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The Journal of Immunology, 2005, 175: 6959-6967.
Copyright © 2005 by The American Association of Immunologists

A Mimic of p21WAF1/CIP1 Ameliorates Murine Lupus1

Claire Goulvestre2,*, Christiane Chéreau2,*, Carole Nicco*, Luc Mouthon{dagger}, Bernard Weill* and Frédéric Batteux3,*

* Laboratoire d’Immunologie and {dagger} Service de Médecine Interne, Faculté de Médecine René Descartes et Hôpital Cochin, Université Paris 5, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 x 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic 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 x 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 x NZB)F1 or (NZB x 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 lymphocyte activation and proliferation in (NZB x 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 lupus-like 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 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 x 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Lupus-prone female (NZB x 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

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 membrane-translocating 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.

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 µM solution of biotinylated peptide was preincubated with 100 µM FITC-labeled streptavidin at room temperature for 30 min in Opti-MEM I. Peptide complexes (1 µM) were added to washed splenocytes (2 x 105 cells) in Opti-MEM I at 37°C for 4 h. After 3 h, 1 µg of PE-labeled anti-B220 Ab (BD Pharmingen) and 1 µg 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 x 105 cells) were cultured in complete medium for 48 h in the presence of 50 µg/ml histone H2A, H2B, H3, or H4 subunits (Boehringer Mannheim) or with 10 µg/ml anti-CD3 mAb (BD Pharmingen). Cell proliferation was determined by pulsing the cells with [3H]thymidine (1 µCi/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 x 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 x 105 cells) were cultured in complete medium for 48 h with 50 µg/ml LPS and 5 ng/ml mouse rIL-4 (Tebu), with 2 µg/ml anti-CD40 mAb (BD Pharmingen) and 5 ng/ml mouse rIL-4 (Tebu), or with 50 µg/ml histone H2A, 5 ng/ml mouse rIL-4 (Tebu), and 2 µg/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 µg/ml), anti-mouse IgM (Sigma-Aldrich; 10 µg/ml), calf thymus DNA (Sigma-Aldrich; 5 µg/ml), cardiolipin (Sigma-Aldrich; 50 µg/ml), or 10 µg/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 (anti-dsDNA), 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 PE-conjugated anti-B220, FITC-conjugated anti-CD80, FITC-conjugated anti-CD86, 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 µg/ml LPS and 5 ng/ml mouse rIL-4 (Tebu) or with 2 µg/ml anti-CD40 mAb (BD Pharmingen) and 5 ng/ml mouse rIL-4 (Tebu). T cells were stimulated with 10 µg/ml anti-CD3 mAb (BD Pharmingen) or with 3 µg/ml ionomycin (Sigma-Aldrich) and 250 ng/ml PMA (Sigma-Aldrich). 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 anti-Phospho-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 x 105 cells) were incubated for 24 h with either medium alone or a combination of 10 µg/ml anti-CD3 mAb and 10 µg/ml anti-CD28 mAb (BD Pharmingen) or 10 µM of the p21WAF1/CIP1 peptide, or a combination of 10 µg/ml anti-CD3 mAb and 10 µg/ml anti-CD28 mAb (BD Pharmingen) along with 10 µM concentrations of the p21WAF1/CIP1construct. Splenocytes (5 x 105 cells) treated as described above were also incubated with 200 µM concentrations of the caspase-8 inhibitor (IETD-CHO) or with 200 µM 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 µM 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 µM paclitaxel (100% apoptosis).

Assessment of autoimmune disease in (NZB x NZW)F1 mice

Ten-week-old (NZB x NZW)F1 mice were injected i.v. six times every 15 days either with 100 µl of saline (control group, 10 mice), or 100 µg of p21WAF1/CIP1 construct in 100 µl saline (p21WAF1/CIP1 construct group, 10 mice), or 100 µg of p21WAF1/CIP1 peptide lacking the fusogenic part of the peptide in 100 µl 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 anti-cardiolipin 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+ = ≤30 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 µg/ml FITC-conjugated 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.

Statistical analysis

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Flow cytometric analysis of p21WAF1/CIP1 construct uptake

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).



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  		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 x 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 µM 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 µM (Fig. 2, middle).



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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 µM) of the p21WAF1/CIP1 peptide fused with the membrane-translocating 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. *, p < 0.05; **, p < 0.02; ***, p < 0.01; ****, p < 0.001 for the comparison of cells treated with p21WAF1/CIP1 construct vs control peptide. {ddagger}, p < 0.05; {ddagger}{ddagger}, p < 0.02; {ddagger}{ddagger}{ddagger}, p < 0.01; {ddagger}{ddagger}{ddagger}{ddagger}, p < 0.001 for the comparison of cells treated with control peptide vs saline (gray bars).

 
The T-independent proliferation of splenic B cells from (NZB x NZW)F1 mice stimulated by LPS + IL-4 was significantly and dose-dependently reduced by 2.5, 5, and 10 µM of the p21WAF1/CIP1 construct (p < 0.02, p < 0.001, p < 0.001, respectively). B cell proliferation upon T-dependent stimulation by anti-CD40 + IL4 with or without H2A, was significantly inhibited by the p21WAF1/CIP1 construct. The IC50 was <5 µM (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 T-dependent than upon T-independent stimulations. Indeed, adding 5 µM p21WAF1/CIP1 construct to B cells, decreased the production of anti-dsDNA IgM Abs induced by LPS + IL-4 x 19% (p < 0.05), compared with 45 and 40% upon stimulation with anti-CD40 + IL-4 (p < 0.01) or anti-CD40 + IL-4 + histones H2A (p < 0.01), respectively.



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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. *, p < 0.05; **, p < 0.02; ***, p < 0.01; ****, p < 0.001 for the comparison of cells treated with the p21WAF1/CIP1 construct vs control peptide. {ddagger}, p < 0.05 for the comparison of cells treated with control peptide vs saline (gray bars).

 
The p21WAF1/CIP1 construct dose-dependently decreased the T-dependent 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 µM construct (p < 0.001; Fig. 4).



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  		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. *, p < 0.05; **, p < 0.02; ***, p < 0.01; ****, p < 0.001 for the comparison of mice treated with the p21WAF1/CIP1 construct vs mice treated with control peptide. {ddagger}, p < 0.05 for the comparison of mice treated with control peptide vs untreated mice.

 
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).


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  		Table I. Effect of the p21WAF1/CIP1 construct on ex vivo T and B cell proliferative responsesa

 
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).



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  		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 anti-CD40 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.

 
Treatment of (NZB x 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.


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  		Table II. Effect of the p21WAF1/CIP1 construct on ex vivo T and B cell numbers in the spleensa

 
Treatment of (NZB x NZW)F1 mice with the p21WAF1/CIP1 construct inhibits the development of murine lupus

We then investigated whether the bimonthly treatment of 10-week-old (NZB x 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 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).



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  		FIGURE 6. In vivo effects of the p21WAF1/CIP1 construct on AAb levels and on renal functions. Ten-week-old (NZB x NZW)F1 mice were injected i.v. four times every 15 days either with 100 µl of saline (control group, 10 mice) or with 100 µg of p21WAF1/CIP1 construct in 100 µl of saline (p21WAF1/CIP1 construct group) or with 100 µg of control peptide lacking the fusogenic part peptide in 100 µl 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 anti-dsDNA 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 peptide-treated mice; E and F, p21WAF1/CIP1 construct-treated mice. *, p < 0.05; **, p < 0.02; ****, p < 0.001 vs untreated controls.

 
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.



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  		FIGURE 7. Effects of the p21WAF1/CIP1 construct on in vitro spleen cells apoptosis. Splenocytes were incubated either with medium alone or with anti-CD3 and anti-CD28 mAbs, or with the p21WAF1/CIP1 construct, or with a combination of anti-CD3 and anti-CD28 mAbs along with the p21WAF1/CIP1 construct. Splenocytes treated as described above were also incubated with the caspase-8 inhibitor (IETD-CHO) or with the caspase-3 inhibitor (DEVD-CHO). Apoptosis was assessed spectrofluorimetrically by YO-PRO-1 staining. Results are expressed as percent of apoptotic cells ± SEM vs cells in culture medium alone (0% apoptosis) and cells treated with 40 µM paclitaxel (100% apoptosis). **, p < 0.02; ***, p < 0.01; ****, p < 0.001 for each treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
For the first time, a cell cycle inhibitor peptide has been used to abrogate the progression of lupus-like syndrome in female (NZB x 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 histone-specific 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 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, 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 x 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 x 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 x NZW)F1 mice generally progresses despite the use of 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 short-lived plasmablasts that represent <60% of the splenic Ab-secreting cells in (NZB x 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 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 x NZW)F1 mice.

In vitro, we have observed that the p21WAF1/CIP1 construct triggers apoptosis of mouse splenocytes in culture and potentiates anti-CD3-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 x 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-independent productions of IgG AAbs by B cells. We have also shown that treating (NZB x 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 Benot Malassagne. Back

2 C.G. and C.C. contributed equally to this work. Back

3 Address correspondence and reprint requests to F. Batteux, Laboratoire d’Immunologie, Pavillon Hardy-Hôpital Cochin, 75679 Paris cedex 14, France. E-mail address: frederic.batteux{at}cch.ap-hop-paris.fr Back

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. Back

Received for publication February 17, 2005. Accepted for publication August 18, 2005.


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 Materials and Methods
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 Disclosures
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