Brain Advance Access originally published online on December 20, 2007
Brain 2008 131(3):785-799; doi:10.1093/brain/awm295
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The blood–brain barrier induces differentiation of migrating monocytes into Th17-polarizing dendritic cells
Neuroimmunology Research Laboratory, Center for the Study of Brain Diseases, CHUM-Hôpital Notre-Dame, Université de Montréal, Montréal, Québec, Canada
Correspondence to: Dr Alexandre Prat, MD, PhD, Department of Medicine (Neurology) and Immunology, Multiple Sclerosis Clinic and Neuroimmunology Research Laboratory, CHUM-Hôpital Notre-Dame and CHUM Research Center, Université de Montréal, 1560 Sherbrooke East, Montréal, Québec, Canada H2L 4M1 E-mail: a.prat{at}umontreal.ca
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Summary |
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Trafficking of antigen-presenting cells into the CNS is essential for lymphocyte reactivation within the CNS compartment. Although perivascular dendritic cells found in inflammatory lesions are reported to polarize naive CD4+ T lymphocytes into interleukin-17-secreting-cells, the origin of those antigen-presenting cells remains controversial. We demonstrate that a subset of CD14+ monocytes migrate across the inflamed human blood–brain barrier (BBB) and differentiate into CD83+CD209+ dendritic cells under the influence of BBB-secreted transforming growth factor-β and granulocyte-macrophage colony-stimulating factor. We also demonstrate that these dendritic cells secrete interleukin-12p70, transforming growth factor-β and interleukin-6 and promote the proliferation and expansion of distinct populations of interferon-
-secreting Th1 and interleukin-17-secreting Th17 CD4+ T lymphocytes. We further confirmed the abundance of such dendritic cells in situ, closely associated with microvascular BBB-endothelial cells within acute multiple sclerosis lesions, as well as a significant number of CD4+ interleukin-17+ T lymphocytes in the perivascular infiltrate. Our data support the notion that functional perivascular myeloid CNS dendritic cells arise as a consequence of migration of CD14+ monocytes across the human BBB, through the concerted actions of BBB-secreted transforming growth factor-β and granulocyte-macrophage colony-stimulating factor.
Key Words: blood–brain barrier; dendritic cells; multiple sclerosis; IL-17; CNS
Abbreviations: BBB, blood–brain barrier; MHC, major histocompatibility complex; EAE, experimental autoimmune encephalomyelitis; TNF, tumour necrosis factor
Received July 18, 2007. Revised November 5, 2007. Accepted November 8, 2007.
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Introduction |
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Immune reactions occurring within the CNS take on a distinctive character given the ability of the blood–brain barrier (BBB) to control passage of leukocytes from the peripheral blood (Pachter et al., 2003
). In addition, there is a limited ability to deploy local immune reactions, likely due to the low basal expression of major histocompatibility complex (MHC) molecules by local antigen-presenting cells, at least in the context of immune homoeostasis. While capillary endothelial cells of the BBB are generally regarded as being protective for CNS immune homoeostasis, they are the first cells encountered by leukocytes migrating to the brain. Hence, leukocyte interaction with the BBB could represent a crucial step during which this barrier shapes immune responses.
Multiple sclerosis is a prototypic CNS-directed inflammatory disease characterized by multi-focal perivascular infiltration of mononuclear cells with a relative breakdown in BBB integrity (Sospedra and Martin, 2005). In multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), disease pathogenesis is attributed to the presence of myelin-reactive T lymphocytes within the parenchyma (Owens and Sriram, 1995; Sospedra and Martin, 2005). While interferon (IFN)--secreting T lymphocytes (Th1) have long been considered the auto-aggressive and disease-inducing CD4+ T lymphocyte subset, recent evidence from EAE studies suggest that interleukin (IL)-17-secreting T lymphocytes (Th17) are responsible for disease initiation (Cua et al., 2003; Langrish et al., 2005; Komiyama et al., 2006; Chen et al., 2006). The cytokine profile of CD4+ T lymphocytes is dictated by the ability of antigen-presenting cells to secrete either IL-12p70, favouring Th1 lymphocytes, or conversely the combination of transforming growth factor (TGF)-β and IL-6, favouring a Th17 lymphocyte phenotype. IL-23 (p19/p40) is now recognized as a dendritic cell-secreted molecule which favours the expansion, rather than the differentiation of Th17 lymphocytes (Mangan et al., 2006; Weaver et al., 2006; Bettelli et al., 2006; Veldhoen et al., 2006a).
Dendritic cells are considered the most potent antigen-presenting cells. While immature myeloid dendritic cells have the capacity to capture CNS antigens and migrate to secondary lymphoid tissues (de Vos et al., 2002), mature ones up-regulate MHC class II, CD80, CD86 and CD40, and acquire the ability to present antigen to naive myelin-specific CD4+ T lymphocytes, directly in the CNS at the inflammatory site (McMahon et al., 2005; Bailey et al., 2007). Karman et al. and Serafini et al. elegantly demonstrated that mature dendritic cells were involved in CNS immunity and were present in EAE lesions (Serafini et al., 2000; Karman et al., 2004). Using human material, Serafini et al. subsequently demonstrated the presence of CD209+ dendritic cells in close association with lymphocytes within active multiple sclerosis lesions (Serafini et al., 2006). Moreover, both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells were shown to be enriched in the cerebrospinal fluid of multiple sclerosis patients, especially at the time of acute relapse (Pashenkov et al., 2001).
In non-CNS organs, peripheral blood CD14+ monocytes are known for their unique ability to differentiate into tissue macrophages or dendritic cells (Randolph et al., 1998, 2002; Fabriek et al., 2005). This differentiation process depends on characteristics of both the inflammatory milieu they are invading, as well as the epithelial (Chomarat et al., 2000; Zhang et al., 2004) or endothelial (Randolph et al., 1998) barriers they cross to enter the target organ. In the CNS, recent evidence suggests that CD11c+ myeloid dendritic cells originate from peripheral blood monocytes (Greter et al., 2005) and are required for naive CD4+ T-cell expansion and for development of EAE lesions (McMahon et al., 2005; Greter et al., 2005; Bailey et al., 2007). These CNS perivascular dendritic cells were shown to express CD209 (Greter et al., 2005; Serafini et al., 2006), CD83 (Serafini et al., 2006) and CD11b (Bailey et al., 2007).
Despite histopathological and functional evidence that dendritic cells are involved in lesion formation in EAE, the exact source and functional characteristics of those CNS perivascular dendritic cells remains elusive, especially in humans. In this study, we demonstrate that human BBB-endothelial cells favour the recruitment of peripheral blood-derived CD14+ monocytes, as well as their differentiation into functional dendritic cells. We show that through secretion of TGF-β and granulocyte-macrophage colony-stimulating factor (GM-CSF), human BBB-endothelial cells promote the differentiation of a subset of peripheral blood CD14+ monocytes into CD83+ (myeloid) dendritic cells which express CD209 and secrete IL-12p70, TGF-β and IL-6, favouring the differentiation of distinct CD4+ T lymphocyte populations into IFN-- or IL-17-secreting cells.
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Materials and Methods |
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Isolation and culture of BBB-endothelial cells and astrocytes
CNS tissue was obtained from temporal lobe resection specimens from young adults undergoing surgery for the treatment of intractable epilepsy. Informed consent and ethical approval were given prior to surgery (ethic approval number HD04.046). BBB-endothelial cells were isolated from non-epileptic material according to a published protocol (Prat et al., 2000
; Biernacki et al., 2001; Prat et al., 2002). As previously demonstrated, these cells express factor VIII, von Willebrand factor, Ulex Agglutenens Europaensis-1-binding sites, endothelial antigen HT-7; and are susceptible to tumour necrosis factor (TNF)-
-induced CD54 and CD106 up-regulation. Immunoreactivity for glial fibrillary acidic protein and -myosin could not be detected, confirming the absence of contaminating astrocytes and smooth muscle cells, respectively. We also confirmed the absence of monocytes and macrophages by immunostaining with anti-CD14 and anti-CD11c antibodies. BBB-endothelial cells were grown in medium composed of M199 (Invitrogen, Burlington, ON, Canada) supplemented with 10% fetal bovine serum (FBS), 20% normal human serum (HS), endothelial cell growth supplement (5 µg/ml) and insulin-selenium-transferrin premix on 0.5% gelatin-coated tissue culture plates (all reagents from Sigma, Oakville, ON, Canada).
For astrocyte culture, human cerebral hemispheres from fetuses of 17–23 weeks of gestation were obtained from the Human Fetal Tissue Repository (Albert Einstein College of Medicine, Bronx, NY) following approved guidelines from the Canadian Institutes of Health Research (CIHR). Astrocytes were cultured as previously described (Jack et al., 2005; Wosik et al., 2007) in complete DMEM media (Invitrogen) supplemented with 10% FBS. Astrocyte-conditioned media (ACM) was harvested once a week from confluent flasks and added to the BBB-endothelial cell-culture media when specified.
Monocyte isolation and culture
Eighty millilitres venous blood samples were obtained from consenting healthy donors, in accordance with the institutional guidelines. Peripheral blood mononuclear cells (PBMCs) were isolated from ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood using standard Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. CD14+ monocytes were purified from PBMCs using magnetic cell sorting (MACS; Miltenyi Biotec, Toronto, ON, Canada) according to the manufacturer's instructions. Monocyte purity was shown to be >97% as assessed by flow cytometry using anti-CD3-fluorescein isothiocyanate (FITC), anti-CD14-R-phycoerythrin (PE) and anti-CD19-PE-Cychrome (Cy) 5 (BD Biosciences, Mississauga, ON, Canada). Monocytes were cultured in RPMI 1640 supplemented with 5% HS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma).
To generate in vitro dendritic cells, monocytes were cultured in fresh complete medium supplemented with 1000 U/ml GM-CSF (specific activity 4.5 x 108 U/mg, BD Biosciences) and 500 U/ml IL-4 (specific activity 1.25 x 108 U/mg, BD Biosciences). After 6 days, 100 ng/ml lipopolysaccharide (LPS from Escherichia coli 0111:B4 lyophilized and sterilized by -irradiation; Sigma) was added for 48 h to complete the maturation process.
Migration of CD14+ monocytes across BBB-endothelial cells
BBB-endothelial cells grown in primary cultures were used to generate an in vitro model of the human BBB, as previously published (Biernacki et al., 2001; Alter et al., 2003). BBB-endothelial cells were plated on gelatin-coated 3 micron pore size Boyden chambers (Collaborative Biomedical Products, Bedford, MA) at a density of 2 x 104 cells/well in endothelial cell-culture media supplemented with 40% (v:v) ACM for 96 h, in order to allow them to reach confluency. The formation of a confluent monolayer was confirmed by haematoxylin–eosin (H&E) staining, as well as soluble tracer diffusion (Ifergan et al., 2006). The media was removed, replaced with fresh endothelial cell media and when applicable, supplemented with 40% ACM. Freshly purified human CD14+ monocytes were then added to the upper chamber and allowed to migrate for 48 h across BBB-endothelial cells either untreated or pre-activated for 24 h with 100 U/ml IFN- (1 U corresponding to 2 ng/ml) and 100 U/ml TNF- (1 U corresponding to 0.05 ng/ml) (Biosource-Invitrogen, Carlsbad, CA). When applicable, migration experiments were performed in the presence of anti-intercellular adhesion molecule (ICAM)-1 and anti-vascular cell adhesion molecule (VCAM)-1 blocking antibodies (10 µg/ml each), or neutralizing antibodies against GM-CSF (0.15 µg/ml) and/or TGF-β1,2,3 (0.02 µg/ml) (all from R&D Systems, Minneapolis, MN). All antibodies were applied to the upper chamber 30 min prior to the addition of monocytes. After 48 h, three cell subsets were collected. The first subset was composed of monocytes that had completely migrated through the BBB and could be recovered from the lower chamber. The second subset was composed of non-migrated monocytes that were collected by vigorous washes of the BBB-endothelial cell monolayer. The third subset was composed of monocytes that had migrated through but were still attached to the BBB-endothelial cells. Those were collected by treating BBB-endothelial cells with trypsin (Invitrogen) and 2 mM EDTA (Sigma) in order to dissociate the adherent sub-endothelial monocyte population. This third subset was named endothelial-associated dendritic cells (eDCs) and would represent cells that could be detected in the perivascular zone of human CNS. CD83+ cells were purified from the BBB-endothelial cell-sub-endothelial monocyte mixture using anti-CD83-R-PE antibodies and anti-R-PE immunobeads (MACS; Miltenyi Biotec). Purity of positively selected cells was consistently >97% as confirmed by flow cytometry.
Flow cytometric analyses
Transmigrated and sub-endothelial monocytes (eDCs) (100 000 cells per stain) were incubated with normal mouse IgG (Caltag Laboratories, Burlingame, CA) to prevent non-specific binding of subsequent antibodies. Cells were phenotyped using FITC, PE, PE-Cy5-conjugated antibodies (10 µg/ml) specific for human CD1a, CD11c, CD14, CD16, CD40, CD54, CD80, CD83, CD86, CD106, CD123, CD209 (DC-SIGN), human leukocyte antigen (HLA)-DR or corresponding isotype controls (all from BD Biosciences). Cells were stained for 30 min at 4°C, washed with phosphate-buffered saline (PBS) containing 1% FBS and then fixed in 2% paraformaldehyde (Sigma). Positive cells were acquired on a FACScan and LSRII (BD Biosciences) and analysed using WinMDI or FACSDiva softwares.
For intracellular cytokine staining (ICS), CD4+ lymphocyte and the corresponding antigen-presenting cell co-cultures were activated for 18 h with 1 µg/ml ionomycin and 20 ng/ml phorbol 12-myristate 13-acetate (PMA) in the presence of 2 µg/ml brefeldin A (Sigma) for the last 6 h of co-culture. Cells were stained for surface markers and were then fixed and permeabilized in 4% (w/v) paraformaldehyde with 0.1% (w/v) saponin in Hank's Balanced Salt Solution for 10 min at room temperature. Since CD4 is dramatically reduced in ICS assays, we stained for CD45RO and gated cells with this marker. After co-culture with eDCs, the percentage of CD45RA+ cells was below 10%. Intracellular staining was performed by incubating cells with antibodies against IFN-, IL-10 (BD Biosciences) or IL-17 (eBioscience, San Diego, CA) (1 mg/ml) for 30 min on ice in PBS buffer containing 0.1% (w/v) saponin, 1% FBS, 0.1% (w/v) NaN3, followed by two washes and resuspended in FACS buffer [1% (v/v) FBS, 0.1% (w/v) NaN3 in PBS]. Cells were acquired on a BD LSRII and analysed using BD FACSDiva software.
Endocytosis assay
Monocytes before migration and eDCs were incubated with 100 µg/ml of FITC-dextran (molecular weight 40 000, Sigma) for 60 min at 37°C. The endocytosis was stopped by washing the cells three times with cold FACS buffer. After staining with anti-CD11c-PE-Cy5, cells were acquired on a LSRII and analysed using FACSDiva software. As a control, we also conducted the experiment at 4°C.
RNA isolation and RNase protection assay for cytokine determination
Quantitative mRNA changes in cytokine production by BBB-endothelial cells were analysed by RNase Protection Assay (RPA), as previously described (Ifergan et al., 2006). In brief, 24 h following treatment, BBB-endothelial cells were lysed in TRIZOL (Invitrogen) and RNA was collected according to the manufacturer's instructions. Following hybridization of the [32P]-labelled probe mix with 10 µg of RNA per condition, probe-RNA duplexes were electrophoresed on a denaturing polyacrylamide/urea gel and exposed overnight on a Kodak autoradiographic film. RPA probe kits (hCK-3 and hCK-4, BD Biosciences) included cytokines TNF-β, lymphotoxin (LT)-β, TNF-, IFN- TGF-β1,-β2, and -β3, IL-3, IL-7, GM-CSF, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), IL-6, leukaemia inhibitory factor, stem cell factor (SCF) and oncostatin-M.
Cytokine determination
Quantitative changes in cytokine production were assessed from culture supernatants by enzyme-linked immunosorbent assay (ELISA) (Pharmingen BD Biosciences), according to the manufacturer's protocol. Plates were read using a Bio-Tek EL800 96-well plate reader at a 450 nm wavelength and analysed using KC Junior program (Bio-Tek, Mississauga, ON, Canada). Because of the low-sensitivity threshold of the available IL-17 ELISA kit, and to better detect differences in IL-17 production between the different antigen-presenting cell subsets, antigen-presenting cell-T-cell co-cultures were stimulated for the last 72 h with 10 µg/ml of anti-human CD3 (clone OKT3; eBioscience). Levels of TGF-βs present in serum-containing culture media were subtracted from each value.
Mixed leukocyte reaction
Irradiated (3000 rads) CD83+ eDCs and migrated CD14+ cells were plated into 96-well plates. Allogeneic CD4+ T lymphocytes were isolated from PBMCs using CD4 beads (MACS, Miltenyi Biotec) according to the manufacturer's instructions. Purified CD4+ T lymphocytes (1 x 105 cells/well) were added to the different antigen-presenting cells at various ratios (5:1; 10:1) and cells were incubated for 5 days in presence of RPMI 1640 supplemented with 5% HS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. CD4+ T lymphocytes cultured with autologous monocytes in presence or absence of phytohemagglutinin (PHA; 2 µg/ml, Sigma) were included as internal proliferative controls. One µCi of [3H-methyl]-thymidine (ICN Biomedical Research Products, Costa Mesa, CA) was added to each well, for the last 18 h. Cells were harvested with a cell harvester and counted with a Beta-counter (LKB Wallac, Turku, Finland). Data are expressed as counts per min (cpm).
Proliferation was also assessed using the vital dye 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR). CD4+ lymphocytes were resuspended at a concentration of 5 x 106 cells/ml in warm RPMI 1640 and incubated for 10 min at 37°C with 5 µM CFSE. Labelling was quenched with HS. After two washes, cells were resuspended in complete media and put in culture with the isolated antigen-presenting cells for 6 days. Cells were then surface-stained for CD4 as described earlier and analysed by flow cytometry.
Immunohistochemistry on human multiple sclerosis brains
Luxol Fast Blue (LFB) and H&E stainings (Wosik et al., 2007) were performed on human brain tissue specimens obtained from four multiple sclerosis patients (autopsy). Post-mortem brain tissue blocks were obtained from the pathology department of the CHUM-Notre-Dame Hospital. Ethic approval has been granted by the CHUM Research Center scientific and ethic committees for brain and peripheral blood samples obtained from multiple sclerosis patients, other neurological and non-neurological disease patients for neuroimmunology-related studies (BH 07.001). Sections showing acute demyelinating lesions and active perivascular mononuclear cell infiltration were selected (8 to 12 blocks per multiple sclerosis donor), and compared to normal-appearing white matter from the same donors (8 blocks per donor) and to non-neurological disease controls (3 donors; 9–11 blocks per donor). Mean age was 49 ± 6 years and disease duration ranged from 3 to 23 years. The causes of death were pneumonia (2), urosepsis (1) and barbiturate intoxication (1). Paraffin-embedded blocks were cut into 3 µm thick sections and de-paraffinized using standard techniques, as described in Wosik et al. 2007. Antigen retrieval was done in sodium citrate (100°C, 20 min). Sections were stained with mouse monoclonal antibodies specific for CD83 (IgG1, 1/100, Serotec, Oxford, UK) or CD209 (IgG2b, 1/100, BD Biosciences) for 1 h at 4°C. Biotin-conjugated polyclonal rabbit anti-mouse antibody (1/400) was incubated for 30 min and followed by FITC-conjugated streptavidin (1/1000, DakoCytomation, Glostrup, Denmark) for 30 min. Image acquisition was performed on a Leica DM6000 epifluorescence microscope and analysed using imageQuant software.
For IL-17 and CD4 immunostainings, frozen CNS material from age- and sex-matched multiple sclerosis patients (n = 4) and healthy donors (n = 4, non-neurological disease controls, traumatic death) was obtained after autopsy. Time of death to snap freezing of the blocks varied from 1 to 2.5 h. These blocks are distinct from the paraffin-embedded material used for dendritic cell stainings. Ten micrometres sections were cut, fixed for 20 min in 4% paraformaldehyde and permeabilized with 1% triton X-100 for 5 min. Sections were blocked with serum for 60 min at room temperature, followed by overnight incubation at 4°C with primary antibodies against IL-17 (1/20, mouse anti-human, eBioscience) and CD4 (1/10, allophycocyanine-conjugated mouse anti-human, BD Biosciences). After several washes, IL-17 stains were amplified by adding biotin-conjugated goat anti-mouse antibody (1/300) followed by streptavidin–FITC (1/300 for 30 min at room temperature; DakoCytomation). CD4 stains were visualized with rabbit anti-allophycocyanine antibody (1/100) followed by goat anti-rabbit coupled to Cy3 (1/300, Jackson ImmunoResearch). Nuclei were stained with TO-PRO3 (1/300 in PBS for 15 min, pre-treatment with 100 µg/ml RNase A for 30 min, Molecular Probes). All control stainings were performed omitting the primary antibody, in which case no immunopositive cells could be detected. Staining was visualized using Leica SP5 confocal microscope and analysed with Leica LAS AF software.
Statistical analyses
Statistical analyses were performed using PRISM GraphpadTM software and included one-way ANOVA (analysis of variance) followed by Student's t-test or Dunnett post hoc test, depending on the number of comparisons to controls. Only P-values <0.05 were considered significant.
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Results |
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Migration of CD14+ monocytes across the human BBB favours a phenotypic change into dendritic cell-like antigen-presenting cells
Migration of monocytes across resting BBB-endothelial cells
Monocytes have been shown to migrate across human BBB-endothelial cells in response to monocyte chemoattractant protein (MCP)-1/CCL2 produced by BBB-endothelial cells (Seguin et al., 2003
). To determine whether the migration process could influence the phenotype of human monocytes, we used the modified Boyden chamber model in which human BBB-endothelial cells separate the upper and lower chambers. From the CD14+ monocytes originally seeded on BBB-endothelial cells, 40% failed to migrate after 48 h and 15% transmigrated completely to the lower chamber. These cells retained high expression of CD14 and CD16, and expressed low levels of HLA-DR, CD40, CD83, CD80, CD86 and CD123, as previously published (Seguin et al., 2003) (data not shown). Another population of migrated cells representing 
45% of the initial seeded population was identified in the sub-endothelial space, closely associated with BBB-endothelial cells. These cells differed from the migrated (lower chamber) population by their significantly lower expression of CD16 (data not shown) and did not constitute a homogenous population as 5% of cells acquired CD83, CD123, CD209, HLA-DR, CD80, CD86 and CD40, suggestive of a partial dendritic cell phenotype (Fig. 1A left panel and Table 1, column untreated BBB-endothelial cells). None of these markers were detected on the peripheral blood CD14-selected monocyte population prior to migration. The presence of ACM had no effect on the phenotype (data not shown). Given the sub-endothelial location of this dendritic cell-like population, they were named endothelial-associated dendritic cells (eDCs).
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Migration of monocytes across TNF-
- and IFN--activated BBB-endothelial cellsUsing the Boyden chamber migration assay, we next analysed the phenotype of migrating monocytes, in the context of an inflamed BBB, as would presumably occur during lesion development in multiple sclerosis. Confluent BBB-endothelial cells were cultured overnight in the presence of TNF-
and IFN- (both 100 U/ml), two cytokines reported to partake in multiple sclerosis pathogenesis (Sospedra and Martin, 2005) and known to activate endothelial cells (Calabresi et al., 2001; Biernacki et al., 2004). After several washes to remove residual cytokines, CD14+ monocytes obtained from healthy donors were added for 48 h on these activated BBB-endothelial cells. Upon migration across IFN-- and TNF--activated BBB-endothelial cells, a large proportion of sub-endothelial monocytes (namely eDCs) acquired the dendritic cell markers CD83 (21%), CD123 (54%), CD209 (40%), HLA-DR (78%), CD80 (76%) and CD86 (88%) (Fig. 1A right panel and Table 1, column activated BBB-endothelial cells), when compared to cells that migrated across resting BBB-endothelial cells (Fig. 1A and Table 1, column untreated BBB-endothelial cells). Addition of astrocyte media to endothelial cells did not affect the dendritic cell phenotype (data not shown). As shown in Fig. 1B, these eDCs remained in close contact with BBB-endothelial cells and adopted a typical elongated (arrowhead) and ramified (arrow) dendritic cell morphology, comparable to dendritic cells generated in vitro from peripheral blood CD14+ monocytes (Fig. 1B). These observations suggest that activated BBB-endothelial cells have the capacity to induce differentiation of peripheral blood CD14+ monocytes into dendritic cells.
Endothelial-associated dendritic cells are potent phagocytes
As dendritic cells are known for their capacity to capture tissue antigens, we tested whether CD83+ eDCs have the capacity to phagocytose FITC-labelled dextran. Figure 1C shows that at 37°C, CD83+ eDCs collected from the sub-endothelial space have a higher capacity to capture FITC-labelled dextran (open histogram) when compared to ex vivo monocytes (shaded histogram), confirming that eDCs behave as traditional dendritic cells (n = 4). In order to validate this assay, the experiment was also conducted at 4°C. The endocytic activity of eDCs was markedly reduced.
BBB-endothelial cells are potent producers of TGF-β, GM-CSF and IL-6
We elected to identify the soluble mediator(s) secreted by BBB-endothelial cells which could induce monocyte differentiation, looking specifically at molecules that have previously been described to influence the formation of dendritic cells or macrophages in vitro (Chomarat et al., 2000; Chen et al., 2002; Zhang et al., 2004). RNA isolated from resting and activated BBB-endothelial cells were analysed by RPA. As shown in Fig. 2A, an up-regulation of IL-6, TGF-βs, G-CSF and GM-CSF mRNAs was detected upon BBB-endothelial cell activation. Equal loading was confirmed by comparable expression of housekeeping genes L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) between samples (gels shown are representative of four different RPAs using five distinct BBB-endothelial cell preparations).
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We further investigated the cytokines secreted by BBB-endothelial cells in supernatants collected 24 h after inflammatory stimulation. As shown in Fig. 2B, a significant up-regulation in the secretion of GM-CSF and TGF-βs by activated BBB-endothelial cells was confirmed at the protein level. However, while levels of IL-6 were high in resting BBB-endothelial cells, they remained unchanged in supernatants from those that were activated. G-CSF protein could not be detected in activated or resting BBB-endothelial cell supernatants.
TGF-β and GM-CSF secreted by BBB-endothelial cells contribute to the differentiation of monocytes into eDCs
To determine whether the increase in GM-CSF and TGF-β secretion by activated BBB-endothelial cells plays a role in the differentiation of peripheral blood monocytes into eDCs, we used blocking antibodies directed at GM-CSF and/or TGF-β1,2,3 during the transmigration assay. As shown in Table 2, simultaneously blocking GM-CSF and TGF-β significantly decreased the number of cells expressing CD83 (from 27% to 7%), CD123 (from 52% to 16%), CD209 (from 43% to 15%) (all, P < 0.01). Furthermore, inactivating GM-CSF and TGF-β allowed monocytes to retain CD14 (79% as compared to 19%) while levels of CD11c were unaffected. Surprisingly, the eDC phenotype was not altered when blocking antibodies were tested separately, suggesting that both cytokines are required to achieve monocyte maturation into eDCs (Table 2).
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To further determine whether soluble mediators of activated BBB-endothelial cells were sufficient to induce monocyte maturation into eDCs, CD14+ monocytes were cultured in the presence of culture media conditioned with TNF-
- and IFN--activated BBB-endothelial cells. Under these experimental culture conditions, we did not observe a change of CD14+ monocytes into dendritic-like cells (data not shown). When migration was performed in the presence of anti-ICAM-1 and anti-VCAM-1 blocking antibodies, the number of CD14+ monocytes that reached the sub-endothelial space was significantly reduced, but still sufficient to allow for phenotypic analysis. When sub-endothelial cells were collected from ICAM-1 and VCAM-1 blocking experiments, we did not observe a phenotypic change of CD14+ monocytes into eDCs (Table 2). These results suggest that in our model, contact-mediated mechanisms are also required to induce the phenotypic differentiation of CD14+ monocytes into eDCs. Our data hence demonstrate that an activated BBB favours the differentiation and maturation of CD14+ monocytes into dendritic cells, through the secretion of GM-CSF and TGF-β, and through yet undetermined contact-dependent mechanisms.
Endothelial-associated dendritic cells have the ability to secrete IL-6, IL-12p70 and TGF-β1
As cytokines produced by antigen-presenting cells have been shown to play a critical role in modulating multiple immune responses, we analysed the eDC cytokine profile focusing particularly on cytokines known to induce CD4+ Th cell polarization. Purified ex vivo CD14+ monocytes, migrated CD14+ cells and eDCs were assessed for their cytokine secretion by ELISA. As shown in Fig. 3, eDCs secreted significantly higher levels of IL-12p70 than ex vivo CD14+ monocytes and migrated CD14 cells (P < 0.01, n = 3 in duplicate). In addition, eDCs secreted elevated levels of TGF-β1 and IL-6, as compared to ex vivo CD14+ monocytes and migrated CD14 cells (P < 0.01, n = 3 in duplicate). Levels of IL-23 (p19) were below detection for all three cell subtypes (data not shown). In these assays, eDCs and monocytes were not stimulated or activated.
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Endothelial-associated dendritic cells behave as professional antigen-presenting cells
We evaluated the functional capacity of the eDCs to activate peripheral blood CD4+ T lymphocytes. Ex vivo CD14+ monocytes, migrated CD14 cells and CD83+ eDCs were purified, irradiated and co-cultured for 5 days with allogeneic CD4+ T lymphocytes, at various ratios. As shown in Fig. 4A, eDCs induced a significant proliferation of allogeneic CD4+ T lymphocytes, as assessed by thymidine incorporation, and confirmed by CFSE assay (Fig. 4B). Such eDC-induced proliferation was optimal at a ratio of 1 eDC to 5 CD4+ T lymphocytes and was comparable to the strong proliferative response induced by fresh autologous CD14+ monocytes in the presence of PHA (
18 000 cpm; positive control). Stimulation indices comparing proliferation of CD4+ T lymphocytes in the absence of antigen-presenting cells (>600 cpm) with CD4+ T lymphocytes co-cultured with eDCs were consistently greater than 30 (mean = 42 ± 2.7).
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Endothelial-associated dendritic cells favour the production of IL-17 or IFN-
by CD4+ T lymphocytesTo determine whether eDCs could promote the polarization of CD4+ T lymphocytes, we evaluated, by ICS and ELISA, the cytokine profile of CD4+ T lymphocytes after co-culture with allogeneic CD83-expressing eDCs. As shown in Fig. 5A, eDCs promoted the expansion of a greater number of IFN-
-producing Th1 lymphocytes, as compared to migrated CD14 cells and ex vivo CD14+ monocytes. These results were confirmed by ELISA (Fig. 5B). Furthermore, we noted that CD83+eDCs could induce IL-17 production by a distinct subset of CD4+ T lymphocytes (29.8–31.6% of all CD45RO T lymphocytes, Fig. 5A), most of which did not express IFN-. Only 10% of IL-17+ CD45RO lymphocytes co-expressed IFN-. Such IL-17-producing T lymphocytes were undetectable in the co-cultures with migrated CD14 cells and with PHA, probably as a consequence of higher IFN- levels (Harrington et al., 2005; Park et al., 2005) or possibly because of high levels of IL-2, as recently reported (Laurence et al., 2007). When eDCs were co-cultured with CD4+ T lymphocytes, IL-10 secretion remained low (Fig. 5B), and IL-10+ CD4+ T lymphocytes could not be detected by ICS (Fig. 5A). Levels of additional Th2 cytokines (IL-4 and IL-5) were consistently below threshold, when analysed by ICS or ELISA (data not shown). From these observations, we conclude that CD83+eDCs induce CD4+CD45RO+ T lymphocyte proliferation, and polarize T cells towards distinct Th17 and Th1 phenotypes, probably depending on their initial differentiation profile.
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Presence of endothelial-associated dendritic cells in active and demyelinating multiple sclerosis lesions
To validate the biological and clinical relevance of these in vitro observations, we performed in situ immunostaining for CD83 and CD209 using four multiple sclerosis brain specimens showing intense immune cell infiltration within confluent areas of demyelination (by LFB and H&E), two important criteria used to define active multiple sclerosis lesions. As shown in Fig. 6, CD83 (upper left panel) and CD209 (upper right panel) immunopositive cells were detected within perivascular cuffs of immune cells in demyelinated areas (2–26 cells per field, mean = 6, in n = 14 plaques and 25 vessels). CD83+ and CD209+ dendritic cells exhibited a ramified and elongated morphology, as one would expect for dendritic cells. Such cells could only be seen in infiltrated and demyelinated areas, and were not detected in normal-appearing white matter or chronic inactive plaques (data not shown). We did not observe any difference in the number of dendritic cells, whether there were one or more vessels per plaque. IgG isotype controls are shown in the lower panels.
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Infiltrating CD4+ T cells express IL-17 in multiple sclerosis lesions
To validate our in vitro findings, we performed double immunohistofluorescence staining for CD4 and IL-17 on active multiple sclerosis lesions. While no CD4+ T lymphocytes could be detected in CNS material obtained from non-multiple sclerosis controls (traumatic death, Fig. 7A left panels), numerous CD4+ T lymphocytes (red) expressing IL-17 (green) were seen in active multiple sclerosis lesions (n = 18 lesions from four multiple sclerosis samples, Fig. 7A right panels and Fig. 7B).
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dendritic cells are known as the most potent antigen-presenting cells. Although they have long been considered to be absent from the animal and human CNS, several groups have recently demonstrated that they play an important role in the development of CNS inflammation in animals (Karman et al., 2004
; Greter et al., 2005; McMahon et al., 2005; Bailey et al., 2007) and are present in multiple sclerosis lesions (Plumb et al., 2003; Serafini et al., 2006). While previous reports showed that dendritic cells can migrate across brain endothelial cells and access the CNS in vivo (Karman et al., 2004, 2006; Zozulya et al., 2007), our current study supports the notion that perivascular dendritic cells within the human CNS also arise from the migration of a subpopulation of peripheral blood monocytes across the inflamed BBB. We further demonstrate that this process is dependent on the secretion of GM-CSF and TGF-β by activated BBB-endothelial cells and provide evidence that such perivascular dendritic cells can induce CD4+ T lymphocyte proliferation and polarize cytokine secretion towards either a Th17 or a Th1 phenotype.
Our data demonstrate that upon migration across human BBB-endothelial cells, freshly ex vivo and non-manipulated peripheral blood CD14+ monocytes acquire dendritic cell markers and functions. We show that following inflammatory challenge, BBB-endothelial cells up-regulate their secretion of GM-CSF and TGF-β. Both cytokines were also previously shown to be secreted by other CNS cells including astrocytes, oligodendrocytes and microglia, suggesting that additional sources of these cytokines in situ can act in a coordinate fashion to promote dendritic cell differentiation and possibly maturation (Benveniste, 1988; Malipiero et al., 1990; Pratt and McPherson, 1997). With regard to GM-CSF, elevated levels of this cytokine are known to correlate with the active phase of multiple sclerosis (Carrieri et al., 1998). Moreover, GM-CSF–/– mice are resistant to EAE, display decreased antigen-specific proliferation of splenocytes and fail to sustain immune cell infiltrates in the CNS (McQualter et al., 2001). These observations combined with the current report, suggest that GM-CSF plays a significant role in the development of inflammatory and demyelinating lesions in the CNS. Although the influence of GM-CSF on the differentiation of monocytes into dendritic cells is well established in the periphery, our report is the first to suggest that a similar process can also occur within the human CNS, and more specifically under the influence of the BBB.
TGF-β is widely known for its immunosuppressive and anti-inflammatory effects (Weinberg et al., 1992; Santambrogio et al., 1993; Sharma et al., 1996). Whereas initial EAE studies suggested that increased peripheral TGF-β levels could suppress the disease (Kuruvilla et al., 1991; Santambrogio et al., 1993), more recent studies demonstrate that local TGF-β expression within the CNS parenchyma can enhance immune cell infiltration and augment CNS damage following peripherally triggered autoimmune responses (Feldmann et al., 1996; Wyss-Coray et al., 1997; Veldhoen et al., 2006b; Li et al., 2007). Furthermore, in conjunction with IL-6 and/or IL-4, TGF-β can induce the differentiation of peripheral blood-derived CD34+ progenitor cells into Langerhans cells (Strobl et al., 1996; Caux et al., 1999; Jaksits et al., 1999; Ginhoux et al., 2006). In this context, our data explain why most dendritic cells detected within the CNS are closely associated with the BBB, as reported in human CNS-inflammatory diseases, in EAE (Boven et al., 2000; Williams et al., 2001; Greter et al., 2005; Serafini et al., 2006), and in the current study. We believe that the vessel-associated and sub-endothelial CNS dendritic cells, that we call eDCs, differentiate from the migrating peripheral blood CD11c+CD14+ monocytes under the influence of GM-CSF and TGF-β secreted by inflamed BBB-endothelial cells. We also demonstrate that this differentiation process is intimately associated with adhesion and migration, since anti-ICAM-1 or anti-VCAM-1 treatment reduced both monocyte migration and acquisition of dendritic cell characteristics. Several groups have already demonstrated the important role of myeloid cell-expressed integrins, such as CD11b (Shi et al., 2004) and CD49d (Puig-Kroger et al., 2000) in the differentiation of monocytes into macrophages or dendritic cells. Data presented in the current study support the notion that integrin-mediated signalling promotes the differentiation and/or maturation of migrating monocytes into more specialized cells such as dendritic cells and confirm the important role for BBB-endothelial cell-expressed ICAM-1 and VCAM-1 in this biological process.
In this study, we further demonstrate the presence of CD83+, CD209+ dendritic cells in situ, specifically within active multiple sclerosis lesions. Our data are in agreement with studies published by Aloisi (Serafini et al., 2006) and McQuaid (Plumb et al., 2003), and extend these studies by providing the biological mechanism by which monocytes adopt a dendritic cell phenotype upon migration. Although we suspect that perivascular dendritic cells found in multiple sclerosis lesions arise from migrating peripheral blood CD14+ monocytes, we cannot exclude the possibility that these dendritic cells would originate from CNS resident cells, such as microglia, or from peripheral blood dendritic cells that have gained access to the CNS through the BBB.
Several groups have demonstrated that dendritic cells partake in the development of CNS inflammation in animals whether autoimmune (Serafini et al., 2000; Plumb et al., 2003; Karman et al., 2004; Greter et al., 2005) or induced by viruses (Lauterbach et al., 2006). Our report provides evidence that perivascular CD83+ CD209+ eDCs are able to secrete TGF-β and IL-6, thus favouring the differentiation of CD4+ T lymphocytes into IL-17-secreting cells (Th17). Moreover, these eDCs are also able to secrete IL-12 and allow differentiation of CD4+ T lymphocytes into IFN--secreting (Th1) cells. These findings are in agreement with previously published studies (Saint-Vis et al., 1998; Morelli et al., 2001; Fonteneau et al., 2003; Bettelli et al., 2006; Weaver et al., 2006; Mangan et al., 2006). Although there is still controversy on the exact phenotype of CD4+ T lymphocytes involved in autoimmune CNS inflammatory diseases, especially in humans, both Th17 and Th1 lymphocytes have been reported to be implicated in CNS inflammatory lesion formation. In a recent publication, Sallusto's group demonstrated that IL-1β and IL-6 are required for the differentiation of naive CD4+CD45RA+ human T cells into Th17 lymphocytes (Acosta-Rodriguez et al., 2007). They also provided evidence that addition of TGF-β prevents the differentiation of naive T lymphocytes into Th17 cells, a phenomenon that was previously reported for Th1 and Th2 lymphocytes (Sad and Mosmann, 1994). In the current study, we have used peripheral blood CD4+ T lymphocytes, containing both CD45RO and CD45RA lymphocytes, to generate Th17 cells upon co-culture with eDCs, and we demonstrate that the vast majority of Th17 lymphocytes are of the CD45RO memory phenotype. We speculate that such a population arises from the initial pool of CD45RO memory lymphocytes, as these cells are more prone to be polarized into Th17 lymphocytes (Kebir et al., 2007).
The in vitro and in situ data presented in this study, combined with our recent observation that Th1 and Th17 lymphocytes avidly migrate across the human and mouse BBB, suggest that the presence of Th1 and Th17 lymphocytes within the CNS reflects both a preferential migration of these cells across the BBB and their local expansion within the perivascular space, under the influence of eDCs. Our study provides strong evidence that in situ expansion of Th17 lymphocytes can occur directly within the human brain, and expands the initial in vivo observations by Greter et al. (2005) and Bailey et al. (2007) showing that CD11c+ perivascular dendritic cells partake in lymphocyte reactivation within the mouse CNS.
By restricting leukocyte migration to the CNS and limiting immune reactions and inflammatory processes occurring within the brain, the BBB has long been considered to promote the CNS immune privileged characteristics. However, this concept has recently been challenged by numerous observations, including the demonstration that (i) BBB-endothelial cells secrete chemokines in vitro and in situ (Simpson et al., 1998; McManus et al., 1998; Biernacki et al., 2001; Prat et al., 2002), (ii) adhesion molecules of the BBB play a critical role in the recruitment of immune cells to the CNS in animals (Archelos et al., 1993; Engelhardt et al., 1998) and (iii) anti-very late antigen (VLA)-4 therapy reduces CNS inflammation in humans (O’Connor et al., 2004). These studies, combined with the current report, suggest that although the BBB partakes in CNS homoeostasis, it can also shape auto-aggressive immune processes occurring early in the course of CNS inflammation.
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Acknowledgements |
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This study was supported by the Multiple Sclerosis Society of Canada (MSSC), the Canadian Institutes of Health Research (CIHR, MOP-81088) and by the Canadian Foundation for Innovation. I.I., H.K., A.D.-D. and R.C. hold studentships from the MSSC. K.W. holds a fellowship from the MSSC. N.A. holds a CIHR Senior Research Fellowship Phase 2. A.P. is a research scholar from the Fonds de la Recherche en Santé du Québec, and holds the Donald Paty Career Development Award of the MSSC. We thank Janet Laganière for her excellent technical assistance on the confocal microscope and Jack P. Antel for providing assistance and human tissue.
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