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Soluble CD40 ligand disrupts the blood–brain barrier and exacerbates inflammation in experimental autoimmune encephalomyelitis
Journal of Neuroimmunology xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Soluble CD40 ligand disrupts the blood–brain barrier and exacerbates inflammation in experimental autoimmune encephalomyelitis ⁎
Hiroki Masudaa, Masahiro Moria, , Kenta Umeharab, Tomomi Furihatab,c, Tomohiko Uchidaa, Akiyuki Uzawaa, Satoshi Kuwabaraa a
Department of Neurology, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan c Department of Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan b
A R T I C L E I N F O
A B S T R A C T
Keywords: Blood–brain barrier Experimental autoimmune encephalomyelitis Multiple sclerosis Soluble CD40 ligand
Serum soluble CD40 ligand (sCD40L) has been reported to positively correlate with the albumin quotient, a marker of blood–brain barrier (BBB) breakdown, in patients with multiple sclerosis (MS). To clarify the mechanisms of sCD40L in MS pathophysiology, sCD40L was administered to experimental autoimmune encephalomyelitis (EAE) mice and a human brain microvascular endothelial cell (HBMEC)-based BBB model. The high-dose sCD40L group showed a worse EAE score than the low-dose and control groups. BBB permeability was increased by administering sCD40L in a HBMEC-based BBB model. Thus, sCD40L induces more severe inflammation in the central nervous system by disrupting the BBB.
1. Introduction Multiple sclerosis (MS) is a demyelinating and neurodegenerative disease of the central nervous system (CNS). A feature of MS is dissemination in time and space. Although the etiology of MS remains unclear, both T and B cells have been reported to play important roles in the pathogenesis of MS (Hartung et al., 2014). Moreover, blood–brain barrier (BBB) breakdown has been recognized as a critical step in MS pathology (Absinta et al., 2015; Spencer et al., 2018). Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS in which T cells expressing interleukin-17 (IL-17) infiltrate the CNS at the early stage of EAE. Therefore, T helper 17 (Th17) cells are considered a key factor in the pathogenesis of EAE (Kurschus, 2015). CD40 ligand (CD40L) is a transmembrane protein that plays an important role in the immune system. The main function of CD40L is to produce high-affinity B cells (Elgueta et al., 2009). Recent studies have suggested that CD40L increases the permeability of the BBB (Davidson et al., 2012; Ishikawa et al., 2005). Soluble CD40L (sCD40L) has been reported to have the same function as CD40L (Vakkalanka et al., 1999). In the peripheral blood, sCD40L has been found to be produced only by shedding from the surface of activated platelets or activated T cells and
exists in trimers (Aloui et al., 2014). The molecular weight of sCD40L is 18 kDa, which is much smaller than albumin (66 kDa) (Becker, 2004; Pietravalle et al., 1996). CD40L works by binding to CD40, which is expressed on the surface of B cells and other cells, including macrophages, dendritic cells, smooth muscle cells, microglia, astrocytes, and endothelial cells. Astrocytes and endothelial cells are also the components of the BBB. Previously, we reported elevated serum sCD40L levels in patients with MS and a positive correlation between serum sCD40L levels and the albumin quotient (Qalb) (Masuda et al., 2017). Qalb gives the cerebrospinal fluid (CSF)/serum albumin ratio, which is a marker of BBB dysfunction. Meanwhile, CD40–CD40L interaction has been reported to be critical for Th17 differentiation (Iezzi et al., 2009). We hypothesized that sCD40L could cause BBB breakdown or severe inflammation through the upregulation of Th17 cells in MS pathogenesis. Therefore, we investigated the function of sCD40L in EAE mice and an in vitro study using human brain microvascular endothelial cells (HBMEC).
Abbreviations: BBB, blood–brain barrier; EAE, experimental autoimmune encephalomyelitis; CD40L, CD40 ligand; CNS, central nervous system; HAND, HIV-associated neurocognitive disorder; HBMEC, human brain microvascular endothelial cells; IL-6, interleukin-6; IQR, interquartile range; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; NMO, neuromyelitis optica; NMOSD, neuromyelitis optica spectrum disorder; Qalb, albumin quotient (CSF/serum albumin ratio); sCD40L, soluble CD40L ⁎ Corresponding author. E-mail addresses: [email protected] (H. Masuda), [email protected] (M. Mori), [email protected] (K. Umehara), [email protected] (T. Furihata), [email protected] (T. Uchida), [email protected] (A. Uzawa), [email protected] (S. Kuwabara). https://doi.org/10.1016/j.jneuroim.2018.01.001 Received 4 December 2017; Received in revised form 21 December 2017; Accepted 2 January 2018 0165-5728/ © 2018 Published by Elsevier B.V.
Please cite this article as: Masuda, H., Journal of Neuroimmunology (2018), https://doi.org/10.1016/j.jneuroim.2018.01.001
Journal of Neuroimmunology xxx (xxxx) xxx–xxx
H. Masuda et al.
2. Materials and methods
2.7 mM KCl, 1.5 mM KH2PO4, 10 mM NaH2PO4, 25 mM glucose, and 10 mM Hepes; pH 7.4). Twenty-four hours before the initiation of the assay, human sCD40L was administered to the insert (apical side). The recombinant human sCD40L was resuspended in sterile water with 0.5% BSA. The concentration of sCD40L was 100 ng/mL based on the previous research (Ramirez et al., 2010). The assay was initiated by adding Na-F (Sigma) (150 ng/insert) to the insert at 37 °C. Immediately after the initiation (0 min) and after incubation for 30, 60, and 90 min, the medium was collected from the well (basolateral side). The fluorescence of Na-F in each medium was determined using an ARVO-SX (PerkinElmer, Waltham, MA, USA) with wavelengths ex/em¼485 nm/ 535 nm. The permeability coefficients (Pe, cm/min) were calculated. The experiment was performed five times.
2.1. EAE induction in mice Ten-week-old C57BL/6 female mice (18–25 g) were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were housed in specific pathogen-free facilities at Chiba University with a maximum of four animals per cage, with free access to water and standard rodent chow. EAE was induced using immunization with myelin oligodendrocyte glycoprotein (MOG). A total of 200 mg MOG peptide 35–55 in complete Freund's adjuvant containing 400 mg of killed Mycobacterium tuberculosis H37Ra was injected subcutaneously at two sites. On the days of immunization (days 0 and 1), 200 ng pertussis toxin was injected intraperitoneally using Hooke kits (EK-0105; Hooke Laboratories, Lawrence, MA, USA). EAE was scored on the following scale: 0 = no clinical signs; 1 = partial paralysis of tail; 2 = flaccid tail; 3 = limp tail and partial weakness of hind legs; 4 = limp tail and complete weakness of hind legs; 5 = limp tail, complete weakness of hind legs and partial weakness of front legs; and 6 = complete hind and front legs paralysis. The intermediate condition of each score was assessed as 0.5. Dr. Uzawa immunized the mice, and EAE clinical scores were assessed blindly by Dr. Masuda to avoid subjective bias.
2.5. Statistical analysis Continuous data were compared using the Mann-Whitney U test with a Bonferroni correction. To consider the multiple testing problem, we applied the Bonferroni correction on the computed P values to reduce type I errors. A P value of < 0.05 was considered statistically significant. Statistical tests were conducted using SPSS version 24.0 (IBM Corporation, Armonk, NY, USA).
2.2. Treatment with sCD40L
2.6. Ethics
Recombinant mouse sCD40L was obtained from Abnova (P4590; Taipei, Taiwan). Mice were injected intraperitoneally on days 0 and 1 with recombinant mouse sCD40L resuspended in phosphate buffered saline with 0.1% bovine serum albumin (BSA) at the same time with Pertusis Toxin (PT) administration. Injection side of sCD40L was opposite from the injection side of PT. EAE mice were divided into three groups, the high sCD40L group (0.2 mg/g body weight), the low sCD40L group (0.05 mg/g body weight), and the control group (only BSA was administered). Each group comprised 12 mice. The concentration of sCD40L was determined based on previous research (Davidson et al., 2012).
The study procedure in animal experiments was approved by the ethics committee of Chiba University School of Medicine (No. A27209). 3. Results 3.1. EAE severity was increased by sCD40L administration The result of EAE study is shown in Fig. 1. The result showed the high sCD40L group exacerbated EAE clinical score compared with the other two groups. The high sCD40L group showed the worse clinical score on days 18, 21–25 (P < 0.05), and days 23 and 26 (P < 0.01) compared with the control group. Meanwhile, the result showed a
2.3. Serum Th17 cytokine assays in EAE mice To assess sCD40L affecting Th17 cells in the early phase of EAE, we measured serum Th17 cytokines including IL-17E, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, macrophage inflammatory protein (MIP-3a), IL-1b, IL-2, IL-4, IL-5, IL-6, IL-21, IL-22, IL-28B, IL-10, IL-23, IL-12p70, IL-27, IL-13, IL-15, IL-17A, IL-17F, IL-33, IL-31, tumor necrosis factor (TNF)β, TNFα, and CD40L on day 14 (at the early phase of the peak). All serum samples were centrifuged at 3000 rpm for 10 min. Just after centrifugation all serum samples were immediately stored at −80 °C until analysis. Cytokine measurements were performed using a multiplexed fluorescent magnetic bead-based immunoassay (Merck Millipore, Darmstadt, Germany), according to the manufacturer's instructions. Cytokine levels were calculated with reference to a standard curve for each cytokine. 2.4. BBB permeability assay Recombinant human sCD40L was obtained from Enzo Life Sciences (ALX-522-015-2010; Farmingdale, NY, USA). We performed sodium fluorescein (Na-F) permeability assay using the HBMEC/ci18-based in vitro BBB method (Kitamura et al., 2017). Ci18 cell line is a sister clone of HBMEC/ciβ, and the assay was performed as reported previously (Kamiichi et al., 2012). Briefly, HBMEC/ci18 were seeded at 4.0 × 105 cells/mL on a membrane filter of an insert culture system (polyethylene terephthalate, 0.4-mm high-density pores, and 0.3 cm2, BD Falcon, Franklin Lakes, NJ, USA), and then cultured for 3 days. Before the assay, the cells were pre-incubated over 30 min at 37 °C with Ringer-Hepes buffer (136 mM NaCl, 0.9 mM CaCl2, 0.5 mM MgCl2,
Fig. 1. Effects of sCD40L administration to EAE mice. The straight line shows the high sCD40L EAE group (0.2 mg/g body weight of sCD40L and BSA). The dotted line shows the low sCD40L EAE group (0.05 mg/g body weight of sCD40L and BSA). The long-short dashed line shows the control EAE group (BSA without sCD40L). Compared with the control EAE group, the high sCD40L EAE group showed higher clinical EAE scores on days 18, 21–25 (P < 0.05) and days 23 and 26 (P < 0.01). Results are mean ± standard error of the mean. *P < 0.05 and †P < 0.01 by MannWhitney U test between the high sCD40L EAE group and the control EAE group. EAE, experimental autoimmune encephalomyelitis; sCD40L, soluble CD40 ligand.
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neurocognitive disorder (HAND). Although there may be various mechanisms in BBB disruption in HAND (Anesten et al., 2016), a recent study about HAND suggested that sCD40L increased the permeability of the BBB (Davidson et al., 2012). Previously, we reported elevated serum sCD40L levels at the relapse phase in patients with MS and the positive correlation between serum sCD40L levels and Qalb (Masuda et al., 2017). This result may suggest that sCD40L could cause BBB disruption during relapse in patients with MS. However, as noted in the Introduction, the molecular weight of sCD40L is much lower than that of albumin (Pietravalle et al., 1996) (Becker, 2004). Therefore, the possibility wherein sCD40L was increased passively due to the BBB breakdown by neuro-inflammation was not ruled out in our previous report. However, this study, applying the HBMEC/ci18-based in vitro BBB model, revealed that sCD40L administration showed higher Pe, which suggests that sCD40L could increase the permeability of the BBB. CD40, the receptor of CD40L, is expressed on the endothelial cells. Therefore, sCD40L may be involved in the disruption of the BBB by binding to CD40 on the endothelial cells. Our study has some limitations. First, the administration of sCD40L at the initiation of immunization could affect not only endothelial cells but also other cells, such as B cells and astrocytes on which CD40 expresses. Therefore, sCD40L could have multiple functions more than BBB disruption in the pathogenesis of EAE. Second, the timing of BBB opening was not identified in our study in vivo. A previous study revealed increased permeability of the BBB was already observed 5 days after immunization (Bergman et al., 1978). Therefore, administration of sCD40L at immunization could accelerate the onset of EAE mice. However, in this study, the days from immunization to onset were not different between the high sCD40L group and the control group. Increased dosages of sCD40L or number of mice may clarify this discrepancy. Finally, there is a time discrepancy about sCD40L effect between in vivo study and in vitro study in our study. In vivo study, EAE mice severity was increased two weeks after sCD40L administration at the immunization. On the other hand, enhanced BBB permeability was observed 24 h after the administration of sCD40L in vitro study. However, similar result was obtained in a recent study about EAE (Bennett et al., 2010). The study reported PT increased BBB permeability in vitro. In EAE study, PT is administered at the initiation and causes EAE peak after two weeks later. Moreover, the previous study showed PT increased BBB permeability maximally just before the peak (Bergman et al., 1978). Therefore, administration of sCD40L at immunization could also increase BBB permeability just before the peak as PT. In conclusion, our results show sCD40L administration exacerbated the clinical severity of EAE mice and increased the permeability of the endothelial cells, which is a component of the BBB. Previously, thromboembolism was caused by anti-CD40L antibody in patients with systemic lupus erythematosus (Sidiropoulos and Boumpas, 2004). However, new anti-CD40L antibody without the adverse effect of thromboembolism was reported (Shock et al., 2015). Therefore, blocking sCD40L by these antibodies or eliminating sCD40L by plasmapheresis could be a new therapeutic option for patients with MS.
Fig. 2. Na-F permeability assay in using the HBMEC/ci18-based in vitro BBB method. Administration of sCD40L increased Pe compared with the control (P = 0.032). Results are mean ± standard deviation of the mean. HBMEC, human brain microvascular endothelial cells; Na-F, sodium fluorescein; sCD40L, soluble CD40 ligand.
worse clinical score in the high sCD40L group compared with the low sCD40L group on days 20, 22–26 (P < 0.05). No differences were found between the low sCD40L group and the control group. Maximum scores and days from immunization to the onset were not different among the three groups. The days from immunization to the onset or to the peak were not different among the three groups (data not shown). 3.2. Serum Th17 cytokines were not different among the high sCD40L group, the low sCD40L group, and the control group at peak The correct amount of serum was not obtained in five mice (one in the high sCD40L group, two in the low sCD40L group, and two in the control group). The result showed no differences in all the measured cytokines among the three groups (Supplementary Table). 3.3. BBB permeability was increased by the administration of sCD40L The result of BBB permeability study is shown in Fig. 2. Pe in the sCD40L group was higher than Pe in the control group (median: 2.43 vs 1.78 · 10− 3 cm/min, IQR: 0.85 vs 0.64, P = 0.032). 4. Discussion This study revealed the following points: 1) Treatment with sCD40L exacerbated EAE clinical scores; 2) Th17 cytokines at the early phase of the peak were not deviated by sCD40L administration at the induction of EAE mice; 3) Increased BBB permeability was observed by the administration of sCD40L in the HBMEC/ci18 -based in vitro BBB model. A previous study reported that CD40-CD40L cross-talk was important for Th17 cell development (Iezzi et al., 2009). Our study showed that the administration of sCD40L at the induction of EAE exacerbated EAE severity. Therefore, we hypothesized that administered sCD40L promoted Th17 cells differentiation, causing increased severity in EAE mice, because Th17 cells were reported to be a key factor in the pathogenesis of EAE (Kurschus, 2015). However, Th17 cytokines at peak in EAE mice were not different with or without sCD40L administration in our study. Previously, pathogenic role for B cells and antibodies in the onset of EAE was reported (Lyons et al., 2002; Lyons et al., 1999). A recent study suggested B cells transverse the intact BBB more easily than T cells (Liu et al., 2012). Meanwhile, CD40L produces highaffinity B cells (Elgueta et al., 2009). Therefore, sCD40L administration may activate not Th17 cells but B cells in our study. Further investigation is needed to address this hypothesis. Increased BBB permeability was also reported in HIV-associated
Conflicting interests The authors declare that there is no conflict of interest.
Funding None.
Acknowledgements This study was supported by JSPS KAKENHI (Grant Number 17K16109). 3
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Appendix A. Supplementary data
Kamiichi, A., Furihata, T., Kishida, S., Ohta, Y., Saito, K., Kawamatsu, S., Chiba, K., 2012. Establishment of a new conditionally immortalized cell line from human brain microvascular endothelial cells: a promising tool for human blood-brain barrier studies. Brain Res. 1488, 113–122. Kitamura, K., Umehara, K., Suzuki, S., Chiba, K., Anzai, N., Akita, H., Furihata, T., 2017. Functional characterization of a new clone of conditionally immortalized human brain microvascular endothelial cells, HBMEC/ci18. In: 12th International Conference on Cerebral Vascular Biology, Australia, pp. 51. Kurschus, F.C., 2015. T cell mediated pathogenesis in EAE: molecular mechanisms. Biom. J. 38, 183–193. Liu, G., Muili, K.A., Agashe, V.V., Lyons, J.A., 2012. Unique B cell responses in B celldependent and B cell-independent EAE. Autoimmunity 45, 199–209. Lyons, J.A., San, M., Happ, M.P., Cross, A.H., 1999. B cells are critical to induction of experimental allergic encephalomyelitis by protein but not by a short encephalitogenic peptide. Eur. J. Immunol. 29, 3432–3439. Lyons, J.A., Ramsbottom, M.J., Cross, A.H., 2002. Critical role of antigen-specific antibody in experimental autoimmune encephalomyelitis induced by recombinant myelin oligodendrocyte glycoprotein. Eur. J. Immunol. 32, 1905–1913. Masuda, H., Mori, M., Uchida, T., Uzawa, A., Ohtani, R., Kuwabara, S., 2017. Soluble CD40 ligand contributes to blood-brain barrier breakdown and central nervous system inflammation in multiple sclerosis and neuromyelitis optica spectrum disorder. J. Neuroimmunol. 305, 102–107. Pietravalle, F., Lecoanet-Henchoz, S., Blasey, H., Aubry, J.P., Elson, G., Edgerton, M.D., Bonnefoy, J.Y., Gauchat, J.F., 1996. Human native soluble CD40L is a biologically active trimer, processed inside microsomes. J. Biol. Chem. 271, 5965–5967. Ramirez, S.H., Fan, S., Dykstra, H., Reichenbach, N., Del Valle, L., Potula, R., Phipps, R.P., Maggirwar, S.B., Persidsky, Y., 2010. Dyad of CD40/CD40 ligand fosters neuroinflammation at the blood-brain barrier and is regulated via JNK signaling: implications for HIV-1 encephalitis. J. Neurosci. 30, 9454–9464. Shock, A., Burkly, L., Wakefield, I., Peters, C., Garber, E., Ferrant, J., Taylor, F.R., Su, L., Hsu, Y.M., Hutto, D., Amirkhosravi, A., Meyer, T., Francis, J., Malcolm, S., Robinson, M., Brown, D., Shaw, S., Foulkes, R., Lawson, A., Harari, O., Bourne, T., Maloney, A., Weir, N., 2015. CDP7657, an anti-CD40L antibody lacking an Fc domain, inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study. Arthritis Res. Ther. 17 (234). Sidiropoulos, P.I., Boumpas, D.T., 2004. Lessons learned from anti-CD40L treatment in systemic lupus erythematosus patients. Lupus 13, 391–397. Spencer, J.I., Bell, J.S., DeLuca, G.C., 2018. Vascular pathology in multiple sclerosis: reframing pathogenesis around the blood-brain barrier. J. Neurol. Neurosurg. Psychiatry 89, 42–52. Vakkalanka, R.K., Woo, C., Kirou, K.A., Koshy, M., Berger, D., Crow, M.K., 1999. Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera. Arthritis Rheum. 42, 871–881.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jneuroim.2018.01.001. References Absinta, M., Nair, G., Sati, P., Cortese, I.C., Filippi, M., Reich, D.S., 2015. Direct MRI detection of impending plaque development in multiple sclerosis. Neurol. Neuroimmunol. Neuroinflam. 2, e145. Aloui, C., Prigent, A., Sut, C., Tariket, S., Hamzeh-Cognasse, H., Pozzetto, B., Richard, Y., Cognasse, F., Laradi, S., Garraud, O., 2014. The signaling role of CD40 ligand in platelet biology and in platelet component transfusion. Int. J. Mol. Sci. 15, 22342–22364. Anesten, B., Yilmaz, A., Hagberg, L., Zetterberg, H., Nilsson, S., Brew, B.J., Fuchs, D., Price, R.W., Gisslen, M., 2016. Blood-brain barrier integrity, intrathecal immunoactivation, and neuronal injury in HIV. Neurol. Neuroimmunol. Neuroinflam. 3, e300. Becker, G.J., 2004. Which albumin should we measure? Kidney Int. Suppl. S16–7. Bennett, J., Basivireddy, J., Kollar, A., Biron, K.E., Reickmann, P., Jefferies, W.A., McQuaid, S., 2010. Blood-brain barrier disruption and enhanced vascular permeability in the multiple sclerosis model EAE. J. Neuroimmunol. 229, 180–191. Bergman, R.K., Munoz, J.J., Portis, J.L., 1978. Vascular permeability changes in the central nervous system of rats with hyperacute experimental allergic encephalomyelitis induced with the aid of a substance from Bordetella pertussis. Infect. Immun. 21, 627–637. Davidson, D.C., Hirschman, M.P., Sun, A., Singh, M.V., Kasischke, K., Maggirwar, S.B., 2012. Excess soluble CD40L contributes to blood brain barrier permeability in vivo: implications for HIV-associated neurocognitive disorders. PLoS One 7, e51793. Elgueta, R., Benson, M.J., de Vries, V.C., Wasiuk, A., Guo, Y., Noelle, R.J., 2009. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol. Rev. 229, 152–172. Hartung, H.P., Aktas, O., Menge, T., Kieseier, B.C., 2014. Immune regulation of multiple sclerosis. Handb. Clin. Neurol. 122, 3–14. Iezzi, G., Sonderegger, I., Ampenberger, F., Schmitz, N., Marsland, B.J., Kopf, M., 2009. CD40-CD40L cross-talk integrates strong antigenic signals and microbial stimuli to induce development of IL-17-producing CD4 + T cells. Proc. Natl. Acad. Sci. U. S. A. 106, 876–881. Ishikawa, M., Vowinkel, T., Stokes, K.Y., Arumugam, T.V., Yilmaz, G., Nanda, A., Granger, D.N., 2005. CD40/CD40 ligand signaling in mouse cerebral microvasculature after focal ischemia/reperfusion. Circulation 111, 1690–1696.
4
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Soluble CD40 ligand disrupts the blood–brain barrier and exacerbates inflammation in experimental autoimmune encephalomyelitis ⁎
Hiroki Masudaa, Masahiro Moria, , Kenta Umeharab, Tomomi Furihatab,c, Tomohiko Uchidaa, Akiyuki Uzawaa, Satoshi Kuwabaraa a
Department of Neurology, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan c Department of Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan b
A R T I C L E I N F O
A B S T R A C T
Keywords: Blood–brain barrier Experimental autoimmune encephalomyelitis Multiple sclerosis Soluble CD40 ligand
Serum soluble CD40 ligand (sCD40L) has been reported to positively correlate with the albumin quotient, a marker of blood–brain barrier (BBB) breakdown, in patients with multiple sclerosis (MS). To clarify the mechanisms of sCD40L in MS pathophysiology, sCD40L was administered to experimental autoimmune encephalomyelitis (EAE) mice and a human brain microvascular endothelial cell (HBMEC)-based BBB model. The high-dose sCD40L group showed a worse EAE score than the low-dose and control groups. BBB permeability was increased by administering sCD40L in a HBMEC-based BBB model. Thus, sCD40L induces more severe inflammation in the central nervous system by disrupting the BBB.
1. Introduction Multiple sclerosis (MS) is a demyelinating and neurodegenerative disease of the central nervous system (CNS). A feature of MS is dissemination in time and space. Although the etiology of MS remains unclear, both T and B cells have been reported to play important roles in the pathogenesis of MS (Hartung et al., 2014). Moreover, blood–brain barrier (BBB) breakdown has been recognized as a critical step in MS pathology (Absinta et al., 2015; Spencer et al., 2018). Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS in which T cells expressing interleukin-17 (IL-17) infiltrate the CNS at the early stage of EAE. Therefore, T helper 17 (Th17) cells are considered a key factor in the pathogenesis of EAE (Kurschus, 2015). CD40 ligand (CD40L) is a transmembrane protein that plays an important role in the immune system. The main function of CD40L is to produce high-affinity B cells (Elgueta et al., 2009). Recent studies have suggested that CD40L increases the permeability of the BBB (Davidson et al., 2012; Ishikawa et al., 2005). Soluble CD40L (sCD40L) has been reported to have the same function as CD40L (Vakkalanka et al., 1999). In the peripheral blood, sCD40L has been found to be produced only by shedding from the surface of activated platelets or activated T cells and
exists in trimers (Aloui et al., 2014). The molecular weight of sCD40L is 18 kDa, which is much smaller than albumin (66 kDa) (Becker, 2004; Pietravalle et al., 1996). CD40L works by binding to CD40, which is expressed on the surface of B cells and other cells, including macrophages, dendritic cells, smooth muscle cells, microglia, astrocytes, and endothelial cells. Astrocytes and endothelial cells are also the components of the BBB. Previously, we reported elevated serum sCD40L levels in patients with MS and a positive correlation between serum sCD40L levels and the albumin quotient (Qalb) (Masuda et al., 2017). Qalb gives the cerebrospinal fluid (CSF)/serum albumin ratio, which is a marker of BBB dysfunction. Meanwhile, CD40–CD40L interaction has been reported to be critical for Th17 differentiation (Iezzi et al., 2009). We hypothesized that sCD40L could cause BBB breakdown or severe inflammation through the upregulation of Th17 cells in MS pathogenesis. Therefore, we investigated the function of sCD40L in EAE mice and an in vitro study using human brain microvascular endothelial cells (HBMEC).
Abbreviations: BBB, blood–brain barrier; EAE, experimental autoimmune encephalomyelitis; CD40L, CD40 ligand; CNS, central nervous system; HAND, HIV-associated neurocognitive disorder; HBMEC, human brain microvascular endothelial cells; IL-6, interleukin-6; IQR, interquartile range; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; NMO, neuromyelitis optica; NMOSD, neuromyelitis optica spectrum disorder; Qalb, albumin quotient (CSF/serum albumin ratio); sCD40L, soluble CD40L ⁎ Corresponding author. E-mail addresses: [email protected] (H. Masuda), [email protected] (M. Mori), [email protected] (K. Umehara), [email protected] (T. Furihata), [email protected] (T. Uchida), [email protected] (A. Uzawa), [email protected] (S. Kuwabara). https://doi.org/10.1016/j.jneuroim.2018.01.001 Received 4 December 2017; Received in revised form 21 December 2017; Accepted 2 January 2018 0165-5728/ © 2018 Published by Elsevier B.V.
Please cite this article as: Masuda, H., Journal of Neuroimmunology (2018), https://doi.org/10.1016/j.jneuroim.2018.01.001
Journal of Neuroimmunology xxx (xxxx) xxx–xxx
H. Masuda et al.
2. Materials and methods
2.7 mM KCl, 1.5 mM KH2PO4, 10 mM NaH2PO4, 25 mM glucose, and 10 mM Hepes; pH 7.4). Twenty-four hours before the initiation of the assay, human sCD40L was administered to the insert (apical side). The recombinant human sCD40L was resuspended in sterile water with 0.5% BSA. The concentration of sCD40L was 100 ng/mL based on the previous research (Ramirez et al., 2010). The assay was initiated by adding Na-F (Sigma) (150 ng/insert) to the insert at 37 °C. Immediately after the initiation (0 min) and after incubation for 30, 60, and 90 min, the medium was collected from the well (basolateral side). The fluorescence of Na-F in each medium was determined using an ARVO-SX (PerkinElmer, Waltham, MA, USA) with wavelengths ex/em¼485 nm/ 535 nm. The permeability coefficients (Pe, cm/min) were calculated. The experiment was performed five times.
2.1. EAE induction in mice Ten-week-old C57BL/6 female mice (18–25 g) were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were housed in specific pathogen-free facilities at Chiba University with a maximum of four animals per cage, with free access to water and standard rodent chow. EAE was induced using immunization with myelin oligodendrocyte glycoprotein (MOG). A total of 200 mg MOG peptide 35–55 in complete Freund's adjuvant containing 400 mg of killed Mycobacterium tuberculosis H37Ra was injected subcutaneously at two sites. On the days of immunization (days 0 and 1), 200 ng pertussis toxin was injected intraperitoneally using Hooke kits (EK-0105; Hooke Laboratories, Lawrence, MA, USA). EAE was scored on the following scale: 0 = no clinical signs; 1 = partial paralysis of tail; 2 = flaccid tail; 3 = limp tail and partial weakness of hind legs; 4 = limp tail and complete weakness of hind legs; 5 = limp tail, complete weakness of hind legs and partial weakness of front legs; and 6 = complete hind and front legs paralysis. The intermediate condition of each score was assessed as 0.5. Dr. Uzawa immunized the mice, and EAE clinical scores were assessed blindly by Dr. Masuda to avoid subjective bias.
2.5. Statistical analysis Continuous data were compared using the Mann-Whitney U test with a Bonferroni correction. To consider the multiple testing problem, we applied the Bonferroni correction on the computed P values to reduce type I errors. A P value of < 0.05 was considered statistically significant. Statistical tests were conducted using SPSS version 24.0 (IBM Corporation, Armonk, NY, USA).
2.2. Treatment with sCD40L
2.6. Ethics
Recombinant mouse sCD40L was obtained from Abnova (P4590; Taipei, Taiwan). Mice were injected intraperitoneally on days 0 and 1 with recombinant mouse sCD40L resuspended in phosphate buffered saline with 0.1% bovine serum albumin (BSA) at the same time with Pertusis Toxin (PT) administration. Injection side of sCD40L was opposite from the injection side of PT. EAE mice were divided into three groups, the high sCD40L group (0.2 mg/g body weight), the low sCD40L group (0.05 mg/g body weight), and the control group (only BSA was administered). Each group comprised 12 mice. The concentration of sCD40L was determined based on previous research (Davidson et al., 2012).
The study procedure in animal experiments was approved by the ethics committee of Chiba University School of Medicine (No. A27209). 3. Results 3.1. EAE severity was increased by sCD40L administration The result of EAE study is shown in Fig. 1. The result showed the high sCD40L group exacerbated EAE clinical score compared with the other two groups. The high sCD40L group showed the worse clinical score on days 18, 21–25 (P < 0.05), and days 23 and 26 (P < 0.01) compared with the control group. Meanwhile, the result showed a
2.3. Serum Th17 cytokine assays in EAE mice To assess sCD40L affecting Th17 cells in the early phase of EAE, we measured serum Th17 cytokines including IL-17E, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, macrophage inflammatory protein (MIP-3a), IL-1b, IL-2, IL-4, IL-5, IL-6, IL-21, IL-22, IL-28B, IL-10, IL-23, IL-12p70, IL-27, IL-13, IL-15, IL-17A, IL-17F, IL-33, IL-31, tumor necrosis factor (TNF)β, TNFα, and CD40L on day 14 (at the early phase of the peak). All serum samples were centrifuged at 3000 rpm for 10 min. Just after centrifugation all serum samples were immediately stored at −80 °C until analysis. Cytokine measurements were performed using a multiplexed fluorescent magnetic bead-based immunoassay (Merck Millipore, Darmstadt, Germany), according to the manufacturer's instructions. Cytokine levels were calculated with reference to a standard curve for each cytokine. 2.4. BBB permeability assay Recombinant human sCD40L was obtained from Enzo Life Sciences (ALX-522-015-2010; Farmingdale, NY, USA). We performed sodium fluorescein (Na-F) permeability assay using the HBMEC/ci18-based in vitro BBB method (Kitamura et al., 2017). Ci18 cell line is a sister clone of HBMEC/ciβ, and the assay was performed as reported previously (Kamiichi et al., 2012). Briefly, HBMEC/ci18 were seeded at 4.0 × 105 cells/mL on a membrane filter of an insert culture system (polyethylene terephthalate, 0.4-mm high-density pores, and 0.3 cm2, BD Falcon, Franklin Lakes, NJ, USA), and then cultured for 3 days. Before the assay, the cells were pre-incubated over 30 min at 37 °C with Ringer-Hepes buffer (136 mM NaCl, 0.9 mM CaCl2, 0.5 mM MgCl2,
Fig. 1. Effects of sCD40L administration to EAE mice. The straight line shows the high sCD40L EAE group (0.2 mg/g body weight of sCD40L and BSA). The dotted line shows the low sCD40L EAE group (0.05 mg/g body weight of sCD40L and BSA). The long-short dashed line shows the control EAE group (BSA without sCD40L). Compared with the control EAE group, the high sCD40L EAE group showed higher clinical EAE scores on days 18, 21–25 (P < 0.05) and days 23 and 26 (P < 0.01). Results are mean ± standard error of the mean. *P < 0.05 and †P < 0.01 by MannWhitney U test between the high sCD40L EAE group and the control EAE group. EAE, experimental autoimmune encephalomyelitis; sCD40L, soluble CD40 ligand.
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neurocognitive disorder (HAND). Although there may be various mechanisms in BBB disruption in HAND (Anesten et al., 2016), a recent study about HAND suggested that sCD40L increased the permeability of the BBB (Davidson et al., 2012). Previously, we reported elevated serum sCD40L levels at the relapse phase in patients with MS and the positive correlation between serum sCD40L levels and Qalb (Masuda et al., 2017). This result may suggest that sCD40L could cause BBB disruption during relapse in patients with MS. However, as noted in the Introduction, the molecular weight of sCD40L is much lower than that of albumin (Pietravalle et al., 1996) (Becker, 2004). Therefore, the possibility wherein sCD40L was increased passively due to the BBB breakdown by neuro-inflammation was not ruled out in our previous report. However, this study, applying the HBMEC/ci18-based in vitro BBB model, revealed that sCD40L administration showed higher Pe, which suggests that sCD40L could increase the permeability of the BBB. CD40, the receptor of CD40L, is expressed on the endothelial cells. Therefore, sCD40L may be involved in the disruption of the BBB by binding to CD40 on the endothelial cells. Our study has some limitations. First, the administration of sCD40L at the initiation of immunization could affect not only endothelial cells but also other cells, such as B cells and astrocytes on which CD40 expresses. Therefore, sCD40L could have multiple functions more than BBB disruption in the pathogenesis of EAE. Second, the timing of BBB opening was not identified in our study in vivo. A previous study revealed increased permeability of the BBB was already observed 5 days after immunization (Bergman et al., 1978). Therefore, administration of sCD40L at immunization could accelerate the onset of EAE mice. However, in this study, the days from immunization to onset were not different between the high sCD40L group and the control group. Increased dosages of sCD40L or number of mice may clarify this discrepancy. Finally, there is a time discrepancy about sCD40L effect between in vivo study and in vitro study in our study. In vivo study, EAE mice severity was increased two weeks after sCD40L administration at the immunization. On the other hand, enhanced BBB permeability was observed 24 h after the administration of sCD40L in vitro study. However, similar result was obtained in a recent study about EAE (Bennett et al., 2010). The study reported PT increased BBB permeability in vitro. In EAE study, PT is administered at the initiation and causes EAE peak after two weeks later. Moreover, the previous study showed PT increased BBB permeability maximally just before the peak (Bergman et al., 1978). Therefore, administration of sCD40L at immunization could also increase BBB permeability just before the peak as PT. In conclusion, our results show sCD40L administration exacerbated the clinical severity of EAE mice and increased the permeability of the endothelial cells, which is a component of the BBB. Previously, thromboembolism was caused by anti-CD40L antibody in patients with systemic lupus erythematosus (Sidiropoulos and Boumpas, 2004). However, new anti-CD40L antibody without the adverse effect of thromboembolism was reported (Shock et al., 2015). Therefore, blocking sCD40L by these antibodies or eliminating sCD40L by plasmapheresis could be a new therapeutic option for patients with MS.
Fig. 2. Na-F permeability assay in using the HBMEC/ci18-based in vitro BBB method. Administration of sCD40L increased Pe compared with the control (P = 0.032). Results are mean ± standard deviation of the mean. HBMEC, human brain microvascular endothelial cells; Na-F, sodium fluorescein; sCD40L, soluble CD40 ligand.
worse clinical score in the high sCD40L group compared with the low sCD40L group on days 20, 22–26 (P < 0.05). No differences were found between the low sCD40L group and the control group. Maximum scores and days from immunization to the onset were not different among the three groups. The days from immunization to the onset or to the peak were not different among the three groups (data not shown). 3.2. Serum Th17 cytokines were not different among the high sCD40L group, the low sCD40L group, and the control group at peak The correct amount of serum was not obtained in five mice (one in the high sCD40L group, two in the low sCD40L group, and two in the control group). The result showed no differences in all the measured cytokines among the three groups (Supplementary Table). 3.3. BBB permeability was increased by the administration of sCD40L The result of BBB permeability study is shown in Fig. 2. Pe in the sCD40L group was higher than Pe in the control group (median: 2.43 vs 1.78 · 10− 3 cm/min, IQR: 0.85 vs 0.64, P = 0.032). 4. Discussion This study revealed the following points: 1) Treatment with sCD40L exacerbated EAE clinical scores; 2) Th17 cytokines at the early phase of the peak were not deviated by sCD40L administration at the induction of EAE mice; 3) Increased BBB permeability was observed by the administration of sCD40L in the HBMEC/ci18 -based in vitro BBB model. A previous study reported that CD40-CD40L cross-talk was important for Th17 cell development (Iezzi et al., 2009). Our study showed that the administration of sCD40L at the induction of EAE exacerbated EAE severity. Therefore, we hypothesized that administered sCD40L promoted Th17 cells differentiation, causing increased severity in EAE mice, because Th17 cells were reported to be a key factor in the pathogenesis of EAE (Kurschus, 2015). However, Th17 cytokines at peak in EAE mice were not different with or without sCD40L administration in our study. Previously, pathogenic role for B cells and antibodies in the onset of EAE was reported (Lyons et al., 2002; Lyons et al., 1999). A recent study suggested B cells transverse the intact BBB more easily than T cells (Liu et al., 2012). Meanwhile, CD40L produces highaffinity B cells (Elgueta et al., 2009). Therefore, sCD40L administration may activate not Th17 cells but B cells in our study. Further investigation is needed to address this hypothesis. Increased BBB permeability was also reported in HIV-associated
Conflicting interests The authors declare that there is no conflict of interest.
Funding None.
Acknowledgements This study was supported by JSPS KAKENHI (Grant Number 17K16109). 3
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Appendix A. Supplementary data
Kamiichi, A., Furihata, T., Kishida, S., Ohta, Y., Saito, K., Kawamatsu, S., Chiba, K., 2012. Establishment of a new conditionally immortalized cell line from human brain microvascular endothelial cells: a promising tool for human blood-brain barrier studies. Brain Res. 1488, 113–122. Kitamura, K., Umehara, K., Suzuki, S., Chiba, K., Anzai, N., Akita, H., Furihata, T., 2017. Functional characterization of a new clone of conditionally immortalized human brain microvascular endothelial cells, HBMEC/ci18. In: 12th International Conference on Cerebral Vascular Biology, Australia, pp. 51. Kurschus, F.C., 2015. T cell mediated pathogenesis in EAE: molecular mechanisms. Biom. J. 38, 183–193. Liu, G., Muili, K.A., Agashe, V.V., Lyons, J.A., 2012. Unique B cell responses in B celldependent and B cell-independent EAE. Autoimmunity 45, 199–209. Lyons, J.A., San, M., Happ, M.P., Cross, A.H., 1999. B cells are critical to induction of experimental allergic encephalomyelitis by protein but not by a short encephalitogenic peptide. Eur. J. Immunol. 29, 3432–3439. Lyons, J.A., Ramsbottom, M.J., Cross, A.H., 2002. Critical role of antigen-specific antibody in experimental autoimmune encephalomyelitis induced by recombinant myelin oligodendrocyte glycoprotein. Eur. J. Immunol. 32, 1905–1913. Masuda, H., Mori, M., Uchida, T., Uzawa, A., Ohtani, R., Kuwabara, S., 2017. Soluble CD40 ligand contributes to blood-brain barrier breakdown and central nervous system inflammation in multiple sclerosis and neuromyelitis optica spectrum disorder. J. Neuroimmunol. 305, 102–107. Pietravalle, F., Lecoanet-Henchoz, S., Blasey, H., Aubry, J.P., Elson, G., Edgerton, M.D., Bonnefoy, J.Y., Gauchat, J.F., 1996. Human native soluble CD40L is a biologically active trimer, processed inside microsomes. J. Biol. Chem. 271, 5965–5967. Ramirez, S.H., Fan, S., Dykstra, H., Reichenbach, N., Del Valle, L., Potula, R., Phipps, R.P., Maggirwar, S.B., Persidsky, Y., 2010. Dyad of CD40/CD40 ligand fosters neuroinflammation at the blood-brain barrier and is regulated via JNK signaling: implications for HIV-1 encephalitis. J. Neurosci. 30, 9454–9464. Shock, A., Burkly, L., Wakefield, I., Peters, C., Garber, E., Ferrant, J., Taylor, F.R., Su, L., Hsu, Y.M., Hutto, D., Amirkhosravi, A., Meyer, T., Francis, J., Malcolm, S., Robinson, M., Brown, D., Shaw, S., Foulkes, R., Lawson, A., Harari, O., Bourne, T., Maloney, A., Weir, N., 2015. CDP7657, an anti-CD40L antibody lacking an Fc domain, inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study. Arthritis Res. Ther. 17 (234). Sidiropoulos, P.I., Boumpas, D.T., 2004. Lessons learned from anti-CD40L treatment in systemic lupus erythematosus patients. Lupus 13, 391–397. Spencer, J.I., Bell, J.S., DeLuca, G.C., 2018. Vascular pathology in multiple sclerosis: reframing pathogenesis around the blood-brain barrier. J. Neurol. Neurosurg. Psychiatry 89, 42–52. Vakkalanka, R.K., Woo, C., Kirou, K.A., Koshy, M., Berger, D., Crow, M.K., 1999. Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera. Arthritis Rheum. 42, 871–881.
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