Peripheral T Cells Derived From Alzheimer's Disease Patients Overexpress CXCR2 Contributing To Its Transendothelial Migration, Which Is Microglial TNF-α Dependent

PurposeThe blood-brain barrier, composed of parenchyma microvascular endothelia cells, basement membrane and foot processes of astrocytes, restricts the free passage of cells and molecules from systemic compartment into the central nervous system (CNS). Because of the complex construct of BBB and the shortage of lymphatic vessel, the central nervous system was been described as immunological privileged. However, several lines evidences suggest that, even under physiologic conditions, immune surveillance is ongoing process within CNS and is achieved preferentially by lymphocytes with an activated phenotype to clear the possible pathogen.Alzheimer's disease (AD) is the most common form of age-related cognitive failure and presents a rising incidence accompanied with lengthen of the human life span. Most commonly, patients die from continued infection and systems failure in the 5-6 years after onset.AD is characterized by deposition of amyloid beta and plaque formation. In AD pathogenesis, inflammatory responses may play an important role that pathological evidence suggests existence of Aβ-reactive T cells in the AD patients' brains compared with age-matched controls. However, how circulating T cells are activated and penetrate the blood-brain barrier (BBB) that mainly consists of endothelial cells is not clear. We have previously observed that AD patients' T cells present a stronger capability of crossing through monolayer of human brain microvasculr endothelial cells (HBMECs) in vitro than age-matched controls. cDNA array analysis showed that a few genes were significantly overexpressed in AD patients' T cells, including CXCR2, a member of CXC chemokines family, who contributes to the migration of lymphocytes, monocytes, macrophages and neutrophils by reacting with its ligand IL-8. However, the role of CXCR2 played in the process of T lymphocytes crossing BBB remains unknown.Based on these results, the aim of this study is to identify the determinants contributed to T cells migration from blood to brain in AD, especially the role of CXCR2 in this process, by mimicking the microenvironment of AD brain.Methods1. The role of CXCR2 in the process of AD patients' T cell transendothelial migration is investigated(1) Real-time PCR analysis of CXCR2 expression in AD patients' T cell.(2) To mimic AD brain's microenvironment, a microglia-containing in vitro BBB model was constructed to investigate the transmigration of AD patients' T cell, Jurkat, 6T-CEM. In some neutralization experiments, anti-CXCR2 neutralization antibody was applied.2. The role of IL-8 in HBMECs induced by Aβstimulated microglia in the process of T cell transendothelial migration is investigated(1) The Real-time PCR and ELISA analysis of IL-8 expression in HBMECs induced by Aβstimulated BV2 microglia.(2) Adhesion assay and Transwell assay analysis of AD patients' T cell, Jurkat, 6T-CEM performed by anti-IL-8 neutralization antibody or si-IL-8 HBMECs.(3) Perpetration a model of Aβ1-42 deposition in rat brain and analysis of CXCR2 expression in peripheral T cell and colocalization between CINC-1 expression on brain microvascular endothelial cells by Real-time PCR and immunofluorescence, respectively.(4) Blockage of T cells entry into rat parenchyma by administration of special antagonist SB332235-Z against CXCR2.3. The role of TNF-αderived from Aβreactived microglia in the CXCR2/IL-8 dependent transendothelial migration is investigated(1) TNF-αderived from Aβstimulated BV2 and U937 was detected by ELISA(2) After the treatment of Aβstimulated BV2 supernatant with anti-TNF-αneutralization antibody, and detected IL-8 level by ELISA.(3) Adhesion and Transwell assay of T cell was performed using rhTNF-αor Aβstimulated BV2 supernatant. In some neutralization experiments, anti-CXCR2, anti-IL-8, anti- TNF-αneutralization antibody was applied.(4) TNF-αantagonist Enbrel was injected into the lateral cerebral ventricle of the rat model of Aβ1-42 deposition. Analysis of CXCR2 expression in peripheral T cell and colocalization between CINC-1 expression on brain microvascular endothelial cells was taken by Real-time PCR and immunofluorescence, respectively. T cells entry into rat parenchyma by administration using immunofluorescence.Results1. The ability of AD patients' T cell transendothelial migration is increased by CXCR2(1) CXCR2 expression level is up-regulated in AD patients' T cells.(2) Anti-CXCR2 neutralization antibody can reduce the transendothelial migration ratio of T cell.2. The IL-8 in HBMECs is induced by Aβ-reactived microglia and promoted the process of CXCR2/IL-8 dependent T cell transendothelial migration.(1) The IL-8 expression level of HBMECs was up-regulated by Aβstimulated BV2 supernatant.(2) Anti-CXCR2 neutralization antibody and si-IL-8 can effectively block the CXCR2/IL-8 adhesion and transmigration of AD patients' T cells and Jurkat T cells.(3) In the rat model of Aβ1-42 deposition, CXCR2 and CINC-1 expression was up-regulated in peripheral T cell and brain microvascular endothelial cells, respectively. The intrahippocampus injection of Aβ1-42 induced T cell crossing of BBB at a CXCR2-dependent manner.3. TNF-αderived from Aβ-reactived-microglia promoted the CXCR2 /IL-8 dependent T cell transendothelial migration.(1) Aβ1-42 promoted microglial TNF-αsecretion and induced the up-regulation of IL-8 in HBMECs.(2) Anti-TNF-αneutralization antibody reduced the Aβ1-42 stimulated microglia promoted transmigration. Anti-CXCR2 and anti-IL-8 neutralization antibody diminished the TNF-αpromoted transmigration.(3) Enbrel abolished the upregulation of CXCR2 in peripheral T cells, CINC-1 in brain endothelial cells and the increased T cells occurrence in the brain induced by Aβinjection in rat hippocampusConclusionsPeripheral T cells derived from Alzheimer's disease patients overexpress CXCR2 contributing to its adhesion and transendothelial migration through an endothelial CXCR2/IL-8 pathway, and is microglial TNF-αdependent.