Journal of Neuroimmunology 309 (2017) 88–99

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Journal of Neuroimmunology
journal homepage: www.elsevier.com/locate/jneuroim

B cells from patients with multiple sclerosis induce cell death via apoptosis MARK
in neurons in vitro
Robert P. Lisaka,b,⁎,1, Liljana Nedelkoskaa, Joyce A. Benjaminsa,b,1, Dana Schalkc,2,
Beverly Bealmeara, Hanane Touild,e,3, Rui Lid,e,3, Gillian Muirheadd, Amit Bar-Ord,e,⁎⁎,1,3
a
Department of Neurology, Wayne State University School of Medicine, Detroit, MI, USA
b
Department of Microbiology, Immunology and Biochemistry, Wayne State University School of Medicine, Detroit, MI, USA
c
Department of Oncology, Wayne State University School of Medicine and Karmanos Cancer Institute, Detroit, MI, USA
d
Department of Neurology, McGill University, Montreal Neurological Institute, Montreal, Quebec, Canada
e
Department of Neurology and Center for Neuroinflammation and Neurotherapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

A R T I C L E I N F O A B S T R A C T

Keywords: B cells mediate multiple sclerosis (MS) pathogenesis by mechanisms unrelated to immunoglobulin (Ig). We
B cells reported that supernatants (Sup) from cultured B cells from blood of relapsing remitting MS (RRMS) patients, but
Cell death not normal controls (NC), were cytotoxic to rat oligodendrocytes (OL). We now show that RRMS blood B cells,
Cytokines not stimulated in vitro, secrete factor/s toxic to rat and human neurons. Cytotoxicity is independent of Ig and
Multiple sclerosis
multiple cytokines, not complement-mediated, and involves apoptosis. The factor/s have an apparent mw
Neurons
Oligodendrocytes
of > 300 kDa. B cells could contribute to damage within the central nervous system by secreting molecules toxic
to OL and neurons.

1. Introduction
Studies in multiple sclerosis (MS) have continued to highlight the
complexities of the pathogenesis of this disorder, particularly mechan-
isms underlying relentless progressive disease. MS was often described
as an inflammatory demyelinating disease of the central nervous system
(CNS) mainly affecting focal perivascular white matter regions, invol-
ving microglial activation, astrocyte proliferation and hyperplasia;
secondary axonal changes were thought to occur as a late phenomenon.
We have relearned that damage to axons occurs in the earliest stages
even of relapsing remitting MS (Ferguson et al., 1997; Trapp et al.,
1998). Gray matter involvement including the cerebral cortex was also
viewed as involved late in disease, yet we now know that cortical gray
matter damage occurs not only in chronic disease but also early in the
course of the disease (Absinta et al., 2011; Bo et al., 2007; Calabrese
et al., 2009; Kutzelnigg et al., 2005; Lucchinetti et al., 2011; Peterson
et al., 2001; Rocca et al., 2005). The subpial pattern of cortical
involvement appears to be a major component of gray matter injury
highlighted in recent years as likely representing a major pathologic
substrate of (non-relapsing) progressive MS, and correlating with both
motor and cognitive decline better than the focal perivascular deep
white matter lesions (Fisher et al., 2008; Fisniku et al., 2008; Rudick
and Trapp, 2009). This subpial pathology is not perivascular and
involves injury of both oligodendrocytes (OL) and neurons, evidenced
in part by apoptosis of these cells in the superficial layers of cortex (Bo
et al., 2006; Kutzelnigg et al., 2005; Peterson et al., 2001; Serafini et al.,
2004). Of growing interest has been the potential contribution to such
injury by immune cell collections in the meninges, some of which are

Abbreviations: CNS, central nervous system; DMEM, Dulbecco's modified essential medium; DMT, disease modifying therapy; EBV, Epstein-Barr virus; FGF, fibroblast growth factor; GM-
CSF, granulocyte macrophage-colony stimulating factor; IFN, interferon; Ig, immunoglobulin; IL, interleukin; kDa, kilodalton; LT, lymphotoxin; MIP, macrophage inflammatory protein;
MMP, matrix metalloproteinase; MS, multiple sclerosis; mw, molecular weight; NC, normal control; OL, oligodendrocyte; OPC, oligodendrocyte precursor cell; PBS, phosphate buffered
saline; PPMS, primary progressive multiple sclerosis; RRMS, relapsing remitting multiple sclerosis; SPMS, secondary progressive multiple sclerosis; Sup, supernatant; TGF, transforming
growth factor; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl dUTP nick end labeling
⁎
Correspondence to: R.P. Lisak, Department of Neurology, Wayne State University School of Medicine, 8D, University Health Center 4201 St Antoine, Detroit, MI 48201, USA.
⁎⁎
Correspondence to: A. Bar-Or, Perelman Center for Advanced Medicine (PCAM) South Pavilion, 7th Floor; Rm 7633400 Civic Center Blvd, Philadelphia, PA 19104, USA
E-mail addresses: rlisak@med.wayne.edu (R.P. Lisak), lnedelk@med.wayne.edu (L. Nedelkoska), jbenjami@med.wayne.edu (J.A. Benjamins),
DLS2DA@hscmail.mcc.virginia.edu (D. Schalk), bbealmea@med.wayne.edu (B. Bealmear), hanane.touil@mail.med.upenn.edu (H. Touil), rui.li@mail.med.upenn.edu (R. Li),
g.muirhead@gmail.com (G. Muirhead), amitbar@upenn.edu (A. Bar-Or).
1
Drs. Lisak, Benjamins and Bar-Or contributed equally to this study.
2
Current address: Department of Medicine, Division of Hematology/Oncology, University of Virginia, Charlottesville, VA, USA.
3
Current address: Center for Neuroinflammation and Neurotherapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

http://dx.doi.org/10.1016/j.jneuroim.2017.05.004
Received 7 February 2017; Received in revised form 9 May 2017; Accepted 10 May 2017
0165-5728/ © 2017 Elsevier B.V. All rights reserved.
R.P. Lisak et al. Journal of Neuroimmunology 309 (2017) 88–99

described as ‘B cell rich’ (Choi et al., 2012; Kutzelnigg et al., 2005; Table 1
Magliozzi et al., 2007; Serafini et al., 2004). Cytokines and factors assayed in B cell supernatants from NC and RRMS.
We have been interested in the effector role of B cells in causing
Cytokine (14-PLEX) Cytokine (25-PLEX) Individual ELISAs
damage to OL/myelin and neurons/axons independent of involvement
of immunoglobulin (Ig) and/or complement. We previously reported GM-CSF Eotaxin IL-10 CD40
that secretory products from cultures of B cells (including unstimulated IFNα FGF (basic) ILp40p70 CD40 ligand
IL-1β GM-CSF IL-13 Fas
B cells) from blood of untreated patients with RRMS are cytotoxic for
IL-4 IFN-α IL-15 Fas ligand
OL in vitro in mixed rat glial cultures, whereas Sup from B cells from IL-6 IFN-γ IL-17 Granzyme B
normal controls (NC) are not cytotoxic (Lisak et al., 2012). We IL-10 IL-1β IP-10 IL-16
hypothesize that B cells entering the meninges and cerebral spinal IL-12p40 IL-1RA MCP-1 IL-23
fluid (CSF) from the peripheral immune system could secrete factors IL-12p60 IL-2 MIG (CXCL9) LT-α
MIP-1α (CCL3) IL-2R MIP-1α (CCL3) LT-β
that lead to damage characteristic of MS (Fischer et al., 2013; Haider
MIP-1β IL-4 MIP-1β MMP-9
et al., 2016; Lassmann, 2012; Lassmann, 2014) in the underlying TGF-α IL-5 RANTES (CCL5) TGF-β
cortical gray matter. TNF-α IL-6 TNF-α
In the current study we tested whether Sup from blood-derived B TNF-β IL-7 VEGF
VEGF IL-8
cells of patients with RRMS seen at the Montreal Neurological Institute/
McGill University also compromise neuronal viability in comparison to Abbreviations: FGF, fibroblast growth; GM-CSF, granulocyte-colony stimulating factor;
Sup of B cells from NC and examined the mechanism underlying such IFN, interferon; IL, interleukin; IL-1RA, interleukin 1 receptor; IL-2R, interleukin-2
toxicity if observed. We hypothesized that neuronal cell death would be receptor; LT, lymphotoxin; MCP, monocyte chemoattractant protein; MIG, monocyte-
induced by the MS patient-derived B cell Sup and this death would be induced by gamma interferon; MIP, macrophage inflammatory protein; MMP-9, matrix
due to apoptosis. We examined the effects of B cell Sup from RRMS metalloproteinase; TGFβ, transforming growth factor-alpha; TNF, tumor necrosis factor;
RANTES, regulated on activation, normal T cell expressed and secreted; VEGF, vascular
patients and NC on both rodent and human neurons in vitro. We
endothelial growth factor.
measured levels of IgM and IgG and an expanded number of cytokines
and related protein secretory products of inflammatory cells in the B
2.3. Human neuronal cultures
cell culture Sup.

Coverslips previously coated with poly-D-lysine (1 mg/10 ml) were

2. Methods
2.1. Materials
Human neurons (#1520, medium (#1521), growth supplement
(#1562), and penicillin streptomycin solution (#0503) were purchased
from Sciencell (San Diego CA). Poly-D-lysine (P7405) and laminin
(L2020) were from Sigma (St. Louis MO).
ELISA kits were obtained as follows: 14-PLEX magnetic bead custom
array (HCYTOMAG60K-14) from EMD Millipore; 25-PLEX non-mag-
netic cytokine array (LHC00009) from Life Technologies/Invitrogen;
transforming growth factor-β (TGF-β), LEGEND MAX (436707) and
human interleukin (IL)-35 heterodimer, LEGEND MAX (439507) from
BioLegend; lymphotoxin-α (LT-α) (MBS268485) and lymphotoxin-β
(LT-β) (MBS261430) from MyBiosource; IL-16 and IL-23 2-PLEX
(HCYP2MAG-62K-02) from EMD Millipore; and matrix metalloprotei-
nase-9 (MMP-9) (DMP900) from R & D. ELISA assays for Fas (apo-1;
ab100513); Fas ligand (ab100515); CD40 (ab119516); CD40 ligand
(ab196268) and granzyme B (ab46142) were from Abcam. The
complete list of cytokines and biological modifier proteins examined
are listed in Table 1.
2.2. Rat neuronal cultures
Neurons were cultured from neonatal rat brain based on the method
of Eide and McMurray (Eide and McMurray, 2005), modified as
described (Lisak et al., 2011). For each explant, three forebrains were
used; each half forebrain was minced in 2 ml of HEPES buffered saline
containing 2 mg/ml of papain, then combined with the other tissue for
the subsequent steps. The final cell pellet was triturated 5–7 times in
3 ml of plating medium; 50 μl were plated on each coverslip, with 10
coverslips per 60 mm dish. After overnight attachment at 370C, 2 ml of
plating medium with 100 ng/ml nerve growth factor was added to each
dish. Cytosine arabinoside, final concentration 1 × 10− 5 M, was added
the next day; after 5 days, the medium was changed to B-27 feeding
medium containing NGF and cytosine arabinoside. Neuronal cultures
were used to test toxicity of B cell Sup between 5 and 7 days after
explant. Cultures were 85–90% neurons, with the remaining cells
astrocytes and microglia, and no detectable OL based on phenotypic
markers (Lisak et al., 2011; Lisak et al., 2012).
placed in a 24 well plate and coated overnight with laminin (10 μg/ml);
the excess solution was removed just prior to plating the cells. One vial
of cryopreserved primary human neurons (Sciencell) containing
1 × 106 cells was thawed and suspended in 14 ml of complete neuronal
medium, per the supplier's instructions (Sciencell), then plated in 700 μl
aliquots. After 4 days in culture, fresh medium was added, and the
neurons were used the next day to test the B cell Sup.
2.4. Rat mixed glial cell cultures
Some of the B cell supernatants from RRMS patients and NC used in
this study were tested previously for capacity to kill OL in rat mixed
glial cell cultures (Lisak et al., 2012). We tested additional newly
produced supernatants from RRMS patients (n = 6) and NC (n = 6) to
ascertain whether these supernatants were also toxic to OL. Cultures
enriched in differentiated OL were prepared from neonatal rat brain,
using a modification of the shakeoff method (McCarthy and de Vellis,
1980); cultures from the 3rd–5th shakeoffs were used, resulting in
cultures containing on average 35–40% OL, 35–40% astroglia and 15%
microglia. The methods for preparation of cultures have been pre-
viously described in detail (Lisak et al., 2006; Lisak et al., 2012).
2.5. B cell cultures
Blood was obtained with informed consent from patients with RRMS
and age matched controls at the Montreal Neurological Institute/McGill
University. B cells were obtained by positive selection for CD19 from
peripheral blood of consenting patients with RRMS (Polman et al.,
2011) and from NC, as previously described (Bar-Or et al., 2010; Bar-Or
et al., 2001; Duddy et al., 2007; Duddy et al., 2004) and as approved by
the Ethics Review Board of the Montreal Neurological Institute and
McGill University. B cells were isolated by density centrifugation using
Ficoll (GE Healthcare). CD19 beads (Miltenyi Biotech) were used to
positively select B cells according to the manufacturer's protocol. Purity
of the cells was assessed using flow cytometry with an enrichment
of > 98%. B cells were then cultured without any stimulation for 3 days
(Bar-Or et al., 2010; Bar-Or et al., 2001; Duddy et al., 2004; Lisak et al.,
2012); Sup were harvested and frozen at -80° C until testing for
cytotoxicity studies performed at Wayne State University School of

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Medicine.
RRMS patients consisted of 9 women and 4 men with a mean age of
39.8 years of age (range 18–60 years), and the healthy NC were 8
women and 5 men with mean age of 35.5 years of age (range
22–63 years). All MS patients had RRMS (at least one relapse in the
prior 12 months) with the exception of one patient who presented as
clinically isolated syndrome (and was subsequently confirmed to have
RRMS). At the time of blood studies, 10 patients were stable (no new
symptoms or signs to suggest relapse in the prior 3 months). None had
received corticosteroids for at least 3 months, none had received
disease modifying therapy (DMT) for at least 24 months and 9 had
never received any DMT. Of the 13 NC and 13 MS Sup analyzed for
their effects on neurons in this study, 7 NC and 7 MS were used in our
previous study that assessed OL toxicity (Lisak et al., 2012).
2.6. Cytotoxicity assays
Sup from B cell cultures were diluted with either rat or human
neuronal medium (1:4). The diluted B cell Sup were incubated with
neuronal cultures for 72 h. As control medium, we used the B cell
culture medium (not conditioned with B cells), diluted 1:4 with the
respective neuronal medium. Neuronal death was assessed by uptake of
trypan blue as we employed for OL, oligodendrocyte precursors (OPC)
and neurons (Benjamins et al., 2014; Lisak et al., 2012; Lisak et al.,
2011). For all experiments with rat neuronal cultures, the cultures were
analyzed in a blinded fashion. Experiments were performed with mixed
glial cell cultures to assess whether newly obtained B cell supernatants
(6 NC and 6 MS) were also toxic for OL; Sup were diluted 1:4 with glial
cell culture medium as previously reported (Lisak et al., 2012). All
bovine sourced serum used in culture medium for B cells, neurons and
glia were heat inactivated as done in past experiments.
2.7. Detection of apoptosis
Attached cells on coverslips were fixed and stained to assess
apoptosis utilizing the Apoptag In Situ Apoptosis Kit (EM Millipore,
Billerica MA) to visualize terminal deoxynucleotidyl dUTP nick end
labeling (TUNEL), as described previously (Skoff et al., 1998). We
quantitated the percentage of TUNEL positive rat neurons at 24, 48 and
72 h of incubation with supernatants from B cell cultures from 3 NC and
4 RRMS. Additional supernatants from NC and RRMS were tested on
both rat neurons and human neurons at 72 h. Surface appearance of the
apoptotic marker annexin was used to assess apoptosis in OL. Live cells
on coverslips were treated for 15 min at room temperature with
Alexa488-labeled annexin V (Thermo-Fisher, Waltham MA), diluted
1:20 with annexin-binding buffer. Coverslips were washed once gently
in Dulbecco's minimal essential medium (DMEM), fixed, then mounted
for subsequent viewing by fluorescence microscopy. For immunostain-
ing of activated caspase-3, cells were fixed, permeabilized with 0.05%
Triton for 20 min, blocked with 10% bovine serum albumin for 30 min,
then incubated overnight at 4° C with a 1:200 dilution of antibody to
activated caspase 3 (Thermo-Fisher, Waltham MA). Antibody binding
was visualized with FITC-labeled donkey anti-rabbit IgG.
2.8. Immunoglobulins
Human specific IgG and IgM enzyme-linked immunosorbent (ELISA)
assays were performed in the immunoanalysis core of the Bone Marrow
Transplant/Immunotherapy Program of the Karmanos Cancer Institute
and Wayne State University Department of Oncology, using a method
developed by the core. ELISA plates were coated with goat anti-human
IgG (Invitrogen H10000) or goat anti-human IgM (Invitrogen H15000).
The blocking solution was 3% bovine serum albumin + 5% fetal bovine
serum in 1× phosphate buffered saline (PBS), and the washing solution
was 1× PBS + 0.05% Tween 20. The standards were human IgG,
whole molecule (Pierce 31154) or human IgM, whole molecule (Pierce
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Journal of Neuroimmunology 309 (2017) 88–99
31146). Detection was carried out with goat anti-human IgG horse
radish peroxidase conjugate (Invitrogen H10007) or goat anti-human
IgM horse radish peroxidase conjugate (Invitrogen H15007) and TMB
substrate (Thermo 34021).
2.9. Cytokines and related proteins
The human specific cytokines and related protein biological modi-
fiers examined are listed in Table 1.
2.10. Estimation of apparent molecular weight of toxic factors
B cell medium and B cell Sup from NC and RRMS were analyzed
using centrifugal filtration units with molecular weight cutoff mem-
branes. The membranes were pre-washed, 250 μl samples added and
units centrifuged according to manufacturers' instructions. Units with
membrane cutoffs of 30 kilodalton (kDa) and 100 kDa were purchased
from EMD Millipore (Temecula CA), and 300 kDa units (NanoSep
300 K) from Pall Corporation (Ann Arbor, MI).
2.11. Statistical analysis
B cell Sup were tested for cell death or apoptosis on duplicate
coverslips in each experiment; the means of the values for NC and
RRMS were analyzed using the Students' t-test or using one way ANOVA
with Tukey post test, as indicated in the figure legends.
3. Results
3.1. Impact of B cell secreted products on neuronal and glial cell survival
Our prior publication reported OL cytotoxic effects of Sup from 7
RRMS, with little to no toxicity by Sup from 3 out of 4 NC (Lisak et al.,
2012). We have now expanded our findings to an additional 15 RRMS
and 16 NC Sup and find similar results. Data for OL toxicity of the 13
RRMS and 13 NC Sup that we subsequently tested for neuronal toxicity,
are shown in Fig. 1. Substantiating our previous observations, B cell Sup
from all RRMS patients (but not from NC) induced on average 58% OL
death compared to 4% for NC B cells Sup. None of the B cell Sup
appeared to have toxic effects on astrocytes or microglia within the
mixed glial cultures.
We next observed that Sup from the unstimulated B cell cultures of
all 13 patients with RRMS (at a 1:4 dilution with neuronal culture
medium) induced cytotoxicity of rat neurons, while similarly diluted
Sup from 13 NC induced little or no neuronal death above background
(Fig. 2A, B). Background neuronal cell death induced by the control
medium alone at 1:4 ranged between 11 and 14% neuronal death and
was subtracted for each experiment from total trypan blue positive
cells. The mean cell death above background for RRMS patient B cell
Sup was 47.5% ± 2.7% S.E.M compared to 9.6% ± 2.4% S.E.M. for
cell death induced by Sup from NC B cells (Fig. 1C).
To examine whether the cytotoxic potential of MS patient-derived B
cell Sup was limited to rodent neurons, we assessed the effects of B cell
Sup derived from RRMS patients and NC on human-derived neurons. B
cell Sup (at 1:4 dilution with human neuronal medium) from 4 patients
with RRMS were toxic to human neurons while similarly diluted Sup
from 3 NC induced little or no neuronal death (Fig. 3A). In each
experiment, background neuronal cell death induced by control med-
ium alone at 1:4 was assayed as was done for rat neurons and
subtracted as above from total trypan blue positive cells for RRMS
and NC. The mean cell death above background for RRMS patient B cell
Sup was 55.9% ± 1.9% S.E.M. compared to 8.8% ± 0.8% S.E.M cell
death induced by Sup from NC (Fig. 3B).
R.P. Lisak et al. Journal of Neuroimmunology 309 (2017) 88–99

Fig. 1. Secreted products of B cells from RRMS patients are cytotoxic to rat OL compared to those from normal individuals. B cell Sup in addition to those previously tested (Lisak et al.,
2012) were assayed for OL toxicity prior to assays on neuronal cultures, mixed glial cultures were treated for 3 days with Sup from B cells (not stimulated in vitro) from 9 NC and 12
RRMS, diluted 1:4 with rat OL medium; and OL death was measured by trypan blue uptake; OL death in 1:4 B cell medium alone averaged 13%; this value was subtracted from NC and
RRMS values. A. OL death in cultures treated with 1:4 B cell Sup from 9 NC; NC 1, 2 and 4 were tested previously; B. OL death in cultures treated with 1:4 B cell supernatants from 12
RRMS patients; MS 3, 4, 6 and 7 were tested previously (Lisak et al., 2012); C. Comparison of mean % OL death induced by B cell supernatants from NC versus RRMS: NC,
4.1 ± 2.6 S.E.M; RRMS, 58.1 ± 3.6 S.E.M.; p < 0.0001, Student's t-test.

3.2. The impact of MS B cell supernatants on rat and human neuronal and 3.3. Levels of immunoglobulins, cytokines and related proteins in B cell
OL survival involves apoptotic cell death culture supernatants

Sup from B cell cultures of 3 patients with RRMS and 3 NC were
tested to determine if apoptosis contributed to death of rat neurons.
When stained with antibodies to NF-H, the neurons exposed to the MS B
cell Sup appeared damaged and showed process retraction compared to
neurons exposed to control Sup (Fig. 4A). At different time points in
vitro as assessed by percent of neurofilament-H (NF-H)+ neurons that
stained with TUNEL, B cell Sup from RRMS patients induced significant
apoptosis at 72 h with little if any apoptosis induced by Sup from NC
(Fig. 4B). Less TUNEL staining was observed at 24 h and 48 h.
Additional experiments were carried out with 3 more NC and 3 more
RRMS Sup at 72 h (Fig. 4C). The conclusion that TUNEL staining
represents apoptosis rather than DNA fragmentation due to DNA repair
is consistent with the trypan blue results and our observation that 64%
of OL treated for 3 days with Sup from RRMS are positive for annexin V
staining compared to only 15% for NC Sup; similar results were found
for immunostaining for activated caspase 3 (data not shown).
Cell death induced in the human neurons by Sup of B cells from
RRMS patients also involved apoptosis. The human neurons exposed to
the MS B cell Sup appeared damaged, exhibiting greater process
retraction (Fig. 4D) and significantly greater apoptosis by TUNEL
staining (Fig. 4E–F) as compared to human neurons treated with B cell
Sup from NC B cells.
We found very low levels of IgG and IgM in these short-term
cultured B cell Sup, and no significant difference in the mean levels
between the NC and RRMS samples. Average levels ± S.E.M. for IgG
were 0.060 ± 0.016 mg/dl in NC and 0.052 ± 0.016 mg/dl for MS,
p < 0.70. Average levels ± S.E.M. for IgM were 0.017 ± 0.006 mg/dl
in NC and 0.014 ± 0.003 mg/dl for MS, p < 0.63. There was no
correlation between cytotoxicity and the IgG or IgM levels in either
group (Fig. 5A, B).
Levels of most cytokines and chemokines were low or undetectable
in the Sup of these unstimulated B cells, both from NC and RRMS.
Analysis of the values obtained using a 14-PLEX magnetic array (Tables
1 and 2) showed no statistical differences between the concentrations of
any of the cytokines/chemokines/biologic modifiers in the Sup ob-
tained from B cells of NC and RRMS, with the exception of LT-β which
appeared reduced in RRMS B cell Sup. There was a trend for an
increased level of the chemokine MIP-1α in Sup of RRMS patients, as
well a trend for decreased IL-6 compared to the B cell Sup of NC. There
was no correlation between the concentrations of any of these proteins
and neuronal cell death mediated by these respective B cell Sup.
Examination of values obtained using a 25-PLEX non-magnetic im-
munoarray analysis (Tables 1 and 2) showed that only 12 of the
cytokines assayed were detectable in Sup from either NC, RRMS or both
for some of the samples, while 15 of the cytokines were absent or below
detectable levels. There was no significant difference in values for any

Fig. 2. Secreted products of B cells from RRMS patients are cytotoxic to rat neurons compared to those from normal individuals. Neuronal cultures were treated for 3 days with Sup from B
cells (not stimulated in vitro) from NC or RRMS, diluted 1:4 with rat neuronal medium; and neuronal death was measured by trypan blue uptake; neuronal death in 1:4 B cell medium
alone averaged 13%; this value was subtracted from NC and RRMS values. A. Neuronal death in cultures treated with 1:4 B cell Sup from 13 NC. B. Neuronal death in cultures treated with
1:4 B cell Sup from 13 RRMS patients. C. Comparison of mean neuronal death induced by B cell Sup from NC versus RRMS. NC, 9.6% ± 2.4% S.E.M.; RRMS, 47.5% ± 2.7% S.E.M.;
p < 0.001, Student's t-test.

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Fig. 3. Secreted products of B cells from RRMS patients are cytotoxic to human neurons compared to Sup of B cells obtained from normal individuals. A. Neuronal cultures were treated
for 3 days with Sup from B cells (not stimulated in vitro) from 3 NC samples and 4 RRMS samples, each diluted 1:4 with human neuronal medium; neuronal death was measured by trypan
blue uptake. Neuronal death in 1:4 B cell medium alone averaged 12%; this value was subtracted from RRMS values. B. Comparison of mean neuronal death induced by B cell Sup from
NC versus RRMS. NC, 8.8% ± 0.8% S.E.M.; RRMS, 55.9% ± 1.9% S.E.M.; p < 0.0001, Student's t-test.

of the detected cytokines between NC and RRMS B cell Sup.
Six cytokines of potential interest that were not represented on the
arrays were analyzed by individual ELISAs (Tables 1 and 2), with low
levels of MMP-9, TGF-β and IL-16 detected in some samples. There was
no difference in the levels of MMP-9 or TGF-β in the B cell Sup of NC
and RRMS patients. There was also no difference between NC and
RRMS B cell Sup levels of IL-16 when results were analyzed with the
one NC outlier included (p = 0.33); with the outlier removed, mean
levels of IL-16 were slightly increased in RRMS compared to NC
(p < 0.04). Five additional proteins of interest, related to B cell
biology, were also assayed: CD40, CD40 ligand, Fas, Fas ligand and
granzyme B (Table 1); these proteins were not detectable in most of the
samples, and again, there were no differences between NC and MS
samples (data not shown).
When levels of IL-16, LT-α, LT-β, TNF-α (14-PLEX), MIP-1α (14-
PLEX), MIP-1β (14-PLEX) and GM-CSF are compared with neuronal cell
death induced by B cell.
Sup from RRMS and/or NC (Fig. 5C–I), there were no correlations
between any given cytokine concentration and death of neurons. We
performed similar analyses for MMP-9, IL-6 (values from both cytokine
assays), interferon (IFN)-α, IFN-γ, ΤΝF-α, and fibroblast growth factor
(FGF), and there was no correlation between protein levels in the Sup

Fig. 4. Apoptosis is prominent in neurons treated with secreted products of B cells from RRMS patients compared to normal individuals. A. Representative fields of rat neuronal cultures
treated for 3 days with B cell Sup from NC or RRMS, immunostained for NF-H (green) or TUNEL (red). B. Time course shows % of apoptotic rat neurons at 1, 2 and 3 days for 3 NC and 4
MS Sup. *p < 0.01, NC 3d vs. RRMS 3d. C. Apoptosis in rat neurons was measured by immunostaining for TUNEL and NF-H after 3 days of treatment with 1:4 B cell Sup from an
additional 3 NC and 3 RRMS. *p < 0.001, NC 3d vs. RRMS 3d. D. Representative fields of human neuronal cultures treated for 3 days with B cell Sup from NC or RRMS, immunostained
for NF-H (green) or TUNEL (red). E. Apoptosis in human neurons was measured by immunostaining for TUNEL and NF-H after 3 days of treatment with B cell Sup from 4 NC and 4 RRMS.
F. Comparison of mean apoptotic human neuronal death induced by B cell Sup from NC versus RRMS. NC, 5.5% ± 1.6%; RRMS, 53.3% ± 1.0% S.E.M.; p < 0.0001, Student's t-test.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Levels of human IgG, IgM and cytokines are similar in supernatants from RRMS and NC, and do not correlate with death of cultured rat neurons. A. Comparison of % neuronal
death with the levels of IgG in the B cell Sup showed no significant correlation for either NC or RRMS samples, as analyzed by Pearson's correlation coefficient. NC vs. IgG levels, r = 0.39,
p < 0.0.30; RRMS vs, IgG levels, r = 0.44, p < 0.20. B. Comparison of % neuronal death with the levels of IgM in the B cell Sup showed no significant correlation for either NC or RRMS
samples. NC vs. IgM levels, r = 0.41, p < 0.27; RRMS vs, IgM levels, r = 0.46, p < 0.50. C - I. Comparison of % neuronal death with the concentration of levels of GM-CSF, IL-16, LT-α,
LT-β, MIP-1α, MIP-1β, or TNF-α in the B cell Sup showed no significant correlation for either NC or RRMS samples, as analyzed by Pearson's correlation coefficient.

and neuronal cell death (data not shown). In addition, combining levels
of ‘proinflammatory proteins’ or of ‘downregulatory anti-inflammatory’
proteins did not suggest any combination that correlated with neuronal
cell death in our system (data not shown).
3.4. Toxic factor/s in RRMS B cell supernatants are enriched in high
molecular weight fractions
Our experiments determining levels of cytokines and related
proteins as well as Ig levels did not identify a likely toxin or toxins.
Therefore as the next step in identifying the molecule/s responsible for
toxicity, control medium and B cell Sup from NC and RRMS were
analyzed using centrifugal filtration units with molecular weight cutoff
membranes. We used both neurons and OL as targets in these assays.
After filtration, the filtrate and retentate, each in a final volume of
250 μl of B cell medium, were diluted 1:4 with either neuronal or OL
medium and tested for toxicity on neuron or OL cultures, respectively.
Cell death was analyzed at the end of 3 days using trypan blue uptake.
Initial experiments with OL using 30 kDa and 100 kDa cutoff mem-
branes showed that the toxic activity was retained in the higher
molecular weight fraction of RRMS Sup, with no activity in the RRMS
filtrate. The filtrates and retentates from the control medium alone, or
the NC Sup showed no toxicity. In subsequent experiments B cell Sup
from 6 NC and 6 RRMS were fractionated with a 300 kDa cutoff
membrane filter. The 300 kDa retentates from RRMS Sup induced OL
death whereas retentates from control medium or NC Sup did not
(Fig. 6A). None of the filtrates showed toxicity to OL. After centrifuga-
tion of the RRMS > 300 kDa fraction at 2,000 × g for 30 min to remove
cell debris, the toxic factor/s remained in the supernatant. In subse-
quent experiments, > 300 kDa retentates and filtrates from NC and
RRMS Sup were tested for toxicity on neuronal cultures. As with OL, the
toxic factors were concentrated in the > 300 kDa fractions from RRMS
Sup (Fig. 6B); the filtrates from RRMS Sup also exhibited a low but
statistically significant toxicity compared to filtrates from the NC Sup.
4. Discussion
The role of B cells, their progeny (plasmablasts and plasma cells)
and secreted Ig have been the subject of study in MS for many years
(Cross and Wu, 2010; Kabat et al., 1942; Link and Huang, 2006; Miller
et al., 1983). Studies of serum, cerebrospinal fluid (CSF) and eluates of
Ig from CNS of MS patients attempting to identify one or more
antibodies to specific target antigens (either CNS constituents or
specific microbial antigens using increasingly sophisticated techni-

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R.P. Lisak et al. Journal of Neuroimmunology 309 (2017) 88–99

Table 2
Cytokines and other factors detected in B cell supernatants from NC and RRMS.

Cytokine Controls Mean ± S.E.M. Detected/assayed RRMS Mean ± S.E.M. Detected/assayed p Value
pg/ml pg/ml (t-test)

FGF, basicb 0–1.86 0.50 ± 0.27 5/6 0–0.70 0.16 ± 0.12 2/6 ns p = 0.20
GM-CSFa 0–8.9 2.21 ± 1.02 6/9 0–3.5 0.79 ± 0.39 5/10 ns p = 0.25
IFN-αb 0–10.6 5.31 ± 1.94 4/6 0–10.6 3.5 ± 1.77 3/6 ns p = 0.52
IFN-γb 3.7–9.0 0.89 ± 0.28 4/6 0–0.90 0.89 ± 0.28 3/6 ns p = 1.0
IL-4a 0–32.2 1.14 ± 0.99 2/9 0–9.1 1.48 ± 0.99 4/10 ns p = 0.81
IL-6a 0–151 27.8 ± 16.8 5/9 0–5.36 1.81 ± 0.68 5/10 ns p = 0.11
IL-6b 0–2.34 1.19 ± 0.37 5/6 0.68–3.4 1.48 ± 0.45 6/6 ns p = 0.62
IL-12p60a 0–2.76 0.71 ± 0.39 3/9 0–3.70 1.13 ± 0.48 5/10 ns p = 0.52
IL-16c 89–361 153.4 ± 29 9/9 102–272 189 ± 20 10/10 ns p = 0.33
(p = 0.045)⁎
LT-αc 0–39 19.4 ± 6.0 7/9 0–53 20.3 ± 5.4 6/9 ns p < 0.9
LT-βc 0–29 12.4 ± 2.7 8/9 0–13.3 4.8 ± 1.3 9/10 p = 0.018
MCP-1b 3.0–101 19.3 ± 16.3 2/6 0–80.7 24.1 ± 12.3 5/6 ns p = 0.82
MIP1αa 0–108 16.3 ± 12.2 2/9 0–332 83.7 ± 31.7 8/10 ns p = 0.074
MIP1β a 0–548 122 ± 63 6/9 0–424 162 ± 43 9/10 ns p = 0.69
MIP1βb 8–87.1 29.1 ± 11.9 6/6 8–62.6 29.3 ± 8.4 6/6 ns p = 0.98
MMP-9c 0–3895 830 ± 634 3/6 0–3570 800 ± 639 5/6 ns p = 0.97
TGFβc 0–1031 245 ± 173 2/6 0–1250 231 ± 204 3/6 ns p = 0.96
TNFαa 0–100 21.1 ± 10.9 8/9 2.8–26.8 11.1 ± 2.4 10/10 ns p = 0.36

a
B cell Sup from 6 to 9 NC and 6–10 RRMS were analyzed on a Luminex 14-plex magnetic immunoarray.
b
B cell Sup from 6 NC and 6 RRMS were analyzed on a 27-plex non-magnetic immunoarray.
c
B cell Sup from 6 to 9 NC and 6–9 RRMS were analyzed by individual ELISA kits.
⁎
p Value for IL-16 with one NC outlier value removed.

Fig. 6. Cytotoxicity of secretory factors from RRMS B cells is retained in a fraction with > 300 kDa molecular weight. (A) B cell Sup from 3 NC and 3 RRMS were fractionated with a
300 kDa cutoff membrane filter. The > 300 kDa retentates from RRMS Sup induced OL death whereas NC did not, compared to the medium control. Values represent averages of
duplicates. Comparison of mean % OL death induced by > 300 kDa retentates from NC versus RRMS: NC, 7.7 ± 0.7 S.E.M; RRMS, 44.0 ± 4.6 S.E.M.; **p < 0.0015, Student's t-test. No
toxicity above baseline was found in the filtrates or in the pellets (2,000 × g, 30 min) from the retentates for either NC or RRMS samples. The experiment was repeated on the same
samples with similar results. (B) B cell Sup from 5 NC and 5 RRMS were fractionated with a 300 kDa cutoff membrane filter. The > 300 kDa retentates from RRMS Sup induced neuronal
death whereas NC did not, compared to the medium control. Values represent averages of duplicates. Comparison of mean % neuronal death induced by the > 300 kDa retentates from
NC versus RRMS was NC 23.8 ± 1.7 S.E.M., RRMS 67.0 ± 2.5,** p < 0.0001, Student's t-test. Neuronal death with the filtrates was NC 20.4 ± 1.8 S.E.M; RRMS, 31.7 ± 1.6 S.E.M.;
*p < 0.002.

ques), have largely failed to identify disease-implicated target antigens
(Blauth et al., 2015b; Brandle et al., 2016; Genain et al., 1999; Gilden,
2005; Lisak et al., 1968).
Recent clinical trials with agents directed against CD20 (expressed
by B cells) have demonstrated substantial benefit in patients with RRMS
(Hauser et al., 2017; Hauser et al., 2008). The onset of therapeutic
effects in these patients was noted early, before any effect on serum
immunoglobulin levels (Bar-Or et al., 2008; Hauser et al., 2017; Hauser
et al., 2008). Notably, such treatment had no effect on the abnormal IgG
present in CSF of these patients (Piccio et al., 2010). Together, these
findings clearly demonstrate an important role for B cells in mediating
new MS disease activity, but also indicate that their contribution to
such disease activity is independent of Ig secretion (Blauth et al., 2015a;
Hauser, 2015). Some therapeutic benefit of anti-CD20 treatment has
also been reported in subsets of patients with primary progressive MS
(PPMS) (Hawker et al., 2009; Montalban et al., 2017).
B cells in MS migrate from the periphery into the meninges, CSF and
the CNS parenchyma of patients (Levinson et al., 1983; Sandberg et al.,
1986), and such trafficking seems to be bidirectional (Stern et al., 2014;
von Budingen et al., 2012), with activation of B cells both outside
(Sandberg-Wollheim et al., 1986; Stern et al., 2014) and within the
neuraxis (Levinson et al., 1983; Sandberg et al., 1986; Monson et al.,
2005; Qin et al., 1998). Cloning techniques show that the same
populations of B cells documented in the CNS of patients can also be
detected in the peripheral immune compartment (Lovato et al., 2011;
Palanichamy et al., 2014; Hauser, 2015). Therefore studying B cells

94
R.P. Lisak et al.
from peripheral blood of patients may contribute to understanding the
role of B cells in the meninges as well as in CSF and CNS parenchyma in
MS pathogenesis.
There is increasing understanding and interest in the complexity of
B cell subtypes and their functions (Mamula, 2013), with active
research on the roles of these different subsets of B cells in the
pathogenesis of MS (Antel and Bar-Or, 2006; Hauser, 2015). B cells
act as antigen presenting cells (APC) to T cells, which has been well
demonstrated using the experimental autoimmune encephalomyelitis
model (Molnarfi et al., 2013; Weber et al., 2011). Molnarfi and
colleagues also demonstrated that B cell APC function and formation
of meningeal B cells aggregates was not dependent on myelin-specific
antibody production (Molnarfi et al., 2013). Among the functions of
particular interest in our studies are the antibody-independent effector
functions of B cells including production of cytokines and other non-Ig
biological modifiers (Li et al., 2015; Lino et al., 2016).
Subpopulations of B cells include effector B cells, some of which can
secrete proinflammatory cytokines such as GM-CSF (Bhattacharya
et al., 2015; Li et al., 2015), as well as what have been described as
‘killer B cells’ which can also serve regulatory functions (Lundy, 2009).
Is it possible that these cells might secrete factors that can injure other
cell types? Additional studies will examine B cells from the blood of
patients with progressive forms of MS to determine if Sup from B cell
cultures from such patients also are cytotoxic to OL and neurons. Other
studies will include testing Sup from B cell cultures of patients with
other immune-mediated neurological diseases including myasthenia
gravis (MG) and chronic inflammatory demyelinating polyneuropathy
(CIDP) and systemic immune-mediated diseases like rheumatoid ar-
thritis (RA) and systemic lupus erythematosis (SLE), and testing B cell
Sup from patients with MS and NC to determine if they produce death
of Schwann cells, the myelin forming cells of the peripheral nervous
system (PNS). Even if B cells from patients with MG, CIDP, RA or SLE
secrete factors that damage OL or CNS neurons, or B cells from patients
with MS damage Schwann cells, this in no way diminishes the potential
relevance of our findings in MS. These B cells from patients with MS
may secrete factors acting locally where B cells are present in the CNS.
Thus the difference might well be that in MS there are chemokine
signals that attract and hold B cells in the CNS including the meninges
and those factors are not present in the PNS or thymus of patients with
MS. It is likely a combination of where the B cells that mediate
cytotoxicity go in which diseases. We are also performing studies to
determine which subpopulations of B cells are producing the putative
toxic factors including testing Sup from memory (CD27+) and naïve
(CD27-) B cells (Duddy et al., 2007).
Studies of cortical pathology in patients with secondary progressive
MS (SPMS) reveal meningeal inflammation that can be rich in B cells
(some of which have been described to have follicle-like properties),
with underlying demyelination and axonal/neuronal loss and a paucity
of inflammatory cells within cortex itself, particularly in (subpial) Type
III lesions (Howell et al., 2011; Kutzelnigg et al., 2005; Magliozzi et al.,
2007; Peterson et al., 2001). There is no evidence of Ig or significant
complement activation (Brink et al., 2005) in the type III lesions, lesions
that involve layers 1–4, although they may merge with deeper Type I
leukocortical lesions. Intense meningeal inflammation containing B
cells has been reported in some patients with primary progressive MS
(PPMS) (Choi et al., 2012) and in biopsies from some patients with very
early RRMS in which B cells may also be found within the cortex
(Lucchinetti et al., 2011). Meningeal inflammation has also been shown
to correlate with underlying cerebellar atrophy (Kutzelnigg et al.,
2007). In each case the degree of damage to the underlying cortical
OL/myelin and neurons/axons is reportedly directly proportional to the
intensity of the overlying active meningeal disease (Calabrese et al.,
2009; Howell et al., 2011; Lucchinetti et al., 2011; Magliozzi et al.,
2010; Popescu and Lucchinetti, 2012), although one group did not
observe this relationship (Kooi et al., 2009). While some have suggested
much of this damage is the result of downstream demyelination and
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Journal of Neuroimmunology 309 (2017) 88–99
axonal damage and death in the white matter (Bjartmar et al., 2003;
Jeffery et al., 2000; Jurgens et al., 2016), it also seems clear that some
of the damage within the cortical gray matter is not directly related to
lesions within the underlying white matter (Bo et al., 2007; Fischer
et al., 2013; Haider et al., 2016; Lassmann et al., 2007). It has been
postulated that some of the subpial the damage results from diffusion of
cytotoxic factors, perhaps cytokines, from the meninges and CSF into
the cortical gray matter (Serafini et al., 2007; Magliozzi et al., 2010;
Howell et al., 2011; Lisak et al., 2012; Lassmann, 2014) and perhaps, by
extension, to the cerebellum as well (Kutzelnigg et al., 2007). The exact
distribution of meningeal inflammatory lesions is still not fully under-
stood in RRMS, SPMS and PPMS and so factors secreted by B cells might
well be able to circulate for short distances in the CSF and cause
damage at short distance from the meningeal inflammatory lesions. In
addition it is clear that inflammation in the meninges is not limited to
areas where the atypical germinal follicles are seen. And of course some
changes in the cortical gray matter may be the result of lesions
damaging axons and neurons downstream and at a distance from the
cortex, as mentioned above (Bjartmar et al., 2003; Jeffery et al., 2000;
Jurgens et al., 2016).
It has been suggested that other cells in the meninges, including
CD8 T cells reacting to activated EBV in B cells, release toxic factors
that contribute to cortical damage (Meier et al., 2012; Serafini et al.,
2007); however, several groups have not been able to demonstrate EBV
in CNS B cells in patients with MS (Lassmann et al., 2011; Sargsyan
et al., 2010; Lassmann et al., 2011; Sargsyan et al., 2010; Willis et al.,
2009). In other studies, in vitro killing of human neurons in culture by T
cells seemed to require direct cell to cell contact (Giuliani et al., 2003),
yet cortical pathology in MS is notable for relative paucity of T cells
within the tissue (Fischer et al., 2013; Howell et al., 2011; Lassmann,
2012; Peterson et al., 2001).
For these reasons, we have considered whether soluble products,
and particularly those secreted by meningeal B cells, may be toxic to the
underlying cortex. We have shown that B cells obtained from patients
with RRMS but not from NC produce one or more factors that cause
death of OL in our rat mixed glial cell cultures, but do not impact
astrocytes or microglia. This killing of OL does not seem to be caused by
IgM or IgG and is independent of complement activation (Lisak et al.,
2012). The lack of apparent involvement of Ig or complement in this in
vitro toxicity to OL parallels the in vivo findings of type III lesions within
the cortical gray matter particularly in progressive forms of MS.
In the current study, we first extend our findings and demonstrate
that secretory products from B cells obtained from patients with RRMS
(not receiving disease modifying therapy or any recent corticosteroids)
and cultured in vitro, produce one or more factors that induce death of
rat CNS neurons, and that the same effect is seen on human neurons in
culture, demonstrating that this is not a toxic effect directed exclusively
at rat cells or even just against rat neurons or rat OL. A recent study
reported in abstract form showed that B cells from the blood of patients
with MS induced apoptosis of neurons in cerebellar slice cultures
(Harlow et al., 2016) supporting our earlier work on OL (Lisak et al.,
2012) and our current study of rat and importantly human neurons.
Using a different assay for IgG and IgM than in our paper on OL
cytotoxicity where we did not detect any measurable IgM and very low
levels of IgG in a minority of tested Sup (Lisak et al., 2012), we detected
IgG and IgM at very low levels in the Sup we tested for neuronal
cytotoxicity. As in our study of induction of OL death, there was no
correlation between neuronal death and levels of IgG or IgM in the Sup
of B cells from patients with RRMS or NC. As was true in our studies of
OL cell death, serum used in B cell cultures and neuronal cultures was
heat inactivated, eliminating complement activity. Death of both rat
and human neurons in our system seems to be due in part to apoptosis
which is not what one would expect with death induced by antigen-
antibody mediated complement activation.
In our previous study of induction of OL death by B cell secretory
products, we measured levels of several cytokines associated with B
R.P. Lisak et al.
cells: TNF-α, LT-α, IL-6, IL-10 and TGF-β (Lisak et al., 2012). We did
not detect a difference in levels of any of these cytokines in super-
natants from either nonstimulated or unstimulated B cells of patients
with RRMS and NC. There was no correlation of OL death with levels of
any single cytokine or with any combination of cytokines including
TNF-α + LT-α + IL-6, so-called ‘proinflammatory’ cytokines or IL-
10 + TGF-β, considered to be ‘anti-inflammatory’ or ‘downregulatory’.
TNF-α and LT-α have been reported to be toxic for OL and cause
demyelination in vitro (Selmaj et al., 1991a; Selmaj et al., 1991b; Selmaj
and Raine, 1988). In this study we examined levels of a larger number
of cytokines and other protein biologic mediators. With the exception of
LT-β which may have been reduced in RRMS Sup, we did not find
significant differences in the levels of any of the cytokines or protein
biological modifiers in the Sup of cultures of the B cells from RRMS
patients compared to NC. The lack of an increase in the levels of pro-
inflammatory molecules secreted from MS vs. NC B cells in our current
study (in contrast to such previously reported differences, Bar-Or et al.,
2010; Lino et al., 2016; Lisak et al., 2012) may reflect the fact that here
we did not examine Sup from B cells stimulated in vitro whereas in
earlier experiments Sup from B cells stimulated in vitro were also
examined (Bar-Or et al., 2010; Lino et al., 2016; Lisak et al., 2012).
Analysis of the 14-PLEX array data (Table 2) showed a trend in Sup
for B cells from NC for increased levels of IL-6, a proinflammatory
cytokine produced by many inflammatory cell types including B cells.
In Sup of B cells from RRMS patients, there was a trend for increased
levels of the chemokine MIP-1α, which primarily results in attraction of
polymorphonuclear cells. That trend was not seen for MIP-1α using the
25-PLEX assay (Table 2), with no differences in levels of MIP-1β. There
were no differences in levels of MCP-1, IFN-α or IFN-γ. IL-16 levels
were modestly increased in Sup from RRMS compared to NC when the
one outlier among the NC is eliminated (Table 2). IL-16 is a proin-
flammatory cytokine, produced by many inflammatory cell types
including B cells, that is important in the pathogenesis of experimental
autoimmune encephalomyelitis and may be involved in the pathogen-
esis of MS (Skundric et al., 2015). B cells from patients with MS have
increased expression of MMP-9 (Aung et al., 2015), a protease that is
important in opening of the blood brain barrier in inflammatory lesions
allowing peripheral immune cells to enter the parenchyma(Stuve et al.,
1996). We did not find an increase in levels of secreted MMP-9 in Sup of
B cells from patients with RRMS compared to NC, and no correlation
between levels of MMP9 and neuronal cell death.
In earlier work we showed that B cells in the CSF of patients with
MS were more activated than B cells in the blood of the same patients
(Levinson et al., 1983). There is also evidence that the blood B cells, not
stimulated in culture, as well as other mononuclear inflammatory cells,
are in a higher state of activation than seen in normal individuals
(Mathias et al., 2016). Thus these more activated B cells may be
producing and secreting greater amounts of one or more B cells
products when they are placed into culture. In our prior study (Lisak
et al., 2012) in vitro stimulation had a variable affect on B cell Sup from
RRMS patients to kill OL when compared to B cells from the same
subject that had not undergone in vitro stimulation. In addition in vitro
stimulation of B cells from NC further reduced any minimal toxic effects
of Sup on OL (Lisak et al., 2012).
We found no correlation between the level of any single cytokine or
other protein tested and induction of cell death of rat neurons in vitro in
these experiments, as illustrated by several examples in Fig. 5. Analysis
of combinations of cytokine levels, either proinflammatory or down-
regulatory, failed to reveal any combinations that showed any correla-
tion with neuronal cell death. These results indicate that none of the
cytokines analyzed are likely candidates for neurotoxic factors present
in the RRMS B cell Sup, or as protective factors, absent in the RRMS
Sup, but present in the NC Sup.
Our studies on the molecular weight of the toxic factor or factors
indicate that they are primarily > 300 kDa in size. In the case of OL, no
significant toxicity was found in the filtrate containing the lower
96
Journal of Neuroimmunology 309 (2017) 88–99
molecular weight components. For neurons, the toxicity in the
> 300 kDa fraction from MS Sup was 47% compared to 11% in the
< 300 kDa fraction, when corrected for the medium control. If
confirmed with a larger sample size, this suggests that MS Sup may
contain low levels of factors < 300 kDa that are toxic to neurons but
not OL. The apparent large size of the toxic factor(s) raises several
possibilities; 1) cytokines or other proteins that are complexed together
in a manner that they cannot be fully quantitated with immunologic
techniques like ELISA assays and Luminex chips, or 2) larger proteins
including IgM or aggregated IgG. This latter possibility seems unlikely
given the evidence for killing, at least in part, by apoptosis, as
determined by three separate assays for apoptosis, and the lack of
activated complement in our system. It does not appear to be simply
cell debris since the pellet containing cell debris when resuspended was
not toxic. If the toxicity is in exosomes or related extracellular vesicles
in the > 300 kDa fraction, then miRNA or lipids in addition to proteins
within the vesicles are potential toxic factors. Extracellular vesicles
including exosomes have been analyzed in serum and CSF of MS
patients. Their roles in MS pathogenesis and usefulness as biomarkers
or therapeutic targets are active areas of investigation (Saenz-Cuesta
et al., 2014; Carandini et al., 2015; Pusic et al., 2014; Selmaj et al.,
2017). For example, proteomic analysis of CSF exosomes identified
distinctive patterns for MS and neuromyelitis optica (Lee et al., 2016).
Lipidomic analysis of exosome-enriched fractions from plasma showed
increased levels of sulfatide in MS samples compared to controls
(Moyano et al., 2016). Profiling of miRNA patterns in plasma and
CSF has revealed multiple differences between MS and controls;
extracellular vesicles are likely the major carriers for these miRNAs
(Gandhi, 2015; Jadot and Davoust, 2016; Miyazaki et al., 2014; Selmaj
et al., 2017). While extracellular vesicles released by normal B cells
have been characterized in detail (Choi et al., 2013; Danger et al., 2014;
McLellan, 2009), little is known about those released by B cells from MS
patients.
The cargo of extracellular vesicles and exosomes may have toxic or
protective effects on neurons and OL. Exosome-like particles can deliver
neurotoxic or misfolded proteins or neuroprotective factors (Janas
et al., 2016; Yuyama and Igarashi, 2016). Some miRNAs can bind to
toll-like receptors and induce regulatory or cytotoxic effects in the
target cells (Chen et al., 2013). Astrocytes (Wang et al., 2012) and
cytokine-treated glioma cells (Podbielska et al., 2016) release ceramide-
enriched exosomes that can induce cell killing. It has been reported that
ceramides are enriched in CSF of MS patients and can cause neuronal
mitochondrial dysfunction trhough oxidative stress and damage to
axons (Vidaurre et al., 2014). Under some conditions, immune cells
release exosomes that promote CNS myelination and regulate inflam-
mation (Pusic and Kraig, 2014; Pusic et al., 2016). Mesenchymal stem
cell-derived exosomes can improve recovery from experimental trau-
matic brain injury (Zhang et al., 2015) and deliver miRNAs that
enhance neuronal differentiation (Lee et al., 2014). Exosomes from
Schwann cells have been reported to regulate neuron/glia communica-
tion and promote axonal regeneration (Ching and Kingham, 2015;
Lopez-Leal and Court, 2016). OL-derived exosome-like particles can
inhibit CNS myelin membrane sheath formation (Bakhti et al., 2011)
but also regulate neuronal firing rate, signal transduction and gene
regulation (Frohlich et al., 2014).
Current studies to identify the toxic factor or factors secreted by B
cells of patients with RRMS are underway, focusing primarily on
the > 300 kDa retentates of the Sup, employing proteomics and other
approaches. It is possible that the toxic factor/s is/are not proteins. It is
also possible that B cells from RRMS patients compared to those from
NC secrete less of one or more factors necessary for OL and neuronal
health in vitro.
5. Conclusions
RRMS B cells secrete factor/s in vitro toxic to neurons and OL
R.P. Lisak et al.
independent of Ig, not complement-mediated, and involving apoptosis.
The factor/s toxic to neurons and oligodendrocytes have an apparent
mw of > 300 kDa and modest neuronal toxicity may also be induced by
factor/s of < 300 kDa. These results provide significant new informa-
tion about a functional difference in B cells from multiple sclerosis
patients compared to controls, and elucidate a novel mechanism by
which B cells could contribute to disease.
Funding
Funding is provided by the National Multiple Sclerosis Society USA,
Grant #4929A2/3 (RPL, JAB, AB-O), the Research Foundation of the
MS Society of Canada, Grant #204209 (AB-O), the Parker Webber Chair
in Neurology Endowment (DMC Foundation/Wayne State University
School of Medicine; RPL) and DMC Foundation, Grant #2015-1242
(JAB). HT is supported by a doctoral studentship from the MS Society of
Canada. RL is supported by a Banque National Translational Research
Fellowship of the Montreal Neurological Institute.
Acknowledgments
We are grateful to all MS patients and controls who provided
samples and would like to thank the members of the Experimental
Therapeutics Program, including Dr. Boli Fan, as well as the Clinical
Research Unit (CRU) team, at the Montreal Neurological Institute for
their support in sample procurement and sample processing. We also
thank Dr. Paul Stemmer, Institute of Environmental Health Sciences,
Director of the Proteomics Core at Wayne State University for helpful
discussions.
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