Research ArticleNEUROBIOLOGY

Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica

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Science Translational Medicine  05 Jul 2017:
Vol. 9, Issue 397, eaai9111
DOI: 10.1126/scitranslmed.aai9111
  • Fig. 1. Markers of brain endothelial cell activation after exposure to IgG from pooled serum specimens or individual NMO patients.

    (A) Confocal three-dimensional (3D) reconstruction images show immunostaining of BMECs for cytoplasmic versus nuclear NF-κB p65 (top) and ICAM-1 (bottom) after exposure to IgG (400 μg/ml) prepared from pooled NMO patient (50 patients) and control sera. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Arrowheads indicate representative cells negative for nuclear NF-κB p65, and arrows indicate representative nuclear NF-κB p65–positive cells. Three independent experiments were performed. Scale bar, 50 μm. (B) Quantification of nuclear NF-κB p65–positive BMECs by high-content imaging after exposure to IgG (400 μg/ml) from pooled NMO and control sera. Data normalized to cultures unexposed to human IgG are shown as means ± SEM from three independent experiments with technical triplicates. P values were assessed with one-way analysis of variance (ANOVA), followed by Tukey multiple comparisons test (***P < 0.001). (C) Quantification of the effect of IgG from individual patients (NMO or SLE) and healthy controls on NF-κB p65 nuclear translocation in BMECs. IgG (400 μg/ml) from patients NMO1 and NMO2 as well as patients SLE1, SLE2, and SLE3 shows significantly greater NF-κB p65 nuclear translocation than control-IgG (***P < 0.001, **P < 0.01, *P < 0.05 versus control, one-way ANOVA followed by Sidak multiple comparison test). Data are shown as means ± SEM from four independent experiments with technical triplicates.

  • Fig. 2. Effects of an NMO patient-derived rAb on brain endothelial cell activation.

    (A) Confocal 3D reconstruction images show immunostaining of BMECs for NF-κB p65 (top) and ICAM-1 (bottom) after exposure to control-rAb or NMO-rAb ON-12-2-46 (50 μg/ml). Nuclei are counterstained with DAPI (blue). Arrowheads indicate representative NF-κB p65 nuclear–negative cells, and arrows indicate representative NF-κB p65-positive cells. Three independent experiments were performed. Scale bar, 50 μm. (B) Quantification of nuclear NF-κB p65–positive BMECs by high-content imaging after exposure to NMO-rAb ON-12-2-46 or isotype-matched control rAbs (50 μg/ml). Data normalized to cultures unexposed to human IgG are shown as means ± SEM from three independent experiments with technical triplicates. P values were assessed with one-way ANOVA followed by Tukey multiple comparisons test (***P < 0.001 and *P < 0.05). (C and D) Quantification of nuclear NF-κB p65–positive BMECs by high-content imaging after exposure to pooled NMO-IgG and pooled control-IgG (C) or NMO-rAb ON-12-2-46 and control-rAb (D). Data normalized to cultures unexposed to human IgG are shown as means ± SEM from three independent experiments with technical triplicates. Statistical significance was assessed with one-way ANOVA followed by Dunnett’s multiple comparisons test [***P < 0.001 and *P < 0.05 versus NMO-IgG (12.5 μg/ml) or NMO-rAb (3.125 μg/ml)].

  • Fig. 3. Effect of NMO-IgG and NMO-rAb ON-12-2-46 on BMEC function.

    (A) Confocal images of claudin-5 (green) and DAPI (blue) in BMECs after exposure to pooled control-IgG (400 μg/ml), pooled NMO-IgG (400 μg/ml), control-rAb (50 μg/ml), or NMO-rAb ON-12-2-46 (50 μg/ml). Scale bar, 50 μm. (B) Claudin-5 area fraction was determined from confocal microscopy images. Data are means ± SEM from three independent experiments with technical triplicates. P values were calculated by one-way ANOVA followed by Sidak multiple comparisons test (***P < 0.001). (C) Effect of NMO-rAb ON-12-2-46 on the permeability of BMECs to 10-kDa dextran. Data are means ± SEM from three independent experiments with technical triplicates. Data are normalized to control-rAb 1. P values were calculated by one-way ANOVA followed by Tukey’s multiple comparisons test (*P < 0.05). (D) Effect of NMO-rAb ON-12-2-46 and control-rAb on the permeability of BMECs to IgG. Data are means ± SEM from three independent experiments with technical triplicates. Data are normalized to control-rAb 1. P values were obtained by Mann-Whitney U test (***P < 0.001).

  • Fig. 4. Identification of GRP78 as the target antigen of NMO-rAbs ON-12-2-46 and ON-07-5-31.

    (A) Proteins isolated from crude membrane lysates of U87MG (glioblastoma) and OL (oligodendroglia) cells were separated by polyacrylamide gel electrophoresis and analyzed by Western blot. Multiple protein bands reacted with the NMO-rAbs. Unlabeled lanes contain molecular weight standards. (B) Schematic detailing the purification scheme (X-linker, cross-linker; DSS, disuccinimidyl suberate). (C) The protein eluted with DTT from rAb cross-linked to the U87MG cells using DTSSP/disuccinimidyl suberate (DSS) was subjected to polyacrylamide gel electrophoresis and immunoblot analysis (lane 1, molecular weight standards; lane 2, protein bound to ON-07-5-31 rAb; lane 3, protein bound to ON-12-2-46 rAb). The blots were probed with the indicated NMO-rAb. (D) Immunofluorescence labeling of U87MG glioblastoma cells with ON-07-5-31 rAb (10 μg/ml) and rabbit anti-GRP78 antisera shows colocalization of the two proteins (merge in yellow). Nuclei are counterstained with DAPI (blue). (E) ON-12-2-46 rAb (20 μg/ml; green channel) and rabbit anti-GRP78 antiserum (red channel) immunoreactivity on U87MG glioblastoma cells after 24 hours of treatment with 0.5 mM thapsigargin (TG) or DMSO (control). The merged image shows colocalization of the two proteins (yellow). (F) Immunoblot analysis of commercial recombinant GRP78 protein purified from transfected human embryonic kidney (HEK) 293 cells (lane 1) or bacteria (lane 2) and probed with rAb or rabbit anti-GRP78 serum demonstrates that NMO-rAbs ON-12-2-46 and ON-07-5-31 recognize GRP78. (G) Binding of ON-12-2-46 rAb (2 μg/ml) (green channel) to the U87MG glioblastoma cells in competition immunofluorescence assay is blocked in the presence of commercial recombinant GRP78 protein (5 μg). DAPI-stained nuclei are depicted in blue.

  • Fig. 5. Removal of GRP78-specific IgG from pooled NMO-IgG reduces NF-κB p65 nuclear translocation in BMECs.

    Numbers of cells positive for nuclear NF-κB p65 translocation were compared in cultures exposed to pooled NMO-IgG (200 and 150 μg/ml) immunoadsorbed against GRP78 or control SNAP25 protein. Data are normalized to control SNAP25-absorbed NMO IgG. Data are means ± SEM from five experiments in triplicate. Statistical significance was assessed by paired two-tailed t test (*P < 0.05).

  • Fig. 6. In vivo effects of ON-12-2-46 on mouse brain vasculature.

    (A) Experimental setup. IV, intravenously; IP, intraperitoneally. 3D images of 20-μm mouse coronal brain sections immunostained for human IgG (rAb ON-7-5-53) and AQP4 (B), human IgG (rAb ON-7-5-53) and mouse fibrinogen (C), or human IgG, mouse albumin, and AQP4 (D) after administration of murinized GRP78-specific NMO rAb ON-12-2-46 or control rAb in combination with human AQP4-specific rAb ON-7-5-53. (E) Measurement of the average diameter of vessels (mean ± SEM) in mice treated with GRP78-specific NMO rAb ON-12-2-46 (six mice) and control rAb (four mice) in two separate experiments. Vessels were quantified from control-rAb–injected (n = 35) and ON-12-2-46–injected (n = 31) 20× high-power fields, and the diameters were measured using AxioVision LE. P values were calculated by Mann-Whitney U test (***P < 0.0001).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/397/eaai9111/DC1

    Fig. S1. Effects of IgG from NMO or SLE patients or healthy controls on the activation of and binding to BMECs.

    Fig. S2. Binding of IgG from NMO or SLE patients or healthy controls to human astrocytes expressing AQP4 or human astrocytes.

    Fig. S3. Effect of NMO-IgG or NMO-rAb on BMEC activation.

    Fig. S4. Binding of NMO-IgG or NMO-rAb to human astrocytes expressing AQP4 or human astrocytes.

    Fig. S5. Effects of non-AQP4 NMO-rAb on other MECs.

    Fig. S6. Target of rAb ON-12-2-46 is widely expressed at both the cellular and tissue level.

    Fig. S7. GRP78 expression on MECs in vitro and in vivo.

    Fig. S8. Effect of commercial GRP78 Abs on BMEC activation.

    Table S1. Effect of serum IgG from each patient with NMO and SLE and healthy controls on BMEC activation.

    Table S2. Effect of pooled IgG and 14 rAbs from NMO patients and healthy controls on BMEC activation.

    Table S3. Primary data (Microsoft Excel format).

  • Supplementary Material for:

    Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica

    Fumitaka Shimizu, Kristin L. Schaller, Gregory P. Owens, Anne C. Cotleur, Debra Kellner, Yukio Takeshita, Birgit Obermeier, Thomas J. Kryzer, Yasuteru Sano, Takashi Kanda, Vanda A. Lennon, Richard M. Ransohoff,* Jeffrey L. Bennett*

    *Corresponding author. Email: richard.ransohoff{at}biogen.com (R.M.R.); bennett{at}ucdenver.edu (J.L.B.)

    Published 5 July 2017, Sci. Transl. Med. 9, eaai9111 (2017)
    DOI: 10.1126/scitranslmed.aai9111

    This PDF file includes:

    • Fig. S1. Effects of IgG from NMO or SLE patients or healthy controls on the activation of and binding to BMECs.
    • Fig. S2. Binding of IgG from NMO or SLE patients or healthy controls to human astrocytes expressing AQP4 or human astrocytes.
    • Fig. S3. Effect of NMO-IgG or NMO-rAb on BMEC activation.
    • Fig. S4. Binding of NMO-IgG or NMO-rAb to human astrocytes expressing AQP4 or human astrocytes.
    • Fig. S5. Effects of non-AQP4 NMO-rAb on other MECs.
    • Fig. S6. Target of rAb ON-12-2-46 is widely expressed at both the cellular and tissue level.
    • Fig. S7. GRP78 expression on MECs in vitro and in vivo.
    • Fig. S8. Effect of commercial GRP78 Abs on BMEC activation.
    • Table S1. Effect of serum IgG from each patient with NMO and SLE and healthy controls on BMEC activation.
    • Table S2. Effect of pooled IgG and 14 rAbs from NMO patients and healthy controls on BMEC activation.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 Primary data (Microsoft Excel format).

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