-1 and ICAM-1 expression in murine primary cerebral endothelial cells in vitro.Conclusions:Our study provides the first evidence that C3a plays a critical role in cerebral endothelial activation and leukocyte recruitment during inflammation in the brain.