Dissociation between Mature Phenotype and Impaired Transmigration in Dendritic Cells from Heparanase-Deficient Mice

To reach the lymphatics, migrating dendritic cells (DCs) need to interact with the extracellular matrix (ECM). Heparanase, a mammalian endo-β-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and ECM. The role of heparanase in the physiology of bone marrow-derived DCs was studied in mutant heparanase knock-out (Hpse-KO) mice. Immature DCs from Hpse-KO mice exhibited a more mature phenotype; however their transmigration was significantly delayed, but not completely abolished, most probably due to the observed upregulation of MMP-14 and CCR7. Despite their mature phenotype, uptake of beads was comparable and uptake of apoptotic cells was more efficient in DCs from Hpse-KO mice. Heparanase is an important enzyme for DC transmigration. Together with CCR7 and its ligands, and probably MMP-14, heparanase controls DC trafficking

Cerebrospinal Fluid (CSF) Exchange with Artificial CSF Enriched with Mesenchymal Stem Cell Secretions Ameliorates Experimental Autoimmune Encephalomyelitis

The complexity of central nervous system (CNS) degenerative/inflammatory diseases and the lack of substantially effective treatments point to the need for a broader therapeutic approach to target multiple components involved in the disease pathogenesis. We suggest a novel approach directed for the elimination of pathogenic agents from the CNS and, in parallel, its enrichment with an array of neuroprotective substances, using a “cerebrospinal fluid (CSF) exchange„ procedure, in which endogenous (pathogenic) CSF is removed and replaced by artificial CSF (aCSF) enriched with secretions of human mesenchymal stem cells (MSCs). MSCs produce a variety of neuroprotective agents and have shown beneficial effects when cells are transplanted in animals and patients with CNS diseases. Our data show that MSCs grown in aCSF secrete neurotrophic factors, anti-inflammatory cytokines, and anti-oxidant agents; moreover, MSC-secretions-enriched-aCSF exerts neuroprotective and immunomodulatory effects in neuronal cell lines and spleen lymphocytes. Treatment of experimental-autoimmune-encephalomyelitis (EAE) mice with this enriched-aCSF using an intracerebroventricular (ICV) CSF exchange procedure (“CSF exchange therapy„) caused a significant delay in the onset of EAE and amelioration of the clinical symptoms, paralleled by a reduction in axonal damage and demyelination. These findings point to the therapeutic potential of the CSF exchange therapy using MSC-secretions-enriched-aCSF in inflammatory/degenerative diseases of the CNS

DC subpopulations in the spleen of wild type (<i>Hpse-</i>WT) and heparanase-deficient mice (<i>Hpse</i>-KO).

<p>The average and standard deviation of four spleens is shown.</p

Cytokine secretion from iDCs and mDCs is comparable in DCs from <i>Hpse</i>-WT and <i>Hpse</i>-KO mice.

<p><i>A</i>, <i>B</i>, <i>C</i> and <i>D</i>. Following DC maturation, supernatant culture was collected and analyzed by ELISA for (<i>A</i>) murine IL-12/IL-23(p40), (<i>B</i>) murine TNFα (<i>C</i>) murine IL-6, and (<i>D</i>) murine IL-10 production. Asterisk indicates p<0.02, t-test.</p

Morphologic parameters of iBMDCs and mBMDCs are comparable in <i>Hpse</i>-WT and <i>Hpse</i>-KO mice.

<p><i>A</i>. Wright staining of BMDCs from <i>Hpse-</i>WT and <i>Hpse</i>-KO mice. <i>B</i><b>.</b> Nomarsky view via confocal microscopy of BMDCs from <i>Hpse-</i>WT <i>and Hpse</i>-KO mice (zoom 60×5, Olympus IX70, Center Valley, PA). <i>C</i>. Representative dot plot analysis of side scatter (SSCH) and forward scatter (FSCH), and yield of iBMDCs. Average from five experiments. <i>D</i> and <i>E</i>. The same for mBMDCs, matured with mTNFα + PGE2 (<i>D</i>), or LPS (<i>E</i>).</p

BMDCs from <i>Hpse</i>-KO mice have a phenotype with increased expression of maturation molecules and CCR7, and phagocytose apoptotic cells more efficiently.

<p><i>A</i>, <i>B</i>, and <i>C</i>. <i>(A)</i> iBMDCs and (<i>B</i>) mBMDCs matured with LPS, and (<i>C</i>) mBMDCs matured with mTNFα + PGE2, from <i>Hpse-</i>WT and <i>Hpse</i>-KO mice, are stained for CD80, CD86, CD40, IA/IE, CCR7, and CD11c (black line), and for isotype control of each marker (gray-filled curves). Mean fluorescence (MF) is the average of five experiments. Asterisk indicates p<0.02, t-test. <i>D.</i> Uptake of apoptotic cells and FITC-labeled beads by iBMDC from <i>Hpse-</i>WT and <i>Hpse</i>-KO mice. <i>Upper panel.</i> Apoptotic cells were stained with DiI before the interaction with iBMDCs. 2 hours after the interaction with apoptotic cells, iBMDCs were stained with CD11c.FITC. Mean fluorescence (MF) of double positive cells is indicated. Phagocytosis was verified using confocal microscopy (not shown). <i>Lower panel.</i> As an additional control, FITC-labeled beads were offered to CD11c. PE-stained iBMDCs (MF) of double positive cells are indicated.</p

Heparanase is needed for in vitro and in vivo transmigration.

<p><i>A</i>. <i>In vitro</i> transmigration of iBMDCs or mBMDCs from <i>Hpse-</i>WT (black bars) and <i>Hpse</i>-KO mice (gray bars). Cells were incubated in transwells coated with Matrigel (15 µg/50 µl DDW) 20–24 h at 37°C. Cells were incubated with the lower chamber medium containing RPMI, RPMI with 25 ng/ml MIP3β or RPMI with 50 ng/ml MIP3β. Representative of three experiments. Asterisk indicates p<0.02, t-test. <i>B. In vivo</i> DC transmigration. Flow cytometry. Dot plot of CD11c⁺ and CFSE⁺ cells from a popliteal lymph node following foot pad injection of PBS, or, 2×10⁶ mBMDCs from <i>Hpse-</i>WT, or 2×10⁶ mBMDCs <i>from Hpse</i>-KO mice. mBMDCs were stained with CFSE and injected SQ into the footpads of <i>Hpse-</i>WT mice. 24 hours or 48 hours after injection, the regional popliteal lymph node was extracted and analyzed for the presence of CFSE+ cells by flow cytometry. CyoCount beads were used to evaluate cells that had migrated to the popliteal lymph nodes. Representative of three experiments, (p<0.001). <i>C. In vivo</i> FITC skin painting. Dot plot of double positive CD11c-PE+ and FITC+ cells from inguinal lymph nodes. The shaved abdomen was painted with FITC solution. 24 hours after the skin painting, the inguinal lymph node was extracted and analyzed for the presence of CFSE+ cells by flow cytometry. <i>D.</i> Indirect immunofluorescence of frozen sections. Regional popliteal lymph node frozen section of the same experiment in <i>C</i>. Confocal microscopy of lymph nodes stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035602#s4" target="_blank">Methods</a>. CFSE-migrating DCs (green), CD11c migrating (yellow merged), local DCs (red), and T cells (blue), are seen.</p

MMP expression.

<p>RT-PCR of MMPs in iBMDCs (black), and mBMDCs (grilled black), from <i>Hpse-</i>WT or in iBMDCs (gray), mBMDCs (grilled gray), from <i>Hpse</i>-KO mice. Maturation of BMDC was achieved with a cocktail of 30 ng/ml recombinant mTNFα and 1 µg/ml PGE2 for 24 hrs. RNA was extracted from iBMDCs and mBMDCs from <i>Hpse-</i>WT and <i>Hpse</i>-KO mice. Primers specific for MMP-2, MMP-9, MMP-25, and MMP-14 were used for RT-PCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035602#s4" target="_blank">Methods</a>). Asterisk indicates p<0.02, t-test.</p