Syndecan-1, a Cell Surface , Negatively Regulates Initial Leukocyte Recruitment to the Brain across the Choroid Plexus in Murine Experimental Autoimmune This information is current as Encephalomyelitis of September 24, 2021. Xueli Zhang, Chuan Wu, Jian Song, Martin Götte and Lydia Sorokin J Immunol 2013; 191:4551-4561; Prepublished online 27 September 2013; Downloaded from doi: 10.4049/jimmunol.1300931 http://www.jimmunol.org/content/191/9/4551 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/09/27/jimmunol.130093 Material 1.DC1 References This article cites 61 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/191/9/4551.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Syndecan-1, a Cell Surface Proteoglycan, Negatively Regulates Initial Leukocyte Recruitment to the Brain across the Choroid Plexus in Murine Experimental Autoimmune Encephalomyelitis

Xueli Zhang,* Chuan Wu,*,1 Jian Song,* Martin Go¨tte,† and Lydia Sorokin*

The cell surface heparan sulfate proteoglycan, syndecan-1, has been reported to be a negative regulator of various inflammatory processes, but its precise mode of action is poorly defined. In this study, we use the murine model of the 35–55 peptide of myelin oligodendrocyte glycoprotein–induced experimental autoimmune encephalomyelitis (EAE), a T –mediated inflamma- tion where the steps in disease development and recovery are well characterized, to decipher how syndecan-1 impacts on the inflammatory reaction. Syndecan-1 knockout (Sdc-12/2) mice show enhanced disease severity and impaired recovery. The use of Downloaded from bone marrow chimeric mice reveals that both an immune cell and a CNS-resident source of syndecan-1 contribute to this phenotype. Epithelial cells of the choroid plexus, where initial CCL20-induced leukocyte recruitment to the brain occurs, are identified as the predominant site of syndecan-1 expression. Syndecan-1 is lost from this site during the course of EAE by shedding into the cerebrospinal fluid, which correlates with loss of epithelial cell surface–bound CCL20 and is associated with the upreg- ulation of IL-6 expression. In Sdc-12/2 mice, early leukocyte recruitment via the choroid plexus is enhanced, and IL-6 is elevated, http://www.jimmunol.org/ which collectively results in higher numbers of the disease inducing Th17 cells in the CNS, thereby contributing to enhanced disease severity. Furthermore, Sdc-12/2 mice have intrinsically elevated numbers and higher myelin oligodendrocyte glycoprotein–specific Ab levels during EAE, which we propose contributes to impaired recovery. Our data identify the choroid plexus epithelium as a novel source of IL-6 in EAE and demonstrate that its expression negatively correlates with syndecan-1 expression at this site. The Journal of Immunology, 2013, 191: 4551–4561.

ell surface heparan sulfate are gaining (extracellular domain), which carries the glycosylation sites, attention because of their large potential to bind a wide a transmembrane domain and a cytoplasmic domain. The trans- C variety of bioactive molecules and thereby influence membrane and cytoplasmic domains are highly conserved among by guest on September 24, 2021 developmental processes but also pathological situations, including the four syndecans, whereas the ectodomain shows the greatest inflammation (1). As highly glycosylated, sulfated molecules and, variability both in sequence and in glycosaminoglycan hence, negatively charged molecules, they have a large capacity to chains (9, 10). Furthermore, the ectodomain of all syndecans have bind to other charged molecules such as cytokines and chemo- been reported to be shed from the cell surface by matrix metal- kines (2) and have been implicated in the presentation of cytokines loproteinases (9, 11, 12), which can then have paracrine or auto- to cells (3–5) and in the activation of integrin receptors (6–8). crine effects or act as competitive inhibitors, introducing a further Syndecans are the best studied class of cell surface heparan sulfate complicating mode by which they can alter cell functions. proteoglycans (9); they exist in four different forms, syndecan 1–4, Broadly speaking, the different syndecans have distinct dis- all of which consist of a core protein containing an ectodomain tributions in vivo, with syndecan-1 occurring on mainly epithelial and plasma cells, syndecan-2 on endothelial cells, syndecan-3 on neural crest–derived cells, and syndecan-4 showing low-level ubiq- 2 2 *Institute for Physiological Chemistry and Pathobiochemistry, University of Muenster, uitous expression (13). Although syndecan-1 knockout (Sdc-1 / ) † 48149 Muenster, Germany; and Department of Gynecology and Obstetrics, University mice show exacerbated disease symptoms in several inflammatory of Muenster, 48149 Muenster, Germany 1 models (14), precisely why this is the case remains unclear. Most Current address: Center for Neurologic Disease, Brigham and Women’s Hospital, 2/2 Harvard Medical School, Boston, MA. studies report enhanced immune cell infiltration in Sdc-1 mice, Received for publication April 8, 2013. Accepted for publication August 26, 2013. which has been correlated with enhanced extravasation from post- capillary venules because of effects ranging from modulation of This work was supported by the German Research Foundation (Grants SO 285/9-1 and SFB 1009 A02), the European Union’s Seventh Framework Programme (Grant integrin activity and interaction with cell adhesion molecules FP7/2007-2013), and Grant Agreement 202213 from the European Stroke Network (14, 15) to modulation of cytokine and chemokine gradients (16). (to L.S.). X.Z. was supported by the Chinese Scholarship Council. Yet, the expression of syndecan-1 on endothelium and immune Address correspondence and reprint requests to Dr. Lydia Sorokin, Institute for cells in vivo is difficult to detect, with the notable exception of Physiological Chemistry and Pathobiochemistry, University of Muenster, Waldeyer- strasse 15, 48149 Muenster, Germany. E-mail address: [email protected] B , which may be partially because of its dynamic The online version of this article contains supplemental material. regulation (9, 17–19). Abbreviations used in this article: CSF, cerebrospinal fluid; DC, ; LN, In this study, we investigate how syndecan-1 impacts on neu- lymph node; MBP, myelin basic protein; MECA32, mouse endothelial cell Ag 32; roinflammation using the murine model of the 35–55 peptide of 2/2 MOG, myelin oligodendrocyte glycoprotein; q-PCR, quantitative PCR; Sdc-1 , myelin oligodendrocyte glycoprotein (MOG )–induced experi- Syndecan-1 knockout; Treg, regulatory ; WT, wild-type. 35–55 mental autoimmune encephalomyelitis (EAE), a T lymphocyte– Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 mediated inflammation where the steps in disease development www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300931 4552 SYNDECAN-1 IN EAE have been well defined and can be readily followed: early re- Bone marrow chimeric mice cruitment of the disease-inducing encephalitogenic Th17 cells to Recipient mice were lethally irradiated with a single dose of 11 Gy and the CNS occurs via the choroid plexus and follow a CCL20- reconstituted by i.v. injection of 107 donor bone marrow cells. CD45.1 dependent gradient, whereas subsequent recruitment of both en- versus CD45.2 allelic markers were used to trace donor versus host cells. cephalitogenic T cells and bystander cells, such as , Animals were analyzed after 6–8 wk by flow cytometry for reconstitution . which contribute to disease severity, occurs via postcapillary ven- of the hematopoietic system. Only mice with 95% donor cell engraft- ments were used in active EAE experiments. ules within the brain parenchyma (20). Following peak recruitment, disease symptoms normally wane, resulting in a recovery phase Flow cytometry characterized by increasing numbers of CD25+Foxp3+ regulatory Mice were perfused with PBS before spleens, LNs, and brains were T cells (Tregs) and reduced numbers of effector Th17 and Th1 cells. harvested, and total cells were isolated by cell straining (70 mmfor Our data demonstrate that the main site of syndecan-1 expression in spleens and LNs, 100 mm for brains). Erythrocytes were lysed in spleen the brain is the choroid plexus epithelium where its expression preparations by incubating the cells on ice for 3 min with lysing buffer correlates with that of CCL20 and where it negatively correlates (BD Pharm Lyse); leukocytes were isolated from blood using a Ficoll gradient (Cedarlane Laboratories); and brain homogenates were sepa- with IL-6 expression, hence, impacting on the initial recruitment of rated into neuronal and leukocyte populations by discontinuous density the encephalitogenic T cells to the brain. We demonstrate that the gradient centrifugation using isotonic Percoll (Amersham Biosciences). choroid plexus epithelium is an important source of IL-6 at early For intracellular cytokine staining, isolated leukocytes were stimulated EAE and that its expression is increased in the absence of syndecan- with PMA (10 ng/ml)/ionomycin (1 mg/ml) (Sigma-Aldrich) in the 1. In the absence of syndecan-1, early leukocyte recruitment via the presence of brefeldin A (Sigma-Aldrich) at 37˚C for 6 h. The intracellular staining (eBioscience) was used to permeablize and fix the cells prior choroid plexus is enhanced, and CNS levels of IL-6 are elevated, to staining for Foxp3 and intracellular cytokines. Flow cytometry anal- Downloaded from which collectively results in higher numbers of the disease-inducing ysis was performed using a FACSCalibur (BD Biosciences) with the Abs Th17 cells and potentially their prolonged survival, thereby con- listed above. tributing to enhanced disease severity. In addition, Sdc-12/2 mice Immunofluorescence staining have intrinsically elevated plasma cell numbers and higher MOG- specific Ab levels during EAE, which we propose contributes to Mice were intracardially perfused with 4% paraformaldehyde in PBS, and samples were dissected and fixed 1.5 h at 4˚C in 4% paraformaldehyde. impaired recovery. Our data work identifies the choroid plexus http://www.jimmunol.org/ Tissues were immersed in 30% sucrose in PBS and subsequently frozen in epithelium as a novel source of IL-6 in EAE and demonstrates that Tissue-Tek (Sakura Fenetek). Eight-micrometer sections were blocked in its expression is negatively correlated with syndecan-1 expression 1% BSA in PBS and incubated with primary Ab overnight at 4˚C and with at this site. secondary Ab for 2 h at room temperature. Because the heparan sulfate chains of syndecans may mask binding sites of primary Abs, unmasking techniques were performed prior to blocking of tissue samples. These Materials and Methods included 0.1 M acetic acid for 5 min at room temperature or 1 U/ml Mice heparitinase III (Sigma-Aldrich) in 50 mM Tris buffer (pH 7.5) for 10 min at room temperature. Secondary Abs included goat anti-rabbit and donkey 2 2 Syndecan-1 knockout (Sdc-1 / ) mice have been previously described anti-rat IgG conjugated with Alexa Fluor 488 or Cy3 (Molecular Probes). + 2/2

(21). Blimp-1-GFP mice (22) were bred with Sdc-1 mice to generate Sections were examined using a Zeiss AxioImager microscope equipped by guest on September 24, 2021 2 2 Sdc-1 / /Blimp-1-GFP+ mice and heterozygous littermate controls. with epifluorescent optics and documented using a Hamamatsu ORCA ER C57BL/6-Ly5.1 (CD45.1) mice were used in passive transfer and bone camera. Images were analyzed using Volocity 6.0.1 software (ImproVi- marrow chimera experiments. All experiments were conducted according sion; PerkinElmer). to German Animal Welfare guidelines. T cell proliferation Abs + In vitro. CD4 T lymphocytes from LNs of MOG35–55-immunized wild- Sdc-12/2 The Abs used in immunofluorescence staining and flow cytometry were as type (WT) or mice were cultured at 37˚C for 3 d with irradiated follows: pan-laminin (455) (23), laminin g1 (24), CD45 (30G.12), CD45.2 splenic dendritic cells (DCs) (30 Gy) (from nonimmunized mice) as APCs (104; eBioscience), CD45.1 (A20; BD Pharmingen), CD11b/MAC-1 (M1/ plus MOG35–55, OVA fragment 329–339 (Ova323–339) (Schafer-N), or with Ab to CD3 and CD28 (BD Pharmingen). T lymphocyte proliferation was 70; BD Pharmingen), CD11c (N418; eBioscience), CD4 (H129.19; BD determined by [3H]thymidine (Amersham Biosciences) incorporation over Pharmingen), CD8 (53-6.7; eBioscience), B220 (RA3-6B2; BD Pharmin- 2 2 2 2 12 h. WT or Sdc-1 / T lymphocytes were cocultured with WT or Sdc-1 / gen), syndecan-1 (281-2; BD Pharmingen), CD19 (1D3; BD Pharmingen), splenic DC in separate experiments. Similar experiments were performed CD25 (7D4; BD Pharmingen), Foxp3 (FJK-16S; eBioscience), IL-17 2 2 Sdc-1 / (TC11-18H10.1; BD Pharmingen), biotin-labeled IL-6 (MP5-32C11; with irradiated and WT B lymphocytes (5 Gy) as APCs. eBioscience), IFN-g (XMG1.2; BD Pharmingen), CD16/CD32 (2.4G2; In vivo. To measure in vivo proliferation of MOG35–55-specific T cells, + + 2/2 BD Pharmingen), CCL20 (MIP-3a) (AB9829; Abcam), CD68 (FA-11; CD45.1 T cell blasts were transferred to CD45.2 WT and Sdc-1 mice, + + BioLegend), MHC class II (M5/114.15.2; eBioscience), metallophilic and BrdU incorporation into the CD45.1 CD4 population was measured. macrophages 1 (BMA Biomedicals) and S100A9 (25), and mouse endo- BrdU was injected i.p. on days 21 and 23 after transfer. LN, spleen, blood, + + thelial cell Ag 32 (MECA32) (26). and CNS were removed 12 h after the last injection, and CD45.1 CD4 T lymphocytes were isolated and analyzed for BrdU incorporation by flow Active EAE cytometry. Analysis of MOG -specific proliferation and cytokine EAE was induced using MOG35–55 as described previously (27, 28). We 35–55 define early EAE as day 10 after immunization, peak EAE as day 17 after responses immunization, and the recovery phase as .day 20 after immunization. For proliferation assays, LN cells or splenocytes (2.5 3 105 cells per 0.2 ml) Passive EAE from EAE mice were cultured in 96-well plates in RPMI 1640 medium (Life Technologies) plus 10% FCS, 10 mM sodium pyruvate, 1% penicillin/ Ten days after MOG35–55 immunization, draining lymph node (LN) cells streptomycin, and 0.05 mM 2-ME in the presence of different concentrations 7 21 were isolated and seeded at 1 3 10 cells ml in IMDM (Life Tech- (0, 1.1, 3.3, 11, 33, and 100 mg/ml) of MOG35–55 at 37˚C in 5% CO2 for 72 h. nologies) plus 10% FCS. The cells were stimulated with 20 mgml21 Cell proliferation was determined by [3H]thymidine incorporation over the 21 6 MOG35–55 peptide for 2 d and then in the presence of 10 ng ml IL-2, 10 last 12 h. For the cytokine responses, LN cells or splenocytes (1 3 10 ng ml21 IL-23, 5 ng ml21 IL-1b, 10 ng/ml IL-6, and 10 mg/ml anti–IFN-g cells per 0.2 ml) from EAE mice were cultured as above in the presence or 7 and anti–IL-4 for 3 d. A total of 2 3 10 cells/mouse were transferred i.v. absence of 20 mg/ml MOG35–55 at 37˚C in 5% CO2 for 72 h. Culture Mice were then injected i.p. with 200 ng pertussis toxin on days 0 and 2 supernatants were harvested, and cytokines were analyzed using the after transfer. The polymorphic lineage determinants (CD45.1 or CD45.2) cytometric bead assay. All proliferation and cytokine response meas- were used for tracking donor versus host immune cells. urements were performed in triplicate. The Journal of Immunology 4553

Cytometric bead assay AGG TCG GTG TGA ACG GAT TTG-39 (sense) and 59-GGG GTC GTT

2/2 GAT GGC AAC A-39 (antisense). CNS samples from EAE WT and Sdc-1 mice were frozen in liquid mRNA expression was normalized to endogenous GAPDH expression in nitrogen and subsequently homogenized on ice in radioimmunoprecipitation the same sample. Quantitative real-time PCR was performed with an assay buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% NaDoC, 0.1% SDS, 50 Applied Biosystems PRISM 7300 Sequence Detection System by using the mM Tris HCl [pH 8], and proteinase inhibitor mixture (Sigma-Aldrich)). default thermal cycling conditions (10 min at 95˚C and 40 cycles of 15 s at 3 After centrifugation (12,000 g for 10 min), supernatants were collected, 95˚C plus 1 min at 60˚C). Relative quantitation was performed using the and cytokine and chemokine concentrations were determined using the comparative cycle threshold method (32). Three to five biological repli- Chemomix kit (Bender MedSystems) and mouse FlowCytomix kits (eBio- cates were used for each point investigated. science). CSF samples were directly analyzed for chemokine and cytokine levels using the same kits. Statistical analyses Measurement of plasma cell numbers and MOG -specific Quantitative data are expressed as means 6 SEM. Significance of cell 35–55 , serum titers numbers was analyzed with an unpaired Student t test. A p value 0.05 was considered significant. Because syndecan-1 (CD138) could not be used as a marker for plasma cells in the Sdc-12/2 mice, they were crossed to the Blimp-1-GFP+ transgenic + Results line to generate mice carrying GFP plasma cells. Blimp-1 is a transcrip- 2/2 tion factor specific for plasma cells (22). Blood, spleen, LN, bone marrow, Sdc-1 mice show increased EAE severity and impaired and CNS of naive and EAE-induced Sdc-12/2/Blimp-1-GFP+ mice were recovery 2 + analyzed by flow cytometry for syndecan-1 GFP . 2 2 Sdc-1 / mice showed enhanced EAE severity and prolonged Parallel analyses of serum titers for MOG35–55-specific Abs was per- formed using SBA Clonotyping System-B6/C57J-HRP kit (Southern recovery compared with WT littermates (Fig. 1A), which corre- Downloaded from Biotechnology Associates) as described previously (29, 30). Briefly, 96- lated with elevated numbers of the disease-inducing Th1 and Th17 well ELISA plates (Nunc) were precoated overnight at 4˚C with 10 mg/ml cells and lower numbers of CD4+CD25+Foxp3+ Tregs at early MOG35–55 peptide. Plates were incubated with serum samples, and bound (day 10) and peak (day 17) stages of the disease but not in the Ig was detected using HRP-conjugated goat anti-mouse Abs to different . Igs. ABTS was used as a color substrate, and OD was measured at 405 nm. recovery phase ( day 20) (Fig. 1B, 1C). Demyelination, which is associated with disease severity, was analyzed by staining for Collection of choroid plexus and cerebrospinal fluid myelin basic protein (MBP) together with CD45 to define sites of WT and Sdc-12/2 mice were sacrificed at different stages of EAE and 2–5 infiltrated leukocytes and pan-laminin or laminin g1 chain to http://www.jimmunol.org/ ml cerebrospinal fluid (CSF) per mouse was collected from the cisterna define cuff borders (23), revealing a similar loss of MBP staining magna as described previously (31). Brains were then isolated and the at sites of leukocyte penetration of the CNS parenchyma in WT positions of the choroid plexus in the fourth ventricle, and the lateral 2 2 and Sdc-1 / mice (Fig. 1D). However, stereological analyses ventricles were determined under a dissecting microscope and isolated. 2/2 The choroid plexus appeared as a vascularized “gelly” membrane floating revealed larger cuff numbers in Sdc-1 brains at peak and, to in the liquid. Tissues were snap-frozen in liquid nitrogen, and tissues and a lesser extent, recovery phases and hence more extensive demye- CSF were stored at 280˚C. lination (Fig. 1E), consistent with the more severe disease phenotype. Dot blots Flow cytometry of the CNS, spleen, LN, and blood for different immune cell populations at peak and recovery phases of the disease by guest on September 24, 2021 To investigate presence of syndecan-1 ectodomain in CSF, 2-ml CSF revealed that CD4+ T lymphocytes and CD11b+ macrophages samples were dot-blotted onto a nitrocellulose membrane (Whatman). The 2/2 membrane was blocked in TBS containing 1% FCS, washed in TBS were elevated in Sdc-1 CNS only at peak disease (Supple- containing 1% FCS and 0.01% Tween 20, and incubated with biotinylated mental Fig 1A, 1B). Numbers of T cells, macrophages, and B cells anti–syndecan-1 ectodomain Ab (1:500; BD Pharmingen). Bound Abs in the circulation (data not shown), LNs, and spleens were not were detected with HRP-conjugated secondary Abs and the ECL system altered in the Sdc-12/2 mice compared with WT mice (Supple- (Pierce). A total of 2 mg purified recombinant syndecan-1 protein (R&D mental Fig. 1A, 1B), and there was no difference between naive Systems) was used as positive control, and secondary Ab alone was used as 2/2 a negative control. WT and Sdc-1 mice in CD4, CD8, B220, CD11b, and CD11c cell populations, as assessed by flow cytometry of the spleens and ELISA LNs (data not shown). Because myeloid cells in the CNS have To investigate whether syndecan-1 binds IL-6 or CCL20, 50 ng purified been associated with enhanced disease severity, different myeloid recombinant syndecan-1 was coated onto Nunc Maxisorb microtitre plates populations were analyzed during the recovery phase, revealing by overnight incubation at 4˚C. Plates were wash with PBS, blocked with a tendency for higher numbers of CD11b+MHCIIhigh, CD11b+ 1% BSA in PBS and incubated with different concentrations of IL-6 (0–10 + + + mg/ml) or CCL20 (1–2 mg/ml). Bound cytokine or chemokine was CD68 , and CD11b S100A9 macrophages, which denotes acti- 2/2 detected with biotin-labeled anti–IL-6 or anti-CCL20, respectively, and vated macrophages in the CNS of Sdc-1 mice, although these HRP-conjugated secondary Abs. Tetramethylbenzidine was used as a color differences were not statistically significant (Supplemental Fig. substrate; OD was measured at 450 nm. 1C). metallophilic macrophages 1+CD11b+ macrophages that have Quantitative PCR been shown to negatively regulate Treg numbers in EAE (33) were not different in WT and Sdc-12/2 mice (Supplemental Fig. 1C). Total RNA from the whole brains, choroid plexuses, LNs, or spleens were 2/2 prepared using the RNeasy kit (Qiagen) and cDNAs were generated (Omni- Passive transfer of Sdc-1 T cells to WT recipients excluded script RT Kit; Qiagen). Quantitative PCR (q-PCR) was performed using the possibility that a T cell source of syndecan-1 contributed to Brilliant SYBR Green QPCR MasterMix, and the primers are listed below this phenotype (Supplemental Fig. 2A, 2B). However, bone marrow according to the manufacturer’s instructions: syndecan-1, 59-GTG GCG chimeric animals—Sdc-12/2 recipients carrying WT bone marrow GCA CTT CTG TCA TC-39 (sense) and 59-GCA CCT GTG GCT CCT (Fig. 2A) and WT recipients carrying Sdc-12/2 bone marrow (Fig. TCG TC-39 (antisense); syndecan-2, 59-TGT GTC CGC AGA GAC GAG AA-39 (sense) and 59-GGA ATC AGT TGG GAT GTT GTC A-39 (anti- 2B)—identified the involvement of both an immune cell and a sense); syndecan-3, 59-ATA CTG GAG CGG AAG GAG GT-39 (sense) CNS-resident cell source of syndecan-1. Adoptive transfer of WT and 59-TTT CTG GTA CGT GAC GCT TG-39 (antisense); syndecan-4, 59- CD45.1 T cells to the CD45.2 Sdc-12/2 or WT recipients permitted AAC CAC ATC CCT GAG AAT GC-39 (sense) and 59-AGG AAA ACG analysis of whether in vivo proliferation of encephalitogenic T cells GCA AAG AGG AT-39 (antisense); IL-6, 59-AAC CAC ATC CCT GAG was promoted in the syndecan-1–negative environment, thereby AAT GC-39 (sense) and 59-AGG AAA ACG GCA AAG AGG AT-39 (an- + tisense); CCL20, 59-TCC AGA GCT ATT GTG GGT TTC A-39 (sense) and accounting for the elevated CD4 T cell numbers. These studies 59-GAG GAG GTT CAC AGC CCT TTT-39 (antisense); and GAPDH, 59- revealed the absence of any difference in the proliferation of WT 4554 SYNDECAN-1 IN EAE

sence of differences between WT and Sdc-12/2 T cell prolifera- tion either in response to anti-CD3 (Supplemental Fig. 2D) or in response to MOG35–55 or Ova323–339 (Supplemental Fig. 2E, 2F), in the presence of Sdc-12/2 or Sdc-1+/+ APCs, which included DCs and B cells. MOG35–55-specific proliferative and cytokine responses were measured from draining LNs, and splenocytes of bone marrow chimeric mice revealed an absence of differences in proliferation but elevated levels of IFN-g, IL-2, IL-6, IL-17, and TNF-a both in mice lacking an immune cell or a resident cell source of syndecan-1 (Supplemental Fig. 2G, 2H), suggesting that syndecan-1 also has peripheral effects. These results suggest that syndecan-1 contributes to regulation of T cell differentiation in the periphery and that the elevated Th1 and Th17 cells numbers in the Sdc-12/2 CNS at early and peak disease were due to enhanced T cell infiltration into the CNS and/ or their prolonged survival, rather than enhanced proliferation. In vivo expression of syndecan-1 by CNS resident and immune cells Downloaded from To precisely define whether immune cells and/or CNS-resident cells express syndecan-1 during EAE, immunofluorescence mi- croscopy was performed on tissue sections, whereas isolated cells were examined by flow cytometry and q-PCR. Triple immuno- fluorescence staining for syndecan-1, pan-laminin to define

the borders of blood vessels and the leptomeninges in the CNS http://www.jimmunol.org/ (28), together with CD45 to define sites of leukocyte infiltration, revealed no detectable staining for syndecan-1 either on the en- dothelium, astrocytes, or immune cells in perivascular cuffs within the brain (Fig. 3A). As the heparan sulfate chains of syndecans may mask Ab binding sites in vivo, several unmasking techniques were performed, including heparitinase treatment to remove heparan sulfate; however, no staining of perivascular cuffs was detectable; rather, there was strong staining of the choroid plexus. Immuno-

fluorescence staining with pan-laminin, to mark all basement mem- by guest on September 24, 2021 branes, together with MECA32 to mark the choroid plexus blood vessels (26), or syndecan-1, revealed strong staining of syndecan-1 on the basolateral surface of the epithelium of the choroid plexus and little or no staining of the choroid plexus blood vessels (Fig. 3B). Syndecan-1 Abs stained only B220+IgMhigh plasma cells in FIGURE 1. Increased EAE disease severity and delayed recovery in the LN and spleen (data not shown). Flow cytometry was per- Sdc-12/2 mice. (A) Active EAE induction in Sdc-12/2 and WT littermates formed on immune cells from naive and EAE mice to investigate showing increased mean clinical scores and maximal disease severity in potentially low levels of syndecan-1 expression, revealing no de- Sdc-12/2. Data are means 6 SEM for four to six independent experiments tectable signal on T cells, macrophages, and and only performed with at least four Sdc-12/2 and WT littermates in each exper- a low-level signal on B220+ B cells (Supplemental Fig. 3A), as iment. (B) Flow cytometry for CD4+IFN-g+ (Th1), CD4+IL-17+ (Th17) reported previously (17). Because endothelium has been reported + + + 2/2 cells, and CD4 CD25 Foxp3 Tregs in CNS of Sdc-1 and WT litter- to express syndecan-1 in other tissues, brain endothelial cells and mates at peak disease severity (day 17), revealing significantly elevated Th1 2/2 intact choroid plexus were isolated, and syndecan-1 expression and Th17 cell numbers and decreased Treg numbers in CNS of Sdc-1 was investigated by q-PCR, revealing a .10-fold lower signal in mice. For Th1 and Th17 cells, gating was on CD45+ living cells. For Tregs, brain endothelial cells than in choroid plexus samples (Supple- gating was on CD45+CD4+ living cells. (C) Bar graphs quantify flow cytometry data at early (day 10), peak (day 17), and recovery stages (.day mental Fig. 3B). Interestingly, several brain endothelial cell lines 20). Data shown are mean cell numbers 6 SEM from three independent and passaged primary cells were found to have a significantly experiments, with at least four mice per stage per experiment. (D) Double higher PCR signal than that measured in choroid plexus (data not staining of serial sections for laminin g1 and MBP and pan-laminin and shown), indicating that syndecan-1 expression on endothelium CD45, showing that areas where leukocytes penetrate the parenchymal border may be regulated by the in situ milieu. To ensure that loss of and gain access to neuronal tissue show loss of MBP staining. Scale bars, syndecan-1 did not result in upregulation of one of the other three 50 mm. (E) Quantification of cuff numbers per square centimeter (mean syndecans, q-PCR was performed on whole brain, LN, and spleen number 6 SEM) of serial sections throughout entire WT or Sdc-12/2 brains 2/2 extracts of peak EAE mice and on isolated choroid plexuses of at peak and recovery phases of EAE. Brains of three WT and three Sdc-1 WT and Sdc-12/2 mice in the naive situation and at early and peak mice for peak EAE and two WT and three Sdc-12/2 mice for the recovery EAE, revealing the absence of upregulation of any of the other stage were sectioned for the quantitative analyses. *p , 0.05, **p , 0.01, ***p , 0.001. syndecans (Supplemental Fig. 3C). Role of syndecan-1 on WT choroid plexus epithelium 2 2 2 2 or Sdc-1 / T cells in the WT or Sdc-1 / CNS (Supplemental Because the choroid plexus was identified as the major CNS re- Fig. 2C). Similarly, in vitro proliferation assays revealed the ab- sident source of syndecan-1 and because initial encephalitogenic The Journal of Immunology 4555

FIGURE 2. An immune cell and a CNS source of syndecan-1 are required for EAE progression. Active EAE was induced in CD45.2+ Sdc-12/2 or WT hosts carrying CD45.1+ WT bone marrow cells (A)and CD45.1+ WT hosts carrying CD45.2+ Sdc-12/2 or WT bone marrow cells (B). Data shown are mean clinical scores 6 SEM from three independent experiments, with five mice per group. **p , 0.01.

T cell recruitment to the brain has been reported to occur at this site (Fig. 5C). Because syndecans can be shed from the cell surface, (20), we focused on this site. CCL20 has been shown to be re- we examined whether the syndecan-1 ectodomain was detectable quired for Th17 cell recruitment across the choroid plexus (20, in the CSF of naive and EAE mice. Because of the very small 34), and its expression has been reported to be influenced by volume of CSF collectable per mouse, dot blots were performed, various cytokines including IL-6 (35, 36), a known major exac- revealing the presence of syndecan-1 ectodomain at both early and erbating factor in EAE (37). We therefore examined syndecan-1, peak stages of EAE but not in the CSF of naive mice (Fig. 5D). It Downloaded from CCL20, and IL-6 expression at the choroid plexus in the naive was not possible to detect CCL20 in the CSF of naive or EAE brain and during the course of EAE. mice either by dot blot or by ELISA, which may be due to the In naive WT brains, immunofluorescence staining revealed reagents available, whereas IL-6 was weakly detectable in the strong expression of CCL20 both within choroid plexus epithelial CSF using the cytometric bead assay only (data not shown). Taken cells and on their surface, which showed partial colocalization with together, the data suggest that syndecan-1 is normally shed from syndecan-1 staining (Fig. 4A). q-PCR revealed that IL-6 mRNA is the choroid plexus epithelia during EAE into the CSF and that this http://www.jimmunol.org/ also expressed at the choroid plexus (Fig. 5A). correlates with loss of CCL20 from the epithelial cell surface and During the course of EAE in WT mice, immunofluorescence an associated upregulation of IL-6 expression. Using both direct staining revealed a marked loss of syndecan-1 and CCL20 from the and indirect ELISA and far Western techniques, we could not choroid plexus epithelial cells (Fig. 4B), yet q-PCR for syndecan-1 demonstrate a direct binding between syndecan-1 and CCL20, mRNA revealed upregulation at early and peak disease stages which again may be due to the reagents available. However, IL-6 (Fig. 5B), which correlated with an increase in IL-6 mRNA (Fig. showed a clear dose-dependent binding to syndecan-1 (Supple- 5A) and corresponding elevated levels of IL-6 in CNS extracts mental Fig. 4A). by guest on September 24, 2021

FIGURE 3. Predominant syndecan-1 expression on choroid plexus epithelium in the brain. (A) Triple immunofluorescence staining of peak EAE brain sections for syndecan-1 (Sdc-1), pan-laminin (Pan LM) to define cuffs borders and CD45+ leukocytes, failed to reveal any detectable syndecan-1 on endothelium or infiltrating immune cells; scale bar, 50 mm. (B) Double staining for Pan LM and MECA32 or Pan LM and syndecan-1: Pan LM staining reveals the basement membranes of the choroid plexus epithelium and of the endothelium of blood vessels within the choroid plexus, whereas MECA32 marks the en- dothelium of choroid plexus but not CNS blood vessels, revealing that syndecan-1 occurs predominantly on the epithelium of the choroid plexus but not choroid plexus endothelium (arrows). Scale bars, 50 mm. Asterisks in higher magnification images mark the lumen of choroid plexus blood vessels. Scale bars, 60 mm. 4556 SYNDECAN-1 IN EAE Downloaded from

FIGURE 4. Syndecan-1 and CCL20 expression at 2/2

the choroid plexus of naive and EAE WT and Sdc-1 http://www.jimmunol.org/ mice. (A) Double staining for syndecan-1 and CCL20 reveals strong expression of both molecules and partial colocalization in naive WT brain, which are signifi- cantly reduced at peak EAE and on choroid plexus epithelium of naive and EAE Sdc-12/2 mice (B). Scale bars in (A) and (B), 20 mm. by guest on September 24, 2021

Lower levels of CCL20 on choroid plexus epithelium in mice at both early and peak EAE (Fig. 5E), suggesting that it was Sdc-12/2 mice and highly elevated IL-6 levels normally produced but not retained on the epithelial cell surface. Compared with WT mice, naive Sdc-12/2 mice showed a mark- By contrast, IL-6 mRNA expression was significantly higher in 2/2 edly reduced CCL20 immunofluorescence signal on the choroid the choroid plexus of Sdc-1 mice at early and peak EAE plexus epithelium, which was only slightly further reduced during compared with WT mice (Fig. 5A), which correlated with 4- to the course of EAE (Fig. 4B). As in WT mice, it was not possible 40-fold higher levels of IL-6 in CNS extracts at early and peak to detect CCL20 in the CSF of Sdc-12/2 mice either by dot blots EAE but not in the recovery phase (Fig. 5C). Because IL-6 can or by ELISA (data not shown). However, q-PCR revealed that be produced by many tissues and its source in EAE remains ill- CCL20 mRNA expression was comparable in WT and Sdc-12/2 defined, q-PCR was performed to compare IL-6 mRNA levels in The Journal of Immunology 4557

FIGURE 5. Syndecan-1 is shed from choroid plexus epithelium into the CSF at early and peak EAE and is negatively correlated with IL-6 expression. (A) q-PCR reveals IL-6 mRNA in choroid plexus samples from WT and Sdc-12/2 mice and its upregulation at early and peak EAE, with significantly higher levels mea- sured in Sdc-12/2 samples, in particular at early EAE. Ctrl is only primers without template. M is marker. (B) q-PCR for Sdc-1 mRNA in the choroid plexus of WT mice reveals upregulation at early and peak EAE compared with the naive situation. (C) IL-6 levels as measured by cytometric bead assay in the CNS ho- mogenates from naive and EAE WT and Sdc-12/2 mice, revealing significantly higher levels in Sdc-12/2 Downloaded from samples. (D) Dot blot reveals the presence of synde- can-1 ectodomain in WT CSF collected at early and peak EAE but not in the CSF of naive mice. (E) q-PCR reveals no changes in the relative expression of CCL20 mRNA in naive or EAE choroid plexus samples from WT or Sdc-12/2 mice. Data shown in (A)–(C) and (E) are from a minimum of three experiments with at least http://www.jimmunol.org/ three mice per stage per experiment. (D) is one repre- sentative experiment. *p , 0.05, **p , 0.01, ***p , 0.001. by guest on September 24, 2021

brain, spinal cord, and the choroid plexus of WT and Sdc-12/2 chemokines that have been implicated in EAE, including CCL2, mice, with or without EAE. We found that IL-6 mRNA levels CCL3, CCL4, CCL5, CCL7, CXCL1, CXCL2, and CXCL10, in were increased in brain, spinal cord, and choroid plexus during the CSF and also in the whole brain tissue. Several chemokines early and peak stages of EAE compared with naive situation (Sup- were elevated both in the CNS and in the CSF at early and peak plemental Fig. 4B), but there were no differences between brains EAE in Sdc-12/2 compared with WT mice, with CCL2 and and spinal cords of WT and Sdc-12/2 mice. Only in the choroid CXCL10 being particularly high in the CSF (Supplemental Fig. plexus were higher levels of IL-6 mRNA measured in Sdc-12/2 4C, 4D), both of which carry heparan sulfate binding domains (39), mice compared with WT. This suggests that although IL-6 can be raising the possibility that syndecan-1 control of chemokine gra- produced by other cells in the CNS, only the choroid plexus dients may not be restricted to CCL20. source is altered in the Sdc-12/2 mice, thereby, accounting for the higher IL-6 protein levels measured in the Sdc-12/2 brains. Immune cell source of syndecan-1 and recovery phase of EAE Taken together, this suggests that shedding of syndecan-1 from Because only B cells show clear syndecan-1 expression and be- choroid plexus epithelium at early and peak EAE correlates with cause syndecan-1 is used as a marker of plasma cells, which have elevated IL-6 secretion at the choroid plexus, contributing to el- been implicated in prolonged recovery in EAE (40), we examined evated IL-6 protein level in the CNS. Because of the small amount plasma cell numbers in Sdc-12/2 mice. Because we could no of CSF that could be collected, we were not able to measure longer use syndecan-1 as a marker of plasma cells, we crossed the immune cell numbers in the CSF by flow cytometry. However, Sdc-12/2 mice with the Blimp-1-GFP reporter mouse (41) and immunofluorescence staining of choroid plexus sections revealed performed flow cytometry analyses of spleen, LN, bone marrow, higher numbers of CD45+ leukocytes in the CSF of Sdc-12/2 com- and CNS of naive and EAE mice to determine plasma cell num- pared with WT mice at the onset of EAE symptoms (days 9–10) bers. Fig. 7A shows that syndecan-1+CD45+ plasma cells are also (Fig. 6) that was no longer apparent at peak EAE (data not shown), Blimp-1-GFP+. Our data reveal that plasma cell numbers were suggesting enhanced initial recruitment of leukocytes via the cho- elevated in the spleen, LN, and blood but not the bone marrow of roid plexus in Sdc-12/2 mice. both naive (data not shown) and EAE Sdc-12/2 mice (Fig. 7B), Because several chemokines that play a role in leukocyte re- which, in EAE mice, correlated with increased circulating levels cruitment have been shown to bind to syndecan-1 (14, 38) and of MOG35–55-specific IgG (but not IgM levels) (Fig. 7C). How- because chemokines other than CCL20 may also be expressed at ever, we did not detect Blimp-1-GFP+ plasma cells in CNS of EAE the choroid plexus, ELISA was used to measure levels of other WT and Sdc-12/2 mice. Also, immunofluorescence microscopy 4558 SYNDECAN-1 IN EAE

FIGURE 6. Initial recruitment of CD45+ leukocytes to the choroid plexus is enhanced in Sdc-12/2 mice. Double immunofluorescence staining of Sdc-12/2 or WT mice brains at the onset of EAE symptoms (day 9) with pan-laminin Abs (Pan LM), to define the confines of the choroid plexus epithelium and blood vessels, and anti-CD45 to mark leukocytes, reveals larger numbers of CD45+ cells within the CSF of Sdc-12/2 mice, whereas most CD45+ cells in WT mice are still retained within the confines of the choroid plexus blood vessels. Scale bar, 100 mm. Downloaded from http://www.jimmunol.org/ performed at peak EAE and in recovery phases failed to reveal the migration of the CCR6+ Th17 cells into the CSF. It is important to presence of Blimp-1-GFP+ plasma cells in the brains of Sdc-12/2 note that the detection of CCL20 in direct binding assays and in or WT mice or syndecan-1+B220+ plasma cells in the brains of CSF is likely to be handicapped by the tools currently available for WT mice (data not shown). its detection. The increased levels of several other chemokines that carry heparan sulfate–binding motifs in the CSF of the Sdc-12/2 Discussion mice, in particular CCL2 and CXCL10, raises the possibility that Our data reveal that syndecan-1 has effects at two distinct stages of syndecan-1 modulation of chemokine gradients at the choroid EAE; one effect is at the initial recruitment of encephalitogenic plexus may not be restricted to CCL20. Importantly, the effects of T cells to the brain via the choroid plexus, which is due to syndecan-1 syndecan-1 loss on CCL20 at the choroid plexus is independent of by guest on September 24, 2021 expression on choroid plexus epithelium, and the second effect is at the reported CCR6 priming functions at other sites (43, 44) where the recovery phase, which is mainly because of syndecan-1 effects syndecan-1 is not expressed, such as the LN and spleen. on immune cells. In both cases, syndecan-1 has immune-suppressive In addition to the association between syndecan-1 and the effects, and in its absence, disease severity is enhanced and the re- CCL20 localization on choroid plexus epithelium, our data dem- covery phase is prolonged. onstrate that the absence of syndecan-1 associated with increased The choroid plexus is the first site of entry of the EAE-inducing IL-6 secretion at the choroid plexus. Sdc-12/2 mice have inher- Th17 cells into the brain (20, 34). It consists of villous structures ently higher CNS levels of IL-6 compared with WT mice, which is that extend into the CSF-filled ventricular spaces that form a further exacerbated during EAE and which we show is associated blood–CSF barrier at the level of tight junctions between epithelial with enhanced initial leukocyte recruitment into the CSF via the cells of the choroid plexus. A CCL20 gradient across the choroid choroid plexus and increased numbers of Th1 and Th17 cells in plexus epithelium has been previously shown to be required for the CNS. q-PCR of the spinal cord, brains, and choroid plexuses the migration of the initial disease-inducing CCR6+ Th17 cells from naive and EAE WT and Sdc-12/2 mice showed that IL-6 into the CSF from where they disseminate to the meningeal and expression increases at all sites in EAE but only at the choroid perivascular spaces to further recruit leukocytes via the CNS pa- plexus were higher levels of IL-6 mRNA measured in Sdc-12/2 renchyma postcapillary venules (20, 34). We demonstrate strong mice compared with WT. This suggests that although IL-6 can be expression of syndecan-1 on choroid plexus epithelium, which produced by other cells in the CNS, only the choroid plexus ep- decreases with EAE development, because of its shedding into the ithelial cell source is abnormal in the Sdc-12/2 mice, accounting CSF and correlates with reduced levels of CCL20 on the choroid for the higher IL-6 protein levels in the Sdc-12/2 brains. Data epithelial cell surface. We could not detect direct binding of from both WT and Sdc-12/2 mice showed a correlation between CCL20 to syndecan-1, despite previous reports on the interaction loss of syndecan-1, in the case of WT mice by shedding into the of this chemokine with glycosaminoglycans (42) and on the in- CSF during EAE, and upregulation of IL-6 mRNA expression at teractions of syndecan-1 with various chemokines, including the choroid plexus that was paralleled by elevated levels of IL-6 in RANTES/CCL5, eotaxin/CCL11, TARC/CCL17, MARC/CCL7, the CNS, which we proposed may further contribute to enhanced IL-8/CXCL8, and KC/CXCL1 (14, 38). In addition, we could survival of the encephalitogenic Th1 an Th17 cells in the CNS as not detect CCL20 in the CSF of EAE mice, but the absence of previously shown by others (45). differences in q-PCR levels of CCL20 mRNA throughout EAE Loss of syndecan-1 has been shown to be associated with ele- and between WT and Sdc-12/2 choroid plexuses supports its con- vated levels of tissue or serum levels of IL-6 in several inflam- tinual expression and subsequent lack of retention at the choroid matory (14, 46) and tumor models (8, 47); however, the converse plexus epithelium surface, which we propose increases the steep- has also been reported (48, 49). In vitro silencing of syndecan-1 ness of gradient across the choroid plexus epithelium and promotes expression has been reported to both upregulate (47) and down- The Journal of Immunology 4559

FIGURE 7. Increased plasma cell numbers in the periphery and increased MOG-specific Ab levels in serum of Sdc-12/2 mice during active EAE. Sdc-12/2 Downloaded from mice were crossed to Blimp-1-GFP mice to detect plasma cells in Sdc-12/2 mice by using Blimp-1-GFP as a marker. (A) Flow cytometry of bone marrow and spleen, gated on CD45+syndecan-1+ plasma cells, showing that 95% of the syndecan-1+ plasma cells are Blimp-1-GFP+. Data shown are one representative http://www.jimmunol.org/ experiment. (B) Flow cytometry for Blimp-1-GFP+ cells at EAE peak, revealed elevated plasma cell numbers in the spleen, LN, and blood but not bone marrow of Sdc-12/2-Blimp-1-GFP mice. No Blimp-1- GFP+ cells were detected in the CNS of either WT- or Sdc-12/2-Blimp-1-GFP mice. Gating was on CD45+ living cells. (C) MOG-specific total Ig, IgG and IgM levels in the serum of WT and Sdc-12/2 mice were quantified by ELISA at peak and recovery stages of

EAE. Data shown are mean values 6 SEM from three by guest on September 24, 2021 independent experiments performed with at least three mice per stage per experiment. *p , 0.05, ***p , 0.001.

regulate (48) IL-6 mRNA in endometrial stromal cells and epi- responses to IL-6 (8), whereas IL-6 downregulates syndecan-1 in a thelial cells, respectively. In mesothelioma cells, upregulation of variety of cell types, including cells of liver origin (50), osteosarcoma syndecan-1 expression is associated with increased IL-6 expres- cell lines (51), and B lymphoid cells (52), suggesting an interde- sion (49), suggesting that the effects of syndecan-1 on IL-6 are pendency between syndecan-1 and IL-6 regulatory pathways. likely to be influenced by factors such as cell activity or patho- IL-6 is known to specifically bind to heparan sulfate glycos- physiological state. How such effects are mediated is not clear but aminoglycan chains (53, 54), and we show in this study that IL-6 may be due to the high growth factor binding potential of syndecan-1 binds to syndecan-1, which has been proposed to provide a mode (3), which subsequently impacts on IL-6 expression. In breast cancer of retaining IL-6 close to its site of secretion, thus favoring a cells, downregulation of syndecan-1 also results in reduced cellular paracrine mode of action (53). IL-6 can both inhibit or stimulate 4560 SYNDECAN-1 IN EAE its own secretion depending on cell type; it is possible therefore Sdc-12/2 mice. The fact that Sdc-12/2 mice had inherently higher that in the naive situation syndecan-1–bound IL-6 acts locally at numbers of plasma cells indicate that syndecan-1 is not only the choroid plexus epithelium to inhibit its own secretion and that a marker for plasma cells but is also a negative regulator of plasma this inhibition is lifted when syndecan-1 is shed during EAE or is cell formation, a novel finding that requires further investigation. absent as in the Sdc-12/2 mouse. This theory requires in vitro That plasma cell numbers were elevated in the secondary lym- testing that, however, is likely to be difficult given the variabil- phoid tissues but not the bone marrow of Sdc-12/2 mice suggests ity noted here in the expression of syndecan-1 in in vitro–cultured that short-lived plasma cells that appear in the secondary lym- (endothelial) cells. phoid tissue shortly after Ag exposure rather than long-lived IL-6 plays several roles in inflammation (55), and its signifi- plasma cells that reside in the bone marrow are principally af- cance in EAE is reflected in the complete resistance to disease of fected by loss of syndecan-1. mice lacking IL-6 (37, 56). However, the critical source of IL-6 in In summary, we demonstrate immunosuppressive effects for EAE has remained difficult to define. DCs have recently been syndecan-1 in EAE and provide new information on molecular shown to be an important source of IL-6 during EAE, but only processes important for initial encephalitogenic T cell recruitment transiently during the early T cell activation stages (57). Our data to the brain via the choroid plexus and for disease recovery. Our suggest that IL-6 expression at the choroid plexus occurs subse- data support a role for syndean-1 in modulation of the immune quent to T cell activation, principally at the stage of early re- response by contributing to cytokine and potentially also chemo- cruitment of T cells via the choroid plexus and up to peak EAE. To kine gradients, interestingly at the level of the choroid plexus our knowledge, this is the first description of IL-6 expression at epithelial barrier rather than endothelial barrier, as well as on the choroid plexus and, together with other published data (57), plasma cell development. Downloaded from supports the concept that IL-6 is produced by different cell types at different stages of EAE. Acknowledgments Our studies revealed that the endothelial cells of perivascular We thank Stefan Liebner (University of Frankfurt, Frankfurt, Germany) for cuffs in the brain did not express detectable amounts of syndecan-1. the gift of brain-derived endothelial cells; Johanna Breuer, Maik Worlitzer, Even though the mRNA for syndecan-1 was detectable in freshly Jens Swanborn, and Nicholas Schwab (University of Muenster) for teach-

. http://www.jimmunol.org/ isolated brain endothelial cells, levels were 10-fold lower than in ing and helping us collect CSF; Stephen Nutt and David Tarlington (Walter choroid plexus epithelium, and the protein was not detectable by and Eliza Hall Institute, Melbourne, VIC, Australia) for the generous gift immunofluorescence staining, regardless of unmasking techniques of the Blimp-1-GFP mice; and Rupert Hallmann for critical reading of the used. This is in contrast to others who have reported syndecan-1 manuscript. We also thank Merton Bernfield (Boston Children’s Hospital, 2 2 expression on endothelium, albeit in tissues other than brain (6, Boston, MA) who provided Sdc-1 / mice. 19, 58). Past studies have also focused on in vitro–cultured en- dothelial cell lines, which we found to have significantly higher Disclosures levels of syndecan-1 mRNA than freshly isolated primary en- The authors have no financial conflicts of interest. dothelial cells, suggesting that the in vivo milieu may control the syndecan-1 expression on endothelium and that this varies with by guest on September 24, 2021 tissue type. This is supported by reports that syndecan-1 can be References induced or repressed in a transient manner during development 1. Parish, C. R. 2006. The role of heparan sulphate in inflammation. Nat. Rev. and in a variety of pathological processes (9). Immunol. 6: 633–643. The bone marrow chimeric studies suggest a role for an immune 2. Proudfoot, A. E. 2006. The biological relevance of chemokine-proteoglycan interactions. Biochem. Soc. Trans. 34: 422–426. cell source of syndecan-1 in the recovery phase of EAE; however, 3. Forsten-Williams, K., C. L. Chu, M. Fannon, J. A. Buczek-Thomas, and 2/2 Th1, Th17, and Treg numbers were similar in the WT and Sdc-1 M. A. Nugent. 2008. Control of growth factor networks by heparan sulfate mice during the recovery phase, and IL-6 CNS levels were also proteoglycans. Ann. Biomed. Eng. 36: 2134–2148. 4. Yayon, A., M. Klagsbrun, J. D. Esko, P. Leder, and D. M. Ornitz. 1991. Cell not significantly different. Rather, the slower recovery for EAE surface, heparin-like molecules are required for binding of basic fibroblast 2 2 symptoms observed in the Sdc-1 / mice may be partially accounted growth factor to its high affinity receptor. Cell 64: 841–848. for by the slightly elevated numbers of macrophages (albeit not 5. Rapraeger, A. C., A. Krufka, and B. B. Olwin. 1991. Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Sci- significant). In addition, the low syndecan-1 expression detected ence 252: 1705–1708. on B cells prompted us to also invest a role for B cells. B cells 6. Beauvais, D. M., B. J. Ell, A. R. McWhorter, and A. C. Rapraeger. 2009. Syndecan-1 regulates avb3 and avb5 integrin activation during angiogenesis and have been proposed to have an Ag-presenting role in EAE (29); is blocked by synstatin, a novel peptide inhibitor. J. Exp. Med. 206: 691–705. 2/2 for this reason, we tested the ability of B cells from the Sdc-1 7. Morgan, M. R., M. J. Humphries, and M. D. Bass. 2007. Synergistic control of mice to act as APCs, revealing no differences to WT B cells. cell adhesion by integrins and syndecans. Nat. Rev. Mol. Cell Biol. 8: 957–969. 2/2 8. Hassan, H., B. Greve, M. S. Pavao, L. Kiesel, S. A. Ibrahim, and M. Go¨tte. 2013. The prolonged recovery phase in Sdc-1 mice correlated with Syndecan-1 modulates b-integrin-dependent and -6-dependent func- higher numbers of MOG-specific plasma cells in the spleen, LNs, tions in breast cancer cell adhesion, migration, and resistance to irradiation. and circulation but not in the brain and with elevated serum titers FEBS J. 280: 2216–2227. 9. Bernfield, M., M. Go¨tte, P. W. Park, O. Reizes, M. L. Fitzgerald, J. Lincecum, of MOG33–55 Ab, which has been previously reported to correlate and M. Zako. 1999. Functions of cell surface heparan sulfate proteoglycans. with enhanced demyelination and slower EAE recovery (29, 40). Annu. Rev. Biochem. 68: 729–777. 10. Rapraeger, A. C. 2000. Syndecan-regulated receptor signaling. J. Cell Biol. 149: How MOG specific Abs can prolong recovery in EAE is not 995–998. clearly defined; in mice, inflammation in the CNS can result in 11. Park, P. W., O. Reizes, and M. Bernfield. 2000. 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