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• W2136174903 abstract "Proinflammatory circulating monocytes have important roles in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Yet there is limited information on their accumulation in blood during disease, the mechanisms that regulate their infiltration into the central nervous system (CNS), and whether medications affect their biology. We found a significant and prolonged elevation of CD11b+CCR2+Ly6Chigh proinflammatory monocytes in the blood of mice by the second day of immunization for EAE. At onset of clinical signs, levels of proinflammatory monocytes plummeted to those in naive mice. At day 16, when the majority of mice were at peak disease severity, clinical scores were inversely correlated to the proportion of proinflammatory monocytes in blood, and directly correlated with that in the spinal cord. Treatment with the MS medication laquinimod prevented EAE, correspondent with retention of proinflammatory monocytes in blood. The reduced entry of proinflammatory monocytes into the CNS by laquinimod was attributed to reduction of their levels of CD62L and matrix metalloproteinase-9. Moreover, the spinal cord of laquinimod-treated mice did not have elevated levels of CCR2 and CCL2, which provide chemotactic cues for monocytes. These results shed light on the important role of the trafficking of proinflammatory monocytes into the CNS to promote disease activity, and they identify a mechanism of action of laquinimod in MS. Proinflammatory circulating monocytes have important roles in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Yet there is limited information on their accumulation in blood during disease, the mechanisms that regulate their infiltration into the central nervous system (CNS), and whether medications affect their biology. We found a significant and prolonged elevation of CD11b+CCR2+Ly6Chigh proinflammatory monocytes in the blood of mice by the second day of immunization for EAE. At onset of clinical signs, levels of proinflammatory monocytes plummeted to those in naive mice. At day 16, when the majority of mice were at peak disease severity, clinical scores were inversely correlated to the proportion of proinflammatory monocytes in blood, and directly correlated with that in the spinal cord. Treatment with the MS medication laquinimod prevented EAE, correspondent with retention of proinflammatory monocytes in blood. The reduced entry of proinflammatory monocytes into the CNS by laquinimod was attributed to reduction of their levels of CD62L and matrix metalloproteinase-9. Moreover, the spinal cord of laquinimod-treated mice did not have elevated levels of CCR2 and CCL2, which provide chemotactic cues for monocytes. These results shed light on the important role of the trafficking of proinflammatory monocytes into the CNS to promote disease activity, and they identify a mechanism of action of laquinimod in MS. Multiple sclerosis (MS) is an inflammatory condition in which a variety of immune subsets enter the central nervous system (CNS) to produce demyelination, axonal injury, and oligodendrocyte and neuronal loss. The immune abnormalities in MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have focused largely on the adaptive immune response, and both B cells and CD4+ T cells of the proinflammatory subsets, T helper 1 (Th1) and Th17, have been highlighted.1Berer K. Wekerle H. Krishnamoorthy G. B cells in spontaneous autoimmune diseases of the central nervous system.Mol Immunol. 2011; 48: 1332-1337Crossref PubMed Scopus (33) Google Scholar, 2El-behi M. Rostami A. Ciric B. Current views on the roles of Th1 and Th17 cells in experimental autoimmune encephalomyelitis.J Neuroimmune Pharmacol. 2010; 5: 189-197Crossref PubMed Scopus (179) Google Scholar, 3Lovett-Racke A.E. Yang Y. Racke M.K. Th1 versus Th17: are T cell cytokines relevant in multiple sclerosis?.Biochim Biophys Acta. 2011; 1812: 246-251Crossref PubMed Scopus (174) Google Scholar Abnormalities of the innate immune system, including blood monocytes (that become macrophages in tissues) and CNS-resident microglia, have received renewed attention in MS.4Gandhi R. Laroni A. Weiner H.L. Role of the innate immune system in the pathogenesis of multiple sclerosis.J Neuroimmunol. 2010; 221: 7-14Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 5Heppner F.L. Greter M. Marino D. Falsig J. Raivich G. Hovelmeyer N. Waisman A. Rulicke T. Prinz M. Priller J. Becher B. Aguzzi A. Experimental autoimmune encephalomyelitis repressed by microglial paralysis.Nat Med. 2005; 11: 146-152Crossref PubMed Scopus (611) Google Scholar This attention is deserved given that monocytoid populations constitute the predominant immune subset in active MS brain lesions,6Lucchinetti C. Bruck W. Parisi J. Scheithauer B. Rodriguez M. Lassmann H. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination.Ann Neurol. 2000; 47: 707-717Crossref PubMed Scopus (2688) Google Scholar, 7Prineas J.W. Wright R.G. Macrophages, lymphocytes, and plasma cells in the perivascular compartment in chronic multiple sclerosis.Lab Invest. 1978; 38: 409-421PubMed Google Scholar and that the areas of their accumulation display demyelination and axonal injury.8Kornek B. Storch M.K. Weissert R. Wallstroem E. Stefferl A. Olsson T. Linington C. Schmidbauer M. Lassmann H. Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions.Am J Pathol. 2000; 157: 267-276Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, 9Nikic I. Merkler D. Sorbara C. Brinkoetter M. Kreutzfeldt M. Bareyre F.M. Bruck W. Bishop D. Misgeld T. Kerschensteiner M. A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis.Nat Med. 2011; 17: 495-499Crossref PubMed Scopus (511) Google Scholar The renewed focus has also been helped by the recent discovery of at least two subsets of monocytes/macrophages (the distinction for microglia is currently less clear): the classically activated M1 cells that are largely proinflammatory, and the alternately activated M2 cells with anti-inflammatory/regulatory properties.10Auffray C. Sieweke M.H. Geissmann F. Blood monocytes: development, heterogeneity, and relationship with dendritic cells.Annu Rev Immunol. 2009; 27: 669-692Crossref PubMed Scopus (1176) Google Scholar, 11Martinez F.O. Helming L. Gordon S. Alternative activation of macrophages: an immunologic functional perspective.Annu Rev Immunol. 2009; 27: 451-483Crossref PubMed Scopus (2079) Google Scholar, 12Murray P.J. Wynn T.A. Protective and pathogenic functions of macrophage subsets.Nat Rev Immunol. 2011; 11: 723-737Crossref PubMed Scopus (3419) Google Scholar, 13Prinz M. Priller J. Sisodia S.S. Ransohoff R.M. Heterogeneity of CNS myeloid cells and their roles in neurodegeneration.Nat Neurosci. 2011; 14: 1227-1235Crossref PubMed Scopus (525) Google Scholar A number of other properties differentiate these two populations, including inducible nitric oxide synthase and CCR2+Ly6Chigh expression in proinflammatory cells, and arginase and CX3CR1+Ly6Clow in M2 cells.14Charo I.F. Macrophage polarization and insulin resistance: pPARgamma in control.Cell Metab. 2007; 6: 96-98Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 15Gordon S. Taylor P.R. Monocyte and macrophage heterogeneity.Nat Rev Immunol. 2005; 5: 953-964Crossref PubMed Scopus (3864) Google Scholar, 16Mildner A. Schmidt H. Nitsche M. Merkler D. Hanisch U.K. Mack M. Heikenwalder M. Bruck W. Priller J. Prinz M. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.Nat Neurosci. 2007; 10: 1544-1553Crossref PubMed Scopus (821) Google Scholar, 17Mills C.D. Kincaid K. Alt J.M. Heilman M.J. Hill A.M. M-1/M-2 macrophages and the Th1/Th2 paradigm.J Immunol. 2000; 164: 6166-6173Crossref PubMed Scopus (2022) Google Scholar A predominance of proinflammatory M1 over M2 cells is reported to promote relapses in EAE.18Mikita J. Dubourdieu-Cassagno N. Deloire M.S. Vekris A. Biran M. Raffard G. Brochet B. Canron M.H. Franconi J.M. Boiziau C. Petry K.G. Altered M1/M2 activation patterns of monocytes in severe relapsing experimental rat model of multiple sclerosis Amelioration of clinical status by M2 activated monocyte administration.Mult Scler. 2011; 17: 2-15Crossref PubMed Scopus (306) Google Scholar, 19Valerio M. Liu H.B. Heffner R. Zivadinov R. Ramanathan M. Weinstock-Guttman B. Awad A.B. Phytosterols ameliorate clinical manifestations and inflammation in experimental autoimmune encephalomyelitis.Inflamm Res. 2011; 60: 457-465Crossref PubMed Scopus (38) Google Scholar Other studies further suggest the important roles of circulating monocytes in EAE and MS. Their peripheral depletion by clodronate liposomes reduced the severity of EAE20Huitinga I. van Rooijen N. de Groot C.J. Uitdehaag B.M. Dijkstra C.D. Suppression of experimental allergic encephalomyelitis in Lewis rats after elimination of macrophages.J Exp Med. 1990; 172: 1025-1033Crossref PubMed Scopus (468) Google Scholar and prevented the accumulation of T cells and macrophages in the parenchyma of the spinal cord.21Tran E.H. Hoekstra K. van Rooijen N. Dijkstra C.D. Owens T. Immune invasion of the central nervous system parenchyma and experimental allergic encephalomyelitis, but not leukocyte extravasation from blood, are prevented in macrophage-depleted mice.J Immunol. 1998; 161: 3767-3775Crossref PubMed Google Scholar Mice deficient for the CCR2 receptor, which facilitates the trafficking of monocytes into inflamed tissues, were protected from EAE.16Mildner A. Schmidt H. Nitsche M. Merkler D. Hanisch U.K. Mack M. Heikenwalder M. Bruck W. Priller J. Prinz M. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.Nat Neurosci. 2007; 10: 1544-1553Crossref PubMed Scopus (821) Google Scholar, 22Izikson L. Klein R.S. Charo I.F. Weiner H.L. Luster A.D. Resistance to experimental autoimmune encephalomyelitis in mice lacking the CC chemokine receptor (CCR)2.J Exp Med. 2000; 192: 1075-1080Crossref PubMed Scopus (521) Google Scholar King et al23King I.L. Dickendesher T.L. Segal B.M. Circulating Ly-6C+ myeloid precursors migrate to the CNS and play a pathogenic role during autoimmune demyelinating disease.Blood. 2009; 113: 3190-3197Crossref PubMed Scopus (326) Google Scholar (2009) reported that granulocyte-macrophage colony-stimulating factor mobilized CD11b+Ly6Chigh proinflammatory monocytes from the bone marrow to produce an earlier onset and increased severity of EAE. Using a combination of irradiation and parabiosis, the infiltration of monocytes into the CNS was found to be necessary to drive the progression of clinical severity of EAE from mild to paralysis.24Ajami B. Bennett J.L. Krieger C. McNagny K.M. Rossi F.M. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool.Nat Neurosci. 2011; 14: 1142-1149Crossref PubMed Scopus (771) Google Scholar Despite the now extensive evidence for the crucial role of monocytoid cells in MS and EAE, there is a dearth of information on the temporal mobilization of proinflammatory monocytes from the bone marrow into blood, and on the mechanisms that facilitate their eventual migration from blood into the CNS to produce EAE disease. Also unknown is whether and how MS medications may alter these features to attenuate neuroinflammation and pathology. Laquinimod is an oral medication being considered for potential use in patients with MS.25Comi G. Jeffery D. Kappos L. Montalban X. Boyko A. Rocca M.A. Filippi M. Placebo-controlled trial of oral laquinimod for multiple sclerosis.N Engl J Med. 2012; 366: 1000-1009Crossref PubMed Scopus (321) Google Scholar In phase III clinical trials, laquinimod reduced the annualized relapse rate and brain volume loss, and slowed progression of disability in patients with the relapsing-remitting form of MS.25Comi G. Jeffery D. Kappos L. Montalban X. Boyko A. Rocca M.A. Filippi M. Placebo-controlled trial of oral laquinimod for multiple sclerosis.N Engl J Med. 2012; 366: 1000-1009Crossref PubMed Scopus (321) Google Scholar, 26Comi G. Pulizzi A. Rovaris M. Abramsky O. Arbizu T. Boiko A. Gold R. Havrdova E. Komoly S. Selmaj K. Sharrack B. Filippi M. Group L.A.Q.S. Effect of laquinimod on MRI-monitored disease activity in patients with relapsing-remitting multiple sclerosis: a multicentre, randomised, double-blind, placebo-controlled phase IIb study.Lancet. 2008; 371: 2085-2092Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar Laquinimod was shown to interfere with the NF-κB signaling network27Gurevich M. Gritzman T. Orbach R. Tuller T. Feldman A. Achiron A. Laquinimod suppress antigen presentation in relapsing-remitting multiple sclerosis: in-vitro high-throughput gene expression study.J Neuroimmunol. 2010; 221: 87-94Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar and, thus, to reduce proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFNγ), and interleukin-17 (IL-17).28Wegner C. Stadelmann C. Pfortner R. Raymond E. Feigelson S. Alon R. Timan B. Hayardeny L. Bruck W. Laquinimod interferes with migratory capacity of T cells and reduces IL-17 levels, inflammatory demyelination and acute axonal damage in mice with experimental autoimmune encephalomyelitis.J Neuroimmunol. 2010; 227: 133-143Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar The number of infiltrated T cells and macrophages in the CNS was reduced by laquinimod treatment.28Wegner C. Stadelmann C. Pfortner R. Raymond E. Feigelson S. Alon R. Timan B. Hayardeny L. Bruck W. Laquinimod interferes with migratory capacity of T cells and reduces IL-17 levels, inflammatory demyelination and acute axonal damage in mice with experimental autoimmune encephalomyelitis.J Neuroimmunol. 2010; 227: 133-143Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 29Yang J.S. Xu L.Y. Xiao B.G. Hedlund G. Link H. Laquinimod (ABR-215062) suppresses the development of experimental autoimmune encephalomyelitis, modulates the Th1/Th2 balance and induces the Th3 cytokine TGF-beta in Lewis rats.J Neuroimmunol. 2004; 156: 3-9Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar Additionally, demyelination and axonal loss was decreased by both preventive and therapeutic laquinimod treatment regimens.28Wegner C. Stadelmann C. Pfortner R. Raymond E. Feigelson S. Alon R. Timan B. Hayardeny L. Bruck W. Laquinimod interferes with migratory capacity of T cells and reduces IL-17 levels, inflammatory demyelination and acute axonal damage in mice with experimental autoimmune encephalomyelitis.J Neuroimmunol. 2010; 227: 133-143Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar More recently, laquinimod was found to increase the levels of brain-derived neurotrophic factor (BDNF) in the serum of patients with MS; in EAE-afflicted mice, laquinimod treatment altered monocytes into those that secrete anti-inflammatory cytokines, apparently through a BDNF-dependent mechanism.30Thone J. Ellrichmann G. Seubert S. Peruga I. Lee D.H. Conrad R. Hayardeny L. Comi G. Wiese S. Linker R.A. Gold R. Modulation of autoimmune demyelination by laquinimod via induction of brain-derived neurotrophic factor.Am J Pathol. 2012; 180: 267-274Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Laquinimod was also reported to induce a type II myeloid cell population in EAE that inhibited the differentiation of proinflammatory T helper 1 or 17 lymphocyte subsets.31Schulze-Topphoff U. Shetty A. Varrin-Doyer M. Molnarfi N. Sagan S.A. Sobel R.A. Nelson P.A. Zamvil S.S. Laquinimod, a quinoline-3-carboxamide, induces type II myeloid cells that modulate central nervous system autoimmunity.PloS One. 2012; 7: e33797Crossref PubMed Scopus (82) Google Scholar Given the dearth of information on the generation, kinetics, and mechanisms of proinflammatory monocytes in EAE, we have investigated their accumulation in the blood and CNS of EAE mice prior to and following the onset of clinical signs. We have also determined whether laquinimod alters these kinetics. Our results shed light on the important role of the trafficking of proinflammatory monocytes into the CNS to promote disease activity, and they identify the perturbation of the accumulation of proinflammatory monocytes in the CNS by laquinimod. EAE was induced in female C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME), aged 7 to 8 weeks, by injecting subcutaneously 50 μg of myelin oligodendrocyte glycoprotein (MOG)35–55 in complete Freund's adjuvant (Fisher Scientific, Waltham, MA) supplemented with 4 mg/mL Mycobacterium tuberculosis on day 0.32Giuliani F. Metz L.M. Wilson T. Fan Y. Bar-Or A. Yong V.W. Additive effect of the combination of glatiramer acetate and minocycline in a model of MS.J Neuroimmunol. 2005; 158: 213-221Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 33Goncalves DaSilva A. Yong V.W. Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation.Am J Pathol. 2009; 174: 898-909Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Intraperitoneal pertussis toxin (300 ng/200 μL; List Biological Laboratories, Hornby, ON, Canada) was administered on days 0 and 2. Laquinimod (25 mg/kg body weight) was given every day by oral gavage in 100 μL of saline, whereas 100 μL of saline was used as the vehicle control. Treatment was initiated at day 5 post MOG immunization. Animals were assessed daily using a 15-point disease score scale previously described.32Giuliani F. Metz L.M. Wilson T. Fan Y. Bar-Or A. Yong V.W. Additive effect of the combination of glatiramer acetate and minocycline in a model of MS.J Neuroimmunol. 2005; 158: 213-221Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 33Goncalves DaSilva A. Yong V.W. Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation.Am J Pathol. 2009; 174: 898-909Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar The 15-point scale is the sum of the disease state for the tail (scored from 0 to 2) and all 4 limbs (each limb is scored from 0 to 3). All animals were handled in accordance with the policies outlined by the Canadian Council for Animal Care and the University of Calgary. Following a sublethal dose of ketamine/xylazine, blood was drawn by cardiac puncture and collected in heparin-containing tubes. Spleens and CNS were dissected out, and single-cell suspensions were isolated from these tissues by sieving through a 70-μm nylon filter. From blood and spleen, mononuclear cells were obtained following Ficoll gradient centrifugation. Cells from the spinal cords were further separated into neural and leukocyte populations by discontinuous density gradient centrifugation using isotonic Percoll (GE Healthcare, Little Chalfont, UK).34Agrawal S.M. Silva C. Tourtellotte W.W. Yong V.W. EMMPRIN: a novel regulator of leukocyte transmigration into the CNS in multiple sclerosis and experimental autoimmune encephalomyelitis.J Neurosci. 2011; 31: 669-677Crossref PubMed Scopus (79) Google Scholar Blood, spleen, and CNS cell samples were prepared at 4°C in buffer solution [PBS or fluorescence-activated cell sorting (FACS) buffer] and stained with antibodies against CD45, CD11b, and CD62L (L-selectin) (BD Pharmingen; all from BD Biosciences, San Jose, CA) or CCR2.35Mack M. Cihak J. Simonis C. Luckow B. Proudfoot A.E. Plachy J. Bruhl H. Frink M. Anders H.J. Vielhauer V. Pfirstinger J. Stangassinger M. Schlondorff D. Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice.J Immunol. 2001; 166: 4697-4704Crossref PubMed Scopus (351) Google Scholar Isotype controls were purchased from eBioscience (San Diego, CA) and BD Biosciences. To detect CCR2, cells were incubated with 5 μg/mL monoclonal antibody for mouse CCR2 (MC-21) for 60 minutes on ice. Isotype controls were always included in the assays. After three washing steps, the cells were incubated for 1 hour on ice with a biotin-labeled anti-rat polyclonal antibody (eBioscience) followed by phosphatidylethanolamine-labeled streptavidin (BD Biosciences) and a combination of directly conjugated antibodies. Data acquisition was performed on a flow cytometer (FACSCalibur or FACSAria; BD Biosciences) and analyzed with FlowJo (TreeStar, Ashland, OR) or with CellQuest Pro (BD Biosciences) software. Following a lethal overdose of ketamine/xylazine, mice were perfused with ice-cold PBS through the left ventricle of the heart. Spinal cords were removed and fixed in 4% buffered formalin, and then embedded in paraffin. For histology for each mouse, five longitudinal sections, 100 μm apart, were stained with hematoxylin & eosin and Luxol Fast Blue, and then scored. Each section was analyzed bilaterally and at rostral, middle, and caudal portions, and the histological score then averaged as described previously.33Goncalves DaSilva A. Yong V.W. Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation.Am J Pathol. 2009; 174: 898-909Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Sections were also stained with Bielschowsky silver stain for axonal integrity. For immunostaining, sections were blocked for 30 minutes with skimmed milk, incubated for 1 hour at room temperature or at 4°C overnight with Iba-1 (1:500) antibody diluted in skim milk, followed by 1 hour at room temperature with secondary antibodies. Sections were mounted in Mowiol (Calbiochem; EMD Millipore, Billerica, MA) and stored at 4°C in the dark. Lumber sacral parts of spinal cord were flash frozen in liquid N2 and stored at −80°C before use. The tissue samples for RNA were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) using, successively, 20G and 25G needles and stored at −80°C before use.33Goncalves DaSilva A. Yong V.W. Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation.Am J Pathol. 2009; 174: 898-909Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar RNA was extracted using the RNeasy Mini Kit columns (Qiagen, Mississauga, ON, Canada), and for quantitative real-time PCR, RNA was first treated with DNase (Promega, Madison, WI) and reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen). The resulting cDNA was used as a template for the Bio-Rad iCycler detection system (Bio-Rad, Hercules, CA) and 2× SYBR Green Master Mix (Qiagen). Every primer (10× QuantiTect Primer Assay) that was used was purchased from Qiagen, and details can be found on their homepage. Expression of gene transcripts was normalized against the housekeeping gene GAPDH. Relative expression levels for our genes of interest were determined using the formula 2−ΔCT, where ΔCT = CT (gene of interest) − CT (housekeeping gene). From the venous blood of healthy volunteers subjected to Ficoll centrifugation, cells were passed through a CD14 antibody–coated magnetic bead separation process as previously described.36Bar-Or A. Nuttall R.K. Duddy M. Alter A. Kim H.J. Ifergan I. Pennington C.J. Bourgoin P. Edwards D.R. Yong V.W. Analyses of all matrix metalloproteinase members in leukocytes emphasize monocytes as major inflammatory mediators in multiple sclerosis.Brain. 2003; 126: 2738-2749Crossref PubMed Scopus (293) Google Scholar Cells were then cultured in RPMI medium containing 20% human serum. Monocyte migration assays were conducted in Boyden chambers coated with fibronectin, as previously described.36Bar-Or A. Nuttall R.K. Duddy M. Alter A. Kim H.J. Ifergan I. Pennington C.J. Bourgoin P. Edwards D.R. Yong V.W. Analyses of all matrix metalloproteinase members in leukocytes emphasize monocytes as major inflammatory mediators in multiple sclerosis.Brain. 2003; 126: 2738-2749Crossref PubMed Scopus (293) Google Scholar Briefly, each chamber (5-μm pore-size membrane) precoated with fibronectin (BD Biosciences) was inserted into a well of a 24-well plate to create a two-compartment migration system. This assay was performed by seeding cells into the upper chamber of the insert and allowing them to migrate for 24 hours to the other side of the membrane in response to medium containing chemoattractant. At the end of assay, the upper chamber was removed and washed, and any nonmigratory cells on the upper side of the membrane were carefully swabbed away. The migrated cells on the underside of the membrane were stained with hematoxylin for visualization and were counted. Cells that migrated across the membrane and dropped into the medium on the other side were also counted. Cytokine production by human monocytes was assessed after activation of cells with lipopolysaccharide (LPS), with laquinimod added 24 hours before LPS. Cytokines in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol. ELISA kits for matrix metalloproteinase-9 (MMP-9) were purchased from Invitrogen. Data were acquired using a SpectraMax 384 (Molecular Devices Corporation, Sunnyvale, CA) according to the manufacturer's instructions. We treated human monocytes in culture with laquinimod (5 μmol/L) for 24 hours followed by activation with a combination of IFNγ and LPS (iLPS) for two different early time points. Cells were lysed in PhosSTOP and protease inhibitor (Roche, Mannheim, Germany) containing RIPA buffer (Thermo Scientific, Rockford, IL). Ten micrograms of each sample was electrophoresed on polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane. After blocking, the blots were incubated overnight at 4°C with primary antibodies (1:1000) against phospho-p38 MAPK, p38 MAPK, phospho-Jun-N-terminal kinase, Jun-N-terminal kinase, IκB, extracellular regulated kinase (ERK), and phospho-ERK (Cell Signaling Technology, Boston, MA). After extensive washes in PBS–Tween, blots were incubated with appropriate secondary antibodies. The blots were again rinsed in PBS–Tween and processed for development using a chemiluminescence reagent. The images were captured using the ChemiGenius Bioimaging System (Syngene, Cambridge, UK). The blots were stripped and reprobed with anti–β-actin (1:5000; Millipore) to determine equivalent loading of samples. Results were reported as mean ± SEM, and statistical analyses were done by Student's t-test for two groups, or one-way analysis of variance with a Tukey post hoc test for multiple groups. Statistical differences in nonparametric histological scores were evaluated using Mann–Whitney U-test. Differences were considered significant when the P value <0.05. The accumulation of proinflammatory monocytes in blood is poorly understood in EAE. We induced C57BL/6 mice for EAE, and blood was then collected and subjected to Ficoll centrifugation to obtain a mononuclear cell fraction. The proinflammatory monocyte subset was identified on the basis of the CD11b+CCR2+Ly6Chigh phenotype (Figure 1, A and B).37Geissmann F. Jung S. Littman D.R. Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2606) Google Scholar, 38Mildner A. Mack M. Schmidt H. Bruck W. Djukic M. Zabel M.D. Hille A. Priller J. Prinz M. CCR2+Ly-6Chi monocytes are crucial for the effector phase of autoimmunity in the central nervous system.Brain. 2009; 132: 2487-2500Crossref PubMed Scopus (345) Google Scholar The blood of naive mice contained ∼7% of CD11b+ cells that were CCR2+Ly6Chigh. This proportion of proinflammatory monocytes increased by the second day following MOG immunization, was fourfold higher than that of naive mice at day 3, and remained elevated in blood, whereas mice were asymptomatic (Figure 1C). At day 12 of MOG immunization, when most animals displayed the onset of clinical signs (limp tail), the proportion of proinflammatory monocytes in blood returned to naive levels. With increasing evolution to peak clinical signs (tail and hind limb paralysis, and forelimb paresis), at approximately 16 days after MOG immunization in most mice, levels remained at those of naive mice, and remained there at post-peak clinical severity (day 19). The significant drop of proinflammatory monocytes in blood at onset of clinical signs suggests that these cells were entering into the CNS to initiate neuropathology. To address this, we focused on a group of mice at day 16 after MOG immunization, when we expected clinical severity to be at peak for most mice. The proportion of CD11b+ cells that were CCR2+Ly6Chigh was evaluated simultaneously in both the blood and spinal cord. Figure 2A shows the relationship between blood and spinal cord levels of proinflammatory monocytes in individual mice. When these were mapped against clinical disease scores across the group, it was noted that mice with manifestations of clinical disease tended to have low levels of proinflammatory cells in their blood but elevated content in their spinal cord (Figure 2, B and C). Four MOG-immunized mice had unexpectedly shown no clinical signs at day 16 (Figure 2B). However, these mice had weight loss (data not shown), suggesting that clinical EAE was imminent. When examined, these mice had approximately 40% of proinflammatory monocytes in blood, and no detectable levels in the spinal cord (Figure 2, B and C). These results emphasize the importance" @default.
• W2136174903 created "2016-06-24" @default.
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• W2136174903 title "Kinetics of Proinflammatory Monocytes in a Model of Multiple Sclerosis and Its Perturbation by Laquinimod" @default.
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