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Kinetics of Proinflammatory Monocytes in a Model of Multiple Sclerosis and Its Perturbation by Laquinimod

      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.
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      (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.
      • Ajami B.
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      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.
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      and, thus, to reduce proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFNγ), and interleukin-17 (IL-17).
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      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.
      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.
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      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.
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      Laquinimod, a quinoline-3-carboxamide, induces type II myeloid cells that modulate central nervous system autoimmunity.
      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.

      Materials and Methods

      Disease Induction and Assessment of EAE

      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.
      • Giuliani 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.
      • Goncalves DaSilva A.
      • Yong V.W.
      Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation.
      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.
      • Giuliani 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.
      • Goncalves DaSilva A.
      • Yong V.W.
      Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation.
      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.

      Flow Cytometry

      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).
      • Agrawal 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.
      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.
      • Mack 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.
      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.

      Histology and Immunohistochemistry

      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.
      • Goncalves DaSilva A.
      • Yong V.W.
      Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation.
      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.

      Quantitative Real-Time PCR

      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.
      • Goncalves DaSilva A.
      • Yong V.W.
      Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation.
      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).

      Isolation of Human Monocytes and Migration Assay

      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.
      • Bar-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.
      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.
      • Bar-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.
      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.

      ELISA

      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.

      Immunoblots

      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.

      Statistical Analysis

      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.

      Results

      The Proportion of Proinflammatory Monocytes Increases in Blood Promptly after EAE Induction, and Declines to Naive Levels during Onset of Clinical Signs

      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).
      • Geissmann F.
      • Jung S.
      • Littman D.R.
      Blood monocytes consist of two principal subsets with distinct migratory properties.
      • Mildner 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.
      Figure thumbnail gr1
      Figure 1The number of proinflammatory monocytes increases in blood after MOG immunization, and declines during onset of clinical signs. Following Ficoll centrifugation, FACS profiles of CD11b+ cells (A) were obtained. The CD11b+ cells were further gated for CCR2+ and Ly6Chigh expression (B, box in top right quadrant) to detect proinflammatory monocytes. From these, it was noted that levels were high by day 2, maximum at day 3, and remained high at asymptomatic disease; levels were lowered to those of naive mice at onset of clinical signs (day 12), peak clinical disease (day 16), and post peak (day 19) (C). Note that a control group of mice injected with complete Freund's adjuvant, mycobacterium, and pertussis, but without MOG, did not elevate proinflammatory monocytes in blood at day 3 (data not shown). Results are from 4 to 10 mice at each time point. **P < 0.01, ***P < 0.001 compared to naive mice.
      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).

      At Day 16 of EAE, Clinical Scores Are Inversely Correlated with the Proportion of Proinflammatory Monocytes in Blood, and Directly with That in Spinal Cord

      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).
      Figure thumbnail gr2
      Figure 2At day 16 of EAE, clinical scores were inversely related to the number of proinflammatory monocytes in blood, and directly correlated with the number in spinal cord. In mice at day 16 post-MOG immunization, when most animals are expected to be at peak clinical severity, levels of proinflammatory monocytes in blood and corresponding spinal cord (CNS) are displayed (A). When levels in blood were plotted against clinical disease score, an inverse relationship was found (B). By contrast, levels of proinflammatory monocytes in the spinal cord correlated positively with clinical scores (C). The spinal cord was then subjected to discrimination of macrophages from microglia (D), and a direct correlation of macrophage extent in the spinal cord with disease score was found (E). By contrast, the microglia density was not directly correlated with clinical score (F).
      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 of the migration of proinflammatory monocytes into the spinal cord to produce clinical signs of EAE; the mere generation of the proinflammatory monocytes and their accumulation in blood are insufficient for clinical manifestation. Indeed, at time points before the expected occurrence of peak EAE clinical severity, a mouse with a high level of proinflammatory monocytes in blood is likely devoid of EAE symptoms, as monocytes have yet to enter the CNS.
      The monocytoid populations within the inflamed spinal cord can be differentiated into macrophages or microglia based on their high or low expression of CD45, respectively
      • Agrawal 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.
      • Dick A.D.
      • Ford A.L.
      • Forrester J.V.
      • Sedgwick J.D.
      Flow cytometric identification of a minority population of MHC class II positive cells in the normal rat retina distinct from CD45lowCD11b/c+CD4low parenchymal microglia.
      (Figure 2D). Thus, using CD45 and CD11b antibody staining, we addressed whether macrophages or microglia were elevated during EAE and whether their relative numbers corresponded with clinical scores. Figure 2E shows that although the macrophage density in the spinal cord was correlated with clinical scores, the microglia density did not display such statistical correlations (Figure 2F).
      Overall, proinflammatory monocytes accumulate early in blood of mice afflicted with EAE, and a switch then occurs to cause their migration into the CNS correspondent with clinical signs of disease. In mice “protected” from EAE clinical signs at the time of sacrifice, their monocyte numbers in blood would tend to be high, as the cells have yet to acquire the signals and molecules needed for infiltration into the CNS.

      Laquinimod Prevents Manifestation of Clinical Signs of EAE in Mice

      We tested the hypothesis that laquinimod affects the biology of proinflammatory monocytes in the blood of mice immunized for EAE. Laquinimod (25 mg/kg) or saline vehicle was administered by oral gavage from day 5 after MOG immunization. We found that laquinimod prevented mice from succumbing to EAE (Figure 3A). Mice were then sacrificed at day 16 for histological analyses of the spinal cord. Relative to the vehicle-treated EAE mice, laquinimod prevented inflammation, demyelination, and axonal loss (Figure 3, B–D). Finally, using Iba1 immunohistochemistry, we observed a significant increase of Iba1+ macrophages/microglia cells in EAE mice, whereas laquinimod treatment reduced this outcome (Figure 3E).
      Figure thumbnail gr3
      Figure 3Laquinimod prevents manifestation of clinical disease of EAE in mice. Whereas vehicle-treated EAE mice developed progressive clinical signs of EAE, this was prevented by administration of laquinimod (A). This profile is reproduced in four separate experiments involving 8 to 10 mice each time. H&E and Luxol Fast Blue staining showed relative preservation of myelin in the lateral column of the spinal cord in mice treated with laquinimod when mice were sacrificed at day 16, whereas this was severely disrupted in EAE mice treated with vehicle (B). Axonal integrity evaluated using silver stain shows preservation in laquinimod-treated mice and axonal loss in vehicle controls (C). When histological scores were evaluated blind using criteria described previously,
      • Goncalves DaSilva A.
      • Yong V.W.
      Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation.
      laquinimod prevented manifestation of histological alterations (D); **P < 0.01. Iba1 staining for macrophage/microglia in the spinal cord shows an intense up-regulation in EAE, and normal profiles in mice protected with laquinimod (E).

      The Elevation of Proinflammatory Monocytes in Blood of Mice Treated with Laquinimod Corresponds with Reduced Influx into the Spinal Cord

      We investigated the effect of laquinimod on the accumulation of monocytes at day 16 of MOG immunization. When CD11b+ cells were enumerated in blood, we found that there was a trend toward an increase in symptomatic EAE vehicle–treated mice compared to naive controls; in laquinimod-treated mice protected from EAE signs, total blood monocyte counts did not differ from those of EAE vehicle–treated controls (Figure 4A). However, when the proportion of proinflammatory monocytes (CCR2+Ly6Chigh) from the total CD11b+ pool was examined, we found that this was significantly increased in laquinimod-treated mice compared to that of EAE vehicle–treated controls (Figure 4, B and C).
      Figure thumbnail gr4
      Figure 4At day 16 post-MOG immunization, the proportion of proinflammatory monocytes is elevated in laquinimod-treated mice. Although the number of CD11b+ cells in blood did not differ between vehicle or laquinimod-treated EAE animals (A), separation into those that are proinflammatory shows the laquinimod-treated animals had elevated levels of proinflammatory monocytes in blood (B); at this point, all animals are asymptomatic in the laquinimod-treated animals. C: FACS profiles of proinflammatory monocytes (box in top right quadrant) in naive (left), EAE vehicle (middle), or laquinimod-treated (right) mice. Spinal cord infiltration of proinflammatory monocytes in EAE was diminished in laquinimod-treated animals (D). In contrast to their counts in blood, no differential effects were seen in the spleen (E). Panels A, B, D, and E are of six EAE mice in either the vehicle or laquinimod group, and the results were reproduced in another experiment of six mice each. **P < 0.01, ***P < 0.001 compared to naive mice; P < 0.01 compared to the corresponding EAE group.
      In support of the contention that laquinimod treatment prevents proinflammatory monocytes from entering into the CNS, the macrophage population was enumerated in the spinal cord. Figure 4D shows that this was significantly decreased in EAE-immunized mice that were treated with laquinimod.
      Finally, we addressed whether the proinflammatory monocytes in laquinimod-treated mice would be similarly retarded in their migration into other organs. We chose the spleen for analysis, as this is an organ that monocytes migrate to. We found that the spleen did not discriminate trafficking of proinflammatory monocytes in vehicle- or laquinimod-treated EAE mice, as comparable levels were found in both groups (Figure 4E). Thus, the reduced entry of proinflammatory monocytes into the spinal cord appears to be selective for this organ, at least when compared to the spleen.

      Mechanisms of Laquinimod in Reducing the Influx of Proinflammatory Monocytes into the CNS

      The trafficking of leukocytes into the CNS is dependent on several factors, including their adhesion onto endothelial cells of blood vessels, the production of MMPs to migrate across the parenchymal basement membranes separating blood vessels from the CNS parenchyma, and a chemokine gradient to increase cellular attachment and provide directional cues.
      • Agrawal S.
      • Anderson P.
      • Durbeej M.
      • van Rooijen N.
      • Ivars F.
      • Opdenakker G.
      • Sorokin L.M.
      Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis.
      • Greenwood J.
      • Heasman S.J.
      • Alvarez J.I.
      • Prat A.
      • Lyck R.
      • Engelhardt B.
      Review: leucocyte-endothelial cell crosstalk at the blood-brain barrier: a prerequisite for successful immune cell entry to the brain.
      As we observed the accumulation of proinflammatory monocytes in blood and corresponding reduction in CNS, we addressed whether laquinimod affects these factors that regulate leukocyte trafficking. Key molecules that influence the attachment of monocytes onto endothelial cells include CD62L (L-selectin).
      • Xu H.
      • Manivannan A.
      • Crane I.
      • Dawson R.
      • Liversidge J.
      Critical but divergent roles for CD62L and CD44 in directing blood monocyte trafficking in vivo during inflammation.
      We found that CD62L was present on all leukocyte subsets and we focused our attention on the proinflammatory monocytes. We found that when CD11b+CCR2+ly6Chigh monocytes were gated from blood of mice at day 16 following MOG immunization, the percentage of cells expressing CD62L was elevated in symptomatic EAE-vehicle mice compared to naive controls; importantly, the percentage of cells expressing CD62L in asymptomatic laquinimod-treated, MOG-immunized mice was at naive levels (Figure 5A). The amount of CD62L per cell in positive monocytes was not altered from normal levels by laquinimod (data not shown).
      Figure thumbnail gr5
      Figure 5Laquinimod alters the expression of molecules that regulate infiltration of proinflammatory monocytes into the spinal cord. The blood of EAE mice treated with laquinimod, and gated for proinflammatory monocytes at day 16, shows that these cells have reduced expression of CD62L (A). Moreover, when the spinal cord was subjected to PCR analysis, the elevation of CCR2 (B) and CCL2 (C) during EAE is reduced in laquinimod-treated animals. *P < 0.05, **P < 0.01 compared to naive animals; P < 0.05 compared to EAE vehicle animals.
      Next, we determined the levels of CCR2 and its ligand CCL2 in the spinal cord, as these are key chemokine–receptor pairs that regulate the trafficking of monocytes into tissues.
      • Glabinski A.R.
      • Bielecki B.
      • O'Bryant S.
      • Selmaj K.
      • Ransohoff R.M.
      Experimental autoimmune encephalomyelitis: CC chemokine receptor expression by trafficking cells.
      Transcripts encoding these molecules were detected by PCR, and we found that the significant elevation of both CCR2 and CCL2 in the spinal cord of EAE mice was prevented by laquinimod (Figure 5, B and C). However, we note that reduced levels of CCR2 and CCL2 could also be the consequence of suppressed EAE, rather than the reason for the reduced disease.
      Finally, we examined the levels of MMP-9. Attempts to detect MMP-9 in the gated CD11b+CCR2+Ly6Chigh population by flow cytometry were futile due to unreliable staining by a panel of MMP-9 antibodies. We thus resorted to tissue culture studies using human monocytes. Cells were activated with LPS, with different concentrations of laquinimod added 24 hours before LPS. The elevated level of MMP-9 by LPS was reduced by laquinimod to a non-LPS–treated level at concentrations of 0.1 μmol/L and higher (Figure 6A).
      Figure thumbnail gr6
      Figure 6The production of monocyte MMP-9 and cellular transmigration was reduced by laquinimod. Monocytes increase their content of MMP-9 on activation with LPS, and this was reduced by laquinimod (A). ***P < 0.001 compared to LPS. The number of cells that transmigrated a fibronectin-coated membrane was reduced by laquinimod (5 μmol/L), either when cells that were still attached to the under surface of the fibronectin membrane (B) or had dropped off into the culture medium (C) were evaluated. *P < 0.05, **P < 0.01 compared to the LPS group. Each histogram represents quadruplicate cultures, and the results were reproduced in another experiment.
      The transmigratory capacity of monocytes was determined by their protease-dependent migration across a Boyden chamber coated with fibronectin, a model of the blood-brain barrier. Human monocytes were pretreated with laquinimod (5 μmol/L) for 24 hours, and were then activated with LPS to obtain a proinflammatory subset for another 24 hours. The cells were examined for their transmigration over a 24-hour period. Laquinimod reduced the transmigration of monocytes, either when the cells that were still attached to the underside of the fibronectin membrane were evaluated (Figure 6B), or when the number of cells that had detached from the underside and suspended in medium in the lower chamber was counted (Figure 6C). Note that the results of attached cells (Figure 6B) and those that had dropped into the culture medium after transmigration (Figure 6C) are presented separately since the latter quantity is at least two orders of magnitude higher than that of attached cells. In either case, migration of monocytes was reduced by laquinimod treatment.
      In summary, laquinimod treatment reduces molecules that are implicated in the trafficking of leukocytes into the CNS, thereby helping to account for the accumulation of proinflammatory monocytes in blood and their failure to enter the spinal cord.

      Laquinimod Reduces the Activity of Stress-Related MAPK Enzymes and NF-κB

      To investigate further the impact of laquinimod on monocytes that may help account for the above observations, 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). Cells were harvested promptly thereafter since phosphorylation events occur rapidly in cells. Western blot analyses demonstrated a significant inhibition by laquinimod of the phosphorylation state of the stress-related proteins p38 mitogen-activated protein kinase (MAPK) and Jun-N-terminal kinase (JNK), but not in ERK1/2 (extracellular signal-regulated kinase) (Figure 7). Moreover, the NF-κB pathway, whereby activation can be monitored by loss of the inhibitory IκB protein, was activated by iLPS, and this was prevented by laquinimod. Thus, laquinimod has the capacity to prevent cellular signaling induced by potent activators of monocytes, and this may help account for the various effects that it imparts on proinflammatory monocytes.
      Figure thumbnail gr7
      Figure 7The activation of stress-related proteins is attenuated by laquinimod. Human monocytes were left as controls (C), treated with laquinimod alone, or were exposed to interferon-γ and LPS (iLPS) for 10 or 30 minutes, with or without laquinimod. Although laquinimod alone did not alter levels seen in controls, the significant elevation of phospho-p38 MAPK and pJNK induced by iLPS was suppressed by laquinimod. Levels of the nonphosphorylated forms of the stress proteins were used as loading controls, as were levels of β-actin. Laquinimod also reduced NF-κB activation as depicted by the lowered degradation of IκB. The results of this experiment were reproduced in another series.

      Discussion

      A hallmark of MS is the dysfunction of the immune system, and this is emphasized by the recent report
      • Sawcer S.
      • Hellenthal G.
      • Pirinen M.
      • Spencer C.C.
      • Patsopoulos N.A.
      • Moutsianas L.
      • et al.
      Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.
      that of over 50 gene loci associated with increased risk for MS, all of them code for components of immunity. B lymphocytes, T cells (particularly of the Th1 and Th17 subsets), dendritic cells, monocytes, macrophages, and microglia, all have roles in promoting immune reactivity in MS.
      • Berer K.
      • Wekerle H.
      • Krishnamoorthy G.
      B cells in spontaneous autoimmune diseases of the central nervous system.
      • Gandhi R.
      • Laroni A.
      • Weiner H.L.
      Role of the innate immune system in the pathogenesis of multiple sclerosis.
      • Murphy A.C.
      • Lalor S.J.
      • Lynch M.A.
      • Mills K.H.
      Infiltration of Th1 and Th17 cells and activation of microglia in the CNS during the course of experimental autoimmune encephalomyelitis.
      • Weiner H.L.
      The challenge of multiple sclerosis: how do we cure a chronic heterogeneous disease?.
      However, much remains to be uncovered on how one subset interacts with another, how each affects CNS elements to produce neurodegenerative changes, and when they play particularly prominent roles in initiating or promoting a relapse. We have focused in this study on the generation and dynamics of proinflammatory monocytes in EAE as a model of MS.
      Monocytes are produced in the bone marrow and are continuously released into the circulation. We found that on immunization with MOG, the proinflammatory monocyte number characterized by CCR2+ and Ly6Chigh expression
      • Geissmann F.
      • Jung S.
      • Littman D.R.
      Blood monocytes consist of two principal subsets with distinct migratory properties.
      • Mildner 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.
      was expanded rapidly and significantly, and these cells remained elevated in the circulation without significant infiltration into the CNS in asymptomatic animals. At onset of clinical signs of EAE, the blood content of proinflammatory monocytes dropped significantly to the levels in naive mice, and this was coincident with their appearance within the spinal cord. Interestingly, in animals in which the EAE induction appeared to be unsuccessful, as these mice had not succumbed to EAE clinical signs at the expected time point, the numbers of proinflammatory monocytes were significantly elevated in blood; thus, immunization occurred, but cells did not migrate into the CNS. Indeed, the content in blood at around onset of clinical signs was negatively correlated with impending clinical signs. We found that the emerging MS medication laquinimod reduced the trafficking of proinflammatory monocytes into the CNS and prevented the development of clinical signs. Laquinimod itself did not appear to influence the generation of monocytes, since total CD11b+ numbers from the post-Ficoll pool that was devoid of neutrophils did not differ between EAE mice treated with vehicle or with laquinimod. However, the proinflammatory monocytes were retained in blood by laquinimod, likely due to the reduced expression of molecules that regulate the trafficking of monocytes into inflamed tissues: CD62L, CCL2, CCR2, and MMP-9. The decreased migration of proinflammatory monocytes may also be contributed by the laquinimod attenuation of the activity of the stress-related proteins p38MAPK and JNK within monocytes. MAP kinases are implicated in vivo in injury-induced corneal epithelial migration,
      • Okada Y.
      • Saika S.
      • Shirai K.
      • Yamanaka O.
      • Kitano A.
      • Wang Z.
      • Yang H.
      • Reinach P.
      JNK MAPK signaling contributes in vivo to injury-induced corneal epithelial migration.
      in epithelial wound healing in general,
      • Sharma G.D.
      • HE J.
      • Bazan H.E.
      p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades.
      and in the motility of bone marrow–derived mesenchymal stem cells when activated with TNF-α.
      • Fu X.
      • Han B.
      • Cai S.
      • Lei Y.
      • Sun T.
      • Sheng Z.
      Migration of bone marrow-derived mesenchymal stem cells induced by tumor necrosis factor-alpha and its possible role in wound healing.
      Transient activation of NF-κB can be achieved in many cells by treatment with lipopolysaccharide.
      • Sen R.
      • Baltimore D.
      Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism.
      The regulatory IκB proteins sequester NF-κB in cytoplasm, to suppress their DNA binding activities and to release them in response to the appropriate stimuli.
      • Traenckner E.B.
      • Wilk S.
      • Baeuerle P.A.
      A proteasome inhibitor prevents activation of NF-kappa B and stabilizes a newly phosphorylated form of I kappa B-alpha that is still bound to NF-kappa B.
      The activation of NF-κB occurs by phosphorylation and degradation of the IκB protein.
      • Traenckner E.B.
      • Wilk S.
      • Baeuerle P.A.
      A proteasome inhibitor prevents activation of NF-kappa B and stabilizes a newly phosphorylated form of I kappa B-alpha that is still bound to NF-kappa B.
      Laquinimod also reduced NF-κB activity in monocytes, as determined by the lack of loss of IκB level.
      Recently, the data of two phase III clinical trials of laquinimod have been disclosed. In the first (ALLEGRO), the annualized relapse rate reduction was 23% (P < 0.05) compared to placebo, the reduction of risk of disability progression was 36% (P = 0.0122), and the reduction of brain volume loss was 33%.
      • Comi G.
      • Jeffery D.
      • Kappos L.
      • Montalban X.
      • Boyko A.
      • Rocca M.A.
      • Filippi M.
      Placebo-controlled trial of oral laquinimod for multiple sclerosis.
      • Comi G.J.D.
      • Kappos L.
      • Montalban X.
      • Boyko A.
      • Filippi M.
      Allergo Study Group
      Oral medication slows disability progression and reduces severe relapses in the placebo-controlled phase III Allergo trial for treatment of relapsing-remitting multiple sclerosis.
      Recent data from the second trial (BRAVO) reinforced these results.
      • Vollmer TL S.P.
      • Arnold D.L.
      BRAVO study group
      A placebo-controlled and active comparator phase III trial (BRAVO) for relapsing-remitting multiple sclerosis.
      The more pronounced effect on disability and brain atrophy versus relapse rate may suggest that laquinimod has less impact on immune functions and a more prominent role in neuroprotection. Although this remains to be clarified, our results of laquinimod inhibiting the migration of proinflammatory monocytes into the CNS may help explain the reduction in brain atrophy observed in MS subjects. Proinflammatory monocytes become macrophages in tissue, and the local accumulation of macrophages and activated microglia is correspondent with degeneration of axons in EAE and MS lesions.
      • Nikic 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.
      • Bitsch A.
      • Schuchardt J.
      • Bunkowski S.
      • Kuhlmann T.
      • Bruck W.
      Acute axonal injury in multiple sclerosis Correlation with demyelination and inflammation.
      • Trapp B.D.
      • Peterson J.
      • Ransohoff R.M.
      • Rudick R.
      • Mork S.
      • Bo L.
      Axonal transection in the lesions of multiple sclerosis.
      Thus, the reduced accumulation of macrophages in the CNS by laquinimod, suggested from this study, would lead to fewer neurodegenerative processes and thus the reduction of brain atrophy observed in clinical trials.
      Our data guide future immunological studies of laquinimod in MS subjects. In those treated with laquinimod, it would be important to discern whether the proinflammatory subset of monocytes is altered, as simply enumerating the total monocyte numbers in blood would likely not be as informative. Moreover, the reduced levels of CD62L on proinflammatory monocytes, and their production of MMP-9, could be instructive for the effectiveness of laquinimod in treated subjects.
      There remain many unanswered questions on the biology of proinflammatory monocytes in MS. It is unclear why these cells accumulate in the circulation for a number of days before their entry into the spinal cord. Whether the time delay is necessary for proinflammatory monocytes to accumulate molecules necessary for transmigration, including adequate levels of integrins, selectins, and MMPs, or whether the hiatus represents the time required for a chemotactic gradient to develop in the CNS, are reasonable questions to resolve in future studies. Moreover, as we evaluated only proinflammatory monocytes in this study, it clearly would be of interest in future to delineate the dynamics of M2 cells in EAE, and to address whether these are also altered by laquinimod treatment. Another MS medication, glatiramer acetate, promotes the generation of M2 cells in addition to activity on other leukocyte subsets.
      • Weber M.S.
      • Prod'homme T.
      • Youssef S.
      • Dunn S.E.
      • Rundle C.D.
      • Lee L.
      • Patarroyo J.C.
      • Stuve O.
      • Sobel R.A.
      • Steinman L.
      • Zamvil S.S.
      Type II monocytes modulate T cell-mediated central nervous system autoimmune disease.
      In reviewing the literature, it is clear that laquinimod has several immunomodulatory activities, including reducing the generation of proinflammatory T cells,
      • Wegner 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.
      altering the monocyte population such that these produce anti-inflammatory cytokines,
      • Thone 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.
      and the generation of type II antigen-presenting cells that can favor the differentiation of anti-inflammatory/regulatory T cells.
      • Schulze-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.
      It is possible that the alterations of the proinflammatory monocytes observed in this study are secondary to one of these outcomes, and vice versa; this possibility will be examined in future studies.
      In conclusion, our studies have provided novel insights into the kinetics of proinflammatory monocytes that accumulate early in the blood of mice immunized for EAE, with a switch then occurring to cause their migration into the CNS, correspondent with the development of clinical signs of disease. Our results invite investigations into the mechanisms that transform circulating proinflammatory monocytes into migratory cells that then produce CNS pathology. Finally, we provide the novel findings of laquinimod as a medication that modulates the biology and migration of proinflammatory monocytes into the CNS, thereby extending the understanding of the mechanism of action of laquinimod.

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