Amelioration of brain pathology and behavioral dysfunction in mice with lupus following treatment with a tolerogenic peptide.

Abstract

Central nervous system (CNS) involvement in systemic lupus erythematosus (SLE) is manifested by neurologic deficits and psychiatric disorders. The aim of this study was to examine SLE-associated CNS pathology in lupus-prone (NZBxNZW)F1 (NZB/NZW) mice, and to evaluate the ameliorating effects of treatment with a tolerogenic peptide, hCDR1 (human first complementarity-determining region), on these manifestations.
Histopathologic analyses of brains from lupus-prone NZB/NZW mice treated with vehicle, hCDR1, or a control scrambled peptide were performed. The messenger RNA expression of SLE-associated cytokines and apoptosis-related molecules from the hippocampi was determined. Anxiety-like behavior was assessed by open-field tests and dark/light transfer tests, and memory deficit was assessed using a novel object recognition test.
Infiltration was evident in the hippocampi of the lupus-afflicted mice, and the presence of CD3+ T cells as well as IgG and complement C3 complex deposition was observed. Furthermore, elevated levels of gliosis and loss of neuronal nuclei immunoreactivity were also observed in the hippocampi of the mice with lupus. Treatment with hCDR1 ameliorated the histopathologic changes. Treatment with hCDR1 down-regulated the high expression of interleukin-1beta (IL-1beta), IL-6, IL-10, interferon-gamma, transforming growth factor beta, and the proapoptotic molecule caspase 8 in the hippocampi of the mice with lupus, and up-regulated expression of the antiapoptotic bcl-xL gene. Diseased mice exhibited increased anxiety-like behavior and memory deficit. Treatment with hCDR1 improved these parameters, as assessed by behavior tests.
Treatment with hCDR1 ameliorated CNS pathology and improved the tested cognitive and mood-related behavior of the mice with lupus. Thus, hCDR1 is a novel candidate for the treatment of CNS lupus.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 12, December 2009, pp 3744–3754
DOI 10.1002/art.25013
© 2009, American College of Rheumatology
Amelioration of Brain Pathology and Behavioral Dysfunction in
Mice With Lupus Following Treatment With
a Tolerogenic Peptide
Smadar Lapter, Anat Marom, Asher Meshorer, Anat Elmann, Amir Sharabi,
Ezra Vadai, Adi Neufeld, Yejezkel Sztainberg, Shoshana Gil, Dmitriy Getselter,
Alon Chen, and Edna Mozes
Objective. Central nervous system (CNS) involve-
ment in systemic lupus erythematosus (SLE) is mani-
fested by neurologic deficits and psychiatric disorders.
The aim of this study was to examine SLE-associated
CNS pathology in lupus-prone (NZB ? NZW)F1(NZB/
NZW) mice, and to evaluate the ameliorating effects of
treatment with a tolerogenic peptide, hCDR1 (human
first complementarity-determining region), on these
manifestations.
Methods. Histopathologic analyses of brains from
lupus-prone NZB/NZW mice treated with vehicle,
hCDR1, or a control scrambled peptide were performed.
The messenger RNA expression of SLE-associated cyto-
kines and apoptosis-related molecules from the hip-
pocampi was determined. Anxiety-like behavior was
assessed by open-field tests and dark/light transfer
tests, and memory deficit was assessed using a novel
object recognition test.
Results. Infiltration was evident in the hip-
pocampi of the lupus-afflicted mice, and the presence of
CD3? T cells as well as IgG and complement C3
complex deposition was observed. Furthermore, ele-
vated levels of gliosis and loss of neuronal nuclei
immunoreactivity were also observed in the hippocampi
of the mice with lupus. Treatment with hCDR1 amelio-
rated the histopathologic changes. Treatment with
hCDR1 down-regulated the high expression of
interleukin-1? (IL-1?), IL-6, IL-10, interferon-?, trans-
forming growth factor ?, and the proapoptotic molecule
caspase 8 in the hippocampi of the mice with lupus, and
up-regulated expression of the antiapoptotic bcl-xLgene.
Diseased mice exhibited increased anxiety-like behavior
and memory deficit. Treatment with hCDR1 improved
these parameters, as assessed by behavior tests.
Conclusion. Treatment with hCDR1 ameliorated
CNS pathology and improved the tested cognitive and
mood-related behavior of the mice with lupus. Thus,
hCDR1 is a novel candidate for the treatment of CNS
lupus.
Systemic lupus erythematosus (SLE) is an auto-
immune disease characterized by dysregulated T cell and
B cell immune responses that are associated with clinical
manifestations involving multiple organ systems (1). A
synthetic peptide, hCDR1 (human first complementarity-
determining region) (2), based on the CDR1 sequence
of an autoantibody (3), ameliorated lupus manifesta-
tions in models of both spontaneous and induced SLE
(4). Thus, hCDR1 down-regulated anti–double-stranded
DNA autoantibody levels, proteinuria, and the forma-
tion of immune complex deposits in the kidneys, result-
ing in better survival rates for the treated mice (4).
Treatment with hCDR1 reduced production of the
“pathogenic” cytokines (i.e., interleukin-1? [IL-1?], tu-
mor necrosis factor ? [TNF?], interferon-? [IFN?], and
IL-10), whereas expression of the immunosuppressive
cytokine transforming growth factor ? (TGF?) was
up-regulated (4). The mechanisms underlying the bene-
ficial effects of hCDR1 involve inhibition of T cell
receptor signaling following its binding to class II major
histocompatibility complex (5), the induction of
Smadar Lapter, PhD, Anat Marom, MSc, Asher Meshorer,
DVM, PhD, Anat Elmann, PhD, Amir Sharabi, MD, PhD, Ezra Vadai,
Adi Neufeld, MSc, Yejezkel Sztainberg, BSc, Shoshana Gil, BSc,
Dmitriy Getselter, BSc, Alon Chen, PhD, Edna Mozes, PhD: The
Weizmann Institute of Science, Rehovot, Israel.
Dr. Lapter and Ms Marom contributed equally to this work.
Address correspondence and reprint requests to Edna Mozes,
PhD, Department of Immunology, The Weizmann Institute of Science,
Rehovot, 76100 Israel. E-mail: edna.mozes@weizmann.ac.il.
Submitted for publication August 5, 2008; accepted in revised
form August 31, 2009.
3744

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CD4?CD25? Treg cells (6), and the reduction of
apoptosis rates (7,8).
Neuropsychiatric (NP) syndrome is one of the
manifestations reported in patients with SLE (9,10).
Active kidney disease and central nervous system (CNS)
involvement are the most frequently observed causes of
death in patients with SLE (11). Elevated levels of
brain-reactive antibodies were detected in the serum or
cerebrospinal fluid (CSF) of patients with NPSLE
(12,13). Double-stranded DNA–specific antibodies that
cross-react with NR2, a subunit of the N-methyl-D-
aspartate receptor on neuronal cells, were shown to
cause neuronal death in vivo and in vitro (14). Elevated
levels of NR2-specific autoantibodies were also observed
in the sera of patients with SLE (15) and were reported
to cause neuronal damage and memory deficit via access
to the CNS (16). Hippocampal atrophy and biochemical
signs of neuronal and astrocytic damage were reported
in SLE patients with CNS involvement (17,18).
(NZB ? NZW)F1(NZB/NZW) mice in which
lupus develops spontaneously (19) exhibit CNS involve-
ment manifested by the presence of antineuronal anti-
bodies in the brain (20) and changes in cognitive func-
tion (21). Neuropathology in mice with lupus involves
disruption of the blood–brain barrier, consequently al-
lowing infiltration of large molecules and immune cells,
which is normally restricted (22). The latter is accompa-
nied by deposition of immune complexes, complement
activation (23), and induction of proinflammatory cyto-
kines (24) that have been implicated in the pathogenesis
of SLE. Behavioral changes were reported in lupus
models and were found to correlate with hippocampus
aberrations (25–28).
In the present study, we investigated CNS mani-
festations in NZB/NZW mice with lupus and their
association with behavior deficits. Furthermore, we as-
sessed the effects of treatment with the tolerogenic
peptide, hCDR1, on these manifestations. We observed
cell infiltration, immune complex deposits, gliosis, loss of
neuronal nuclei immunoreactivity, and an altered cyto-
kine profile in the hippocampi of mice with lupus. These
manifestations were accompanied by behavioral abnor-
malities. Treatment with hCDR1 ameliorated the CNS
manifestations and improved the behavioral perfor-
mance of the mice with lupus.
MATERIALS AND METHODS
Mice. Female NZB/NZW mice were obtained from
The Jackson Laboratory (Bar Harbor, ME). Mice were han-
dled according to protocols approved by the Weizmann Insti-
tute Animal Care and Use Committee, using international
guidelines.
Peptides and treatment. The synthetic peptide hCDR1
(2), which has a sequence (GYYWSWIRQPPGKGEEWIG)
based on the CDR1 of the human monoclonal autoantibody
(3), was synthesized at Polypeptide Laboratories (Torrance,
CA). A peptide containing the same amino acids as hCDR1,
with a scrambled order, designated scrambled peptide (SKGI-
PQYGGWPWEGWRYEI), was used as a control. Captisol
(sulfobutylether ?-cyclodextrin; CyDex, Lenexa, KS) was used
as a vehicle. Eight-month-old NZB/NZW mice were injected
subcutaneously with hCDR1, scrambled peptide (both at a
dose of 50 ?g per mouse), or vehicle alone, once weekly for 10
weeks.
Brain histology. The brain hemispheres of the mice
were fixed with 4% paraformaldehyde. Serial sagittal sections
from the lateral 1.08–1.68-mm area were prepared. Frozen
cryostat (20 ?m) or paraffin-embedded (4 ?m) sections were
used. All treatment experiments consisted of 6–8 mice/group;
2 sections from each mouse were analyzed. All regions within
the sections were examined. All data presented are based on
3–5 independent experiments. Histopathology was evaluated
by 2 examiners who were blinded to the treatment groups.
Hematoxylin and eosin (H&E ) staining. Sections were
stained with Mayer’s hematoxylin solution (Finkelman, Yehud,
Israel). Slides were analyzed using a light microscope (Nikon
Eclipse E800). The infiltration index, determined by H&E
staining, was graded as follows: 0 ? no infiltration, 1 ? low
level of infiltration, 2 ? moderate level of infiltration, and 3 ?
high level of infiltration.
Staining for CD3? T cells. Sections were incubated
with a rat anti-mouse CD3? antibody (Serotec, Raleigh, NC)
and then incubated with biotin-conjugated goat anti-rat IgG
(Chemicon, Temecula, CA) followed by incubation with Cy3-
conjugated streptavidin (Jackson ImmunoResearch, West
Grove, PA) and Hoechst stain (added in all immunofluores-
cence procedures) (Molecular Probes, Eugene, OR). Slides
were mounted in Aqua-Poly/Mount (Polysciences, War-
rington, PA) and analyzed with a fluorescence microscope
(Nikon Eclipse E800), using Nikon ACT-1 software.
IgG and complement C3 immune complex deposits.
Sections were incubated with fluorescein isothiocyanate
(FITC)–conjugated goat anti-mouse IgG (Jackson Immu-
noResearch) or with complement C3 FITC-conjugated goat
anti-mouse IgG (ICN Pharmaceuticals, Costa Mesa, CA). The
stained sections were graded as follows: 0 ? no deposits, 1 ?
minimal level of deposits, 2 ? moderate level of deposits, and
3 ? high level of deposits.
Neuronal nuclei and glial fibrillary acidic protein
(GFAP) staining. Sections were incubated with mouse antineu-
ronal nuclei (Chemicon) or with rabbit anti-GFAP antibodies
(Dako, Glostrup, Denmark). The secondary antibodies used
were Cy3-conjugated goat anti-rabbit antibodies (Jackson Im-
munoResearch) for GFAP or biotin-conjugated anti-mouse
IgG (Vector, Burlingame, CA) for neuronal nuclei. FITC-
conjugated streptavidin (Jackson ImmunoResearch) was
added thereafter. A semiquantitative gliosis score based on
GFAP staining was graded as follows: 0 ? no gliosis, 1 ? low
level of gliosis (up to 10% of the area evaluated), 2 ?
moderate level of gliosis (up to 50% of the area evaluated),
and 3 ? high level of gliosis (?50% of the evaluated area). A
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semiquantitative score for neuronal nuclei loss was graded as
follows: 0 ? no loss, 1 ? minimal loss (up to 30% of the brain
sections), 2 ? moderate loss (30–90% of the brain sections),
and 3 ? massive loss of neuronal nuclei staining (?90% of the
brain sections). In addition, identical fields from the same area
of the brain sections were photographed and computed using
Image-Pro Plus software (Media Cybernetics, Silver Spring,
MD). Ten different square areas (600 ?m2each) were mea-
sured in the dentate gyrus region of each section. The intensity
threshold was set to exclude nonspecific background fluores-
cence and was applied to all sections analyzed. The integrated
optical density (IOD) was determined using the following
equation: average intensity ? area.
Electron microscopy. The mouse brain hemispheres
were fixed with 3% paraformaldehyde and 2.5% glutaralde-
hyde. Ultrathin sections (70 nm) were prepared, analyzed
under 120 kV using a Tecnai 12 transmission electron micro-
scope (FEI, Hillsboro, OR), and digitized with a MegaView III
CCD camera (Olympus, Lake Success, NY) using AnalySIS
software (Nikon, Dusseldorf, Germany). Electron microscopy
was performed at the Irving and Cherna Moskowitz Center
for Nano and Bio-Nano Imaging at the Weizmann Institute of
Science.
Flow cytometry. Splenocytes (1 ? 106) were incubated
with anti-CD4–specific antibodies (Southern Biotechnology,
Birmingham, AL), anti-TGF?–specific antibodies (IQ Prod-
ucts, Groningen, The Netherlands), or anti-IFN?–specific anti-
bodies (eBioscience, San Diego, CA) and analyzed by flow
cytometry. For intracellular staining, cells were fixed and
permeabilized.
Real-time reverse transcription–polymerase chain re-
action (RT-PCR). Brain hippocampi were isolated from mice
in the different groups. Total RNA was prepared using TriRe-
agent (Molecular Research Center, Cincinnati, OH). Comple-
mentary DNA was prepared, and real-time RT-PCR was
performed using the LightCycler system (Roche, Basel, Swit-
zerland), according to the manufacturer’s instructions. The
following primer sequences were used (forward and reverse,
respectively): for IL-1?, 5?-GAGAACCAAGCAACGAC-3?
and 5?-GTGCTGATGTACCAGT-3?; for IL-6, 5?-CCAC-
GGCCTTCCCTAC-3? and 5?-AAGTGCATCATCGTTGT-
3?; for IFN?, 5?-GAACGCTACACACTGC-3? and 5?-CTGG-
ACCTGTGGGTTG-3?; for IL-10, 5?-ACCTCGTTTG-
TACCTCT-3? and 5?-CACCATAGCAAAGGGC-3?; for
TGF?, 5?-AGCGGACTACTATGCTAAAG-3? and 5?-
GTAACGCCAGGAATTGT-3?; for Bcl-xL, 5?-GGACC-
GCGTATCAGAGC-3? and 5?-GCATTGTTCCCGTACCC-
3?; for caspase 8, 5?-ACATAACCCAACTCCGAA-3? and
5?-GTGGGATAGGATACAGCAGA-3?; for GFAP, 5?-
AGCTAACGACTATCGCC-3? and 5?-GGCCTTCTGA-
CACGGA-3?; for ?-actin, 5?-GTGACGTTGACATCCG-3?
and 5?-CAGTAACAGTCCGCCT-3?. Levels of ?-actin were
used for normalizing the expression levels of other genes.
Results are presented relative to the levels in the vehicle-
treated group (considered as 100%).
Western blot analysis. Lysates extracted from the
hippocampi were separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis, as described previously (8).
The membranes were incubated with antineuronal nuclei
(Chemicon), anti–caspase 8 (Alexis Biochemicals, San Diego,
CA), anti–Bcl-xL(Santa Cruz Biotechnology, Santa Cruz, CA),
and antitubulin antibodies (Sigma-Aldrich, Poole, UK). Mem-
branes were incubated with the second antibody coupled to
horseradish peroxidase. Detection was performed using the
enhanced chemiluminescence method. Protein expression was
determined by photodensitometry, using the Image program
(National Institutes of Health, Bethesda, MD).
Behavioral analyses. Behavioral analyses were per-
formed at the end of treatment, before the mice were killed.
One week before the behavior analyses, mice were placed in a
system space with 12 hours of dark during daytime and 12
hours of light during the night. During experiment days, the
mice were brought to the testing room for 1 hour of habitua-
tion before being subjected to the tests. Testing was conducted
during the dark phase of the light/dark cycle.
Open-field test. The open-field apparatus consisted of
a white plexiglass box (50 ? 50 ? 22 cm) with 16 squares
painted on the floor. A lamp provided 120-lux illumination of
the floor. Mice were placed individually in one corner of the
open field for a 10-minute test session. Inner squares were
defined as the center; the time spent in the center, the number
of entrances to the center, and the total distance traveled were
quantified manually or using a video tracking system (Video-
Mot2; TSE Systems, Bad Hamburg, Germany). The results
presented were obtained from the video tracking system.
Light/dark transfer test. The light/dark transfer test
was conducted in a square white plexiglass arena (50 ? 50 ? 22
cm) separated into 2 compartments. The “light compartment”
was constructed of white plexiglass illuminated with 120 lux,
and the “dark compartment” was made of black plexiglass and
covered with black-topped plexiglass. Individual mice were
placed into the dark compartment and allowed to explore the
apparatus for 5 minutes. The transition between the light
compartment and the dark compartment and the time spent in
the illuminated side were measured by a video tracking system.
Novel object recognition test. The novel object recog-
nition test was performed in the open-field apparatus (50 ?
50 ? 22 cm). Two days before the test, each mouse was
subjected to a habituation session in the open field and
explored the area without the presence of objects for 10
minutes. One day before the test, each mouse explored the
area in the presence of 2 identical objects. On the experiment
day, a novel (N) object replaced 1 of the 2 identical familiar (F)
objects presented the previous day. Mice were placed in the
field for 5 minutes, and the total time spent in exploration of
each object was determined. The recognition index (RI) was
determined as follows: RI ? (N – F)/(N ? F) ? 100. Two
unbiased observers recorded the data.
Statistical analysis. Statistical analysis was performed
using the Mann-Whitney U test, the Kruskal-Wallis nonpara-
metric test, Student’s t-test, and Spearman’s rank correlation
test. P values less than 0.05 were considered significant.
RESULTS
Down-regulation of cell infiltration and immune
complex deposition in the brains of mice with lupus
following treatment with hCDR1. To investigate cell
infiltration in the brains of NZB/NZW mice with lupus,
the brains were extracted from mice that had been
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treated for 10 weeks with vehicle, hCDR1, or the control
scrambled peptide (see Materials and Methods), stained
with H&E, and analyzed for cell infiltration. Figure 1A
shows representative images of brain sections from mice
in the different groups. A prominent reduction in brain
infiltration can be seen in the section from an hCDR1-
treated mouse as compared with brain sections from
vehicle-treated and scrambled peptide–treated mice
(Figure 1A, parts a–c). Electron microscopy analysis
revealed immune-cell infiltration of mononuclear cells
(Figure 1A, part a, lower left inset). Figure 1A part e
shows the results of analyses of infiltration in all brains
tested. A significant decrease (P ? 0.0001) in brain
infiltration in hCDR1-treated mice in comparison with
Figure 1. Treatment with hCDR1 (human first complementarity-determining re-
gion) down-regulates cell infiltration in the brains of (NZB ? NZW)F1mice with
lupus. A, Representative hematoxylin and eosin–stained brain sections from a,
vehicle-treated mice, b, hCDR1-treated mice, c, scrambled peptide–treated mice,
and d, young control mice. Arrowheads denote the infiltrated zones, which are shown
at higher magnification (40?) in the insets at the top right in a and c and the lower
right in b. Inset at the lower left in a shows an electron microscope image of a single
infiltrating cell. Bar ? 200 ?m. e, Infiltration index for vehicle-treated mice (n ? 29),
hCDR1-treated mice (n ? 31), and scrambled peptide–treated mice (n ? 17). B,
Staining for CD3? T cells in mice with the same treatments described in A.
Arrowheads in a and c indicate the regions that are shown at higher magnification
(40?) in the lower left insets. Insets in the lower right show nuclear staining. Bar ?
200 ?m. e, Numbers of CD3? cells in the dentate gyrus regions for vehicle-treated
mice (n ? 3), hCDR1-treated mice (n ? 4), and scrambled peptide–treated mice
(n ? 3). Bars show the mean and SEM. ? ? P ? 0.0001; ?? ? P ? 0.03, versus
scrambled peptide.
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the vehicle-treated and scrambled peptide–treated mice
was observed. Young, disease-free mice were excluded
from the analysis, because no infiltration was observed
in their brains (Figure 1A, part d).
In order to determine whether T cells were
present in the brain infiltrates, immunohistochemical
analysis, using an anti-CD3 antibody, was performed.
Positively stained cells were counted (Figure 1B, part e).
Figure 1B, parts a and c, show positively stained cells
that were identified in the dentate gyrus area of the
experimental mice that were treated with either vehicle
or the scrambled peptide. The brains of hCDR1-treated
mice were almost free of CD3? T cells (Figure 1B, part
b), as were the brains of the young control mice (Figure
1B, part d). The numbers of CD3? cells in the hip-
pocampi of the NZB/NZW mice, as shown in Figure 1B,
part e, confirm the significant reduction in the number
of CD3? cells following treatment with hCDR1.
Figure 2 shows IgG and complement C3 complex
deposits in representative brain sections from NZB/
NZW mice in the different groups. Positive staining for
IgG (Figure 2, parts a and c) and complement C3
complex deposits (Figure 2, parts e and g) can be seen in
the brains of the vehicle-treated and scrambled peptide–
treated mice, respectively. Treatment with hCDR1 re-
duced IgG (Figure 2, part b) and complement C3
(Figure 2, part f) complex deposition to levels shown for
the young, disease-free mice (Figure 2, parts d and h).
Scores for the levels of IgG and complement C3 complex
deposits (Figure 2, parts i and j, respectively) in brain
sections from all experimental mice indicated a signifi-
cant reduction in immune complex deposition following
treatment with hCDR1.
Reduced gliosis and protection against neuronal
nuclei immunoreactivity loss in the brains of mice with
lupus following treatment with hCDR1. The inflamma-
tory events in the brains of NZB/NZW mice might affect
the status of glial cells and neurons. We therefore
investigated expression of the astrocytic marker GFAP
in the brains of mice with lupus in comparison with that
in the brains of mice treated with hCDR1. Figure 3A,
parts a, c, e, and g, and parts b, d, f, and h, show GFAP
staining and the matching nuclear images, respectively.
Representative brain sections from young control mice
served as a baseline for normal GFAP distribution.
Increased expression of GFAP was observed in the
brains of vehicle-treated and scrambled peptide–treated
mice (Figure 3A, parts a and e, respectively). Treatment
with hCDR1 resulted in reduced GFAP expression
(Figure 3A, part c) to levels similar to those seen in the
young mice (Figure 3A, part g). Reduced gliosis in the
hippocampi following hCDR1 treatment is shown as a
reduction in the mean IOD (Figure 3A, part i), the mean
semiquantitative gliosis score (Figure 3A, part j), and the
Figure 2. Treatment with hCDR1 (human first complementarity-
determining region) down-regulates IgG and complement C3 immune
complex deposition in the brains of lupus-afflicted (NZB ? NZW)F1
mice. a–h, Representative IgG (a–d) and complement C3 (e–h)
immune complex deposits in brain sections from vehicle-treated mice
(a and e), hCDR1-treated mice (b and f), scrambled peptide–treated
mice (c and g), and young control mice (d and h). The corresponding
insets show nuclear (Hoechst) staining. Bar ? 200 ?m. i and j, levels
of IgG and complement C3 deposits, respectively, in the brains from
6–7 mice in each group. Bars show the mean and SEM. ? ? P ? 0.004;
?? ? P ? 0.03, versus scrambled peptide.
3748LAPTER ET AL

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mean GFAP gene expression, as measured by real-time
RT-PCR (Figure 3B).
To determine the effect of lupus on the status of
neurons in the brains of NZB/NZW mice, we stained
brain sections with an antibody to neuronal nuclei and
analyzed them for neuronal nuclei immunoreactivity.
Figure 3C shows representative brain sections stained
with neuronal nuclei–specific antibodies (parts a, c, e,
and g) and nuclear staining (parts b, d, f, and h). A loss
or decrease in neuronal nuclei immunoreactivity was
observed in the brains of vehicle-treated and scrambled
peptide–treated mice (Figure 3C, parts a and e, respec-
tively), as compared with the brain of a young mouse
(Figure 3C, part g). The loss of neuronal nuclei immu-
noreactivity was observed mainly in the hippocampus.
Treatment with hCDR1 restored the immunoreactivity
to neuronal nuclei (Figure 3C, part c). Double staining
for neuronal nuclei and Hoechst staining (Figure 3C,
parts a and b and parts e and f, respectively) revealed
that the nuclei of the neuronal cells in the pyramidal cell
layer and in the dentate gyrus were intact, although their
hippocampi were negative for neuronal nuclei staining.
Figure 3C, parts i and j, which present the mean IOD of
neuronal nuclei staining and mean semiquantitative
score for neuronal nuclei loss, respectively, confirm the
significant differences between mice treated with
hCDR1 and the control group.
To determine whether the reduction in neuronal
nuclei immunoreactivity was attributable to the loss of
neuronal nuclei protein, we analyzed neuronal nuclei
expression by Western blotting. Figure 3D shows West-
ern blots for neuronal nuclei in brain hippocampi of the
different groups of mice. Although a moderate reduc-
tion can be seen in neuronal nuclei protein levels in
hippocampi from vehicle-treated and scrambled
peptide–treated mice, this reduction cannot explain the
significant decrease in neuronal nuclei staining in the
brains of mice from these groups.
Immunomodulation of proinflammatory cyto-
kines and apoptosis-related molecules in the brains of
NZB/NZW mice treated with hCDR1. Because cytokines
play an important role in SLE, and because hCDR1 was
shown to immunomodulate the cytokine profile (4), it
was of interest to determine the status of the relevant
cytokines in the brains of mice with lupus following
treatment with hCDR1. Because most of the morpho-
logic alterations in the brains of NZB/NZW mice with
lupus (Figures 1–3) were mapped to the hippocampus,
we extracted RNA from the hippocampi of mice from
the different groups. Gene expression was determined
by real-time RT-PCR. As shown in Figure 4A, the
mRNA levels of IL-1?, IL-6, IFN?, IL-10, and TGF?
were elevated in the hippocampi of vehicle-treated and
Figure 3. Treatment with hCDR1 (human first complementarity-
determining region) diminishes gliosis and protects against loss of
neuronal nuclei (NeuN) immunoreactivity. A, Glial fibrillary acidic
protein (GFAP) staining in brain sections from a, vehicle-treated mice
(n ? 22), c, hCDR1-treated mice (n ? 21), e, scrambled peptide–
treated mice (n ? 11), and g, young control mice (n ? 14). Nuclear
staining of brain sections from these mice is shown in b, d, f, and h,
respectively. Insets show higher-magnification views (40?) of the
boxed areas. Bar ? 200 ?m. i and j, Integrated optical density (IOD)
of GFAP staining and semiquantitative gliosis scores, respectively. B,
GFAP mRNA expression. C, Neuronal nuclei staining of brain sec-
tions, as described in A. i and j, Integrated optical density of neuronal
nuclei staining and semiquantitative neuronal nuclei loss score, respec-
tively. D, Western blot for neuronal nuclei, with results expressed
quantitatively as densitometric units, indicating the neuronal nuclei–
to-tubulin ratio. Bars show the mean and SEM. ? ? P ? 0.0001; ?? ?
P ? 0.005; ??? ? P ? 0.05, versus scrambled peptide.
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scrambled peptide–treated mice compared with the
young control mice. Treatment with hCDR1 reduced
the levels of these cytokines to those determined for the
young control mice.
We sought to determine whether the down-
regulation of all of the above-mentioned cytokines
tested in the hippocampi of hCDR1-treated mice was
attributable to a decrease in the number of immune cells
or was a result of cytokine immunomodulation by
hCDR1. No significant differences were observed be-
tween the total numbers of spleen-derived CD4? cells
of vehicle-treated mice (mean ? SEM 27 ? 106? 0.2)
and hCDR1-treated mice (28 ? 106? 0.8). However, as
shown in Figure 4B, analysis of intracellular cytokine
expression in spleen-derived CD4? cells indicated that
treatment with hCDR1 resulted in a significant increase
in the expression of TGF?, whereas the levels of IFN?
were reduced as compared with those in vehicle-treated
mice.
It has been established that apoptosis plays a role
in the pathogenesis of SLE (29). We therefore measured
the mRNA expression of the proapoptotic molecule
caspase 8, and that of the antiapoptotic molecule Bcl-xL,
in the hippocampi of the experimental mice (Figure 4C).
Protein levels of the apoptotic-related molecules were
also measured (Figure 4D). The mRNA (Figure 4C) and
protein (Figure 4D) levels of caspase 8 were up-
regulated in vehicle-treated and scrambled peptide–
treated mice as compared with the healthy controls,
whereas treatment with hCDR1 diminished the expres-
sion of caspase 8 to levels similar to those observed in
the young mice. In contrast, the gene expression (Figure
4C) and protein (Figure 4D) levels of Bcl-xLwere
up-regulated in hCDR1-treated mice compared with
vehicle-treated and scrambled peptide–treated mice.
Improvement in the behavior parameters of
NZB/NZW mice with lupus following hCDR1 treatment.
We were interested in investigating whether the his-
topathologic findings in the hippocampi of the diseased
mice were associated with impaired behavior, and
whether treatment with hCDR1 ameliorated such be-
havior. To this end, after 10 weeks of treatment, female
NZB/NZW mice were subjected to behavioral studies.
First, in the open-field test, we monitored NZB/NZW
mice that were treated with vehicle, hCDR1, or the
scrambled peptide. The mean ? SEM total distances
traveled were determined to be 1,313 ? 199 cm, 1,681 ?
316 cm, and 1,521 ? 216 cm for vehicle-treated, hCDR1-
treated, and scrambled peptide–treated mice, respec-
tively, suggesting no significant differences in the loco-
motion of the different groups. Young, disease-free mice
were excluded, because their traveled distances were
extremely different (data not shown).
Figure 5A shows that the mean percentage of
time spent in the center (part a) and the mean number
of entrances to the center (part b) in the open-field test
were significantly higher among the hCDR1-treated
mice (mean ? SEM 4.78 ? 1.50% and 7.07 ? 1.46,
respectively) compared with the vehicle-treated mice
(1.90 ? 0.51% and 3.44 ? 0.63, respectively) and the
scrambled peptide–treated mice (0.69 ? 0.27% and
2.47 ? 0.69, respectively). Representative spontaneous
performances in the open-field test by individual mice
Figure 4. Treatment with hCDR1 (human first-complementarity-
determining region) immunomodulates cytokines and apoptosis-
signaling molecules in hippocampi of (NZB ? NZW)F1mice with
lupus. A, Messenger RNA expression of cytokine genes, as determined
by real-time reverse transcription–polymerase chain reaction. B, Cy-
tokine expression in spleen-derived CD4? cells. C and D, Messenger
RNA expression and Western blot analysis, respectively, of the
apoptosis-related molecules caspase 8 and Bcl-xL. Lysates were ex-
tracted from the hippocampi of mice from the different groups. A
tubulin-specific antibody was used to determine the total protein
loaded. Densitometric units indicate the ratio of caspase 8 and Bcl-xL
to tubulin. Bars show the mean and SEM of 6–8 mice per group.
IL-1? ? interleukin-1?; IFN? ? interferon-?; TGF? ? transforming
growth factor ?. ? ? P ? 0.05 versus vehicle.
3750LAPTER ET AL

Page 8

from the different groups are shown in Figure 5A, parts
c–e. Furthermore, as shown in Figure 5B, an inverse
correlation was observed between the mean time spent
in the center during the open-field test (Figure 5B, part
a) and the severity of infiltration in the hippocampi, as
shown by the infiltration index (Figure 5B, part b) (r ?
?0.48, P ? 0.006). Representative images of the hip-
pocampi of individual mice from the different groups
and the performance of these mice in the open-field test
are shown in Figure 5C.
An additional test to assess anxiety-like behavior,
namely, the light/dark transfer test, was performed. As
can be seen in Figure 5D, hCDR1-treated mice spent
more time in the light compartment of the box (mean ?
SEM 4.41 ? 1.46% of the time) in comparison with
vehicle-treated mice (1.87 ? 0.58%) and scrambled
peptide–treated mice (1.65 ? 0.57%). Learning and
memory performances of the experimental mice were
assessed by the novel object recognition test. Figure 5E
shows a significant reduction in the recognition index of
vehicle-treated mice (mean ? SEM ?27.55 ? 8.55) and
scrambled peptide–treated mice (?1.45 ? 11.43) in
comparison with hCDR1-treated mice (30.07 ? 8.76).
DISCUSSION
The main findings of the present study are that
treatment with hCDR1 significantly ameliorated the
brain pathology and behavioral abnormalities that de-
veloped in NZB/NZW mice with lupus. To the best of
our knowledge, this is the first study showing significant
beneficial effects on CNS manifestations in mice with
lupus following treatment with a tolerogenic peptide.
Cell infiltration, including that of CD3? T cells,
was observed in the brains of the NZB/NZW mice with
lupus, mainly in the hippocampi (Figure 1). Indeed,
histopathologic findings in the brain that involved
perivascular lymphocytic infiltrates were reported in
patients with SLE (30) as well as in lupus-prone mice
(23,31). Cell infiltration into the CNS could be attribut-
able to the observed increased expression of adhesion
molecules such as intercellular adhesion molecule 1 and
vascular cell adhesion molecule 1 (32). In agreement
with those observations, treatment with hCDR1 down-
regulated elevated levels of vascular cell adhesion mol-
ecule 1 in the hippocampus (Lapter S, et al: unpublished
observations).
IgG deposition was reported in the choroid
plexus of patients with SLE (33) and in the brains of
MRL/lpr mice (23). Furthermore, elevated levels of
complement C3 and C4 were observed in the CSF of
patients with CNS lupus (34). Similarly, we demon-
strated increased IgG and complement C3 deposition in
the brains of NZB/NZW mice with lupus (Figure 2).
Treatment with hCDR1 significantly down-regulated the
deposition of both IgG and complement C3 immune
Figure 5. Treatment with hCDR1 (human first complementarity-
determining region) affects anxiety-like behavior and memory deficit.
A, Open-field test, showing spontaneous locomotion of mice treated
with c, vehicle, d, hCDR1, and e, scrambled peptide. B, Inverse
correlation between open-field test performance and the infiltration
index. C, Representative brain sections from a, vehicle-treated mice, b,
hCDR1-treated mice, and c, scrambled peptide–treated mice. Arrows
indicate cell infiltration. Bar ? 250 ?m. D, Light/dark transfer test,
showing the time spent in light. E, Novel object recognition test,
showing the recognition index. Bars show the mean and SEM. All tests
involved 20 vehicle-treated mice, 20 hCDR1-treated mice, and 17
scrambled peptide–treated mice. ? ? P ? 0.02; ?? ? P ? 0.002; ??? ?
P ? 0.05; # ? P ? 0.06; ## ? P ? 0.0001, versus scrambled peptide.
TREATMENT WITH hCDR1 FOR CNS LUPUS IN NZB/NZW MICE3751

Page 9

complex in the brains of the treated mice, as previously
demonstrated in the kidneys of diseased mice (4).
Upon their activation, astrocytes express elevated
levels of GFAP, which is considered to be a marker for
astrogliosis (35). Indeed, activated astrocytes and micro-
glia were observed in the substantia nigra of lupus-prone
NZM88 mice (36), and augmented levels of GFAP were
detected in the CSF of patients with CNS lupus (17). In
the present study, high levels of gliosis were observed in
the hippocampi of the NZB/NZW mice with lupus, and
treatment with hCDR1 significantly down-regulated the
gliosis to levels similar to those observed in young
control mice (Figure 3).
Upon their activation, astrocytes produce cyto-
kines such as IL-6 (37). In accordance, in the present
study, increased expression of the pathogenic cytokines
IL-1?, IL-6, IL-10, and IFN? was observed in the
hippocampi of the diseased mice. Treatment with
hCDR1 reduced the gene expression of those cytokines
to the levels observed in young healthy mice (Figure
4A). Similar to our findings in NZB/NZW mice, IL-1?
and IL-6 were shown to be up-regulated in the hip-
pocampi of lupus-prone MRL/lpr mice (24) and in the
CSF of SLE patients with neuropsychiatric involvement
(38).
We previously reported that, whereas treatment
with hCDR1 resulted in diminished secretion and ex-
pression of the pathogenic cytokines INF?, IL-1?,
TNF?, and IL-10 in the spleens and lymph node cells of
mice with lupus, expression of the immunosuppressive
cytokine TGF? was up-regulated by treatment (4). In
the present study, elevated expression of TGF? was
observed in the hippocampi of the diseased mice (Figure
4A). Similarly, high levels of TGF? were detected in the
CSF of patients with neuropsychiatric SLE (39). Treat-
ment with hCDR1 significantly down-regulated the ex-
pression of this cytokine in the brains of the treated
mice. It is noteworthy that expression of TGF? protein
and mRNA was elevated in other target organs, such as
the kidneys, of the mice with lupus (40). Enhanced
expression of TGF? in target organs may lead to dys-
regulated tissue repair, progressive fibrogenesis, and
eventually end-organ damage (40).
A significant observation was the loss of immu-
noreactivity for neuronal nuclei in the hippocampi of the
mice with lupus and its restoration following treatment
with hCDR1 (Figure 3C). It is noteworthy that nuclear
staining (Hoechst) in negative neuronal nuclei neurons
indicated that their nuclei were intact. In addition, no
significant differences in the neuronal nuclei protein
content were detected in the hippocampi of the different
treatment groups (Figure 3D). The neuronal nuclei
antigen was used as a marker to identify neuronal cell
loss under various pathologic conditions (41,42), and a
decrease in neuronal nuclei immunoreactivity was sug-
gested to be predictive of delayed neuronal degenera-
tion (42). To the best of our knowledge, this is the first
study showing the loss of neuronal nuclei immunoreac-
tivity in the brains of mice with lupus.
Apoptosis has been shown to play a role in
various autoimmune diseases, including SLE (43). In-
creased expression of mRNA and protein of the pro-
apoptotic molecule caspase 8 and diminished expression
of the antiapoptotic molecule Bcl-xLwere observed in
the hippocampi of the diseased mice. Up-regulated
expression of caspase 3 in the brains of MRL/lpr mice
suggested a caspase-dependent mechanism for apoptosis
in these brains (44). Treatment with hCDR1 down-
regulated the levels of caspase 8 and up-regulated
expression of Bcl-xL. In addition, amelioration of the
lupus manifestations in the NZB/NZW mice following
treatment with hCDR1 was associated with diminished
apoptosis, which was manifested by a decrease in
caspase 8 expression and an increase in Bcl-xLexpres-
sion (7,8,45).
SLE-induced hippocampal damage was reported
to lead subsequently to behavioral dysfunction (46). In
addition, structural hippocampal atrophy, as demon-
strated by magnetic resonance imaging, was associated
with the presence of cognitive dysfunction, especially
memory, in patients with lupus (18). As in the case of
patients with SLE, a link between neurodegeneration,
behavioral dysfunction, and memory impairment was
observed in lupus-prone mice (21,46). In the present
study, we observed increased anxiety-like behavior and
memory impairment in the mice with lupus. Treatment
with hCDR1 significantly improved the performance of
the mice in all behavior tests in association with amelio-
ration of the histopathologic findings in their brains
(Figure 5).
Thus, treatment with hCDR1 led to significant
amelioration of brain pathology in lupus-prone NZB/
NZW mice, including improvement in the cognitive
behavior of the treated mice. The beneficial effects of
hCDR1 were specific, because treatment with a control
scrambled peptide did not affect the diseased mice.
Therefore, hCDR1 is a potential candidate for the
specific treatment of CNS lupus.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
3752 LAPTER ET AL

Page 10

the final version to be published. Dr. Mozes had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Lapter, Marom, Meshorer, Elmann,
Sharabi, Vadai, Neufeld, Sztainberg, Gil, Getselter, Chen, Mozes.
Acquisition of data. Lapter, Marom, Meshorer, Elmann, Sharabi,
Vadai, Neufeld, Sztainberg, Gil, Getselter, Chen, Mozes.
Analysis and interpretation of data. Lapter, Marom, Meshorer, El-
mann, Sharabi, Vadai, Neufeld, Sztainberg, Gil, Getselter, Chen,
Mozes.
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