Transmigration Of Peripheral Blood Mononuclear Cells Research Articles

An In Vitro Model of the Blood-Brain Barrier to Study Alzheimer's Disease: The Role of β-Amyloid and Its Influence on PBMC Infiltration.

The blood-brain barrier (BBB) is a highly specialized structure, constituted by endothelial cells that together with astrocytes and pericytes provide a functional interface between the central nervous system and the periphery. Several pathological conditions may affect its functions, and lately BBB involvement in the pathogenesis of Alzheimer's disease has been demonstrated. Both endothelial cells and astrocytes can be differentially affected during the course of the disease. In vitro BBB models present a powerful tool in evaluating the effects that β-amyloid (Aβ), or other pathogenic stimuli, play on the BBB at cellular level. In vitro BBB models derived from human cell sources are rare and not easily implemented. We generated two conditionally immortalized human cell lines, brain microvascular endothelial cells (TY10), and astrocytes (hAST), that, when co-cultured under appropriate conditions, exhibit BBB-like characteristics. This model allowed us to evaluate the transmigration of peripheral blood mononuclear cells (PBMCs) through the in vitro barrier exposed to Aβ and the role played by astrocytes in the modulation of this phenomenon. We describe here the methodology used in our lab to set up our in vitro model of the BBB and to carry out a PBMC transmigration assay.

Caspase-1 has a critical role in blood-brain barrier injury and its inhibition contributes to multifaceted repair

BackgroundExcessive inflammation might activate and injure the blood-brain barrier (BBB), a common feature of many central nervous system (CNS) disorders. We previously developed an in vitro BBB injury model in which the organophosphate paraoxon (PX) affects the BBB endothelium by attenuating junctional protein expression leading to weakened barrier integrity. The objective of this study was to investigate the inflammatory cellular response at the BBB to elucidate critical pathways that might lead to effective treatment in CNS pathologies in which the BBB is compromised. We hypothesized that caspase-1, a core component of the inflammasome complex, might have important role in BBB function since accumulating evidence indicates its involvement in brain inflammation and pathophysiology.MethodsAn in vitro human BBB model was employed to investigate BBB functions related to inflammation, primarily adhesion and transmigration of peripheral blood mononuclear cells (PBMCs). Caspase-1 pathway was studied by measurements of its activation state and its role in PBMCs adhesion, transmigration, and BBB permeability were investigated using the specific caspase-1 inhibitor, VX-765. Expression level of adhesion and junctional molecules and the secretion of pro-inflammatory cytokines were measured in vitro and in vivo at the BBB endothelium after exposure to PX. The potential repair effect of blocking caspase-1 and downstream molecules was evaluated by immunocytochemistry, ELISA, and Nanostring technology.ResultsPX affected the BBB in vitro by elevating the expression of the adhesion molecules E-selectin and ICAM-1 leading to increased adhesion of PBMCs to endothelial monolayer, followed by elevated transendothelial-migration which was ICAM-1 and LFA-1 dependent. Blocking caspase-8 and 9 rescued the viability of the endothelial cells but not the elevated transmigration of PBMCs. Inhibition of caspase-1, on the other hand, robustly restored all of barrier insults tested including PBMCs adhesion and transmigration, permeability, and VE-cadherin protein levels. The in vitro inflammatory response induced by PX and the role of caspase-1 in BBB injury were corroborated in vivo in isolated blood vessels from hippocampi of mice exposed to PX and treated with VX-765.ConclusionsThese results shed light on the important role of caspase-1 in BBB insult in general and specifically in the inflamed endothelium, and suggest therapeutic potential for various CNS disorders, by targeting caspase-1 in the injured BBB.

Conserved structure and function of chemokine CXCL8 between Chinese tree shrews and humans

Chemokines represent a superfamily of small secretion proteins that functionally mediate immune cell transmigration in normal or inflammatory conditions. Although anatomic and polygenetic evidence suggests that tree shrews are primate-like species, understanding of the structure and function of tree shrew chemokines has only just commenced. In this study, we cloned tree shrew chemokine CXCL8 and its cognate receptors. Predicted three-dimensional (3D) structures showed that binding domains in CXCL8 and CXCR1/2 were highly conserved between tree shrews and humans. We found that the human CXCL8 (hCXCL8) protein induced migration of tree shrew peripheral blood mononuclear cells (PBMCs) expressed by CXCR1/2 (tsCXCR1/2). Blocking interaction between hCXCL8 and tsCXCR1/2 with allosteric antagonists (reparixin and SB265610) significantly decreased tree shrew PBMC transmigration. Over-expressing tree shrew CXCR1 in human HEK 293 T cells further enhanced cellular in vitro transmigration. Similar to primate species, our findings suggest that CXCL8 and CXCR1/2 constitute a structurally- and functionally-conserved chemotaxis responsible for tree shrew immune activities.

Longitudinal chemokine profile expression in a blood-brain barrier model from Alzheimer transgenic versus wild-type mice

BackgroundAlzheimer’s disease is widely described since the discovery of histopathological lesions in Mrs. Auguste Deter in 1906. However to date, there is no effective treatment to deal with the many cellular and molecular alterations. The complexity is even higher with the growing evidence of involvement of the peripheral blood mononuclear cells (PBMCs). Indeed, monocytes and T cells are shown in the cerebral parenchyma of AD patients, and these cells grafted to the periphery are able to go through the blood-brain barrier (BBB) in transgenic mouse models. It is known that BBB is disrupted at a late stage of AD. Chemokines represent major regulators of the transmigration of PBMCs, but many data were obtained on AD animal models. No data are available on the role of AD BBB in a healthy brain parenchyma. Therefore, the purpose of this study was to analyze the longitudinal chemokine profile expression in a BBB model from AD transgenic mice versus wild-type (WT) mice.MethodsA primary mouse BBB model was used with a luminal compartment either AD or WT and an abluminal compartment WT consisting of astrocytes and microglia. PBMCs were extracted by a ficoll gradient and incubated in the transwell with a direct contact with the luminal side, including the endothelial cells and pericytes. Then, the complete BBB model was incubated during 48 h, before supernatants and cell lysates were collected. Chemokines were quantified by X-MAP® luminex technology.ResultsAbluminal CX3CL1 production increased in 12-month-old AD BBB while CX3CL1 levels decreased in luminal lysates. CCL3 in luminal compartment increased with aging and was significantly different compared to AD BBB at 12 months. In addition, abluminal CCL2 in 12-month-old AD BBB greatly decreased compared to levels in WT BBB. On the contrary, no modification was observed for CCL4, CCL5, and CXCL10.ConclusionThese first findings highlighted the impact of AD luminal compartment on chemokine signature in a healthy brain parenchyma, suggesting new therapeutic or diagnostic approaches.

Mo1710 Regulation of Smad7 Translation by microRNA-195 Affects Steroid Resistance of Ulcerative Colitis

G A A b st ra ct s during 24h with agonists and antagonists of the Hh pathway, inflammatory cytokines and bacterial products. Supernatants were analyzed for cytokine production by ELISA, and caspase-3 activity was assayed in epithelial cells isolated from mucosal samples. Chemoattraction of peripheral blood mononuclear cells (PBMC) was analyzed in a transwell system. Primary human fibroblasts were used for analyzing migration and BrDU incorporation. Results: Hh-proteins were basically expressed at the superficial epithelium, and a marked reduction was observed in CD compared to UC (p=0.04), and controls (p=0.001). In the lamina propria, Gli-1 predominantly co-localizes with non-immune cells, such as alphasmooth muscle actin-, vimentin-, and tubulin-positive cells, with lower levels in CD. Levels of Indian and Sonic Hh mRNA, Ptch-1, and the downstream transcription factors Gli-1 and Gli-2 were remarkably lower in CD (p<0.04). In CD colon explants, treatment with Shh peptide resulted in a significant decrease in the levels of TNF-alpha (p=0.04), IL-17 (p= 0.01), and TGF-beta (p=0.03). Caspase-3 activity was higher in CD epithelial cells and significantly decreased after blockade of Hh with anti-Shh antibody, cyclopamine, and Gant61 (p<0.04), in contrast to UC and controls. Levels of Gli-1 mRNA negatively correlated to caspase-3 activity (CC -0.75, p=0.01). PBMC transmigration significantly increased after treatment with Gant61 (p<0.04). Primary fibroblasts incorporated significantly more BrdU (p<0.04), and migrated less (p<0.03) after treatment with Gant61. Conclusion: Loss of Hhsignaling is associated with disruption of the epithelial barrier and pro-inflammatory cytokine production in CD intestinal mucosa. In vitro studies indicate that blocking the Hh pathway induces mononuclear cell chemo-attraction and fibroblast proliferation, but reduces fibroblast migration. Taken together, these data support the notion of a reduced intestinal Hh-signaling underlying the pathogenesis of CD.

Increase in multiple sclerosis activity after assisted reproduction technology

Our objective was to evaluate risk of exacerbations in multiple sclerosis (MS) patients undergoing assisted reproduction technology (ART) infertility treatment. Sixteen patients with relapsing-remitting MS subjected to 26 ART treatment cycles receiving gonadotropin-releasing hormone (GnRH) agonists and recombinant follicle-stimulating hormone were studied prospectively. The baseline study period encompassed 12 months prior to the first cycle and 9 months after final ART cycle. Neurological examinations, brain magnetic resonance imaging (MRI), and immunology testing were conducted every 3 months. Anti-myelin-oligodendrocyte glycoprotein (MOG) antibody production, interleukin (IL)-4, IL-8, IL-10, IL-12, IL-17, interferon (IFN)-γ, and transforming growth factor (TGF)-β secretion by myelin basic protein- and MOG-peptide-specific T cells, as well as ex vivo isolated peripheral blood mononuclear cells (PBMCs), were studied using enzyme-linked immunospot. vascular endothelial growth factor (VEGF) production by PBMCs was assessed using enzyme-linked immunosorbent assay. ART was associated with a 7-fold increase in risk of MS exacerbation, and a 9-fold increase in risk of enhanced disease activity on MRI. Worsening was associated with higher number of cells producing IL-8, IL-12, IFN-γ, and TGF-β, as well as increased VEGF production by CD4(+) T cells and CXCL-12 plasma levels, all GnRH-mediated effects. A rise in 17-β estradiol production associated with ART increased anti-MOG antibody titers, as well as B-cell survival factor BAFF (B-cell activating factor) and antiapoptotic molecule Bcl-2 levels from purified CD19(+) B cells. Finally, ART facilitated PBMC transmigration across an in vitro blood-brain barrier model, an effect mediated by IL-8, VEGF, and CXCL-12. Results indicate a significant increase in MS disease activity in patients receiving ART, a risk that neurologists should be aware of. Reproductive hormones appear to exert an important role in regulating immune responses during the course of autoimmune diseases.

Ephrin-B2–Activated Peripheral Blood Mononuclear Cells From Diabetic Patients Restore Diabetes-Induced Impairment of Postischemic Neovascularization

We hypothesized that in vitro treatment of peripheral blood mononuclear cells (PB-MNCs) from diabetic patients with ephrin-B2/Fc (EFNB2) improves their proangiogenic therapeutic potential in diabetic ischemic experimental models. Diabetes was induced in nude athymic mice by streptozotocin injections. At 9 weeks after hyperglycemia, 105 PB-MNCs from diabetic patients, pretreated by EFNB2, were intravenously injected in diabetic mice with hindlimb ischemia. Two weeks later, the postischemic neovascularization was evaluated. The mechanisms involved were investigated by flow cytometry analysis and in vitro cell biological assays. Paw skin blood flow, angiographic score, and capillary density were significantly increased in ischemic leg of diabetic mice receiving EFNB2-activated diabetic PB-MNCs versus those receiving nontreated diabetic PB-MNCs. EFNB2 bound to PB-MNCs and increased the adhesion and transmigration of PB-MNCs. Finally, EFNB2-activated PB-MNCs raised the number of circulating vascular progenitor cells in diabetic nude mice and increased the ability of endogenous bone marrow MNCs to differentiate into cells with endothelial phenotype and enhanced their proangiogenic potential. Therefore, EFNB2 treatment of PB-MNCs abrogates the diabetes-induced stem/progenitor cell dysfunction and opens a new avenue for the clinical development of an innovative and accessible strategy in diabetic patients with critical ischemic diseases.

Adhesion molecules are promising candidates to establish surrogate markers for natalizumab treatment

Background: Natalizumab is the first monoclonal antibody therapy approved for multiple sclerosis (MS). Its therapeutic mechanism is the blockade of the α4-integrin subunit of the adhesion molecule (AM) very late activation antigen-4 (VLA-4), which leads to an inhibition of immune cell extravasation into the central nervous system (CNS). Methods: We investigated changes in the expression levels of unblocked α4-integrin and further AM (intercellular adhesion molecule-1, -2, -3 (cICAM-1, -2, -3), leukocyte function associated antigen-1 (LFA-1)) on peripheral blood mononuclear cells (PBMC) determined by flow cytometry from 25 patients with MS before the first natalizumab infusion and before the fourth infusion. In 15 MS patients AM expression was evaluated every 3 months over 1 year. Results:We found a significant decrease (p < 0.0001) of unblocked α4-integrin cell surface expression on all investigated PBMC subsets (T cells −61.7%, B cells −69.1%, monocytes/macrophages −46.4%) in the blood of MS patients after 3 months of natalizumab treatment. Moreover, a continuous decrease (p < 0.05) of unblocked α4-integrin expression levels was seen after 3, 6, 9, and 12 months. As a secondary effect, expression levels of the other investigated AM were differentially affected. Conclusions:Results show a sustained decrease of unblocked α4-integrin expression not only in all patients but also in all investigated PBMC subsets. This probably results in a continuously decreasing transmigration of PBMC into the CNS and may explain the improved clinical efficacy in the second treatment year and also the increasing risk of progressive multifocal leukoencephalopathy during long-term natalizumab therapy. We conclude that AM expression profiles are promising candidates for the development of a biomarker system to determine both natalizumab treatment response and patients at risk for opportunistic CNS infections.

Endothelial cell suppression of peripheral blood mononuclear cell trafficking in vitro during acute exposure to feline immunodeficiency virus

Trafficking of peripheral blood mononuclear cells (PBMCs) into the brain is a critical step in the initiation of human immunodeficiency virus (HIV)-associated central nervous system disease. To examine potential factors that control trafficking during the earliest stages of infection, PBMC transmigration across a cultured feline brain endothelial cell (BECs) monolayer was measured after selective exposure of various cell types to feline immunodeficiency virus (FIV). Infection of the PBMCs with FIV increased the trafficking of monocytes and CD4 and CD8 T cells. Additional exposure of the BECs to FIV suppressed mean monocyte, CD4 T cell, and CD8 T cell trafficking. B cell trafficking was unaltered by these changing conditions. Subsequent exposure of astrocytes or microglia to FIV altered transmigration of different PBMC subsets in different ways. Treated microglia compared with treated astrocytes decreased monocyte transmigration, whereas B cell transmigration was increased significantly. When both astrocytes and microglia were exposed to FIV, an increase in CD8 T cell transmigration relative to BECs alone, to BECs plus astrocytes, or to BECs plus microglia was demonstrated. Thus, initial exposure of PBMCs to FIV is sufficient to induce a general increase in trafficking, whereas initial exposure of endothelial cells to FIV tends to down-regulate this effect. Selectivity of trafficking of specific PBMC subsets is apparent only after exposure of cells of the central nervous system to FIV in co-culture with the endothelium.

The alveolar epithelial type I-like cell line as an adequate model for leukocyte migration studies in vitro

The lung is unique as leukocytes not only migrate into the bronchoalveolar space but also return to the parenchyma and then via the lymphatics to the draining lymph node. The aim of this study was to investigate the migration of leukocytes via an epithelial monolayer in a Transwell system against a chemokine gradient. Rat type I-like R3/1 alveolar epithelial cells were cultivated on a Transwell polyester membrane (pore diameter 3 μm) for 3 days until a monolayer was formed. The tightness of the monolayer was determined by transepithelial transport of horseradish peroxidase. Isolated human and rat peripheral blood mononuclear cells (PBMC) were placed in the upper chamber, and different concentrations of monocyte chemotactic protein-1 (MCP-1) in the lower chamber. The transmigration of PBMC was quantified and investigated by light and transmission electron microscopy. PBMC migrated through the epithelial cell barrier intercellularly as well as transcellularly. The migration of PBMC against the MCP-1 gradient was dose dependent. The results indicate that this model could help in the study of key events involved in chemokine-induced cell migration from the airways into tissue.

Increased synovial endothelium binding and transendothelial migration of mononuclear cells during Salmonella infection.

To investigate the adhesion and extravasation capacity of peripheral blood mononuclear cells (PBMC) and the transport of bacterial antigens within these cells during Salmonella infection. Thirteen patients who were part of 2 outbreaks of Salmonella enteritidis infection were included in this study. The capacity of PBMC from these patients to bind to vascular endothelium in inflamed synovium was tested using a Stamper-Woodruff-type frozen-section assay. The same cells were studied for the presence of Salmonella antigens by immunofluorescence staining. The transendothelial migration of mononuclear cells containing Salmonella or its components through unstimulated endothelial cell layer was quantified. The capacity of PBMC to adhere to synovial vessels was significantly increased during Salmonella infection (P=0.0003). Monocytes had a transiently high adhesive state between 2 and 5 weeks after the patients had eaten the contaminated food. The cells containing Salmonella antigens were concentrated in the transmigrated population. During acute Salmonella infection the increased binding of PBMC to vascular endothelium in inflamed synovium and enhanced transmigration of PBMC containing Salmonella may be the key factors leading to transport of bacterial antigens through the endothelial barrier and initiation of arthritis in susceptible individuals.