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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neuroimmunol. Author manuscript; available in PMC May 29, 2010.
Published in final edited form as:
PMCID: PMC2696803
NIHMSID: NIHMS102845

α4 Integrin/FN-CS1 Mediated Leukocyte Adhesion to Brain Microvascular Endothelial Cells Under Flow Conditions

Abstract

Insights into sequential leukocyte-endothelial interactions during leukocyte trafficking have been obtained through experiments using human umbilical vein endothelial cells (HUVEC) under flow conditions. To investigate leukocyte-brain endothelial cell interactions, we developed a dynamic in vitro system, using Transfected Human Brain Microvascular Endothelial Cells (THBMEC) and a parallel plate flow chamber. Human peripheral blood mononuclear cells (PBMC) were perfused across confluent THBMEC cultures at a velocity that approximates the rate found in human brain capillaries. Leukocyte-THBMEC interactions were visualized by phase-contrast microscopy, and images were captured on a CCD camera. To simulate inflammatory conditions, we activated THBMEC with the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), which up-regulated chemokine and adhesion molecule expression in THBMEC without affecting the distribution of immunoreactivity for tight junction-associated proteins. PBMC adhesion was enhanced by cytokine-mediated activation of THBMEC. G protein-coupled receptor (GPCR) activation was essential for leukocyte-THBMEC interaction, as pertussis toxin (PTX) treatment of PBMC abrogated PBMC adhesion to activated THBMEC. The anti-α4 integrin antibody, natalizumab, infused into MS patients, significantly reduced the adhesion of their ex-vivo PBMC to activated THBMEC under flow conditions. Further study showed that alternatively spliced fibronectin containing the CS1 region (FN-CS1), but not Vascular Cell Adhesion Molecule type 1 (VCAM-1), was the ligand of α4 integrin on activated THBMEC. Blocking FN-CS1 abrogated PBMC adhesion on activated THBMEC, while anti-VCAM-1 antibodies had no effect. These results established a novel in vitro dynamic BBB model. We also demonstrated the dependence of leukocyte-endothelial interactions in this model on α4 integrins and FN-CS1.

Keywords: chemokines, α4 integrin, leukocyte, blood brain barrier, adhesion, shear stress, Multiple Sclerosis

1. Introduction

Elucidating mechanisms of leukocyte migration across the blood brain barrier (BBB) may be useful for understanding the pathogenesis of disorders including multiple sclerosis (MS), brain and spinal cord trauma, stroke, and HIV infection (Chaudhuri et al., 2008; Danton and Dietrich, 2003; Lossinsky and Shivers, 2004; McCandless et al., 2008; Wang et al., 2008). Components that subserve leukocyte-endothelial cell interactions define the molecular pathways that regulate leukocyte trafficking.

Insights into sequential leukocyte-endothelial interactions have been obtained through experiments using human umbilical vein endothelial cells (HUVEC) under flow conditions (Schreiber et al., 2007; Burns et al., 2000). The step-wise interactions between leukocytes and endothelial cells are governed by adhesion molecules, chemokines, and chemokine receptors. Chemokine engagement of cognate G protein-coupled receptor (GPCRs) activates leukocyte integrins through inside-out signaling, resulting in the leukocyte adhesion to endothelial cells (Doring et al., 2007; Hartmann et al., 2008; Luster et al., 2005; Man et al., 2008; Schreiber et al., 2007; Shulman et al., 2006; Woolf et al., 2007). Unexpectedly, flow conditions promote, rather than antagonize, leukocyte-endothelial interactions (Ransohoff et al., 2007). As one example, T cells encountering apically-presented CXCL12 underwent robust LFA-1-dependent transmigration across HUVEC toward abluminal CCL5 under shear stress, in contrast to negligible transmigration under static conditions (Schreiber et al., 2007).

Leukocytes entering the CNS parenchyma must cross the BBB (Bechmann et al., 2007; Engelhardt and Ransohoff, 2005). The barrier function of the BBB relies on highly specialized brain microvascular endothelial cells, which lack pinocytotic vesicles and fenestrae but possess intercellular tight junctions. These features limit transcellular and paracellular movement of cells and molecules (Man et al., 2007). Mechanisms of leukocyte migration across the BBB are incompletely understood. Transwell cultures of brain endothelial cells have been used to study leukocyte trafficking into the brain (Callahan et al., 2004; Kraus et al., 2004; Luster et al., 2005; Mahad et al., 2006; Man et al., 2008; Ubogu et al., 2006). However, the lack of shear stress limits the interpretation of the results of such studies. The current report describes our progress in developing an in vitro BBB model using human brain microvascular cells and a parallel plate flow chamber to study leukocyte-brain endothelial cell interactions under flow conditions. The current paper focuses in particular on leukocyte adhesion to brain microvascular endothelial cells under flow. Analyses of the earlier stages of leukocyte/endothelial interaction, tethering and rolling, are ongoing.

Anti-α4 integrin antibodies (natalizumab; NTZ) is an FDA approved treatment for MS. Although it was designed to block α4 integrin-mediated leukocyte-endothelial interactions, this effect has not been directly evaluated in MS patients receiving NTZ. Using a static BBB model, we found that NTZ infusion to MS patients inhibited leukocyte migration across BBB, and that this blockade was reversed by removing NTZ with plasmapheresis (Khatri et al., 2008). We extended these results in the present study by examining NTZ effects under flow conditions.

2. Materials and Methods

2.1. Patients

Eligible patients were 18 to 50 years of age, and were diagnosed with relapsing remitting MS (RRMS). All the patients were free of relapse within the last month. MS patients (n=9) were recruited from the Mellen Center for Multiple Sclerosis Treatment and Research at the Cleveland Clinic. Before assay, patients had received at least three courses of NTZ, following the recommendations outlined in the US Food and Drug Administration-mandated Tysabri Outreach Unified Commitment to Health (TOUCH) program, and tested negative for anti-natalizumab antibodies. Four additional MS patients had been treated with interferon β-1a 30 mcg IM weekly for at least 12 months. All patients gave written informed consent, and all study protocols were approved by the Cleveland Clinic Institutional Review Board. Patients and healthy controls had not received corticosteroid within the last three months. Clinical data were collected by an investigator blinded to the leukocyte adhesion assay, with the oversight of an independent monitor (Table 1).

Table 1
Information of patients and healthy control

2.2. Cell culture and PBMC isolation

THBMEC are adult human brain microvascular endothelial cells transfected and immortalized with a plasmid containing simian virus 40 large T antigen (SV40-LT) as previously described (Mahad et al., 2006; Man et al., 2008; Stins et al., 1997). THBMEC were grown in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 10% Nu-Serum, 2 mM glutamine, 1 mM pyruvate, essential amino acids, and vitamins. PBMCs were isolated from fresh whole heparinized blood from healthy volunteers or MS patients by density centrifugation, using Lymphocyte Separation Medium (Mediatech, Herndon, VA) as previously described (Mahad et al., 2006; Man et al., 2008; Stins et al., 1997). PBMCs were resuspended at 5×105 cells/ml in transendothelial migration (TEM) buffer (RPMI 1640 without phenol red + 1% bovine serum albumin). The research protocol was approved by the local institutional review board, and signed informed consent was obtained from all donors studied.

2.3. THBMEC activation (aTHBMEC) and PBMC adhesion assay

THBMEC were cultured on a 35mm collagen-coated Corning tissue culture dish to subconfluence, followed by activation with TNF-α 10 u/ml and IFN-γ 20 u/ml (R&D systems, Minneapolis, MN) or control media for 24 hours. THBMEC cultures were washed with TEM buffer twice, and immediately assembled with a parallel plate flow chamber and mounted on the stage of a Leica DMI3000 B inverted phase contrast microscope (Leica Microsystems Inc. Bannockburn, IL). This allowed for the study of leukocyte-endothelial interactions from a top-down view under a flow rate of 9ml/hr, calculated according to the size of the chamber and the velocity in human brain capillaries, which is 1mm/sec (Hudetz et al., 1996). This velocity was generated by a Medfusion 3010a syringe pump (Smiths Medical, St. Paul, Minnesota). Since the TEM buffer we used has a lower viscosity than the blood, the shear stress we used, which is 0.25dyn/cm2, should be lower than the shear stress in brain capillaries. In pilot studies, higher flow rates decreased adherent cell numbers. The experiments were performed at 37ºC. Cell interactions on a field of 0.15 mm2 were visualized with a 20× objective. Cell images were captured 2 frames per second with a QICAM fast 1394 CCD camera (QImaging, Pleasanton, CA) during 20 minutes of shear application. Cell images were manually quantified by analysis of the video.

For Go/i inhibition assay, PBMC were treated with pre-activated pertussis toxin (PTX) at 100 ng/ml for 10 min, followed by perfusion across aTHBMEC.

For experiments using antibodies to block endothelial adhesion molecules, aTHBMEC were incubated with mouse anti-human FN CS-1 IgM (clone P1F11, Santa Cruz Biotechnology. Santa Cruz, CA), mouse IgM Isotype (clone MOPC 104E, Sigma-Aldrich Inc. St. Louis, MO), neutralizing rat anti-mouse/human VCAM-1 IgG1 (clone M/K-2, Santa Cruz Biotechnology. Santa Cruz, CA) antibody, mouse anti-human VCAM-1 IgG1 (clone 4B2, R&D. Minneapolis, MN) antibody that have been previously proved as functionally neutralizing antibody for human VCAM-1 (Needham et al., 1994; Spertini et al., 1994), or mouse IgG1 isotype (clone 11711.11, R&D. Minneapolis, MN), at 0.05μg/ml for 5min, followed immediately by washing twice with TEM buffer and analysis in the parallel plate flow chamber.

For experiments in which leukocyte adhesion molecules were blocked, PBMC were incubated with recombinant humanized mouse anti-human α4 integrin IgG4k (natalizumab, a gift from Susan Goelz, Biogen Idec. Cambridge, MA), or human IgG4k isotype control (Sigma-Aldrich Inc. St. Louis, MO), at 0.05μg/ml for 10min. Alternatively, PBMC were incubated with FN CS-1 peptide CYLHGPEILDVPST or control peptide (FN-CS1c) DELPQLVTLPHP (gifts from Dr. Mark Newman, Pharmexa-Epimmune Inc. San Diego, CA) at 1.875 μM for 10 min, followed by perfusion across aTHBMEC without washing.

2.4. Image analysis

Images captured during 20 min were made into a movie with QCapture software. The motions of each cell that flowed into the field and became adhesive during the shear stress application period was tracked and quantified. Adhesion includes the following three types of motion: Pure arrest was defined as cells that remained stationary throughout the period; Locomotion was defined as cells that spread and migrated over the endothelial surface without detaching or crossing the endothelial cell barriers, remaining phase bright; Transmigration was defined as cells that underwent phase change from bright to dark, indicating that they transmigrated through the endothelial cell barriers after spreading, migrating for variable distances on the endothelial surface.

2.5. Immunocytochemistry of tight junction associated protein

THBMEC were cultured on collagen-coated glass slides, followed by cytokine stimulation for 24 hours or PBS control treatment. THBMEC slides were assembled on the parallel plate flow chamber, and TEM was perfused across THBMEC at 9ml/hr for 20 min. As a no shear-stress control, THBMEC slides were assembled on the parallel plate flow chamber for 20 min without turning on the flow. THBMEC were fixed by 4% PFA, and ZO-1, JAM-A, and VE-cadherin were detected by indirect immunocytochemistry. Rabbit anti-ZO-1 (Zymed Laboratories. San Francisco, CA), mouse anti-JAM-A antibodies (CloneJ3F.1, Santa Cruz Biotechnology. Santa Cruz, CA), and mouse anti-VE-cadherin (Clone F-8, Santa Cruz Biotechnology. Santa Cruz, CA) were used in combination with Alexa Fluor 594 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG (H+L), and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen Corporation, Carlsbad, CA). Slides were viewed using a Leica Aristoplan laser scanning confocal microscope (Leica Wetzlar, Heidelberg, Germany).

2.6. RT-PCR, subcloning and sequencing

Total RNA was obtained from THBMEC using Trizol (Invitrogen Corporation, Carlsbad, CA). Reverse transcription (RT) and polymerase chain reaction (PCR) were performed according to the manufacturer’s instructions, using Roche first strand cDNA synthesis kit (Roche Diagnostics GmbH. Mannheim, Germany). PCR was performed using Roche Taq DNA polymerase. RNA from HUVEC was used as a control. The primers set for the various conditions were: GAPDH: Forward 5′-GGTGGAGGTCGGA GTCAACG-3′ and Reverse 5′-CAAAGTTGTCATGGATGACC-3′; FH-CS1: Forward 5′-TTCCCCAACTGGT AACCCTT-3′ and Reverse 5′-TTTAAAGCCTGATTCAGAC AT TCG-3′; Fibronectin: Forward 5′-GTGCCACTTCCCCTTCCTAT-3′ and Reverse 5′-ATCCCACTGATC TCCAATGC-3′. VCAM-1 Forward 5′-ATGACATGCTTGAGC CAGG-3′ and reverse 5′-GTGTCTCCTTCTTTGACACT-3′. PCR product of FN-CS1 was subcloned with pCR II TOPO-TA expression vector (Invitrogen Corporation, Carlsbad, CA), and the resulting clone was proven by sequencing to have an insert that was 100% identical to the target gene.

2.7. Chemokine antibody arrays

We measured a total of 36 chemokines produced by aTHBMEC, compared with resting THBMEC, using chemokine antibody arrays according to the manufacturer’s instructions (Raybiotech Inc. Norcross GA). Briefly, THBMEC were cultured and activated with TNF-α and IFN-γ for 24 hours as outlined above. After supernatant was harvested, THBMEC were washed, and protein was abstracted using cell lysis buffer provided by the kit. Total protein from cell lysate and supernatant was carefully quantified using Bio-Rad protein assay dye reagent (Life Science Research, Hercules, CA), and diluted into a working concentration of 1 mg/ml. Nitrocellulose membranes, each containing 36 different antibodies in duplicate spots, were blocked for 1 hour, and incubated with 1ml of each sample at 4°C overnight. Membranes were washed, and incubated with a cocktail of biotin-conjugated antibodies specific for the different proteins. Membranes were developed with streptavidin-conjugated peroxidase and ECL chemiluminescense reagent, and then exposed to Kodak® BioMax™ light film (Sigma-Aldrich Inc., St. Louis, MO). Samples were processed simultaneously to decrease observational error. Experiments were repeated on two different passages of THBMEC. Autoradiographic films were scanned and digitized spots were quantified with Image J software (BioDiscovery Inc.). Local background intensities were subtracted from each spot, and the average of the duplicate spots for each protein was normalized to the average of six positive controls on each membrane.

2.8. Statistical analysis

Each adherent PBMC in a 0.15 mm2 field was tracked for 20 min during shear application manually by an investigator blinded to the experiment. Student’s t-test analyses were performed on total adherent cell number to ascertain statistical significance, after the data were confirmed to be normally distributed using Kolmogorov-Smirnov test. A p-value <0.05 was used to establish significance.

3. Results

3.1. Development of an activated dynamic BBB model to study leukocyte-brain endothelial cell interaction under flow conditions

Intact morphology and tight junction integrity under physiological flow are essential for dynamic BBB models, where the intention is to study leukocyte transmigration. ZO-1 is critical for tight junction function, and is linked to the actin cytoskeleton (Luscinskas et al., 2002). JAM co-distributes with tight junction components at the apical region of the intercellular cleft and plays a role in regulating monocyte transmigration (Martin-Padura et al., 1998). Vascular Endothelium cadherin (VE-cadherin) is an important determinant of microvascular integrity and, together with catenins, forms the complex that mediates early-recognition between ECs. (Navarro, et al., 1998; Vorbrodt and Dobrogowska, 2004). THBMEC maintained cobblestone morphology and immunoreactivity for tight junction-associated proteins ZO-1, JAM-A and VE-cadherin after shear stress with or without cytokine exposure (Fig 1a). In the conditions evaluated, the continuous immunoreactivity of ZO-1, JAM-A and VE-cadherin along the intercellular junctions suggested that tight junctions were intact during shear stress application (Fig 1a). Cytokine exposure did exert biological effects. Among 36 chemokines we measured by chemokine antibody array, chemokines of the GRO family were up-regulated. Using a common antibody to GRO family members CXCL1, CXCL2, and CXCL3, modestly higher levels of GRO peptide were detected both in cell culture media and in cell lysates of aTHBMEC (Fig 1b).

Fig 1
Characterization of the inflammatory dynamic BBB model. A: Neither shear stress nor cytokines cause observable alterations of immunoreactivity for tight junction-associated protein ZO-1, JAM-A, and VE-cadherin. THBMEC culture were activated with TNF-α ...

3.2. Cytokine activation of THBMEC-enhanced PBMC adhesion

In order to explore the impact of THBMEC inflammation on leukocyte adhesion, we compared PBMC adhesion on activated versus resting THBMEC under flow conditions. In the in vitro dynamic BBB system, cytokine mediated activation of THBMEC enhanced PBMC adhesion (Fig. 2a, supplementary movie1 and 2).

Fig. 2
Cytokine activation of THBMEC enhanced PBMC adhesion and GPCR receptor activation is essential for PBMC adhesion to inflammatory THBMEC. A: THBMEC were cultured on 35mm collagen-coated tissue culture dishes to subconfluence, followed by incubation with ...

3.3. Go/i receptor signal is essential for PBMC adhesion to inflammatory THBMEC

Leukocyte adhesion to endothelium under flow conditions is contingent on signaling via Gαi-linked G protein coupled receptors (GPCRs). Pertussis toxin (PTX) almost completely blocked PBMC adhesion (Fig 2b). This observation suggests that GPCR signaling is involved in PBMC adhesion to brain endothelial cells in the present system.

3.4. NTZ infusion inhibited leukocyte adhesion on activated THBMEC under flow conditions

Natalizumab was designed to inhibit α4 integrin-dependent leukocyte-endothelial interaction. IFN β-1a is also used for MS treatment and acts through as yet unknown mechanisms. We evaluated the effect of NTZ or IFN β-1a, using ex vivo PBMC from treated patients. PBMC adhesion to aTHBMEC was reduced by 85 percent in subjects receiving NTZ, compared to MS patients treated with IFN-β 1a (Fig 3a). For control experiments, we used PBMC from healthy subjects, exposed in vitro to NTZ or isotype control immunoglobulins (IgG4k). In vitro exposure of NTZ blocked PBMC adhesion on aTHBMEC, while nonspecific human IgG4k did not exert a significant effect (Fig 3b).

Fig. 3
Natalizumab inhibited PBMC adhesion to inflammatory THBMEC. A: Natalizumab infusion to MS patients inhibited their leukocyte adhesion on inflammatory THBMEC. Nine MS patients were treated with natalizumab for at least three courses (300mg IV). Four MS ...

3.5. FN CS1 is the ligand on brain endothelial cells for alpha 4 integrin-mediated leukocyte adhesion

The relevant α4β1 integrin ligand on brain endothelial cells has remained uncertain. THBMEC expressed FN-CS1 mRNA with no detectable VCAM-1, even though HUVEC expressed both FN-CS1 and VCAM-1 (Fig 4a). We then used FN-CS1 peptide to occupy the leukocyte α4 integrin binding site, and found that FC-CS1 peptide blocked PBMC adhesion to aTHBMEC, compared to control peptide (Fig 4b). Anti-FN-CS1 antibodies were incubated with aTHBMEC, and blocked PBMC adhesion to aTHBMEC, while isotype IgM had no effect (Fig 4c). We used two different anti-VCAM-1 antibodies, neither of which affected PBMC adhesion (Fig. 4c). These results demonstrated that α4 integrin/FN-CS1 mediated PBMC adhesion on brain endothelial cells.

Fig. 4
FN-CS1, not VCAM-1, is the ligand on brain endothelial cells for alpha 4 integrin-mediated leukocyte adhesion. A: FN-CS1 RNA, but not VCAM-1 RNA, was detected on THBMEC. Total RNA was obtained from THBMEC. HUVEC was used as a control. RT-PCR was performed, ...

4. Discussion

The initial objective of this study was to develop an in vitro inflammatory BBB model to visualize sequential leukocyte-brain endothelial interactions under physiological flow conditions. The leukocyte extravasation process, including apical chemokine deposits, integrin engagement, firm adhesion, locomotion, and transendothelial migration, are all dependent on hemodynamic shear, a fundamental physiological feature of all leukocyte-endothelial interactions. The present dynamic in vitro BBB model using parallel plate flow chamber and THBMEC allows us to visualize leukocyte-brain endothelial interactions under a flow rate simulating the velocity in brain capillaries (Hudetz et al., 1996). THBMEC maintained their morphology and the distribution of immunoreactivity for tight junction-associated proteins under flow conditions. These findings indicate that the leukocyte-endothelial interaction was not a passive process caused by mechanical damage to THBMEC.

To simulate inflammatory BBB, we stimulated THBMEC with TNF-α and IFN-γ at concentrations seen in patients with sepsis or systemic inflammatory response syndrome (Brunner et al., 2004; Collighan et al., 2004; Kabir et al., 2003; Watanabe et al., 2005). Cytokines did not cause observable changes in the distribution of immunoreactivity for tight junction-associated proteins. These observations are consistent with our prior report that aTHBMEC maintained a high transendothelial electrical resistance (TEER) and low solute permeability after cytokine exposure at these concentrations (Ubogu et al., 2006). As a positive control, we showed that these inflammatory cytokines increased expression of the chemokine GRO peptides CXCL1-3, indicating a biological response. Compared with resting THBMEC, activation of THBMEC with cytokines was required for robust adhesion of leukocytes under flow conditions (Fig 2a). The relationship between the enhancement of adhesion and up-regulation of chemokine and adhesion molecules is under investigation. When G (i/o) signaling was blocked with PTX, PBMC adhesion was almost completely abrogated. This indicates that PBMC adhesion to inflammatory brain endothelial cells is GPCR-dependent.

The α4 integrins are constitutively expressed on lymphocytes, monocytes, and eosinophils (Hemler, 1990). The interaction between α4β1 integrin on leukocytes and its counter-receptors on the vascular endothelium plays a key role in MS (Rice et al., 2005; Von Andrian and Engelhardt, 2003). It is considered likely that α4β1 integrin mediates activated effector memory T cell and monocyte extravasation across the BBB into inflamed MS lesions (Ransohoff, 2007). NTZ demonstrated efficacy for treatment of MS in two large clinical trials (Polman et al., 2006; Rudick et al., 2006). Using the dynamic activated BBB model, we report that NTZ administration to MS patients significantly reduced PBMC adhesion to aTHBMEC under flow conditions. It is worth mentioning that both natalizumab and IFNβ-1a treatment affect leukocyte composition in MS. Natalizumab releases leukocytes from the bone marrow and leads to an approximate doubling of T cells, B cells, pre-B cells, and monocytes in the peripheral blood (Krumbholz et al., 2008). In MS patients receiving IFN β-1a treatment, the numbers of CD3+, CD8+ T cells, natural killer (NK) T cells per mL blood were slightly lower than in healthy control subjects (Sellebjerg et al., 2005). The changes of leukocyte compositions are not likely to account for effect of these medications on PBMC adhesion under flow, as PBMC from patients receiving IFN β-1a showed adhesive properties equal to those of healthy controls, while NTZ changes the numbers of PBMC but does not drastically alter their relative proportions. Because standardized numbers of PBMC were used for each experiment, we consider it likely that the direct effects of NTZ on cells, rather than flux in cell populations, led to the results reported here.

The brain endothelial ligand for leukocyte α4 integrin has been unclear (Ransohoff, 2007). α4 integrin has at least two potential vascular ligands, VCAM- 1 and FN-CS1 (Elices et al., 1990; Guan and Hynes, 1990). VCAM-1 is expressed by human aortic endothelial cells (Ganji et al., 2008) and HUVEC, forming transmigratory cups around lymphocytes during diapedesis across HUVEC monolayers (Carman and Springer, 2004; Miyake et al., 2008; Engelhardt and Wolburg, 2004). VCAM-1 was involved in leukocyte homing into lung, as well as melanoma and other tissue (Hallgren et al., 2007; Deem et al., 2007). Fibronectin is a high-molecular-weight glycoprotein found in plasma, at the cell surface, and in the extracellular matrix. It is involved in a variety of biological functions, such as cell attachment, spreading, and migration. Fibronectin can be alternatively spliced in at least three regions, generating multiple molecular moieties (Gutman and Kornblihtt, 1987; Ting et al., 2000; Wagner et al., 2000). In autopsy MS brain tissues, FN CS-1 was detected on astrocyte endfeet and astrocytes at the lesion edge (Van et al., 2005). In EAE mice, blocking α4β1 integrin with antibodies or peptides harboring the CS-1 sequence prevented cellular infiltration in the CNS parenchyma (Kent et al., 1995; Van der Laan et al., 2002). FN-CS1 is expressed on THBMEC at rest, with a significant increase in surface expression following cytokine activation (Ubogu et al., 2006). We confirmed that FN-CS1 mRNA was constitutively expressed by THBMEC. The absence of VCAM-1 expression by THBMEC is consistent with previous reports of lack of VCAM-1 immunoreactivity on brain endothelium in autopsy MS tissue sections (Kivisakk et al., 2003; Peterson et al., 2002). Our present and previous results demonstrated that among the two α4 integrin receptors, FN-CS1 is selectively found on THBMEC. When stimulated with higher concentrations of cytokines, THBMEC also exhibited VCAM-1 expression, consistent with previous reports by other groups, using 50–100 fold greater concentrations than those used here (Kallmann et al., 2000; Wong et al., 1999). Cytokine concentration may play a key role in inducing different ligands for α4 integrins.

In order to test the function of FN-CS1, FN-CS1 peptide was introduced to occupy the ligand binding site of leukocyte α4 integrin before the adhesion assay. The result showed that PBMC adhesion to aTHBMEC was abrogated. Function-neutralizing antibodies to FN-CS1 also blocked PBMC adhesion to aTHBMEC. It has been reported that Jurkat T cell adhesion to RA synovium was mediated by the interaction between VLA-4 and FN-CS1, but not VCAM-1 (Elices et al., 1994). Monocyte and T cell migration across in vitro BBB toward CCL5 was mediated by α4β1 integrin and FN-CS1, not VCAM-1 (Ubogu et al., 2006). Collectively, these data suggest that FN-CS1, is the ligand for leukocyte α4 integrin on activated brain endothelial cells in vitro.

In conclusion, we describe a dynamic activated BBB model, combining inflammatory human cytokine exposed THBMEC and parallel plate flow chamber. We found that anti-α4 integrin treatment of MS patients inhibited PBMC adhesion to inflammatory THBMEC, as compared with MS controls that received IFN β1a. We found that the ligand for leukocyte α4 integrin on brain endothelial cells is FN-CS1. In addition to α4 integrin, GPCR signaling is essential for leukocyte adhesion on aTHBMEC. This study provides a tool to study leukocyte-brain endothelial interactions, and contributes to our understanding of the effect of anti-α4 integrin antibodyin MS treatment.

Supplementary Material

01

Movie 1. Interactions between PBMC and resting THBMEC:

THBMEC were cultured on 35mm collagen-coated tissue culture dishes to confluence followed by assembling with a parallel plate flow chamber and mounting on the stage of a Leica inverted phase contrast microscope. PBMC were perfused across THBMEC at 9ml/hr by a Medfusion 3010a syringe pump. Cell interactions on a field of 0.15 mm2 were captured 2 frames per second with a CCD camera. Cell images were manually quantified by analysis of the video. This movie represents 3 minutes from 20 minutes’ record and played at 6 frames per second.

02

Movie 2. Interactions between PBMC and activated THBMEC:

THBMEC were cultured on 35mm collagen-coated tissue culture dishes to subconfluence followed by activation with TNF-α 10 u/ml and IFN-γ 20 u/ml or control media for 24 hours. THBMEC culture were assembled with a parallel plate flow chamber and mounted on the stage of a Leica inverted phase contrast microscope. PBMC were perfused across THBMEC at 9ml/hr by a Medfusion 3010a syringe pump. Cell interactions on a field of 0.15 mm2 were captured 2 frames per second with a CCD camera. Cell images were manually quantified by analysis of the video. This movie represents 3 minutes from 20 minutes’ record and played at 6 frames per second.

Acknowledgments

This research is supported in part by the National Institutes of Health grant P50 NS38667 (to RMR). The authors thank Dr. Grahame Kidd for providing help with the imaging system and Dr. Monique F. Stins for donating THBMEC.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at

Footnotes

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