MIP-1α Interaction With CCR5 On HBMEC To Promote T Cells Crossing Of The Blood Brain Barrier At An Aβ Dependent Manner

The blood-brain barrier, composed of parenchymal 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). It is of a physiologic barrier that is responsible for selective flux of substances between blood and brain. The selective permeability of the BBB is crucial for the maintenance of the CNS homeostasis. Although the central nervous system has been described as immunologically privileged, it is now gradually known that activated T cells can penetrate the BBB, and migrate to CNS^[1,2] .The alterations in the barrier may be involved in many pathophysiological processes, including HIV meningitis, MS and AD etc.After disruption of BBB, immune cell from blood can entry into the parenchyma of brain to participate in the CNS inflammation^[3-5] , which can trigger the immune response.Alzheimer's disease (AD) is the most common form of age-related cognitive failure, which affect strongly the old man's life. So the investigation on the the pathogenesis of AD is important in neuroscience.More and more evidence to date have suggested that inflammation may play an important role in AD process^[6-8] . In 1994, Mc Geer and Rogers firstly provided that degenerative brain disorder in AD may be due to insuitable activation of brain immune responses and inflammation, that is, strong immune responses can attack CNS to induce cell death^[9] . In addition, Togo et al. reported that increased occurrence of T cells in the brains of patients with AD as compared with subjects with non-AD degenerative dementias and aged-matched controls^[10] .But it is unclear how circulating T cells cross the blood-brain barrier (BBB) and participate in the inflammation process in Alzheimer's disease (AD). T cells crossing the blood-brain barrier is a complicated process. The interaction between adhesion molecules and their receptors on the surface of lymphocytes and cerebral vascular endothelium plays a key role during lymphocyte transmigration. In the process of investigating the mechanism of T lymphocyte crossing brain-blood-barrier, we have previously observed over-expression of MIP-1αin circulating T cells from patients with AD, which correlated with increased number of T cells from AD patients crossing through monolayer of human brain microvasculr endothelial cells in vitro^[11] . Then the results from gene chip analysis showed that MIP-1αexpression secreted by AD patients' T lymphocytes significantly increased. However,no evidence to date suggested that MIP-1αis involved in the process of T lymphocytes transendothelial migration.The pathogenesis of AD is specialized by extracellular deposits of highly aggregated Aβ(beta-amyloid protein), NFT (neurofibrillary tangles) and neuron death. Aβaccumulation in the brain is the primary influence driving AD early pathogenesis^[12,13] .Aggregated Aβcan activate resident brain cells such as microglia, and involvement of a broad variety of inflammation-related immune response^[14,15] . Our group has established a model of Aβdeposition in parenchyma by injection of Aβ_1-42 into rat hippocampus and analyzed MIP-1αexpression in circulating T cells and its effects on T cell trafficking to CNS.Based on these results, the aim of this study is to investigate the molecular mechanism of contributing to T cells migrating from blood to brain in AD by 6T-CEM as an AD T lymphocyte model, and further analysis about the crosstalk between microglia and T cell by microglia primary culture, which is useful for AD therapy in the futher.Methods1．The role of MIP-1αin the process of 6T-CEM transendothelial migration is investigated(1) Cell cultureâ‘ HBMEC is cultured in RPMI-1640, supplemented with 10%Nu-serum etc.â‘¡6T-CEM is cultured in RPMI-1640, 10%FBS.(2) Tight junction protein ZO-1 expression displayed by immunofluorescence â‘ ZO-1 distribution displayed after 6T-CEM coculturing with HBMEC at different time.â‘¡ZO-1 distribution displayed after rhMIP-1αcoculturing with HBMEC in a time-and-dose manner.(3) The analysis on the ability of 6T-CEM transendothelial migration and the permeability of HBMEC monolayers.â‘ 6T-CEM cells and different dose rhMIP-1αare added to the upper chamber of Transwell (the BBB model in vitro) for 20h, the cells that had transmigrated into the lower chamber were harvestedand counted in a hemocytometer.â‘¡6T-CEM cells and different dose MIP-1αneutralization antiboly are added to the upper chamber of Transwell for 20h, the cells that had transmigrated into the lower chamber were harvestedand counted in a hemocytometer.Different dose rhMIP-1αare added to the upper chamber of Transwell in several groups.â‘¢TEER is detected after rhMIP-1αare added to the upper chamber of Transwell at a time-or-dose manner. The HRP flux is analyzed by adding HRP to the upper chamber of Transwell.2．The role of CCR5 on HBMEC in the process of 6T-CEM transendothelial migration is investigated(1) The western blot analysis of CCR5 and ROCK expression on HBMEC by different treatments.â‘ The CCR5 expression is detected followed by 6T-CEM cells coculturing with HBMECs at different time.â‘¡The CCR5 expression is detected followed by HBMEC are treated by rhMIP-1αat different dose and time.â‘¢The p-coffilin is detected after HBMEC or 2D7-or Y27632-pretreated-HBMEC are treated by rhMIP-1α.â‘£The CCR5 expression is detected followed by HBMECs are treated by Aβat different dose and time.(2) The construction of eukaryotic expression plasmid and the establish of HBMEC (CCR5+) cell line by stable transfection.A. The construction of pUCmT-CCR5 plasmidâ‘ Designing PCR primers in line with the principle of PCR detection and amplification of CCR5 gene from HBMEC.â‘¡T-A cloning of CCR5 into pUCmT, named as pUCmT-CCR5．③Transforming competent bacterial with pUCmT-CCR5 plasmid and conforming the cloning process by blue-white screening and Pstâ… digestionâ‘£Sequencing pUCmT-CCR5 plasmid.B. The construction of eukaryotic expression plasmidâ‘ Preparation of plasmid DNA of pUCmT-CCR5 and pcDNA3．1/Myc-HisA, directionally clone CCR5 (1．0kb) into pcDNA3．1/Myc-HisA.â‘¡Double digestion to confirm the cloning process, confirm the reading frame of CCR5 by sequencingâ‘¢Midi-preparation of plasmid DNA for transfection using Qiagen Midi Prep KitC. Stable transfectionâ‘ The expression plasmids were transfected into HBMEC cells by Fugene^TM 6 transfection reagent (Roche) according to the manufacturer's instruction. The transfected cells were selected in geneticin for 10-14 days. The resulting single colonies were multiplied as stable cell lines.â‘¡Western blot analysis of CCR5 expression on HBMEC by CCR5 antibody(3) Tight junction protein ZO-1 expression displayed by FITC-ZO-1ZO-1 distribution displayed after 6T-CEM coculturing with HBMEC pretreated by 2D7 (the blocking antibody of CCR5) .(4) The analysis on the ability of 6T-CEM transendothelial migration.â‘ 6T-CEM cells are added to the upper chamber of Transwell (HBMEC (CCR5+)) for 20h, the cells that had transmigrated into the lower chamber were harvestedand counted in a hemocytometer.â‘¡6T-CEM cells are added to the upper chamber of Transwell (pretreated by 2D7, the blocking antibody of CCR5) for 20h, the cells that had transmigrated into the lower chamber were harvestedand counted in a hemocytometer.(5) The colocalization between CCR5 expression on brain microvascular endothelial cells and rhMIP-1αdisplayed by double immunofluorescence.3．The effect on T cells migration into brain induced by Aβ_1-42 deposition is investigated(1) Preparation of a model of Aβ_1-42 deposition in rat brain and sample collection and treatment.A. Preparation of aggregated Aβ_1-42 and Aβ_42-1 , adult male Wistar rats were mounted in a stereotaxic apparatus and received bilaterally injection of 5μl aggregated Aβ_1-42 , Aβ_42-1 or PBS into hippocampus.Stereotaxic coordinates were 3．0 mm lateral, 4．0 mm posterior to the bregma, to 3．2 mm ventral.B. Sample collection and treatment.â‘ Heparinized whole blood was freshly drawn from rat heart.â‘¡Rats were perused transcardially with phosphated-buffered saline (PBS) following by 4% paraformaldehyde. The brains were removed and post-fixed in the fixitave for 2～4 h at 4⁰C.â‘¢Postfixed brains were sank in ice cold 30% sucrose in distilled water about 24 h and cut into 10μm-thick coronary sections using a cryostat.â‘£T cells Isolation from heparinized whole blood by NycoPrep^TM 1．077 A and MagCellect Rat CD3+ T Cell Isolation agent according to manufacturer' s protocol.⑤Preparation of rat T cell total RNA with Trizol agent and reverse transcription of RNA into cDNA.(2) CCR5 expression on brain microvascular endothelia cells and T cell effect in a model of Aβ_1-42 deposition in rat brain displayed by double immunofluorescence.â‘ CCR5 expression on brain microvascular endothelia cells induced by Aβ_1-42 is detected by double immunofluorescenceâ‘¡Occurrence of T cells in rat parenchyma displayed by immunofluorescence.â‘¢Blockage of T cells entry into rat parenchyma by administration of special antibody against MIP-1α.(3) MIP-1αexpression of T cells in a a model of Aβ_1-42 deposition in rat brain by half quantification RT-PCR.4．The effect of MIP-1α-and-Aβ_1-42 on microglia is analyzed(1) Cell cultureâ‘ BV-2 is cultured in DMEM, 10% FBS.â‘¡Rat primary microglia cultured in DMEM, 10% FBS.(2) Cell proliferation assay of microglia.â‘ The primary microglia cells are seeded to 96 well to confluence.â‘¡The different treatment is added to coculturing with microglia according to different group.â‘¢Operate according to the instruction of CyQuent kit.â‘£Detected by Spectrofluorophotometer(3) Nitrite measurementâ‘ The primary microglia cells are seeded to 96 well to confluent.â‘¡The different treatment is added to coculturing with microglia according to different group.â‘¢Operate according to the instruction of Nitrite measurement kit.â‘£Detected by ELIASA.(4) Aβphagocytosis assayâ‘ The Primary microglia grown on glass coverslips in 24 well.â‘¡The different treatments are added to coculturing with microglia according to different group,at the same time, FITC-Aβare added for different time.â‘¢The slips were visualized by confocal laser scanning microscope (LeicaTCS SP2, Heidelberg, Germany), fluorescence content is analyzed.Results1．The ability of 6T-CEM's transendothelial migration is increased by MIP-1α(1) The 6T-CEM cell line as an AD patients' T lymphocyte model cell was established, and disruption of tight junction is induced by HBMEC coculturing with 6T-CEM or rhMIP-1α.(2) rhMIP-1αcan promote 6T-CEM's transendothelial migration, while the ability of 6T-CEM's transendothelial migration decreased by MIP-1αneutralization antibody or MIP-1αsiRNA.2．The CCR5 on HBMEC is involved in the process of 6T-CEM's transendothelial migration(1) CCR5 on HBMEC can overexpressed induced by 6T-CEM or rhMIP-1α.(2) The eukaryotic expression construct pcDNA3．1/Myc-HisA-CCR5 and HBMEC single-clone cell lines stably expressing CCR5 gene are obtained.(3) The ability of 6T-CEM transendothelial migration is enhanced by HBMEC single-clone cell lines stably expressing CCR5 gene as a monolayer. While, the ability of 6T-CEM transendothelial migration is down-regulated by the 2D7 antibody, a specificmonoclonal antibody and antagonist of CCR5, which also maitained tight junction integrity.(4) The MIP-1αcan colocalized with CCR5．(5) The interaction between MlP-1αand CCR5 can promote transendothelial migration of 6T-CEM and disrupts tight junction distribution by ROCK activation.3．Aβdeposition in rat brain can induce T cells' transendothelial migration MIP-1α-dependently(1) CCR5 expression on HBMEC can be up-regulated by Aβ_1-42 .(2) Aβdeposition model is obtained.(3) In the Aβdeposition model, MIP-1αsecreted by T cells and CCR5 on rat brain microvascular cells overexpressed by Aβ_1-42 , and the intrahippocampus injection of Aβ_1-42 induced T cell crossing of BBB at a MIP-1α-dependent manner.4．MIP-1αcan't contribute to the proliferation and Aβphagocytosis of microglia induced by Aβ(1) Obtain primary rat microglia cells and successful culturing of BV2．(2) MIP-1αcan't promote microglia proliferation, microglia-secreted NO level and Aβphagocytosis progress of microglia induced by Aβ.Conclusions1．The interaction between with MIP-1αsecreted by T cells and CCR5 on HBMEC induced the disruption of tight junction by ROCK activation, which promoted T cells crossing of HBMEC monolayer in vitro.2．Aβdeposits in rat parenchyma specifically induced over-expression of MIP-1αin circulating T cells and up-regulated CCR5 expression on brain microvascular endothelia cells, which lead to the MIP-1α-dependent migration into brain of peripheral T cells.3．MIP-1αsecreted by T cells can't promote microglia proliferation induce by Aβ, it might be involved in immune response in brain by interacting with some factor to remove Aβdeposit.