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Interferon-╨Ю╤Цdependent inhibition of B cell activation by bone marrowderived mesenchymal stem cells in a murine model of systemic lupus erythematosus.

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ARTHRITIS & RHEUMATISM
Vol. 62, No. 9, September 2010, pp 2776–2786
DOI 10.1002/art.27560
© 2010, American College of Rheumatology
Interferon-␥–Dependent Inhibition of B Cell Activation by
Bone Marrow–Derived Mesenchymal Stem Cells in a Murine
Model of Systemic Lupus Erythematosus
Francesca Schena,1 Claudio Gambini,1 Andrea Gregorio,1 Manuela Mosconi,1
Daniele Reverberi,2 Marco Gattorno,1 Simona Casazza,3 Antonio Uccelli,4 Lorenzo Moretta,5
Alberto Martini,6 and Elisabetta Traggiai1
Objective. Bone marrow–derived mesenchymal
stem cells (BM-MSCs) are multipotent cells characterized by immunomodulatory properties and are therefore
considered a promising tool for the treatment of
immune-mediated diseases. This study was undertaken
to assess the influence of murine BM-MSCs on the
activation of B cells in (NZB ⴛ NZW)F1 mice as an
animal model of systemic lupus erythematosus (SLE).
Methods. We evaluated the in vitro effects of
BM-MSCs on the proliferation and differentiation to
plasma cells of splenic mature B cell subsets, namely
follicular and marginal zone B cells isolated from
(NZB ⴛ NZW)F1 mice. Lupus mice were also treated
with BM-MSCs, and serum autoantibodies, proteinuria,
histologic changes in the kidney, and survival rates were
monitored.
Results. BM-MSCs inhibited antigen-dependent
proliferation and differentiation to plasma cells of follicular and marginal zone B cells in vitro. This inhibitory effect was dependent on interferon-␥ (IFN␥) and
was mediated by cell-to-cell contact, involving the programmed death 1 (PD-1)/PD ligand pathway. In vivo
treatment with BM-MSCs did not affect the levels of
anti–double-stranded DNA antibodies or proteinuria.
However, a reduction in glomerular immune complex
deposition, lymphocytic infiltration, and glomerular
proliferation was observed.
Conclusion. Our findings indicate that BM-MSCs
affect B cell receptor–dependent activation of both
follicular and marginal zone B cells from lupus mice.
This inhibitory effect is IFN␥-dependent and cell
contact–dependent. MSCs in vivo do not affect the
production of autoantibodies, the level of proteinuria, or
the mortality rates. Nonetheless, the significant improvement in histologic findings in the kidney supports
the potential role of MSCs in the prevention of glomerular damage.
Supported by grants from the Associazione Italiana per la
Ricerca sul Cancro, the Istituto Superiore di Sanità, and the Ministero
della Salute (Ricerca Finalizzata Ministeriale 2005 “Caratterizzazione
delle proprietà di immunomodulazione delle cellule mesenchimali e
possibile applicazione nel trattamento delle malattie autoimmuni”).
Dr. Gregorio is recipient of a Fellowship from the Fondazione Italiana
Neuroblastoma.
1
Francesca Schena, PhD, Claudio Gambini, MD, Andrea
Gregorio, PhD, Manuela Mosconi, PhD, Marco Gattorno, MD, Elisabetta Traggiai, PhD: IRCCS Institute G. Gaslini Hospital, Genoa Italy;
2
Daniele Reverberi, PhD: Tumor Scientific Institute, Genoa, Italy;
3
Simona Casazza, PhD: Advanced Biotechnology Center, Genoa,
Italy; 4Antonio Uccelli, MD, PhD: University of Genoa, Advanced
Biotechnology Center, and Center of Excellence for Biomedical
Research, University of Genoa, Genoa, Italy; 5Lorenzo Moretta, MD,
PhD: Università degli Studi di Genova and IRCCS Institute G. Gaslini
Hospital, Genoa, Italy; 6Alberto Martini, MD: University of Genoa,
and IRCCS Institute G. Gaslini Hospital, Genoa, Italy.
Address correspondence and reprint requests to Elisabetta
Traggiai, PhD, Istituto Giannina Gaslini, Largo Gerolamo Gaslini 5,
16147 Genova, Italy. E-mail: elisabetta.traggiai@gmail.com.
Submitted for publication July 16, 2009; accepted in revised
form May 6, 2010.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of serum
autoantibodies directed against nucleic acids. It is presumed that, analogous to other experimental models as
well as other autoimmune diseases, a defect in T cell
tolerance and/or activation might be at the origin of the
disease (1,2). Nonetheless, several lines of evidence
support a crucial role of B lymphocytes in the pathogenesis of SLE, both in mice and in humans. Autoantibodies
directed to DNA, RNA, or other nuclear and cytoplasmic antigens have been detected in serum from
humans with SLE as well as from strains of lupus-prone
mice, and serum autoantibodies have been correlated
with disease activity in humans with SLE, particularly in
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EFFECT OF BM-MSCs ON B CELL ACTIVATION IN A MURINE MODEL OF SLE
those with lupus nephritis (3). Moreover, results from
clinical studies suggest that B cell depletion with rituximab improves the clinical manifestations of SLE (4).
Bone marrow–derived mesenchymal stem cells
(BM-MSCs) are a heterogeneous population of selfrenewing and multipotent cells. BM-MSCs can be rapidly expanded in vitro and can differentiate into bone
cells, fat cells, and cartilage cells (5). Moreover, they
have been hypothesized to possess immunomodulatory
properties because they affected both the phenotype and
the function of a number of cells belonging to the innate
or adaptive immune system (6). This led to the idea that
these cells could be beneficial in the treatment of
autoimmune diseases (7).
It is presumed that the effect of the interaction
between MSCs and cells of the immune system vary
depending on the microenvironment in which this interaction takes place (8,9). Indeed, we have previously
shown that human BM-MSCs could support and enhance the in vitro polyclonal expansion of circulating
human B cell subsets from both normal donors and SLE
patients (10). However, it has been reported that BMMSCs can also inhibit B cell proliferation and effector
function both in vitro (11) and in vivo (12).
In the present study, we found that murine
BM-MSCs did not affect polyclonal B cell activation
mediated by Toll-like receptor 9 (TLR-9) agonist, while
they inhibited the B cell receptor (BCR)–dependent
proliferation and differentiation to plasma cells of
splenic B cells derived from (NZB ⫻ NZW)F1 mice.
This suppressive effect was cell-dose dependent and was
significantly enhanced by exogenous interferon- ␥
(IFN␥). The capacity of IFN␥ to induce the suppressive
activity of BM-MSCs was not dependent on the enzyme
indoleamine 2,3-dioxygenase (IDO), but was dependent
on cell-to-cell contact mediated by the interaction between programmed death 1 (PD-1) and PD ligand 1
(PDL-1). Injection of BM-MSCs into (NZB ⫻ NZW)F1
mice did not affect the generation and maintenance of
specific serum autoantibodies, but significantly ameliorated the kidney histopathology scores, indicating that
administration of BM-MSCs might be beneficial in
patients with SLE-associated glomerulonephritis.
MATERIALS AND METHODS
Media and reagents. For B cell culture, we used RPMI
1640 medium (BioWhittaker) supplemented with 10% fetal
bovine serum (FBS; defined) (HyClone), 2 mM Glutamax
(Gibco), 10 mg/liter of nonessential amino acids (Gibco), 1
mM pyruvate (Gibco), 50 units/ml of penicillin (Gibco), 50
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units/ml of streptomycin (Gibco), and 5 ⫻ 10 –2 M
2-mercaptoethanol (Sigma).
Generation of BM-MSCs. Briefly, bone marrow was
flushed out of the tibias and femurs of 6–8-week-old C57BL/6J
mice. After 2 washings by centrifugation at 1,500 revolutions
per minute (352g) for 5 minutes in phosphate buffered saline
(PBS), cells were plated in 75-cm2 tissue culture flasks at a
concentration of 0.3–0.4 ⫻ 106 cells/cm2 using murine MesenCult as medium (Stem Cell Technologies). Cells were maintained at 37°C in a humidified incubator with an atmosphere of
5% CO2, and the medium was refreshed every 3–4 days for
about 4–5 weeks. After this time, adherent cells were collected
following a 10-minute incubation at 37°C with 0.05% trypsin
solution (Sigma-Aldrich). The identity of MSCs was confirmed
according to their immunophenotype, based on the expression
of positive markers CD9 and Sca-1 antigen and negative CD45
(all from BD PharMingen). Cells between passages 20 and 25
were used in the experiments.
Isolation of follicular and marginal zone B cells. To
avoid inadvertent activation of B cells, B cells were isolated by
negative selection (CD43 depletion) with the use of CD43
microbeads (Miltenyi Biotec) according to the manufacturer’s
instructions. Following staining for B220 (BD Biosciences),
CD24, CD21, CD23 (eBioscience), follicular, and marginal
zone B cells were sorted with a FACSAria (Becton Dickinson).
The purity of the sorted cells was ⬎95%.
Proliferation assay. B cell subsets were labeled with 0.5
␮M 5,6-carboxyfluorescein diacetate N-succinimidyl ester
(Molecular Probes) for 8 minutes at room temperature. Purified B cells subsets were cultured with mouse BM-MSCs
irradiated at 10,000 rads at different ratios (1:1, 3:1, and 9:1
ratio of B cells to BM-MSCs) in a 96-well flat-bottomed plate
with 2.5 ␮g/ml of CpG-containing oligodeoxynucleotide (CpG
ODN) 1826 (5⬘-TCC-ATG-ACG-TTC-CTG-ACG-TT-3⬘; TIB
Molbiol), 25 ng/ml of soluble CD40L (R&D Systems), 2.5
␮g/ml of F(ab⬘)2 anti-mouse IgM (Jackson ImmunoResearch),
and 1,000 units/ml of interleukin-2 (IL-2) (Proleukin;
Prometheus). The proliferation profile of propidium iodide–
negative viable B220-positive cells was analyzed on day 3 of
culture. IFN␥ was added at a concentration of 5 ng/ml, and the
IDO inhibitor 1-methyl-DL-tryptophan was added at a concentration of 100 ␮M. MSCs were incubated with anti–PD-1 and
anti–PDL-1 (eBioscience) at 2.5 ␮g/ml as well as with 2
irrelevant isotype antibodies, anti-CD4 (BioLegend) and antiCD3 (R&D Systems), which served as controls.
Evaluation of apoptosis. Apoptotic cells were detected
at 20 hours by intracellular staining for annexin V and
7-aminoactinomycin D using an Annexin V–PE Apoptosis
Detection Kit I (BD PharMingen) according to the manufacturer’s instructions.
Differentiation analysis by enzyme-linked immunospot (ELISpot) assay and enzyme-linked immunosorbent assay (ELISA). Plates (96-well flat-bottomed; Greiner) were
coated with isotype-specific goat anti-mouse IgG or IgM
antibodies (SouthernBiotech). Plates were washed and
blocked with PBS–10% FBS for 2 hours at room temperature.
After washing, serial dilutions of culture supernatants were
added, and incubation was continued for 2 hours at room
temperature. Plates were washed again, alkaline phosphatase–
conjugated goat anti-mouse IgG or IgM was added, and plates
were incubated for 2 hours at room temperature. The reaction
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SCHENA ET AL
Figure 1. A, Proliferation of follicular (FO) and marginal zone (MZ) B cells incubated either alone or in combination with bone marrow–derived
mesenchymal stem cells (BM-MSCs) at different ratios. Cultures were stimulated with interleukin-2 and CpG-containing oligodeoxynucleotide 1826,
either alone or in the presence of interferon-␥ (IFN␥), and proliferation was measured on day 3 of culture as the absolute number of B220-positive,
propidium iodide–negative cells which have diluted 5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (numbers at the top of the
histograms). Shown is a representative experiment of 4 experiments performed. B, IgM secretion in culture supernatants, as determined by
enzyme-linked immunosorbent assay, and frequency of immunoglobulin-secreting cells (ISCs), as determined by enzyme-linked immunospot assay
in follicular and marginal zone B cells. Cells were cultured as described in A, in the presence (solid bars) or absence (open bars) of IFN␥, and assays
were performed on day 4 of culture. Values are the mean and SEM of 3 experiments.
was developed with Sigma 104 substrate. Plasma cells secreting
IgG or IgM were detected using an ELISpot assay. Briefly,
96-well plates (Millipore catalog no. MAIPS4510) were coated
with 10 ␮g/ml of purified goat anti-mouse IgG or IgM (SouthernBiotech). After washing and blocking with PBS–1% bovine
serum albumin (BSA) for 30 minutes, serial dilutions of
cultured B cells were added, and plates were incubated
overnight at 37°C. Plates were then washed and incubated
with isotype-specific secondary antibodies, followed by
streptavidin–horseradish peroxidase (Sigma). The assay was
developed with aminoethylcarbazole (Sigma) as a chromogenic
substrate.
Transwell experiments. Transwell chambers with
0.2-␮M pore membranes (Nunc) were used to physically
separate the stimulated B cells from the MSCs. Follicular and
marginal zone B cells (3 ⫻ 104 cells/well) were cocultured in
the lower Transwell chamber, and the C57BL/6J MSCs were
placed in the upper chamber at the same ratios as described
above (1:1, 3:1, and 9:1 ratio of B cells to BM-MSCs).
Protein kinase signaling pathways. The analysis of
phosphoproteins was performed after 16 hours and 24 hours of
culture for marginal zone and follicular B cells, respectively.
Cells were stained for surface marker B220 and then permeabilized and fixed using a Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. Intracellular staining was performed with primary antibodies
against phospho-ERK, phospho-p38, or phospho-Akt (Cell
Signaling Technology), as well as a secondary biotinylated
IgG antibody (Jackson ImmunoResearch). Streptavidin–
phycoerythrin (PE) (BD PharMingen) was used for signal
detection.
In vivo treatment of mice. Female (NZB ⫻ NZW)F1
mice were purchased from Harlan Italy. Mice were used for
isolation of lymphoid organs between the ages of 9 weeks and
33 weeks. Mice were treated intravenously with 1.25 ⫻ 106
BM-MSCs at weeks 27, 28, and 29. Mice were bred and
maintained at the animal facility of the Advanced Biotechnology Center in Genoa. The care and use of the animals were in
compliance with the laws of the Italian Ministry of Health and
with the guidelines of the European Community.
Detection of antinuclear antibodies (ANAs) and anti–
double-stranded DNA (anti-dsDNA) antibodies by immunofluorescence and ELISA. Serum levels of circulating ANAs
were detected using permeabilized HEp-2 cells (Biomed Instruments), and anti-dsDNA antibodies were determined by
immunofluorescence using Crithidia luciliae (Biomed Instruments) and by ELISA. IgG ANAs and anti-dsDNA were
detected with fluorescein isothiocyanate–conjugated goat antimouse IgG (SouthernBiotech) and were scored by an observer
(FS) who was blinded to the experimental group. For antidsDNA ELISAs, polystyrene plates were coated with poly-Llysine and calf thymus DNA (both from Sigma). Plates were
postcoated for 45 minutes with 50 ␮g/ml of polyglutamic acid,
blocked for 45 minutes with PBS–3% BSA, and then serial
EFFECT OF BM-MSCs ON B CELL ACTIVATION IN A MURINE MODEL OF SLE
2779
Figure 2. A, Proliferation of follicular (FO) and marginal zone (MZ) B cells incubated either alone or in combination with bone marrow–derived
mesenchymal stem cells (BM-MSCs) at different ratios. Cultures were stimulated with interleukin-2, CpG-containing oligodeoxynucleotide 1826,
anti-Ig, and CD40L, either alone or in the presence of interferon-␥ (IFN␥), and proliferation was measured on day 3 of culture as the absolute
number of B220-positive, propidium iodide–negative cells which have diluted 5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)
(numbers at the top of the histograms). Shown is a representative experiment of 11 experiments performed. B, Percentage of B220-positive apoptotic
cells (annexin V⫹/7-aminoactinomycin D– [7-AAD–] and annexin V⫹/7-AAD⫹) in follicular and marginal zone B cells cultured as described in A,
in the presence or absence of IFN␥. Values are the mean and SEM of 3 experiments. C, IgM and IgG secretion in culture supernatants (top), as
determined by enzyme-linked immunosorbent assay (top), and frequency of immunoglobulin-secreting cells (ISCs) (bottom), as determined by
enzyme-linked immunospot assay (bottom). Cells were cultured as described in A, in the presence (solid bars) or absence (open bars) of IFN␥, and
assays were performed on day 4 of culture. Values in B and C are the mean and SEM of 6 experiments. Significant comparisons were as follows:
for IgM (␮g/ml) in follicular cells, ⴱⴱ ⫽ P ⫽ 0.0013 and ⴱ ⫽ P ⫽ 0.0177; for IgG (␮g/ml) in follicular cells, ⴱ at a 1:1 ratio ⫽ P ⫽ 0.0165 and ⴱ at
a 3:1 ratio ⫽ P ⫽ 0.0190; for IgM (␮g/ml) in marginal zone cells, ⴱⴱ ⫽ P ⫽ 0.0082 and ⴱ ⫽ P ⫽ 0.0349; for IgG (␮g/ml) in marginal zone cells, ⴱⴱⴱ
⫽ P ⫽ 0.0003 and ⴱ ⫽ P ⫽ 0.0192; for IgM (ISC/well) in follicular cells, ⴱⴱ ⫽ P ⫽ 0.0019 and ⴱ ⫽ P ⫽ 0.0377; for IgG (ISC/well) in follicular cells,
ⴱⴱⴱ ⫽ P ⬍ 0.0001 and ⴱⴱ ⫽ P ⫽ 0.0023; and for IgM (ISC/well) in marginal zone cells, ⴱⴱⴱ ⫽ P ⫽ 0.0005 and ⴱⴱ ⫽ P ⫽ 0.0038; and for IgG (ISC/well)
in marginal zone cells, ⴱⴱⴱ at a 1:1 ratio ⫽ P ⬍ 0.0001, ⴱⴱⴱ at a 3:1 ratio ⫽ P ⫽ 0.0007, and ⴱ ⫽ P ⫽ 0.0138.
dilutions of serum (from 1:100 to 1:3,200) were incubated
overnight. Specific antibodies were detected with alkaline
phosphatase–conjugated goat anti-mouse IgG (SouthernBiotech).
Histopathologic assessment of kidneys. Mice were
killed, and their kidneys were isolated, preserved in 10%
formalin, and embedded in paraffin. Histopathologic assessments were performed on 4-␮m formalin-fixed, paraffinembedded sections that had been stained with periodic acid–
Schiff. A total of 50 sequential glomeruli from the superior,
middle, and inferior cortices of each kidney were scored for the
presence of proliferating glomeruli and were then expressed as
the percentage of proliferating glomeruli of the total number
of glomeruli analyzed. Lymphocytic infiltration of 50 sequential glomeruli from each section was evaluated and scored
using a scale of 0–3⫹, where 0 ⫽ normal, 1 ⫽ mild, 2 ⫽
moderate, and 3 ⫽ severe abnormalities.
Kidney cryosections (4 ␮m) were fixed in ice-cold
acetone, washed with PBS, blocked with normal goat serum,
and then incubated with fluorescein-conjugated goat IgG, goat
anti-mouse IgG, and goat anti-mouse IgM (SouthernBiotech).
The relative fluorescence intensity for IgM and for IgG was
scored separately using a scale of 0–3⫹, where 0 ⫽ no
apparent staining as compared with the isotype control, 1 ⫽
detectable staining, 2 ⫽ moderate staining intensity, and 3 ⫽
maximum staining intensity. Two experienced evaluators (AG
and AM) who were blinded with regard to the experimental
group evaluated the samples.
Assessment of proteinuria. Proteinuria was evaluated
colorimetrically using Albustix (Bayer) at weeks 25, 26, 27, 28,
29, and 32.
Statistical analysis. Proteinuria data were analyzed by
Fisher’s exact test. All the other parameters were analyzed by
Mann-Whitney U test and Student’s t-test. Statistical significance was defined as P ⬍ 0.05. The time to the occurrence
of death was assessed by Kaplan-Meier cumulative survival
functions.
2780
SCHENA ET AL
Figure 3. A, Proliferation of follicular (FO) and marginal zone (MZ) B cells incubated either alone or in combination with bone marrow–derived
mesenchymal stem cells (BM-MSCs) at different ratios. Cultures were stimulated with interleukin-2, CpG-containing oligodeoxynucleotide 1826,
anti-Ig, CD40L, and interferon-␥ (IFN␥), either alone or in the presence of the indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl-DLtryptophan. Proliferation was measured on day 3 of culture as the absolute number of B220-positive, propidium iodide–negative cells which have
diluted 5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (numbers at the top of the histograms). Shown is a representative experiment
of 3 experiments performed. B, IgM and IgG secretion in culture supernatants, as determined by enzyme-linked immunosorbent assay (top), and
frequency of immunoglobulin-secreting cells (ISCs), as determined by enzyme-linked immunospot assay (bottom). Cells were cultured as described
in A, in the presence (solid bars) or absence (open bars) of 1-methyl-DL-tryptophan, and assays were performed on day 4 of culture. Values are the
mean and SEM of 3 experiments.
RESULTS
Requirement of IFN␥ priming for BM-MSC–
mediated inhibition of B cell proliferation and differentiation to plasma cells. The pool of mature murine
splenic B cells is mainly composed of 2 anatomically and
functionally distinct subsets: follicular and marginal zone
B cells. These subsets can be identified by the differential expression of surface markers (13). We isolated B
cells from the spleens of (NZB ⫻ NZW)F1 mice by
negative enrichment with the surface marker CD43 to
avoid the inadvertent activation of B cells. CD43negative cells were then sorted with antibodies specific
for CD21, CD24, and CD23 to purify the follicular
(CD23⫹CD24–CD21⫹) and marginal zone (CD23–
CD24⫹CD21⫹) B cells (14).
We have previously shown that human BMMSCs promote the proliferation and differentiation into
immunoglobulin-secreting cells (ISCs) of human transitional, naive, and memory B cells stimulated with CpG
ODN 1826 (used as an agonist of TLR-9) in the absence
of BCR triggering (10). Thus, in the present study, we
stimulated follicular and marginal zone B cells with CpG
ODN 1826 and IL-2, either alone or in combination with
BM-MSCs. BM-MSCs did not inhibit the proliferative
response of either follicular or marginal zone B cells to
TLR-9 stimulation, even at a 1:1 ratio (Figure 1A).
Thus, the CpG responsiveness of murine splenic B cells
to CpG ODN 1826 was not influenced by BM-MSCs.
Next, we sought to determine whether BM-MSCs
could negatively influence splenic B cell responsiveness.
Since IFN␥ has been previously shown to be crucial for
the immunomodulatory activity of BM-MSCs (8,9), we
added IFN␥ to cocultures of B cells and MSCs. The
addition of IFN␥ to the cocultures did not induce
inhibition of the proliferation of either follicular or
marginal zone B cells (Figure 1A). In addition, the
differentiation to plasma cells, which was measured as
immunoglobulin secretion and as the ISC frequency, was
EFFECT OF BM-MSCs ON B CELL ACTIVATION IN A MURINE MODEL OF SLE
2781
Figure 4. A, Proliferation of follicular (FO) and marginal zone (MZ) B cells incubated either alone or in combination with bone marrow–derived
mesenchymal stem cells (BM-MSCs) at different ratios. B cells labeled with 5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) were
cocultured or were cultured in a Transwell (TW) system with irradiated BM-MSCs at the indicated ratios. For the Transwell culture, B lymphocytes
were placed in the lower chamber and BM-MSCs in the upper chamber. Cultures were stimulated with interleukin-2, CpG-containing
oligodeoxynucleotide 1826, anti-Ig, CD40L, and interferon-␥ (IFN␥), and proliferation was measured on day 3 of culture as the absolute number
of B220-positive, propidium iodide–negative cells which have diluted CFSE (numbers at the top of the histograms). Shown is a representative
experiment of 5 experiments performed. B, IgM and IgG secretion in culture supernatants, as determined by enzyme-linked immunosorbent assay
(top), and frequency of immunoglobulin-secreting cells (ISCs) as determined by enzyme-linked immunospot assay (bottom). Cells were cultured as
described in A, either in a Transwell system (solid bars) or in contact (open bars), and assays were performed on day 4 of culture. Values are the
mean and SEM of 5 experiments. Significant comparisons were as follows: for IgG (␮g/ml) in follicular cells, ⴱⴱ at a 1:1 ratio ⫽ P ⫽ 0.0089 and ⴱⴱ
at a 3:1 ratio ⫽ P ⫽ 0.008; for IgG (ISC/well) in follicular cells, ⴱ ⫽ P ⫽ 0.0421; and for IgG (ISC/well) in marginal zone cells, ⴱⴱ ⫽ P ⫽ 0.0042.
not affected by BM-MSCs, whether they had been left
untreated or had been treated with IFN␥ (Figure 1B).
The production of autoantibodies in systemic
autoimmune diseases is often related to the synergistic
engagement of the BCR and TLR-9 in response to
DNA-containing antigens (15). Thus, we stimulated
follicular and marginal zone B cells with CpG, anti-Ig,
CD40L, and IL-2 in the presence of BM-MSCs. BMMSCs inhibited follicular and marginal zone proliferation at a 1:1 ratio, while this effect was lost at a 9:1 ratio
of B cells to MSCs. Proliferation of both follicular and
marginal zone B cells was strongly impaired in the
presence of IFN␥, and this effect was observed at all
ratios of B cells to MSCs that were tested (Figure 2A).
Remarkably, MSCs did not induce apoptosis in either
follicular or marginal zone B lymphocytes (Figure 2B).
Furthermore, BM-MSCs did not significantly inhibit
IgM or IgG secretion by follicular or marginal zone cells
and did not influence the frequency of ISCs of either
isotype. The addition of IFN␥ induced a significant
inhibition of plasma cell differentiation at a 1:1 and a 3:1
ratio of B cells to MSCs (Figure 2C). Moreover, preincubation of BM-MSCs with IFN␥ displayed comparable
inhibition of follicular and marginal zone B cell proliferation, thus suggesting a direct involvement of IFN␥ in
the modulation of BM-MSC signals (data not shown).
IDO-independent, but cell-to-cell contact–
dependent, inhibition of B cell proliferation and differentiation to plasma cells mediated by BM-MSCs. BMMSC–mediated inhibition of T cell proliferation and
differentiation to cytokine-secreting cells has been reported to be dependent on, among other factors, the
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SCHENA ET AL
Figure 5. A, Phosphorylation of p38, ERK, and Akt in follicular (FO) B cells (measured at 24 hours) and in marginal zone (MZ) B cells (measured at
16 hours) after stimulation with interleukin-2 (IL-2), CpG-containing oligodeoxynucleotide (ODN) 1826, anti-Ig, and CD40L either alone (solid bars) or
in combination with (open bars) bone marrow–derived mesenchymal stem cells (BM-MSCs). Results are expressed as the fold increase in phosphorylation
in stimulated versus nonstimulated (nil) B cells. Values are the mean and SEM of 3 different experiments. B, The 5,6-carboxyfluorescein diacetate
N-succinimidyl ester (CFSE) dilution of follicular and marginal zone B cells stimulated with IL-2, CpG ODN 1826, anti-Ig, CD40L, and interferon-␥
(IFN␥), either alone (left) or with BM-MSCs in the presence (right) or absence (middle) of antibodies specific for programmed death 1 (PD-1) and PD
ligand 1 (PDL-1). Proliferation was measured on day 3 of culture as the absolute number of B220-positive, propidium iodide–negative cells which have
diluted CFSE (numbers at the top of the histograms). Shown is a representative experiment of 3 experiments performed. C, Expression of PD-1 and PDL-1
on resting follicular B cells, marginal zone B cells, and BM-MSCs, as well as PDL-1 expression on follicular and marginal zone B cells stimulated for 24
hours with IFN␥ and on BM-MSCs stimulated for 48 hours with IFN␥ (red histograms). Values shown in each histogram are the percentage of positive
cells. D, Phosphorylation of p38, ERK, and Akt at 24 hours in follicular B cells (left) and at 16 hours in marginal zone B cells (right) cultured without
BM-MSCs (open bars), with BM-MSCs (closed bars), with BM-MSCs in the presence of anti–PD-1 and anti–PDL-1 (striped bars), or in the presence of
irrelevant antibodies (shaded bars). Values are the mean and SEM of 3 experiments. (Color figure can be viewed in the online issue, which is available at
http: //www.arthritisrheum.org.)
expression of IDO, which catalyzes the conversion of
tryptophan to kynurenine. IDO expression in BM-MSCs
has been shown to be regulated by IFN␥ (8). To
determine whether IDO could also be involved in the
inhibition of B cell proliferation and differentiation, we
tested in our assay system a competitive inhibitor of the
IDO pathway, 1-methyl-DL-tryptophan (16). The proliferation and differentiation to plasma cells of both
follicular and marginal zone B cells were not restored in
the presence of IDO inhibitor (Figures 3A and B).
To test whether BM-MSC inhibition was dependent on cell-to-cell contact, we evaluated the proliferative responses of stimulated follicular and marginal zone
B cells in a Transwell system with B lymphocytes in the
lower chamber and BM-MSCs in the upper chamber.
When MSCs and B cells were physically separated, the
follicular and marginal zone B cells proliferated and
differentiated into ISCs to the same extent as in control
cultures. We concluded that direct cell-to-cell contact
with MSCs is required in order to inhibit the response of
follicular or marginal zone B cells to BCR, TLRs, and
CD40L (Figures 4A and B).
We next evaluated the effect of BM-MSCs on 3
of the major signaling axes involved in BCR-mediated
activation of follicular and marginal zone B lymphocytes: the phosphatidylinositol 3-kinase/Akt/mammalian
target of rapamycin pathway, the MEK-1/ERK-1/2 pathway, and the p38 MAPK pathway. We analyzed specific
intracellular phosphoproteins by flow cytometry in stimulated follicular and marginal zone B cells. As shown in
Figure 5A, BCR stimulation of follicular and marginal
zone B cells led to an increased level of p38, ERK, and
Akt phosphorylation at 24 and 16 hours of stimulation.
Coculture with MSCs resulted in inhibition of p38, ERK,
and Akt phosphorylation in follicular and marginal zone
B cells. These findings suggest that the inhibition of
EFFECT OF BM-MSCs ON B CELL ACTIVATION IN A MURINE MODEL OF SLE
2783
Figure 6. A, Histopathologic assessment of kidney sections from mice treated intravenously with 1.25 ⫻ 106 bone marrow–derived mesenchymal
stem cells (BM-MSCs) or with phosphate buffered saline (PBS) at weeks 27, 28, and 29. Photomicrographs of kidney sections from a mouse treated
with BM-MSCs (left) and PBS (right) are shown at the left. Kidney sections were assessed for lymphocytic infiltration, glomerular proliferation, and
IgG immune complex deposition, and the results are shown at the right. Values are the mean and SEM of 10 mice. ⴱ ⫽ P ⫽ 0.045; ⴱⴱⴱ ⫽ P ⫽ 0.0001;
ⴱⴱ ⫽ P ⫽ 0.0088. B, Serum levels of IgG1, IgG2a, IgG2b, and IgG3 anti–double-stranded DNA (anti-dsDNA) antibodies in mice treated with
BM-MSCs (solid line) or with PBS (broken line), as determined by enzyme-linked immunosorbent assay (left). Values are the mean and SEM optical
density (OD) of 10 sera. The percentage of sera positive for IgG antinuclear antibodies (top right) and IgG anti-dsDNA antibodies (bottom right)
in mice treated with BM-MSCs or PBS, as determined by immunofluorescence analysis. C, Proteinuria index from week 25 to week 30 in mice treated
with BM-MSCs (solid line) or PBS (broken line). Proteinuria was scored on a 0–4 scale, where 0 ⫽ ⬍30 mg/dl, 1 ⫽ 30 mg/dl, 2 ⫽ 100 mg/dl, 3 ⫽
300 mg/dl, and 4 ⫽ ⬎2,000 mg/dl. Values are the mean and SEM of 10 mice. D, Kaplan-Meier survival curves in mice treated with BM-MSCs (solid
line) or PBS (dotted line).
BCR signaling by BM-MSCs affected all 3 signaling
pathways, perhaps acting upstream of BCR signaling,
splitting into the 3 axes.
Requirement of PD-1/PDL-1 interaction for BMMSC–mediated inhibition of B cell activation. One of
the molecular mechanisms suggested to be crucially
involved in the inhibition of T cell and B cell activation
by BM-MSCs is the interaction of the inhibitory molecule PD-1 with its ligand PDL-1 (17). PD-1 was expressed on resting follicular and marginal zone B cells at
lower levels than PDL-1 (Figure 5C), whereas murine
BM-MSCs expressed PD-1 and PDL-1 at similar levels,
as previously reported (18) (Figure 5C). We sought to
determine whether IFN␥ treatment could modulate the
expression of PD-1 and PDL-1 on BM-MSCs and on
follicular and marginal zone B lymphocytes. BM-MSCs
displayed a significant increase in PDL-1 expression
after IFN␥ treatment (Figure 5C), whereas no effect on
PD-1 expression was observed (data not shown). Anal-
ogously, IFN␥ affected PDL-1 expression on both follicular and marginal zone B lymphocytes while PD-1
expression was unaltered.
In order to verify whether the inhibitory molecular pathway induced by PD-1/PDL-1 interactions could
be involved in the inhibition of BCR stimulation of
murine B lymphocytes by BM-MSCs, we used specific
monoclonal antibodies against PD-1 and PDL-1 to block
PD-1/PDL-1 interactions. Treatment with monoclonal
antibodies against anti–PD-1 and anti–PDL-1 completely restored p38, ERK, and Akt phosphorylation in
both follicular and marginal zone B cells upon stimulation with anti-Ig and TLR-9 agonist and partially rescued B cell proliferation at a 3:1 ratio of B cells to MSCs
(Figures 5B and D).
These results suggest that PD-1/PDL-1 interactions between MSCs and B lymphocytes inhibit the
signal transduction pathways triggered by productive B
cell stimulation by antigen and affect B cell expansion.
2784
SCHENA ET AL
Lack of effect of in vivo cell therapy with BMMSCs on dsDNA autoantibodies and proteinuria, but
significant improvement in histopathologic changes in
the kidney. We next tested whether BM-MSCs might
have beneficial in vivo effects on B cell activation and
SLE pathogenesis in (NZB ⫻ NZW)F1 mice. Beginning
at age 27 weeks, when IgG anti-dsDNA were detectable
in the serum of (NZB ⫻ NZW)F1 mice, we initiated a
therapeutic protocol consisting of an intravenous infusion of 1.25 ⫻ 106 BM-MSCs, which was administered
weekly for 3 weeks. The following parameters were
evaluated: serum levels of IgG autoantibodies, degree of
proteinuria, histopathologic changes in the kidney, and
percentages of mice surviving over time.
Infusion of BM-MSCs did not significantly affect
serum concentrations of IgG anti-dsDNA or IgG ANAs,
as measured by immunofluorescence (Figure 6B). Moreover, serum levels of IgG1, IgG2a, IgG2b, and IgG3
anti-dsDNA were unaffected by treatment with BMMSCs (Figure 6B). We also measured the frequencies of
total and anti-dsDNA–specific ISCs and found that they
were not modified by treatment with BM-MSCs (data
not shown). In contrast, despite the lack of a significant
impact of BM-MSC infusions on proteinuria and percentage of survival in these mice (Figures 6C and D),
histopathologic analysis of the kidney revealed a significant improvement in glomerular proliferation, lymphocytic infiltration, and IgG immune complex deposition in
mice treated with BM-MSCs (Figure 6A). These results
indicate that BM-MSCs could have a beneficial effect on
kidney function in experimental SLE, independently of
their lack of effect on serologic anti-dsDNA and ANA
immunoglobulins.
DISCUSSION
SLE is a systemic autoimmune disease characterized by the continuous generation of autoantibodyproducing cells (i.e., autoreactive plasma cells) through
mechanisms that are not yet fully understood. Nevertheless, it is well established that both B lymphocytes and T
lymphocytes are critical in the pathogenesis of the
disease through autoantibody-dependent and
-independent mechanisms (19). Steroids and immunosuppressants are currently used to treat many patients
with SLE (20). The efficacy of these agents lies in their
ability to suppress inflammation and to block or partially
reduce abnormal T cell and B cell activation (21).
Therefore, targeting both the B lymphocytes and the T
lymphocytes as well as their interaction could represent
an alternative approach to the currently available pharmacologic methods.
MSCs can interact with cells of both the innate
and adaptive immune system, leading to the modulation
of several effector functions. However, the mechanisms
involved in their function are still a matter of debate and
may be diverse or only partially overlapping (8). With
regard to the effect of MSCs on human B cell activation,
previous studies have yielded contradictory data
(11,12,22,23), despite some evidence of an in vivo effect
on antibody production (13,24). Thus, in order to verify
whether BM-derived MSCs could affect systemic autoimmune responses, we investigated their impact on the
proliferation and differentiation of B cells isolated from
(NZB ⫻ NZW)F1 mice.
We found that BM-MSCs did not influence B cell
activation of either follicular or marginal zone B cells
upon TLR-9 stimulation. In contrast, they inhibited the
proliferation of both follicular and marginal zone B cells
after stimulation with both BCR and TLR-9 agonists if
MSCs were used at high doses, and this inhibitory effect
was increased when exogenous IFN␥ was added to the
cultures. This observation is consistent with previous
studies by Krampera et al (8) and Spaggiari et al (25), in
which they showed that CD4, CD8, and natural killer
cells are inhibited by MSCs through an IFN␥-dependent
mechanism.
Experiments were performed in an attempt to
clarify the mechanisms involved in the suppressive activity of MSCs. First, we established that IFN␥-dependent
inhibition of B cell proliferation and differentiation by
MSCs was dependent on cell-to-cell contact but not on
soluble factors. Second, we found that MSCs were able
to inhibit the activation of both follicular and marginal
zone B cells when BCR was engaged, but not when
TLR-9 alone was triggered. These 2 observations led us
to hypothesize that inhibitory surface molecules could
trigger the IFN␥-dependent suppressive activity of BMMSCs. In this context, a good candidate was the costimulatory molecule PD-1 and its ligand PDL-1, which control
an inhibitory pathway of T cell activation (18). It is
noteworthy that in a previous study, PD-1 was shown to
inhibit BCR signaling in B lymphocytes by recruiting SH2
domain–containing phosphatase 2 to its phosphotyrosine
and by dephosphorylating key signal transducers of BCR
signaling (26). Furthermore, Augello et al (17) reported a
possible involvement of the PD-1/PDL-1 pathway in the
suppressive effect of BM-MSCs on T lymphocytes.
Two lines of evidence from the present study
support our hypothesis. First, IFN␥ treatment increased
the expression of PDL-1 on BM-MSCs and follicular
and marginal zone B lymphocytes. Second, blocking
EFFECT OF BM-MSCs ON B CELL ACTIVATION IN A MURINE MODEL OF SLE
antibodies specific for PD-1 and PDL-1 completely
restored p38, ERK, and Akt phosphorylation and partially restored the proliferation of both follicular and
marginal zone B cells. Thus, our data suggest that IFN␥
is a crucial soluble factor in the induction of the suppressive activity of MSCs on mouse B lymphocytes, and
the interaction of PD-1 with PDL-1 may be one of the
pathways involved in their immunomodulatory activity.
This conclusion is also confirmed by the findings reported by Ren et al (27) in which MSCs generated from
IFN␥-deficient or IFN␥ receptor–deficient mice did not
display immunosuppressive activity. Furthermore, IFN␥
alone or in combination with tumor necrosis factor ␣
(TNF␣) and IL-1␤ stimulated the production of T
cell–attracting chemokines as well as inducible nitric
oxide synthase, which inhibits T cell activation through
the production of nitric oxide by mouse MSCs (27).
These observations support the notion of the importance of the microenvironment in driving the final
outcome of the immunomodulatory activity of MSCs on
cells of the innate or adaptive immune system.
In order to elucidate these microenvironmentdriven effects, we also studied the in vivo impact of
MSCs on the activation of B lymphocytes in the (NZB ⫻
NZW)F1 mouse model of SLE. These mice display
hyperresponsiveness of the T cell effector/memory compartment, with a significant increase in the frequency of
IFN␥- and TNF␣-secreting cells in healthy laboratory
strains (Schena F, et al: unpublished observations). In
this proinflammatory microenvironment, in vitro inhibition of B cell activation by MSCs does not translate to
reduced in vivo production of autoantibodies. This lack
of effect can be related to 2 different mechanisms. First,
it has been proposed that in murine lupus, the production of anti-dsDNA autoantibodies is controlled by
TLR-9 signaling (28). This is consistent with our finding
of a lack of effect of MSCs on in vitro stimulation of both
follicular and marginal zone B lymphocytes with TLR-9.
The second consideration concerns the cellular mechanisms that sustain serum autoantibodies over time.
Hoyer et al (29) demonstrated that in (NZB ⫻ NZW)F1
mice, the number of splenic antibody-secreting cells
increases during ages 1–5 months and becomes stable
thereafter. Thus, serum autoantibodies in our animal
model of lupus could be maintained by a pool of
long-lived plasma cells generated in young mice and not
affected by the presence of MSCs.
Treatment of (NZB ⫻ NZW)F1 mice with allogeneic BM-MSCs did not have an effect on proteinuria.
Two recent studies showed significant amelioration of
immunopathology and a decrease in serum autoantibody
levels in MRL/lpr mice after treatment with human
2785
MSCs (30,31). This discrepancy with our results could be
related to the xenogeneic source of the MSCs used for
the treatments, as well as to the different experimental
models of SLE that were used. Unlike (NZB ⫻ NZW)F1
mice, MRL/lpr mice are homozygous for the Fas mutation and show systemic autoimmunity related to a massive lymphadenopathy due to uncontrolled T cell proliferation and associated with autoantibody production,
glomerulonephritis, and arthritis. Fas is a member of the
TNF receptor superfamily. It is a crucial mediator of
apoptotic cell death and is involved in maintaining T
lymphocyte homeostasis. It is reasonable that in our
model, the effects of MSCs were mainly related to
inhibition of the exaggerated proliferation of T cells,
which in turn, provide help for polyclonal B cell activation and autoantibody production.
Despite the lack of effect on the production of
anti-dsDNA autoantibodies and proteinuria, treatment
with MSCs had a dramatic effect on 3 different histopathologic parameters of the kidney: glomerular proliferation, lymphocytic infiltration, and immune complex
deposition. These effects could be related to a direct
interaction of MSCs with renal cells. Consistent with
these observations, a direct effect of MSCs on the kidney
has been documented in a mouse model of acute kidney
injury, in which MSCs exerted their beneficial effects on
tubular cell repair by producing the mitogenic and
prosurvival factor insulin-like growth factor 1 (32). In a
rat model of acute renal failure, MSCs have also been
shown to exert a protective effect by a paracrine mechanism, tuning down proinflammatory cytokines such as
IL-1␤, TNF␣, and IFN␥ (33).
In conclusion, we have shown that BM-MSCs
inhibited antigen-dependent proliferation and differentiation to plasma cells of both follicular B cells and
marginal zone B cells in vitro. This inhibitory effect was
dependent on IFN␥ and was mediated by cell-to-cell
contact, which involved interactions between PD-1 and
PDL-1. Although BM-MSCs inhibited B cell antigen–
dependent proliferation and differentiation in vitro, the
in vivo treatment with BM-MSCs did not affect levels of
anti-dsDNA antibody or proteinuria. However, we observed a reduction in glomerular IgG immune complex
deposition, lymphocyte infiltration, and in particular,
glomerular proliferation, suggesting a potential role of
mesenchymal stem cells in preventing glomerular damage, an issue that needs further investigation.
ACKNOWLEDGMENT
The authors are thankful to Ennio Albanesi (Core
Facilities, Institute G. Gaslini) for performing the
fluorescence-activated cell sorting.
2786
SCHENA ET AL
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Traggiai 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. Schena, Gattorno, Uccelli, Moretta,
Martini, Traggiai.
Acquisition of data. Schena, Gregorio, Mosconi, Reverberi, Casazza,
Traggiai.
Analysis and interpretation of data. Schena, Gambini, Uccelli, Traggiai.
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