A Mimic of p21WAF1/CIP1 Ameliorates
Murine Lupus
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J Immunol 2005; 175:6959-6967; ;
doi: 10.4049/jimmunol.175.10.6959
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References
Claire Goulvestre, Christiane Chéreau, Carole Nicco, Luc
Mouthon, Bernard Weill and Frédéric Batteux
The Journal of Immunology
A Mimic of p21WAF1/CIP1 Ameliorates Murine Lupus1
Claire Goulvestre,2* Christiane Chéreau,2* Carole Nicco,* Luc Mouthon,† Bernard Weill,* and
Frédéric Batteux3*
S
ystemic lupus erythematosus (SLE)4 is a progressive multiorgan T and B cell-dependent autoimmune disease characterized by the appearance of a variety of autoantibodies
(AAbs), some of which are pathogenic (1). T cells are needed to
initiate and sustain the secretion of Abs, in particular to histones
and dsDNA, the latter contributing to lupus nephritis (2, 3). Early
in the life of (NZB ⫻ NZW)F1 mice that develop a lupus-like
syndrome, T cells primed to nucleosomal Ags can be detected (4).
Moreover, the i.v. administration of nucleosomes to preautoimmune (SWR ⫻ NZB)F1 or (NZB ⫻ NZW)F1 mice accelerates the
development of glomerulonephritis (5), whereas nucleosomes are
ignored by immunocompetent cells in normal mice. Therefore, a
treatment that would inhibit autoreactive T and B cell activation
and proliferation could be of interest to abrogate the development
of the lupus syndrome.
Cell cycle-related proteins (or cyclins) play key roles in cell
activation and proliferation (4). Progression through the cell cycle
is regulated by cyclins, cyclin-dependent kinases (CDKs), and
CDK inhibitors (CDKIs) (6). Disturbances in the interactions between cyclins and their kinases may have significant consequences
for immune responses (7). Thus, it could be worth inhibiting lym-
*Laboratoire d’Immunologie and †Service de Médecine Interne, Faculté de Médecine
René Descartes et Hôpital Cochin, Université Paris 5, Paris, France
Received for publication February 17, 2005. Accepted for publication August
18, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the French Ministry for Research and
Association Benoı̂t Malassagne.
2
C.G. and C.C. contributed equally to this work.
3
Address correspondence and reprint requests to F. Batteux, Laboratoire
d’Immunologie, Pavillon Hardy-Hôpital Cochin, 75679 Paris cedex 14, France. Email address: [email protected]
4
Abbreviations used in this paper: SLE, systemic lupus erythematosus; AAb, autoantibody; AICD, activation-induced cell death; CDK, cyclin-dependent kinase;
CDKI, CDK inhibitor; BUN, blood urea nitrogen.
Copyright © 2005 by The American Association of Immunologists, Inc.
phocyte activation and proliferation in (NZB ⫻ NZW)F1 mice by
administering a CDKI that would interfere with the cyclin-CDK
system. Among the known CDKIs, p21WAF1/CIP1 is expressed in a
variety of terminally differentiating cells where it interacts with its
respective cyclin-dependent kinases and inhibits the transition
from G1 to S phase (8). In addition, p21WAF1/CIP1 can bind to the
proliferating cell nuclear Ag, thereby further inhibiting DNA replication (9), and can also trigger cell apoptosis by affecting key
players of the apoptotic machinery such as p53 and procaspase-3
(10). For all these reasons, p21WAF1/CIP1 has been tried in the
treatment of tumors in animals and in patients (11).
In contrast, the use of p21WAF1/CIP1 to treat autoimmune diseases, and particularly systemic lupus, is also tempting because
female 9- to 12-mo-old p21 knockout mice develop a severe lupuslike disease associated with high levels of anti-dsDNA Abs,
renal involvement, and early mortality (12). Nonfunctional
p21WAF1/CIP1 protein allows excessive activation and proliferation
of otherwise quiescent, low avidity, self-reactive T cells (13). Reduced activation-induced cell death (AICD) can also result from
the absence of p21WAF1/CIP1, a situation analogous to that observed in mice defective in Fas or Fas ligand genes (14). In addition, the lupus-like syndrome associated with the deletion of the
p53-related p21WAF1/CIP1 gene or of the p53 effector gene Gadd45
is worse in mice lacking both p21 and Gadd45 genes (15). Those
data are in line with other observations in humans: decreased expression of p21WAF1/CIP1 in lymphocytes of patients with SLE is
associated with severe forms of the disease including renal involvement (16).
However, the relationship between p21WAF1/CIP1 and the development of SLE is still controversial because a recent report mentioned that the deletion of the p21WAF1/CIP1 gene protects BXSB
mice from autoimmunity through increased susceptibility of activated/memory B and T cells to AICD (17). To avoid any manipulation of the genetic autoimmune background, we chose to use a
new technology that has not been applied to autoimmune diseases
thus far; cell cycle inhibitor peptide therapy allows to silence genes
0022-1767/05/$02.00
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Systemic lupus erythematosus (SLE) is a progressive autoimmune disease characterized by the production of high levels of
affinity-matured IgG autoantibodies to dsDNA and, possibly, visceral involvement. Pathogenic autoantibodies result from the
activation and proliferation of autoreactive T and B lymphocytes stimulated by epitopes borne by nucleosomal histones. To inhibit
the proliferation of autoreactive cells and abrogate the development of SLE, a novel tool, cell cycle inhibiting peptide therapy, was
used. Thus, a peptidyl mimic of p21WAF1/CIP1 that inhibits the interaction between cyclin-dependent kinase 4 and type D cyclins
abrogated the in vitro proliferative response of T cells to histones and T-independent and T-dependent proliferative responses of
B cells. The WAF1/CIP1 mimic also abrogated the in vitro production of total and anti-dsDNA IgG Abs by B cells. Similarly, the
p21WAF1/CIP1 construct inhibited the ex vivo T and B cell proliferative responses to histones and decreased the numbers of
activated/memory B and T spleen cells. The alterations in the balance of spleen cell subsets resulted from proapoptotic effects of
the p21WAF1/CIP1 construct on activated splenocytes. Finally, in vivo, four i.v. injections of the p21WAF1/CIP1 mimic were sufficient
to inhibit the progression of the lupus-like syndrome in (NZB ⴛ NZW)F1 mice. The levels of anti-dsDNA IgG autoantibodies and
the incidence and severity of renal involvement were lower in treated mice than in nontreated mice. Those observations open new
avenues for the treatment of SLE and prompt us to evaluate the potential interest of peptidic therapy in human SLE. The Journal
of Immunology, 2005, 175: 6959 – 6967.
6960
or proteins transiently, and offers the same advantages as small
interfering RNA used to silence target genes (for review, see Ref.
18). To this end, we have designed a peptidyl mimic of p21WAF1/
CIP1 linked to a fusogenic peptide that allows the penetration
through cell membranes and inhibits cell proliferation (19).
This strategy has allowed us to better define the role of p21 in
the lupus-like syndrome of (NZB ⫻ W)F1 mice. In addition, because cell-penetrating peptides are currently tried in phase II clinical trials, our work has also aimed at testing whether such peptide
therapy could be applied to the treatment of patients with SLE.
Materials and Methods
Mice
Lupus-prone female (NZB ⫻ NZW)F1 (H-2d/z) mice (Harlan) were used at
8 –10 wk of age and received humane care in compliance with institutional
guidelines.
Peptides
Flow cytometric analysis of p21WAF1/CIP1 construct uptake
Internalization of the p21WAF1/CIP1 construct complexed with FITC-labeled
streptavidin was performed as described previously (20). A 100 ␮M solution of biotinylated peptide was preincubated with 100 ␮M FITC-labeled
streptavidin at room temperature for 30 min in Opti-MEM I. Peptide complexes (1 ␮M) were added to washed splenocytes (2 ⫻ 105 cells) in OptiMEM I at 37°C for 4 h. After 3 h, 1 ␮g of PE-labeled anti-B220 Ab (BD
Pharmingen) and 1 ␮g of PerCP-labeled anti-CD4 Ab (BD Pharmingen)
were added into the cell suspension. Cells were washed twice in PBS and
used directly for flow cytometric analysis. Internalization of peptide complexes was determined with a FACSCalibur flow cytometer (BD Biosciences); 30,000 cells were analyzed per data point, and all experiments
were conducted in triplicates.
In vitro T cell proliferation and cytokine release
Spleen cells were isolated by gentle disruption of the tissues and the erythrocytes lysed by hypotonic shock in potassium acetate solution. Spleen
cells were cultured in RPMI 1640 supplemented with antibiotics, Glutamax
(Invitrogen Life Technologies), and 10% heat-inactivated FCS (Invitrogen
Life Technologies) (complete medium). The proliferation assay was conducted in 96-well flat-bottom plates. Briefly, spleen cell suspensions (2 ⫻
105 cells) were cultured in complete medium for 48 h in the presence of 50
␮g/ml histone H2A, H2B, H3, or H4 subunits (Boehringer Mannheim) or
with 10 ␮g/ml anti-CD3 mAb (BD Pharmingen). Cell proliferation was
determined by pulsing the cells with [3H]thymidine (1 ␮Ci/well) during the
last 14 –16 h of culture and measuring the radioactivity incorporated by
liquid scintillation counting. The proliferative ratio was calculated as follows: cpm in stimulated T cells vs cpm in unstimulated T cells. IL-2 concentration was quantified using the IL-2-dependent cell line CTLL-2.
Briefly, 4 ⫻ 103 CTLL-2 cells were added to serial dilutions of rIL-2
(Tebu) or test samples in 96-well flat-bottom plates. After 24 h, cell proliferation was determined as above.
B cell in vitro proliferation and AAb production
Spleen cell suspensions isolated as described above were depleted of macrophages by plastic adherence for 3 h at 37°C and of T cells by negative
selection on 100-mm plates (Costar) coated with an anti-Thy-1.2 mAb (BD
Pharmingen). The purity of remaining B cells was higher than 90% as
assessed by FACS using a PE-anti-B220 mAb (BD Pharmingen). The B
cell proliferation assay was conducted in 96-well flat-bottom plates.
Briefly, splenic B cell suspensions (2 ⫻ 105 cells) were cultured in complete medium for 48 h with 50 ␮g/ml LPS and 5 ng/ml mouse rIL-4 (Tebu),
with 2 ␮g/ml anti-CD40 mAb (BD Pharmingen) and 5 ng/ml mouse rIL-4
(Tebu), or with 50 ␮g/ml histone H2A, 5 ng/ml mouse rIL-4 (Tebu), and
2 ␮g/ml anti-CD40 mAb (BD Pharmingen). B cell proliferation was determined as above. For analysis of in vitro production of AAbs, 106 splenic
B cells cultured in 24-well plates were stimulated with LPS and rIL-4, or
anti-CD40 mAb and rIL-4 with or without histone H2A, as above. Supernatants were collected 5 days later, and anti-dsDNA, total IgG, and IgM Ab
levels were determined as described below.
AAb determinations
Levels of total IgG and IgM Abs, levels of anti-dsDNA and anti-cardiolipin
IgG and IgM Abs, and levels of rheumatoid factors were measured using
standard ELISA (21). Anti-mouse IgG (10 ␮g/ml), anti-mouse IgM (Sigma-Aldrich; 10 ␮g/ml), calf thymus DNA (Sigma-Aldrich; 5 ␮g/ml), cardiolipin (Sigma-Aldrich; 50 ␮g/ml), or 10 ␮g/ml human IgG were coated
onto ELISA plates (precoated with protamine sulfate when dsDNA was
used as substrate) overnight at 4°C. Plates were then blocked with PBS-1%
BSA, washed, and 1/50 mouse serum or 1/2 culture supernatants (antidsDNA), or 1/1000 culture supernatants (total IgG or IgM Abs) were added
and allowed to react for 1 h at room temperature. After 5 washes, bound
Abs were detected with alkaline phosphatase-conjugated goat anti-mouse
IgM or IgG (Sigma-Aldrich) and the ELISA was developed by adding the
alkaline phosphatase substrate, p-nitrophenyl phosphate (Sigma-Aldrich).
Optical densities were measured at 405 nm using a Dynatech MR 5000
microplate reader (Dynex Technology). For each determination, optical
densities from blank wells (no Ag coated) were subtracted.
Flow cytometry analysis of splenocyte subsets
Cell suspensions from spleens were prepared after lysis of erythocytes in
potassium acetate solution. Cells were incubated with the appropriate labeled Ab at 4°C for 45 min in PBS with 0.1% sodium azide and 5% normal
rat serum to block nonspecific binding. Cell suspensions were then subjected to two- or three-color analysis on a FACSCalibur flow cytometer
(BD Biosciences). mAbs used in this study (BD Pharmingen) were PEconjugated anti-B220, FITC-conjugated anti-CD80, FITC-conjugated antiCD86, PE-conjugated anti-CD44, FITC-conjugated anti-CD69, and CyChrome-conjugated anti-CD4 Abs.
Inhibition of the CDK4/CDK6-cyclin D pathway by the
p21WAF1/CIP1 construct
Splenic B and T cell suspensions were prepared as above. B cells were
stimulated with 50 ␮g/ml LPS and 5 ng/ml mouse rIL-4 (Tebu) or with 2
␮g/ml anti-CD40 mAb (BD Pharmingen) and 5 ng/ml mouse rIL-4 (Tebu).
T cells were stimulated with 10 ␮g/ml anti-CD3 mAb (BD Pharmingen) or
with 3 ␮g/ml ionomycin (Sigma-Aldrich) and 250 ng/ml PMA (SigmaAldrich). Stimulated cells were treated with the p21WAF1/CIP1 construct or
with the control peptide. Cells were collected after 24 h of stimulation for
flow cytometric analysis of RB phosphorylation at residue Ser807/811.
Briefly, cells were collected and fixed with 1% paraformaldehyde in PBS
at 37°C for 10 min. Cells were then permeabilized with 100% ice-cold
methanol (final concentration, 90%) for 30 min at 4°C. After two washes
in PBS with 0.5% BSA,106 cells were incubated with 1/100 antiPhospho-Rb Ab (Ser807/811) (New England Biolabs) for 30 min at room
temperature. After two washes in PBS, 0.5% BSA, cells were incubated
with 1:100 FITC donkey anti-rabbit IgG (Jackson Immunoresearch) for 30
min at room temperature. After two washes, cells were resuspended in 0.5
ml PBS and analyzed using a FACSCalibur (BD Biosciences) flow cytometer. Forward and side scatters were used to establish size gates and exclude cellular debris from the analysis. The excitation wavelength was 488
nm, and the observation wavelength was 530 nm for green fluorescence.
Cells were examined at a flow rate of 100 to 200 events/s, and 30,000
events were analyzed per sample.
Spectrofluorimetric analysis of apoptosis induced by the
p21WAF1/CIP1 construct
Splenocytes (5 ⫻ 105 cells) were incubated for 24 h with either medium
alone or a combination of 10 ␮g/ml anti-CD3 mAb and 10 ␮g/ml antiCD28 mAb (BD Pharmingen) or 10 ␮M of the p21WAF1/CIP1 peptide, or a
combination of 10 ␮g/ml anti-CD3 mAb and 10 ␮g/ml anti-CD28
mAb (BD Pharmingen) along with 10 ␮M concentrations of the
p21WAF1/CIP1construct. Splenocytes (5 ⫻ 105 cells) treated as described
above were also incubated with 200 ␮M concentrations of the caspase-8
inhibitor (IETD-CHO) or with 200 ␮M concentrations of the caspase-3
inhibitor (DEVD-CHO) or with medium alone (no caspase inhibitor added).
After two washes in PBS, cells were incubated with 2 ␮M YO-PRO-1 (Molecular Probes) for 15 min. Apoptosis was assessed spectrofluorimetrically
(Victor2; PerkinElmer). Results are expressed as the percentage of apoptotic
cells ⫾ SEM vs cells in culture medium alone (0% apoptosis) and cells treated
with 40 ␮M paclitaxel (100% apoptosis).
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The synthetic peptide KRRQTSMTDFYHSKRRLIFS based on the sequence of the 141–160 aa from p21WAF1/CIP1 was synthesized and fused
with a carrier peptide, AAVALLPAVLLALLA, formed by the membranetranslocating hydrophobic sequence derived from the predicted signal peptide sequence of fibroblast growth factor (19) (Neosystem). A peptide
based on the sequence of the 141–160 aa from p21WAF1/CIP1 but lacking the
carrier peptide was used as control.
p21WAF MIMIC AMELIORATES MURINE LUPUS
The Journal of Immunology
6961
Assessment of autoimmune disease in (NZB ⫻ NZW)F1 mice
Results
Ten-week-old (NZB ⫻ NZW)F1 mice were injected i.v. six times every 15
days either with 100 ␮l of saline (control group, 10 mice), or 100 ␮g of
p21WAF1/CIP1 construct in 100 ␮l saline (p21WAF1/CIP1 construct group, 10
mice), or 100 ␮g of p21WAF1/CIP1 peptide lacking the fusogenic part of the
peptide in 100 ␮l of saline (control p21WAF1/CIP1 peptide group, 10 mice).
Animals were killed on day 120 after the first injection, and their spleens
and blood were collected. At the time the mice were killed, serum blood
urea nitrogen (BUN) and creatinine levels were assayed using standard
techniques for laboratory determinations. Serum IgM and IgG anticardiolipin Abs, IgM and IgG anti-dsDNA Abs, and rheumatoid factors
were assayed by ELISA as described previously. Analysis of proliferative
T and B cell responses and flow cytometric analysis of B and T cell subsets
were studied ex vivo in the spleen of each animal.
Proteinuria was evaluated in a blinded manner by testing early morning
urine using Chemistrips (Boehringer Mannheim). Proteinuria was defined
and scored as follows: 0 ⫽ no proteinuria; 1⫹ ⫽ ⱕ30 mg/dl protein;
2⫹ ⫽ 30 –100 mg/dl protein; 3⫹ ⫽ 100 –500 mg/dl protein; 4⫹ ⫽ ⬎500
mg/dl protein (22).
For histological studies, kidneys were removed from mice at the time
they were killed. The left kidney was fixed in 10% neutral buffered formalin, and the right kidney was snap-frozen in OCT compound and stored
at ⫺70°C. The formalin-fixed tissue was sectioned and treated with H&E,
periodic acid-Schiff, and Masson stains. The degree of glomerular damage
was assessed, using the National Institutes of Health activity score system
(23), by an assessor who was blinded with regard to the source of the
sample. Briefly, each sample was assessed on the basis of cellular proliferation, leukocyte infiltration, cellular crescents, and wire loop formation.
Each of these elements was scored 0 (normal), 1 (mild), 2 (moderate), or
3 (severe abnormality). The maximal possible score was 12 points.
Immunofluorescence staining of cryosectioned kidneys was used to
evaluate IgG deposition. Cryosections were fixed in acetone and blocked
with 5% skim milk in PBS. Samples were stained with 10 ␮g/ml FITCconjugated goat anti-mouse IgG (Sigma-Aldrich) in PBS, 0.1% BSA for
1 h at room temperature. Images were viewed and captured using an Olympus IX50 image system (Olympus). IgG deposition was scored on a 0 –3
scale as described previously (22): 0 ⫽ undetectable; 1 ⫽ detectable; 2 ⫽
moderate intensity of staining or ⬎50% of glomeruli with IgG deposits;
and 3 ⫽ severe or ⬎75% of glomeruli with IgG deposits.
The survival rate was studied in sick 31-wk-old mice with anti-dsDNA
Abs and proteinuria. Three groups of five mice were treated as above with
either the p21WAF1/CIP1 construct or the control peptide or remained
untreated.
Flow cytometric analysis of p21WAF1/CIP1 construct uptake
The statistical significance of differences between experimental treated
groups and untreated controls was analyzed by the Mann-Whitney test or
by Student’s t test for comparison of means. p ⬍ 0.05 was accepted as
significant.
FIGURE 1. Flow cytometric analysis of the uptake
of p21WAF1/CIP1 construct. p21WAF1/CIP1 peptides complexed with FITC-labeled streptavidin were added to
splenocytes at 37°C for 4 h. Peptide uptake was evaluated by flow cytometric analysis of B220⫹ B cells and
of CD4⫹ T cells. Experiments were conducted in triplicates. R1, Gated CD4⫹ T cells; R2, gated B220⫹ cells.
Inhibition of in vitro proliferative T and B cell responses by the
p21WAF1/CIP1 construct
The incubation of splenocytes from (NZB ⫻ NZW)F1 mice with
the p21WAF1/CIP1 construct resulted in a dose-dependent decrease
in T cell-proliferative responses to histones H2A and H2B or H3
and H4, with an IC50 of ⬍2.5 ␮M and in the T cell-proliferative
response induced by an anti-CD3 mAb (Fig. 2, left). In contrast,
IL-2 production in response to histone- or anti-CD3 stimulation
was less influenced by the p21WAF1/CIP1 construct than the proliferative responses. The IC50 was always higher than 10 ␮M (Fig. 2,
middle).
The T-independent proliferation of splenic B cells from (NZB ⫻
NZW)F1 mice stimulated by LPS ⫹ IL-4 was significantly
and dose-dependently reduced by 2.5, 5, and 10 ␮M of the
p21WAF1/CIP1 construct ( p ⬍ 0.02, p ⬍ 0.001, p ⬍ 0.001, respectively). B cell proliferation upon T-dependent stimulation by antiCD40 ⫹ IL4 with or without H2A, was significantly inhibited by
the p21WAF1/CIP1 construct. The IC50 was ⬍5 ␮M (Fig. 2, right).
The control p21WAF1/CIP1 peptide lacking the fusogenic region
did not significantly affect the T and B cell-proliferative responses
except at the highest concentrations, probably because of passive
diffusion of the peptide through the cell membranes.
Effect of the p21WAF1/CIP1 construct on in vitro Ab production
Polyclonal stimulation of B cells by LPS ⫹ IL-4, or anti-CD40 ⫹
IL-4, or anti-CD40 ⫹ IL-4 ⫹ histone H2A induced their differentiation into plasma cells as evidenced by the production of IgM
in the supernatants. Whatever the concentrations of stimuli tested,
the p21WAF1/CIP1 construct did not inhibit total IgM production but
dose-dependently decreased the concentration of IgM Abs specific
for dsDNA (Fig. 3). This inhibitory effect was stronger upon Tdependent than upon T-independent stimulations. Indeed, adding 5
␮M p21WAF1/CIP1 construct to B cells, decreased the production of
anti-dsDNA IgM Abs induced by LPS ⫹ IL-4 ⫻ 19% ( p ⬍ 0.05),
compared with 45 and 40% upon stimulation with anti-CD40 ⫹
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Statistical analysis
After 4 h of incubation with the p21WAF1/CIP1 construct, 52 ⫾ 7%
of B220⫹ B cells and 61 ⫾ 8% of CD4⫹ T cells have taken up the
construct (Fig. 1).
6962
p21WAF MIMIC AMELIORATES MURINE LUPUS
IL-4 ( p ⬍ 0.01) or anti-CD40 ⫹ IL-4 ⫹ histones H2A ( p ⬍ 0.01),
respectively.
The p21WAF1/CIP1 construct dose-dependently decreased the Tdependent and the T-independent productions of total IgG and of
anti-dsDNA IgG Abs. The inhibitory effect of the p21WAF1/CIP1
construct was stronger on IgG anti-dsDNA production than on
IgM anti-dsDNA Abs, with an inhibitory effect constantly higher
than 50% when using 5 ␮M construct ( p ⬍ 0.001; Fig. 4).
Inhibition of ex vivo T and B cell proliferative responses by the
p21WAF1/CIP1 construct
The proliferative ratio of splenocytes stimulated by histone H4 was
6.22 ⫾ 0.58 vs nonstimulated cells. In mice treated with four i.v.
injections of p21WAF1/CIP1 construct, the proliferative response of
splenocytes to histone H4 was significantly lower ( p ⬍ 0.01) than
in control mice injected with saline (Table I). Incubation of splenocytes from mice of the various groups with anti-CD3 Abs induced
a polyclonal T cell response that was also reduced after in vivo
treatment with the p21WAF1/CIP1 construct in vivo. The ex vivo
proliferative T cell responses and the T-dependent and T-independent B cell-proliferative responses were significantly abrogated after in vivo treatment with the p21WAF1/CIP1 construct ( p ⬍ 0.01)
(Table I).
Inhibition of the CDK4/CDK6-cyclin D pathway by the
p21WAF1/CIP1 construct
The mitogenic stimulation of splenocytes induced phosphorylation
of pRB fragments at residue Ser807/811. This observation confirmed the involvement of the CDK4/CDK6-cyclin D-dependent
pathway in cell cycle progression (red line) (24, 25). Coincubation
of activated splenocytes with the p21WAF1/CIP1 construct inhibited
phosphorylation of RB (blue line) in all cases (Fig. 5).
Treatment of (NZB ⫻ NZW)F1 mice with the p21WAF1/CIP1
construct depletes splenic activated T and B cell subsets
The p21WAF1/CIP1 construct not only induced a significant decrease in the absolute numbers of spleen cells vs untreated mice
( p ⬍ 0.02) but also altered the balance between cell populations
(Table II). The p21WAF1/CIP1 construct significantly decreased the
total numbers of CD4 T cells ( p ⬍ 0.01) and B cells ( p ⬍ 0.05)
and T cells with an activated/memory phenotype CD4⫹CD69⫹
( p ⬍ 0.001) and CD4⫹CD44high ( p ⬍ 0.01). The ratios of CD69⫹
to CD4⫹ T cells and CD44high to CD4⫹ T cells were 11 and 42%
in treated mice, respectively, vs 18 and 65% in untreated mice,
respectively. The levels B220⫹B7.1⫹ and B220⫹B7.2⫹ activated
B cells also decreased significantly ( p ⬍ 0.01 vs untreated mice)
and were more severely depleted than the nonactivated B cell subsets by the p21WAF1/CIP1 construct.
Treatment of (NZB ⫻ NZW)F1 mice with the p21WAF1/CIP1
construct inhibits the development of murine lupus
We then investigated whether the bimonthly treatment of 10-weekold (NZB ⫻ NZW)F1 mice with the p21WAF1/CIP1 construct for
120 days can affect the severity of SLE. At the time the mice were
killed, IgM Abs to dsDNA, cardiolipin, and rheumatoid factors
were not significantly altered vs nontreated controls (Fig. 6). By
contrast, anti-dsDNA IgG and anti-cardiolipin IgG Abs were significantly lower in mice treated with p21WAF1/CIP1 construct than
in animals treated with the control peptide or saline ( p ⬍ 0.05; p ⬍
0.0.05, respectively). Moreover, BUN, creatinine levels, and proteinuria were significantly reduced after administration of
p21WAF1/CIP1 construct ( p ⬍ 0.02, p ⬍ 0.01, and p ⬍ 0.001, respectively) compared with untreated mice. Renal involvement was
confirmed by a significant decrease in IgG glomerular deposits
and glomerular histological lesions in mice treated with the
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FIGURE 2. In vitro effects of the p21WAF1/CIP1 construct on splenic T and B cell proliferation and IL-2
release. The T cell proliferation assay was conducted in
the presence of histones H2A and H2B, or H3 and H4
subunits, or anti-CD3 mAb. Cells were coincubated
with various amounts (0, 2.5, 5, 10 ␮M) of the
p21 WAF1/CIP1 peptide fused with the membranetranslocating hydrophobic sequence derived from the
predicted signal peptide sequence of fibroblast growth
factor (p21WAF1/CIP1 construct). The same p21WAF1/CIP1
peptide lacking the carrier peptide was used as control
(control peptide). Cell proliferation was determined by
[3H]thymidine incorporation. IL-2 concentration was
quantified in the supernatant of the T cell proliferation
assay using the IL-2-dependent cell line CTLL-2 cells.
The B cell proliferation assay was conducted in the
presence of LPS ⫹ rIL-4, or with anti-CD40 mAb ⫹
rIL-4 with or without histone H2A. B cell proliferation
was determined as above. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.02;
ⴱⴱⴱ, p ⬍ 0.01; ⴱⴱⴱⴱ, p ⬍ 0.001 for the comparison of
cells treated with p21WAF1/CIP1 construct vs control
peptide. ‡, p ⬍ 0.05; ‡‡, p ⬍ 0.02; ‡‡‡, p ⬍ 0.01; ‡‡‡‡,
p ⬍ 0.001 for the comparison of cells treated with
control peptide vs saline (gray bars).
The Journal of Immunology
6963
FIGURE 4. In vitro effects of the p21WAF1/CIP1 construct on the levels
of total IgG and anti-dsDNA IgG Abs produced by splenic B cells. For
analysis of in vitro production of AAbs, 106 splenic B cells cultured in
24-well plates were stimulated with LPS and rIL-4 or anti-CD40 mAb and
rIL-4 with or without histone H2A. Supernatants were collected 5 days
later, and total IgG and IgG anti-dsDNA levels determined by standard
ELISA. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.02; ⴱⴱⴱ, p ⬍ 0.01; ⴱⴱⴱⴱ, p ⬍ 0.001 for the
comparison of mice treated with the p21WAF1/CIP1 construct vs mice treated
with control peptide. ‡, p ⬍ 0.05 for the comparison of mice treated with
control peptide vs untreated mice.
p21WAF1/CIP1 construct ( p ⬍ 0.02 and p ⬍ 0.001, respectively) vs
untreated animals. The p21WAF1/CIP1 construct also reduced the
incidence of renal involvement because histological lesions were
present in 100% of untreated and 100% of control peptide-treated
mice, vs 30% of animals treated with the p21WAF1/CIP1 construct
( p ⬍ 0.01). The survival rate was significantly higher in the group
treated by the p21WAF1/CIP1 construct than in the untreated group
from wk 41 to wk 55 on ( p ⬍ 0.05) (data not shown).
Discussion
The p21WAF1/CIP1 construct mediates apoptosis of splenocytes
Incubation of splenocytes with the p21WAF1/CIP1 construct or with
anti-CD3 ⫹ anti-CD28 mAbs induced cell death as evidenced by
the increase in YO-PRO-1 staining of the treated cells ( p ⬍ 0.01
and p ⬍ 0.001 vs untreated cells, respectively) (Fig. 7). Coincubation of splenocytes with the p21WAF1/CIP1 construct in combination with anti-CD3 ⫹ anti-CD28 mAbs had an additive effect
( p ⬍ 0.001 vs untreated cells). However, whereas AICD mediated
by anti-CD3 ⫹ anti-CD28 mAbs was significantly decreased by
inhibitors of caspase-3 ( p ⬍ 0.001) and ⫺8 ( p ⬍ 0.01), apoptosis
mediated by p21WAF1/CIP1 construct was inhibited only by the
caspase-3 inhibitor DEVD-CHO.
For the first time, a cell cycle inhibitor peptide has been used to
abrogate the progression of lupus-like syndrome in female
(NZB ⫻ NZW)F1 mice. A short peptide, p21WAF1/CIP1, able to
interfere with intracellular signaling pathways and fused with a
hydrophobic carrier peptide, has been used to inhibit cell proliferation and to kill activated/memory B and T cells in mice with
lupus-like syndrome.
Because the production of AAbs that characterize SLE is secondary to the activation of T cells, and particularly of histonespecific T cells (26, 27), we first assessed that the p21WAF1/CIP1
construct dose-dependently interferes with the splenic T cell proliferative response induced either by an anti-CD3 mAb polyclonal
T cell stimulator or by histones. The p21WAF1/CIP1 peptide interacts with the formation of cyclin-D/CDK4 complexes (28) and
thus inhibits Ag-induced and anti-CD3-mediated splenic T cell
proliferation. Cyclin D3, which plays a key role in T cell proliferation (29, 30), is induced by TCR ligation and CD28 costimulation (31, 32) and by chemical or growth factor stimulations (33,
34). Indeed, the immunosuppressive drug rapamycin abrogates
G1-S transition of the cell cycle through the repression of cyclin
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FIGURE 3. In vitro effects of the p21WAF1/CIP1 construct on the levels
of total IgM and anti-dsDNA IgM Abs produced by splenic B cells. For
analysis of in vitro production of AAbs, 106 splenic B cells cultured in
24-well plates were stimulated with LPS ⫹ rIL-4 or anti-CD40 mAb and
rIL-4 with or without histone H2A. Supernatants were collected 5 days
later and total IgM and IgM anti-dsDNA levels determined by standard
ELISA. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.02; ⴱⴱⴱ, p ⬍ 0.01; ⴱⴱⴱⴱ, p ⬍ 0.001 for the
comparison of cells treated with the p21WAF1/CIP1 construct vs control peptide. ‡, p ⬍ 0.05 for the comparison of cells treated with control peptide vs
saline (gray bars).
p21WAF MIMIC AMELIORATES MURINE LUPUS
6964
Table I. Effect of the p21WAF1/CIP1 construct on ex vivo T and B cell proliferative responsesa
Untreated
Control peptide
p21 construct
Histone H4
Anti-CD3
LPS ⫹ IL-4
Anti-CD40 ⫹ IL-4
6.22 ⫾ 0.58
5.14 ⫾ 0.50
4.10 ⫾ 0.33ⴱⴱⴱ
20.15 ⫾ 3.13
17.63 ⫾ 3.20
15.45 ⫾ 1.71
15.80 ⫾ 1.07
13.12 ⫾ 2.47
11.16 ⫾ 0.65ⴱⴱⴱ
8.56 ⫾ 0.65
7.76 ⫾ 0.67
6.00 ⫾ 0.27ⴱⴱⴱ
b
a
Ten-week-old (NZB ⫻ NZW)F1 mice were injected i.v. every 15 days (six times) either with 100 ␮l of saline (control
group, 10 mice) or with 100 ␮g of the p21WAF1/CIP1 construct in 100 ␮l of saline or with 100 ␮g of p21WAF1/CIP1 control peptide
in 100 ␮l of saline. Animals were sacrificed on day 120, and their spleens were collected for analysis of T and B cell proliferative
responses.
b
Proliferative ratio (cpm in stimulated cells:cpm in unsimulated cells). ⴱⴱⴱ, p ⬍ 0.01 vs untreated mice.
FIGURE 5. Inhibition of the CDK4/CDK6-cyclin D
pathway by the p21WAF1/CIP1 construct. Splenic B
cells were stimulated with LPS ⫹ rIL-4 or with antiCD40 mAb ⫹ rIL-4. Splenic T cells were stimulated
with anti-CD3 mAb or with ionomycin (Iono) and
PMA. Stimulated cells were treated by the p21WAF1/CIP1
construct (blue line) or by the control peptide (red line),
whereas control cells remained unstimulated (black
line). Cells were collected after 24 h for flow cytometric
analysis of RB phosphorylation at residue Ser807/811.
One representative experiment of three is shown.
such as thymidine, hydroxyurea, and bromodeoxyuridine, similarly inhibit IgG but not IgM production by mitogen-stimulated B
cells (41).
We then investigated whether the same observations hold true in
(NZB ⫻ NZW)F1 mice in vivo. As usual, those animals developed
a systemic autoimmune disease resembling human SLE in terms of
high levels of anti-dsDNA Abs that led to renal involvement (42).
Other AAbs were also produced, such as anti-cardiolipin Abs and
rheumatoid factors. Treating (NZB ⫻ NZW)F1 mice with the
p21WAF1/CIP1 construct did not affect the levels of anti-dsDNA
IgM Abs, anti-cardiolipin IgM Abs, or IgM rheumatoid factors but
significantly decreased IgG Abs to dsDNA and anti-cardiolipin vs
animals treated with the control peptide or saline. The incidence
of renal involvement is decreased by the treatment with the
p21WAF1/CIP1 construct. As shown by the determination of BUN,
creatinine levels, proteinuria, glomerular lesions, and IgG deposits,
the severity of kidney involvement was also significantly reduced
by the p21WAF1/CIP1 construct vs control peptide or saline. Furthermore, the curative treatment by p21WAF1/CIP1 construct significantly improved the survival rate of clinically sick mice.
As observed in vitro, the administration of the p21WAF1/CIP1
construct resulted ex vivo in the abrogation of T and B cell proliferative responses to nonspecific stimuli and to histone H4. However, this decrease in cell proliferation cannot explain, by itself, the
therapeutic benefits observed. Indeed, the lupus-like syndrome of
(NZB ⫻ NZW)F1 mice generally progresses despite the use of
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D3 levels and the impaired formation of active complexes with
CDK4/6 (35). However, as already observed with rapamycin (36),
the p21WAF1/CIP1 construct minimally affects IL-2 production in
our hands.
Because, in SLE, activated autoreactive T cells stimulate autoreactive B cells to proliferate, the p21WAF1/CIP1 construct also exerts inhibitory effects on T-dependent B cell activation and proliferation in vitro. This phenomenon is linked to the ability of the
p21WAF1/CIP1 construct to inhibit the interaction between cyclin D
and CDK4 as shown by the inhibition of pRb at the Ser807/811
residue. Indeed, cycle progression of normal B cells to S phase,
requires the accumulation of cyclin D2 and CDK4 and, to a lesser
extent, of CDK6 (37, 38). In addition, cyclin D3 can substitute for
cyclin D2 and stimulate mouse cell proliferation induced either by
Ag and CD40 or by LPS (39).
Blocking B cell proliferation by the p21WAF1/CIP1 construct also
inhibited the B cell differentiation process in vitro as evidenced by
the decreased production of Abs. Although the p21WAF1/CIP1 construct had no effect on total IgM Ab levels, it induced a profound
decrease in total IgG Abs and in T-dependent anti-dsDNA IgG
Abs. This particular effect on T-dependent IgG Abs is in line with
previous observations that switch recombinations are dependent on
cell division in conventional B lymphocytes (40). The weak effect
of the p21WAF1/CIP1 construct on IgM Abs is probably related to
the ability of B cells to produce IgM independently of cell proliferation. Indeed, other substances that inhibit cell proliferation,
The Journal of Immunology
6965
Table II. Effect of the p21WAF1/CIP1 construct on ex vivo T and B cell numbers in the spleensa
Untreated
Control peptide
p21 construct
Total
B220⫹
B220⫹B7.1⫹
B220⫹B7.2⫹
CD4⫹
CD4⫹CD69⫹
CD4⫹CD44⫹
120.4 ⫾ 20.0b
101.6 ⫾ 7.2
65.0 ⫾ 5.2ⴱⴱ
59.7 ⫾ 12.0
47.6 ⫾ 4.1
29.1 ⫾ 2.7ⴱ
8.0 ⫾ 1.7
5.0 ⫾ 0.6
2.8 ⫾ 0.4ⴱⴱⴱ
12.3 ⫾ 1.6
9.2 ⫾ 0.6
5.0 ⫾ 0.4ⴱⴱⴱⴱ
34.3 ⫾ 3.9
27.5 ⫾ 2.6
18.3 ⫾ 1.7ⴱⴱⴱ
6.1 ⫾ 1.0
4.7 ⫾ 0.5
2.0 ⫾ 0.2ⴱⴱⴱⴱ
22.1 ⫾ 3.9
17.1 ⫾ 1.6
7.8 ⫾ 0.6ⴱⴱⴱ
a
Ten-week-old (NZB ⫻ NZW)F1 mice were injected i.v. every 15 days (six times) either with 100 ␮l of saline (control group, 10 mice) or with 100 ␮g of the p21WAF1/CIP1
construct in 100 ␮l of saline or with 100 ␮g of p21WAF1/CIP1 control peptide in 100 ␮l of saline. Animals were sacrificed on day 120, and their spleens were collected. B and
T cell subsets were analyzed by flow cytometry.
b
⫻ 106. ⴱ p ⬍ 0.05; ⴱⴱ p ⬍ 0.02; ⴱⴱⴱ p ⬍ 0.01; vs untreated mice.
potent immunosuppressive drugs designed to interfere with cell
proliferation (43). Actually, those drugs target highly dividing
lymphocytes such as activated/memory-effector T cells and shortlived plasmablasts that represent ⬍60% of the splenic Ab-secret-
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FIGURE 6. In vivo effects of the
p21WAF1/CIP1 construct on AAb levels and on renal functions. Tenweek-old (NZB ⫻ NZW)F1 mice
were injected i.v. four times every 15
days either with 100 ␮l of saline
(control group, 10 mice) or with 100
␮g of p21WAF1/CIP1 construct in 100 ␮l
of saline (p21WAF1/CIP1 construct
group) or with 100 ␮g of control peptide lacking the fusogenic part peptide
in 100 ␮l of saline (control peptide
group). Animals were killed on day
120 after the first injection. At the time
the mice were killed, serum BUN and
creatinine levels were assayed using a
standard technique for laboratory determinations. Circulating IgM and IgG
anti-cardiolipin Abs, IgM and IgG antidsDNA Abs, and rheumatoid factors
were assayed by ELISA. Glomerular
IgG deposits, histological scores, and
proteinuria were determined as indicated in Materials and Methods. Renal pathology: A and B, in untreated
mice; C and D, control peptidetreated mice; E and F, p21WAF1/CIP1
construct-treated mice. ⴱ, p ⬍ 0.05;
ⴱⴱ, p ⬍ 0.02; ⴱⴱⴱⴱ, p ⬍ 0.001 vs
untreated controls.
ing cells in (NZB ⫻ NZW)F1 mice. In contrast, antiproliferative
drugs have limited effects on low or nondividing naive T cells, on
central memory T cells, and on long-lived plasma cells (44, 45).
Therefore, a treatment that combines cytostatic and cytotoxic
6966
effects on lymphoid cells will be certainly more effective in curing
autoimmune diseases. Because p21WAF1/CIP1 has already been
shown to induce B cell apoptosis (46), we were prompted to investigate whether the p21WAF1/CIP1 construct retains that proapoptotic
property on diseased lymphoid cells of (NZB ⫻ NZW)F1 mice.
In vitro, we have observed that the p21WAF1/CIP1 construct triggers apoptosis of mouse splenocytes in culture and potentiates antiCD3-mediated AICD. The p21WAF1/CIP1 construct-mediated apoptotic activity is dependent on caspase-3 but not on caspase-8. This
observation confirms not only the role of p21WAF1/CIP1 in lymphocyte apoptosis (46, 47) but also the involvement of the mitochondrial apoptotic pathway in that process, as already described
in p53-mediated apoptosis (48, 49). However, the proapoptotic activity of p21WAF1/CIP1 on lymphocytes and especially on autoreactive lymphocytes remains controversial. Indeed, in a recent
work, Lawson et al. (17) suggested that the deficiency in cyclin
kinase inhibitor p21 does not inhibit but promotes apoptosis of
activated/memory T and B cells, as evidenced by the decreased
number of both CD4⫹CD44high T cells and CD19⫹CD69⫹ B cells
in BXSB p21⫺/⫺ animals vs BXSB p21⫹/⫹ mice. However, the
same group, studying older animals, had previously shown that
CD44highCD4⫹ T cell and CD19⫹CD69⫹ B cell numbers are similar in BXSB p21⫺/⫺ and BXSB p21⫹/⫹ mice (50). In addition,
mice with the p21 gene deleted in the 129/Sv ⫻ C57BL/6 mixed
genetic background had more CD44high, CD4⫹ T cells in their
spleen than p21⫹/⫹ mice (12). These discrepancies may be related
to the use of knockout animals to investigate the involvement of
p21WAF1/CIP1 in autoimmune phenomena (12, 15, 50). Indeed,
backcrosses can have altered the autoimmune genetic background
and led to conflicting data. Our strategy that aims at increasing and
not abrogating the p21 pathway is more in line with the work of
Fotedar et al. (51) who have generated transgenic mice in which
the expression of the p21 transgene is restricted to the T cell lineage. In those mice, T cells are hypersensitive to cell death and are
rescued by the Bcl-2 transgene, thus confirming both the proapoptotic effect of p21 and the major role of the mitochondrial pathway
in p21-mediated cell death.
In summary, we propose cell cycle inhibitor peptide therapy as
a new treatment of SLE. The p21WAF1/CIP1 construct, which prevents the interaction of CDK4 with type D cyclins, inhibits the
proliferation of T and B cells and the T-dependent and T-indepen-
dent productions of IgG AAbs by B cells. We have also shown that
treating (NZB ⫻ NZW)F1 mice with active lupus-like syndrome
by the p21WAF1/CIP1 construct interrupts the course of the disease.
The p21WAF1/CIP1 peptide hits two targets: activated, dividing lymphocytes through its anti-proliferative properties; and nondividing,
long-lived cells through its proaopoptotic properties. Although the
p21WAF1/CIP1 construct acts as an immunosuppressant, no complications caused by immunodeficiency were observed in treated animals, in particular no infection was noted at necropsy. This peculiarity may be explained by the absence of significant reduction
in IL-2 production and the preservation of primary immune response with normal IgM Ab levels. Our observations in mice with
lupus-like syndrome suggest that patients with SLE could benefit
from similar peptide therapy.
Disclosures
The authors have no financial conflict of interest.
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