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TISSUE-SPECIFIC STEM CELLS

Molecular Mechanism of Systemic Delivery of Neural Precursor Cells to the Brain: Assembly of Brain Endothelial Apical Cups and Control of Transmigration by CD44

^aInstitut Cochin, Université Paris Descartes, Paris, France;
^bU567 and
^dU546, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France;
^cCentre National de la Recherche Scientifique (CNRS), UMR 8104, Paris, France;
^eUniversité Pierre et Marie Curie-Paris6, UMRS 546, Paris, France;
^fAssistance Publique–Hôpitaux de Paris, La Pitié-Salpêtrière Hospital, Fédération de Neurologie, Paris, France

Key Words. Neural precursor cells • Migration • Blood-brain barrier • CD44

Correspondence: Dr. Pierre Olivier Couraud, Ph.D., Institut Cochin UMR CNRS 8104/INSERM U 567, Département de Biologie Cellulaire, 22 rue Méchain, 75014, Paris, France. Telephone: (33)1 40 51 64 57; Fax: (33)1 40 51 64 73; e-mail: pierre-olivier.couraud{at}inserm.fr

Received February 8, 2008; accepted for publication March 31, 2008.
First published online in STEM CELLS EXPRESS   May 1, 2008.

	ABSTRACT

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Systemically injected neural precursor cells (NPCs) were unexpectedly shown to reach the cerebral parenchyma and induce recovery in various diffuse brain pathologies, including animal models of multiple sclerosis. However, the molecular mechanisms supporting NPC migration across brain endothelium remain elusive. Brain endothelium constitutes the blood-brain barrier, which uniquely controls the access of drugs and trafficking of cells, including leukocytes, from the blood to the brain. Taking advantage of the availability of in vitro models of human and rat blood-brain barrier developed in our laboratory and validated by us and others, we show here that soluble hyaluronic acid, the major ligand of the adhesion molecule CD44, as well as anti-CD44 blocking antibodies, largely prevents NPC adhesion to and migration across brain endothelium in inflammatory conditions. We present further evidence that NPCs, surprisingly, induce the formation of apical cups at the surface of brain endothelial cells, enriched in CD44 and other adhesion molecules, thus hijacking the endothelial signaling recently shown to be involved in leukocyte extravasation. These results demonstrate the pivotal role of CD44 in the trans-endothelial migration of NPCs across brain endothelial cells: we propose that they may help design new strategies for the delivery of therapeutic NPCs to the brain by systemic administration.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Intracerebral transplantation of neural precursor cells (NPCs) is currently used to partially restore functional neuronal circuits in preclinical models of neurodegenerative pathologies, such as Parkinson's disease or Huntington's disease, or to provide a source of exogenous oligodendrocytes in animal models of genetic disorders, such as the shiverer dysmyelinated mouse [1–4]. However, intraparenchymal transplantation into lesioned brains is not appropriate to address diffuse cerebral pathologies with widespread demyelination and axonal loss, such as multiple sclerosis (MS). Quite recently, NPCs were intravenously injected and appeared to reach the cerebral parenchyma and induce recovery in models of MS [5, 6], epilepsy [7], and Huntington's disease [8], as well as spinal cord injury [9]. Although still controversial [10], these data strongly argue in favor of the capacity of NPCs to cross brain vascular endothelium in inflammatory conditions.

Brain endothelial cells, which constitute the blood-brain barrier (BBB), exhibit unique characteristics that distinguish them from endothelial cells in other organs. In particular, they express numerous intercellular tight junctions that are associated with very low paracellular permeability [11]. Although resting lymphocytes do not breach the BBB, activated T lymphocytes and monocytes can migrate to the inflamed central nervous system (CNS) [12]. Leukocyte trans-endothelial migration, known as extravasation or diapedesis, involves a complex set of adhesion molecules at the surface of leukocytes and vascular endothelial cells. Tethering or rolling of leukocytes is followed by their firm adhesion to endothelium, which precedes extravasation. Although tethering is generally mediated by selectins, it appears to be mediated in brain by the integrins very late antigen (VLA)-4 (4β1) and 4β7 [13]. The endothelial adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and platelet/endothelial cell adhesion molecule (PECAM)-1, belonging to the superfamily of immunoglobulins, actively contribute to the firm adhesion and/or migration of distinct subsets of leukocytes to the CNS through cytokine-activated brain endothelium [14, 15]. Also, CD44, a polymorphic family of related membrane proteoglycans expressed by endothelial cells, leukocytes, metastatic tumor cells, and a variety of other cell types, was shown to play a pivotal role in lymphocyte trafficking [16, 17]. CD44 action is mediated mainly via binding to hyaluronic acid (HA), a glycosaminoglycan composed of multiple disaccharide units of glucuronic acid, N-acetylglucosamine and present in extracellular matrix and on various cell types, including endothelial cells. In addition, chemokines are now well characterized as important signals governing the firm adhesion of leukocytes to the endothelium and their targeted migration toward inflammatory sites. Some chemokines, in particular stromal-derived factor-1 (SDF-1), may also play an important role in controlling the tropism of metastatic tumor cells and neural stem cells [18–20].

Unraveling the molecular mechanisms of leukocyte migration across the BBB was made possible by the production of transgenic mouse models and the development of novel imaging strategies, as well as by the availability of brain endothelial cells and cell lines that stably maintain in vitro the main phenotypic characteristics of brain endothelium [21]. In particular, we recently produced and characterized a human brain endothelial cell line, hCMEC/D3, which has been validated as a unique in vitro model of human BBB [22–25]. Also, the RBE4 rat brain endothelial cell line [26] has been widely used as a model of brain endothelium for biochemical, immunological, and toxicological studies [27–29]. Both cell lines express various leukocyte adhesion molecules in a constitutive or cytokine-inducible manner; these adhesion molecules strictly control the trans-endothelial migration of activated lymphocytes and monocytes [22, 25]. In the present study, we took advantage of these human and rat BBB models to seek the adhesion molecules involved in the extravasation of syngeneic NPCs. We document for the first time that NPCs, like activated leukocytes, induce the formation of apical cups at the surface of brain endothelium. In addition, we demonstrate by in vitro functional assays that CD44 is a key player in rodent and human NPC adhesion to and migration across brain endothelial cells.

	MATERIALS AND METHODS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Reagents
Hyaluronic acid (H1751), hyaluronidase (H3757), poly-L-ornithine hydrobromide (P3655), glucose (G8769), insulin (I5500), basic fibroblast growth factor (bFGF) (F0291), epidermal growth factor (EGF) (E9644), laminin (L2020), and leukemia inhibitory factor (LIF) (L5283) were purchased from Sigma-Aldrich (Saint-Quentin, France, http://www.sigmaaldrich.com). Hyaluronic acid fluorescein-labeled was from Calbiochem (Meudon, France, http://www.emdbiosciences.com). Endothelial Basal Medium (EBM-2) and EGM-2 BulletKit were obtained from Lonza (Walkersville, MD, http://www.lonza.com). Dulbecco's modified Eagle's medium (DMEM), MEM, neurobasal medium, F12 and F10 media, G418, fetal calf serum, basic fibroblast growth factor (bFGF; human, recombinant), L-glutamine, nonessential amino acids, penicillin/streptomycin, sodium pyruvate, HEPES, N2 supplement, and B27 were purchased from Invitrogen (Cergy-Pontoise, France, http://www.invitrogen.com). Rat tail collagen type I was purchased from BD Biosciences (Le Pont-De-Claix, France, http://www.bdbiosciences.com). Tumor necrosis factor (TNF-), interferon (IFN-), and SDF-1 were from AbCys (Paris, http://www.abcysonline.com). 5-Chloromethylfluorescein diacetate (CMFDA) was from Molecular Probes (Eugene, OR, http://probes.invitrogen.com).

Antibodies
Mouse anti-human monoclonal antibodies specific to CD44 (J173 and Bu52) were purchased from Immunotech (Marseille, France, http://www.immunotech.fr) and Biogenesis (Argène, Variles, France, http://www.argene.com), and rat anti-human monoclonal antibody (IM7) from BD Biosciences. Mouse anti-rat monoclonal antibody against CD44 (OX-49) was from BD Biosciences. Antibodies against ICAM-1 were purchased from AbD Serotec (Raleigh, NC, http://www.ab-direct.com) (1A29 mouse anti-rat) and from R&D Systems (Lille, France, http://www.rndsystems.com) (11C81 mouse anti-human). Mouse anti-human monoclonal antibody against VCAM-1 (5110C9) was from BD Biosciences. Mouse monoclonal antibodies specific to LFA-1 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com) (sc-7306 mouse anti-human) and from BD Biosciences (WT.1 mouse anti-rat). Mouse monoclonal antibodies to Mac-1 chain were purchased from Chemicon (Euromedex, Souffelweyersheim, France, http://www.chemicon.com) (MAB2117 mouse anti-human) and from BD Biosciences (WT.5 mouse anti-rat). Antibodies to PECAM-1 were from R&D Systems (9G11 mouse anti-human) and from BD Biosciences (TLD-3A12 mouse anti-rat). Mouse anti-rat and human VLA-4 antibody (HP2/1) was obtained from Chemicon. Cy-2-conjugated anti-mouse and Cy-3-conjugated anti-mouse or anti-rat antibodies were from Jackson Immunoresearch Laboratories (Interchim, France, http://www.jacksonimmuno.com).

Endothelial Cell Cultures
The previously described RBE4 rat brain endothelial cell line [26, 30–32] and the hCMEC/D3 human brain endothelial cell line [25] were used in this study. Briefly, RBE4 cells were grown on type I collagen-coated plates in MEM/F10 medium supplemented with 10% fetal calf serum, bFGF, and G418, and hCMEC/D3 cells were grown on type I collagen-coated plates in EGM-2 MV BulletKit (Lonza) containing EBM-2 (Lonza) and 2.5% fetal bovine serum supplemented with EGM-2 MV SingleQuots (Lonza) at final dilution 4x lower than recommended by the manufacturer and 1 ng/ml bFGF.

NPC Cultures
The RN33B cell line (kindly provided by Drs. C. Lundberg and A. Björklund, Lund, Sweden, and with permission given by S.R. Whittemore, Kentucky Spinal Cord Injury Research Center, Louisville, KY) is a conditionally immortalized neural progenitor cell line, generated from embryonic rat brainstem (embryonic day [E] 12.5) by retroviral transduction of the temperature-sensitive simian virus 40 large T antigen [33, 34]. The RN33B cell line has a remarkable neurogenic capacity in both neonatal and adult recipients and can differentiate into neuron-like and glial cells after intracerebral transplantation. RN33B cells were cultured in polyornithine-coated plates at the permissive temperature (33°C) in DMEM/F12 medium supplemented with 10% fetal calf serum [33, 34].

The HFT13 human neural precursor cell line derived from human fetal week 13 telencephalon was kindly provided by E. Snyder (Burnham Institute, La Jolla, CA). The HFT13 cell line, passages 41–45, was grown as floating clusters in neurobasal medium supplemented with 1% penicillin/streptomycin, L-glutamine, 2% B27, heparin, 20 ng/ml bFGF, and 10 ng/ml LIF [35].

The NR8383 rat monocytic cell line was kindly provided by Dr. E. deVries. Briefly, NR8383 cells were grown in suspension in DMEM/F12 medium supplemented with 20% fetal calf serum at 37°C [36].

Primary rat NPCs were obtained from E14/E16 Dark Agouti embryonic rats (Elevage Janvier, Le Genest-Saint-Isle, France). Brains were dissected free of meninges and enzymatically dissociated using ATV (0.05% trypsin, 0.1% glucose, 0.02% EDTA). Collected cells were resuspended in DMEM/F12 medium (1:1) supplemented with N2 supplements (1%), B27 (0.5%), insulin (25 µg/ml), glucose (6 mg/ml), HEPES (5 mM), bFGF (20 ng/ml), and EGF (20 ng/ml). NPCs, at less than passage 5, were subcultured by dissociating floating neurospheres every 2 weeks. Cultures were fed twice a week.

Human fetuses were obtained after abortion, according to French legislation, with patients' agreement. Primary human NPCs were obtained from the brain of 8.5-week-gestation fetuses, dissected, enzymatically dissociated, and grown as neurospheres in a medium DMEM/F12 (1:1) supplemented with N2 supplement, B27, insulin, glucose, HEPES, EGF (20 ng/ml), bFGF (20 ng/ml), and LIF (10 ng/ml) as described above for rodent NPCs. Cells were used at passages lower than 8.

Flow Cytometry
Fluorescence-assisted cell sorting assay was performed following standard protocols. Briefly, cells were washed, saturated with phosphate-buffered saline (PBS) and 2% fetal calf serum and incubated for 15 minutes at 25°C with the indicated antibodies at 2 µg/ml, followed by Cy2-conjugated secondary antibodies; omission of the first antibodies was used as control. To determine functional activity of CD44-HA binding ability, cells were incubated with HA-fluorescein isothiocyanate (FITC) at 37°C and then washed with fluorescence-activated cell sorting buffer. Nonfluorescent HA was used as control. Acquisitions were performed on an Epics XL cytometer (BD Biosciences).

Adhesion of NPCs to HA
For the adhesion to HA experiment, 24-well plates were coated with 500 µg/ml HA for 3 hours at 37°C, followed by blocking of nonspecific sites with PBS and 3% bovine serum albumin for 2 hours at 37°C. In some cases, HA-coated wells were pretreated with 16 U/ml hyaluronidase for 1 hour at 37°C before cells were added. NPCs were fluorescently labeled with 10 µM CMFDA for 30 minutes at 37°C and were subsequently washed in PBS. NPCs (25 x 10⁴ cells per well) were applied to the wells and were allowed to adhere for 30 minutes at 37°C. When indicated, NPCs were pretreated for 30 minutes at 37°C with various blocking anti-CD44 or control antibodies (20 µg/ml) before the adhesion assay.

Adhesion of NPCs to Brain Endothelial Cells
Adhesion to endothelial cells (10⁵ cells per well) was performed as described previously [37]. RBE4 cells or hCMEC/D3 cells (10⁵ cells per well) were seeded on type I collagen-coated 24-well plates and grown for 2 days. Prior to the assay, monolayers were stimulated or not with 200 U/ml IFN- plus 200 U/ml TNF- for 24 hours and washed twice with PBS. NPCs were fluorescently labeled with 10 µM CMFDA for 30 minutes at 37°C and were subsequently washed in PBS. NPCs (25 x 10⁴ cells per well) were incubated onto endothelial cells and were allowed to adhere for 30 minutes at 37°C. When indicated, NPCs were pretreated for 30 minutes at 37°C with HA (250 µg/ml) before the adhesion assay. After the removal of nonadherent cells with PBS, the adherent cells were hypotonically lysed. The proportion of adherent NPCs was determined by quantification of the amount of fluorescence released using a Fusion fluorescent plate reader (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com) with an excitation wavelength of 492 nm and an emission wavelength of 517 nm.

Visualization of Migration Cups on Brain Endothelial Cells
RBE4 cells or hCMEC/D3 cells (10⁵ cells per well) were seeded on type I collagen-coated 24-well plates and grown for 2 days. Prior to the assay, monolayers were stimulated or not with 200 U/ml IFN- plus 200 U/ml TNF- for 24 hours and washed twice with PBS. NPCs were fluorescently labeled with 10 µM CMFDA for 30 minutes at 37°C and were subsequently washed in PBS. NPCs or rat monocytes (NR8383) (25 x 10⁴ cells per well) were incubated onto endothelial cells and were allowed to adhere for 5 minutes at 37°C in the presence of SDF-1 at 100 ng/ml. After three washes with PBS, cells were fixed with 4% formaldehyde in PBS for 10 minutes, protected with 0.1 M glycine for 15 minutes, and blocked with PBS containing bovine serum albumin (BSA) (2%) for 1 hour. The cells were then incubated for 16 hours at 4°C with monoclonal antibodies against CD44 (2 µg/ml) or VCAM-1 (2 µg/ml); Cy3-conjugated anti-mouse antibodies were used as secondary antibodies.

Immunolabeling was analyzed using multiphoton confocal microscope (TCS SP5 multiphoton resonant scanner; Leica, Heerbrugg, Switzerland, http://www.leica.com), and three-dimensional (3D) reconstructions of acquired pictures were performed using IMARIS software (Bitplane AG, Zurich, Switzerland, http://www.bitplane.com).

Trans-Endothelial Migration of NPCs
Migration through endothelial cells was performed as described previously [37]. RBE4 cells (5 x 10⁴ cells per insert) or hCMEC/D3 cells (3 x 10⁴ cells per insert) were seeded on type I collagen-coated 6.5-mm Transwell culture inserts with a pore size of 8 µm (Corning Enterprises, Corning, NY, http://www.corning.com) and grown for 3 days in 5% CO₂ at 37°C. Prior to the assays, monolayers were stimulated or not with 200 U/ml IFN- plus 200 U/ml TNF- for 24 hours and washed twice with endothelial medium. NPCs were fluorescently labeled with 10 µM CMFDA for 30 minutes at 37°C. Fluorescent NPCs (10⁶ cells per milliliter) in 100 µl of endothelial medium were added to the upper chamber. A chemotactic gradient was created by addition of SDF-1 (100 ng/ml) to the lower chamber. NPCs were allowed to migrate at 37°C and 5% CO₂ for 16–18 hours. When indicated, NPCs were preincubated for 30 minutes at 37°C with HA (250 µg/ml) and various blocking or control antibodies (20 µg/ml) before the migration assay. All experiments were performed in triplicate. After transmigration, cells were fixed with 4% formaldehyde and washed extensively with PBS. To remove nonmigrating cells, cells on the upper face of the filter were gently scraped using a cotton swab, and the migrating NPCs were observed under a confocal microscope (TCS SP2 AOBS; Leica). Using a magnification of x20, central pictures of adjacent fields were taken to represent 1,000 ± 200 migrated counted cells in basal conditions. Fluorescence quantification was made using ImageJ 1.38g (NIH). Migrating cells were manually counted for the more representative experiments to confirm fluorescent quantification.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction Analyses
RNAs were purified by Trizol reagent (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com). Reverse transcription (RT)-polymerase chain reaction (PCR) analyses were performed using exponential amplification. Glyceraldehyde-3-phosphate dehydrogenase cDNA amplification was used as control [38].

Statistical Analysis
Continuous variables are expressed as the mean ± SEM. These variables were compared by using one-way analysis of variance, and thereafter means comparisons were made using Student's t tests adjusted to have an level of .05. All statistical tests were two-tailed. p values that were less than .05 were considered to indicate statistical significance. Statistical analyses were performed with the use of JMP 5.1 (SAS Institute, Cary, NC, http://www.sas.com).

	RESULTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Human and Rat NPCs and Brain Endothelial Cells Express CD44
To identify pairs of adhesion molecules expressed by NPCs and syngeneic brain endothelial cells, which might be involved in the trans-endothelial migration of NPCs, we assessed the expression by flow cytometry and/or by RT-PCR of a panel of adhesion molecules known to be involved in leukocyte extravasation: ICAM-1, PECAM-1, and VCAM-1 of the immunoglobulin superfamily; CD44; and the integrins Mac-1 (Mβ2), LFA-1 (Lβ2), and VLA-4 (4β1). Because in vivo systemically injected NPCs cross the BBB only in an inflammatory situation [5], brain endothelial cells and/or NPCs were used following a 24-hour treatment with the inflammatory cytokines IFN- and TNF-, or without treatment as control. Results (Table 1) indicate that in the panel of adhesion molecules tested, cytokine-treated rat and human primary NPCs and HFT13 human cell line expressed only ICAM-1 and CD44 (except RN33B, which also expressed VLA-4). In addition, CD44 expression was detected in rat and human primary NPCs, as well as cell lines in the absence of cytokine treatment, although at a lower level (except in RN33B cells) than following cytokine treatment (Fig. 1). The human hCMEC/D3 and rat RBE4 brain endothelial cell lines were found to constitutively express ICAM-1, PECAM-1, and CD44, whereas expression of VCAM-1 was induced by cytokine treatment, as previously described [25, 32]. Because endothelial cells do not express ICAM-1 receptors (the integrins Mac-1 and LFA-1), we reasoned that in the panel of adhesion molecules tested, CD44 appeared to be the only candidate to mediate NPC adhesion to and/or extravasation across brain endothelial cells. It is well documented that human CD44 may be expressed under various alternative splicing isoforms, including the standard isoform CD44s and multiple variants, CD44v_1–10, depending on cell types. We observed by RT-PCR, using human isoform-specific primers, that human NPCs and HFT13 cells, together with the human brain endothelial cell line hCMEC/D3, expressed only CD44s transcripts (not shown).

View larger version (43K): [in this window] [in a new window]   Figure 1. Expression of CD44 by rat and human NPCs. Representative fluorescence-activated cell sorting analysis of CD44 expression by RN33B cells, HFT13 cells, and primary rat and human NPCs, in basal culture conditions (light gray) or following pretreatment with tumor necrosis factor- + IFN- (dark gray). Shaded graphs show negative control staining in the absence of primary antibody. Abbreviation: NPC, neural precursor cell.

View larger version (21K): [in this window] [in a new window]   Figure 2. Rat and human neural precursor cell (NPC) cell lines exhibit HA binding. (A, C): FITC-conjugated HA binding by RN33B (A) or HFT13 (C) cells, in basal culture conditions (light gray) or following pretreatment with TNF-+IFN- (dark gray). Shaded graphs show negative control staining in the presence of nonfluorescent HA. (B, D): Interaction of NPCs with immobilized HA mediates cell adhesion under static conditions. Fluorescently labeled RN33B (B) and HFT13 (D) cells were pretreated with TNF-+IFN- or neither. HA immobilized on a microtiter plate was treated or not with Hase. The percentage of bound cells was determined by measuring the fluorescence intensity at 517 nm. Data represent means ± SEM of three distinct experiments, each consisting of quadruplicate determinations. ** represents p < .001. (E): CD44 is implicated in adhesion of HFT13 cells to immobilized HA. Fluorescently labeled HFT13 cells were pretreated with TNF-+IFN- or neither. HFT13 cells were preincubated with blocking anti-CD44 antibodies (Bu52 and IM7) or not (IgG as control). The percentage of bound cells was determined by measuring the fluorescence intensity. Data represent averages ± SEM of two distinct experiments, with each condition in quadruplicate. Abbreviations: BSA, bovine serum albumin; HA, hyaluronic acid; Hase, hyaluronidase; TNF, tumor necrosis factor.

CD44 Is Involved in the Adhesion of NPCs to Brain Endothelial Cells
Because CD44 was previously shown to contribute to the adhesion of activated lymphocytes to vascular endothelial cells, including brain and retinal endothelial cells, we evaluated the contribution of CD44 to the adhesion of RN33B and HFT13 cells to rat (RBE4) and human (hCMEC/D3) brain endothelial cells, respectively, either in basal conditions or following cytokine (IFN- and TNF-) treatment.

As shown in Figure 3A and 3B, adhesion of HFT13 cells to human brain endothelial cells was significantly increased following cytokine pretreatment of HFT13 cells. This cytokine enhancement of cell adhesion was almost completely blocked by soluble HA (250 µg/ml), whereas basal adhesion was not affected. Indeed, basal adhesion of untreated HFT13 was slightly increased by cytokine treatment of hCMEC/D3 endothelial cells, but this increase was not modified in the presence of soluble HA. These observations strongly suggest that CD44 largely contributes to the adhesion of cytokine-treated human NPCs to brain endothelial cells in inflammatory conditions. Higher concentrations of HA did not inhibit further cell adhesion (data not shown). Adhesion of RN33B to RBE4 was also largely inhibited by HA, although basal adhesion was stronger and the cytokine effect was limited (Fig. 3C).

View larger version (15K): [in this window] [in a new window]   Figure 3. CD44 is involved in adhesion of NPCs to endothelial cells. Adhesion assay of NPC cell lines was performed following or not following 24 hours of pretreatment with TNF- and IFN-. RN33B or HFT13 cells were or were not preincubated with soluble HA (250 µg/ml). The percentage of adhesive cells was determined by measuring fluorescence intensity. Data represent means ± SEM of three distinct experiments, with each condition in quadruplicate. * represents p < .05; ** represents p < .001. Abbreviations: CTL, control; EC, endothelial cell; HA, hyaluronic acid; NPC, neural precursor cell; TNF, tumor necrosis factor.

View larger version (42K): [in this window] [in a new window]   Figure 4. NPCs induced CD44-positive trans-migratory cups. (A–E): Visualization of trans-migratory cups after adhesion of rat NPCs: RN33B (B, D, E) or primary NPCs (C) to rat endothelial cells (RBE4) by confocal microscopy after CD44 staining; in control adhesion of rat monocytes: NR8383 (A). (F–J): Visualization of trans-migratory cups after adhesion of human NPCs: HFT13 cells (F, G) or primary NPCs (H) to human endothelial cells (hCMEC/D3) by confocal microscopy after either VCAM-1 (F) or CD44 (G–J) staining. (D, E, I, J): Visualization of trans-migratory cups by CD44 staining (red) surrounding NPCs fluorescently labeled with 5-chloromethylfluorescein diacetate (green): adhesion of rat NPCs (RN33B) to rat endothelial cells (RBE4) (D, E), adhesion of human NPCs (HFT13) to human endothelial cells (hCMEC/D3 cells) (I, J). Images were acquired using confocal microscopy, and three-dimensional reconstruction was performed using IMARIS software. Abbreviations: NPC, neural precursor cell; VCAM, vascular cell adhesion molecule.

View larger version (22K): [in this window] [in a new window]   Figure 5. CD44 is implicated in TEM of NPCs. (A, B): TEM of rat NPCs: RN33B cells (A) or primary rat NPCs (B) across rat endothelial cells (RBE4) after preincubation of NPCs with HA at 250 µg/ml. (C, D): TEM of human NPCs: HFT13 cells (C) or primary human NPCs (D) across human endothelial cells (hCMEC/D3) after preincubation of NPCs with anti-CD44 antibodies (Bu52, IM7 at 20 µg/ml). Migration was performed in the presence of stromal-derived factor-1 in the lower chamber. Values represent means ± SEM of the percentage of the migration under TNF- + IFN- treatment in two (D) or three (A–C) pooled experiments (each condition in triplicate). ** represents p < .001. Abbreviations: HA, hyaluronic acid; NPC, neural precursor cell; TEM, trans-endothelial migration; TNF, tumor necrosis factor.

	DISCUSSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

The membrane proteoglycan CD44 is a multifunctional signaling molecule, required for a variety of cellular activities, including cell-cell adhesion, migration, proliferation, and differentiation. In particular, CD44 expressed by activated lymphocytes has been well documented to play a pivotal role in vivo in their trans-endothelial migration to inflammatory sites [47, 48], including in the CNS in the context of experimental autoimmune encephalomyelitis (EAE) [16]. More recently, CD44 was shown to mediate the initial tethering and subsequent rolling of leukocyte subpopulations either directly, mainly through binding to HA expressed by cytokine-activated vascular endothelial cells, or via formation of a functional complex with the integrin VLA-4 or LFA-1 on the surface of leukocytes [49–51]. In addition, there is now compelling evidence that CD44 is a key mediator of the transendothelial migration of various metastatic tumor cells, notably breast and prostate cancer cells [43, 52]. Also, recent reports clearly established the involvement of CD44 in the homing of leukemic stem cells to their HA-rich bone marrow niche, pointing to CD44 blockade as a potential therapeutic approach in this type of cancer [53, 54]. Moreover, soluble factors may also govern the targeted migration of stem cells and metastatic tumor cells, and several reports document the involvement of chemokines, in particular SDF-1, and various growth factors in the migratory activity of these cells [18, 20, 55, 56].

We observed in the present study that human NPCs (including the HFT13 NPC line) and the rat NPC line RN33B, as well as, to a lower level, rat NPCs constitutively express CD44 in an active (HA binding) conformation. In addition, in line with reports documenting an increase of CD44 expression by inflammatory cytokines [57], expression by the human cell line HFT13 was significantly enhanced by IFN- and TNF-. These data are in agreement with previous studies indicating that rat glial progenitors express a high level of CD44 [58], as well as cells of the human oligodendrocyte lineage [59]. In addition, murine adult neurospheres were also reported to express CD44 [5]. However, although these authors also detected the integrin VLA-4 at the surface of murine NPCs [5], human and rat NPCs (our study) constantly failed to express this integrin (or LFA-1, another leukocyte integrin involved in extravasation), likely pointing to species differences. Among a number of adhesion molecules tested, we could detect only the ubiquitous adhesion molecule ICAM-1 at the surface of human and rat NPCs. Because endothelial cells also express ICAM-1 (but not any counter-receptor of ICAM-1, LFA-1 or Mac-1), these results led us to hypothesize that CD44 might be considered as a candidate adhesion molecule for trans-endothelial migration of NPCs.

Indeed, the main result of the present study is that adhesion of NPCs to brain endothelial cells and their trans-endothelial migration are largely mediated by CD44. This conclusion is based on the following arguments: (a) soluble HA and/or anti-CD44 blocking antibodies are able to partially block NPC adhesion to brain endothelial cells and almost completely prevent their cytokine-induced trans-endothelial migration, and (b) adhesion of human and rat NPCs induces brain endothelial cells to form CD44-enriched membrane protrusions that are highly reminiscent of the transmigratory cups, or docking structures, known to support leukocyte adhesion and trans-endothelial migration.

It has been documented quite recently that the trans-endothelial migration of activated leukocytes is associated with the formation of endothelial microvilli-like protrusions, which partially surround them during the migration process [45, 46]. These so-called transmigratory cups are highly enriched in ICAM-1 and VCAM-1, which are engaged with their counter-receptors LFA-1 or MAC-1 and VLA-4, respectively, at the surface of leukocytes. These cups have been further documented to be enriched in additional adhesion molecules, including CD44, all known to be associated with the cortical actin cytoskeleton via the molecular linkers of the ezrin-radixin-moesin family [45, 60, 61]. Our results indicate that the membrane protrusions formed by cytokine-activated brain endothelial cells in response to NPC adhesion are enriched not only in CD44 but also in VCAM-1 (Fig. 4F), ICAM-1, and ezrin (not shown), thus appearing as bona fide transmigratory cups. Interestingly, the present study documents, for the first time to our knowledge, that formation of transmigratory cups as a mechanism of trans-endothelial migration is not restricted to leukocytes but can be shared by other blood circulating cells. Indeed, preliminary experiments strongly suggest that metastatic cancer cells can also induce the formation of similar endothelial protrusions (not shown).

Is NPC CD44 or endothelial CD44 involved in NPC trans-endothelial migration? Our observation that blocking NPC CD44 by preincubation with soluble HA or CD44 antibodies was sufficient to largely prevent trans-endothelial migration suggests that CD44 present at the surface of NPCs is directly involved in this process. As in the case of leukocyte trafficking and homing of leukemic stem cells, CD44 expressed by NPCs thus likely interacts with HA present at the surface of cytokine-activated endothelial cells [62, 63], associated with various proteoglycans or proteins, including endothelial CD44 [57]. We propose that this engagement of endothelial CD44 may constitute a triggering stimulus for the formation of transmigratory cups underneath NPCs, just as ICAM-1 engagement seems to be responsible for the formation of leukocyte transmigratory cups [46, 64]. In line with this hypothesis, CD44 engagement was reported to activate multiple signaling pathways, including the Rho family GTPases, to recruit ezrin [65] and to induce actin cytoskeleton rearrangements [66], in a way reminiscent of ICAM-1 signaling, which is known to regulate leukocyte trans-endothelial migration [31, 64, 67]. However, we cannot exclude the possibility that other stimuli may also participate in the formation of the endothelial cups observed in response to NPC adhesion. It was very recently reported that β2-, β6-, and β1-integrins can mediate in vitro the initial interaction between human NPCs and human umbilical vein endothelial cells (HUVECs) [68]. This result may reflect the multiple phenotypic differences largely documented between brain microvascular endothelial cells and nonbrain macrovascular HUVECs [69]. Alternatively, putative functional interaction between CD44 and these integrins, as reported between CD44 and VLA-4 [49], might reconcile these findings.

	CONCLUSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
In conclusion, we established here the pivotal role of CD44 in human and rat NPC adhesion and migration across brain endothelial cells in culture. This finding may be particularly important for understanding the mechanisms that mediate the recruitment of systemically injected NPCs to the brain in the context of neuroinflammatory diseases, such as EAE [5, 6]. Recruited NPCs may then either contribute to the replacement of oligodendrocytes in demyelinating lesions, as demonstrated for endogenous neural progenitors [70], or promote neuroprotection by inducing apoptosis of infiltrated T lymphocytes in perivascular spaces [6]. Controlling the expression level and activity of CD44 on NPCs before intravascular injection might then constitute a key issue for their efficient delivery to the brain across the BBB and their therapeutic activity in demyelinating diseases.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
We thank Drs. G. Martino and S. Pluchino (San Raffaele Hospital, Milano, Italy) for helpful discussions. This work was supported by grants from the Association pour la Recherche sur la Sclérose en Plaques (ARSEP), the Centre National de la Recherche Scientifique, and the Institut National de la Santé et de la Recherche Médicale. C.R. and H.T.-L. received a fellowship from ARSEP, and N.W. is a recipient of a fellowship from the Département de la Recherche Clinique et du Développement, Assistance Publique–Hôpitaux de Paris and the Centre National de la Recherche Scientifique. We are grateful to Pierre Bourdoncle (Cell Imaging Platform of the Cochin Institute/Institut Fédératif de Recherche (IFR) Alfred Jost for expert contribution to the confocal microscopy analysis of endothelial transmigratory cups.

	FOOTNOTES

 
Author contributions: C.R., N.W., and C.D.: collection and/or assembly of data, data analysis and interpretation; C.R., N.W., and C.D. contributed equally to this work. N.C. and H.T.-L.: collection and/or assembly of data; F.M. and D.B.: provision of study material or patients; S.C.: conception and design, data analysis and interpretation, final approval of manuscript; A.B.-V.E.: conception and design, final approval of manuscript; P.-O.C.: conception and design, manuscript writing, final approval of manuscript.

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