Peptides based on the complementarity-determining regions of a pathogenic autoantibody mitigate lupus manifestations of (NZB x NZW)F1 mice via active suppression

Heidy Zinger1, Eran Eilat1, Asher Meshorer2 and Edna Mozes1

1 Department of Immunology and 2 Experimental Animal Center, The Weizmann Institute of Science, Rehovot 76100, Israel

Correspondence to: E. Mozes; E-mail: edna.mozes{at}weizmann.ac.il
Transmitting editor: S. Romagnani


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Two peptides based on the complementarity-determining regions (CDR) 1 and 3 (pCDR1 and pCDR3) of a murine monoclonal anti-DNA autoantibody that expresses the common idiotype 16/6Id were shown to down-regulate systemic lupus erythematosus (SLE)-associated T cell responses and to prevent the development of clinical symptoms in the SLE-prone mice, (NZB x NZW)F1. In the present study the ability of the CDR-based peptides to treat an already established disease was tested. Mice were given 10 weekly injections of peptides either i.v. or s.c. The treatment led to a moderate reduction in the anti-DNA autoantibody titer, and a significant decrease in proteinuria and kidney pathology. The CDR-based peptides affected the pathogenic isotypes (IgG2a and IgG3) of the anti-DNA antibodies in the serum and in immune complexes in the kidneys. Both peptides mitigated disease manifestations and prolonged the survival of mice that were treated starting at the age of 7 months when full-blown disease was already developed. Furthermore, some beneficial effects of treatment with the CDR-based peptides could be adoptively transferred to diseased recipients. A reduction in the secretion of IL-2, IFN-{gamma}, IL-4 and IL-10 was detected in supernatants of splenocytes of the treated mice. In contrast, treatment up-regulated the immunosuppresive cytokine-transforming growth factor-ß. Thus the ameliorating effect of the CDR-based peptides on SLE manifestations is at least partially via the immunomodulation of the cytokine profile.

Keywords: cytokine immunomodulation, epitope, experimental systemic lupus erythematosus, immunotherapy, in vivo animal model, peptide


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and defective T cell-mediated responses. The latter are associated with various clinical manifestations including immune complex depositions in the kidneys and other organs (1). Female (NZB x NZW)F1 mice develop spontaneously an SLE-like syndrome. Beginning at the age of ~3–4 months, autoantibodies, including anti-DNA antibodies, are developed in these mice and, by 7–8 months, immune complex deposits form in the kidneys. Mice die of the disease within 1 year (2).

Our previous studies have demonstrated that experimental SLE can be induced in mice of susceptible strains (e.g. BALB/c, SJL, C3H.SW) following their immunization with anti-DNA monoclonal autoantibodies that express a major idiotype designated 16/6Id of either human or mouse origin (3,4). Experimental SLE, although induced in mice that normally do not develop any symptoms of SLE, was found to share features with the disease of (NZB x NZW)F1 mice that develops spontaneously. Thus, high homology was demonstrated between the variable regions coding for the heavy and light chains of anti-DNA mAb isolated from mice afflicted with experimental SLE and the variable regions of anti-DNA mAb from (NZB x NZW)F1 mice (57).

We have previously shown that two peptides based on the sequences of the complementarity-determining regions (CDR) 1 and 3 of a murine anti-DNA 16/6Id+ mAb designated mAb 5G12 (8) were capable of inhibiting specific proliferation of lymph node cells of mice immunized with the same peptide, with the murine mAb 5G12 or with the human mAb anti-DNA, 16/6Id. Furthermore, the CDR-based peptides could prevent autoantibody production in neonatal mice that were immunized later either with the peptide or with the pathogenic autoantibody. We have recently shown that pCDR1 administered in PBS could either prevent or down-regulate experimental SLE induced in naive mice by affecting the production of autoantibodies and the severity of the clinical manifestations, including kidney damage (9). Moreover, in a previous work we have reported that the CDR-based peptides could prevent the spontaneous development of SLE in (NZB x NZW)F1 mice. Both peptides, pCDR1 and pCDR3, given in PBS to 2-month-old (NZB x NZW)F1 mice reduced the levels of anti-DNA autoantibodies and the clinical manifestations, including kidney damage, in the treated mice (10).

The above line of evidence prompted us to investigate whether the CDR-based peptides might be of therapeutic benefit in the treatment of an already established clinical SLE-like disease in (NZB x NZW)F1 mice. We report here that both peptides, pCDR1 and pCDR3, given in PBS to (NZB x NZW)F1 mice with lupus-like manifestations down-regulated autoantibody production as well as clinical symptoms. The beneficial effects of treatment were associated with a diminished production of the Th1-type cytokines (IL-2 and IFN-{gamma}) as well as Th2-type cytokines (IL-4 and IL-10), whereas secretion of the immunosuppressive cytokine transforming growth factor (TGF)-ß was elevated. Further, the beneficial effects of treatment could be transferred to diseased recipients by splenocytes of young (2-month-old) mice that were treated with either pCDR1 or pCDR3. Up-regulated levels of TGF-ß could be detected in supernatants of splenocytes of peptide-treated mice.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female (NZB x NZW)F1 mice (6–8 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained under standard conditions in the animal facility of The Weizmann Institute of Science.

Synthetic peptides
The peptides based on the CDR of the murine anti-DNA, 16/6Id mAb (5G12) were used in the study. The CDR1-based peptide TGYYMQWVKQSPEKSLEWIG (pCDR1) and the CDR3-based peptide YYCARFLWEPYAMDYWGQGS (pCDR3) (the CDR are underlined) were synthesized with an automated synthesizer (model 430A; Applied Biosystems, Weiterstadt, Germany) using the company‘s protocols for the t-BOC strategy (11). A peptide synthesized in the reversed order of pCDR1 (GIWELSKEPSQKVWQMYYGT) was used for control.

Treatment of mice with the CDR-based peptide
Groups of (NZB x NZW)F1 mice at the age of 5–7 months were treated with either pCDR1 or pCDR3. Control groups were treated with either the vehicle, PBS, only or with the control peptide, reversed pCDR1. Various concentrations (100, 250 or 500 µg/mouse) of the peptides were administered into the mice in PBS either i.v. or s.c., once a week for 10 weeks.

Transfer of spleen cells from animals treated with the CDR-based peptides
(NZB x NZW)F1 female mice (8–10 weeks old) received three s.c. injections (every other day) of 300 µg of pCDR1, pCDR3 or the control reversed pCDR1. Thereafter, spleen cells were harvested and injected i.p. (20 x 106/mouse) into 8-month-old (NZB x NZW)F1 recipients. The mice were followed for the presence of dsDNA-specific antibodies, proteinuria and immune complex deposits.

ELISA
For measuring anti-dsDNA antibodies, 96-well Maxisorb microtiter plates (Nunc, Roskilde, Denmark) were coated with poly-L-lysine (Sigma, St Louis, MO). The plates were then washed and coated with {lambda} phage dsDNA (Worthington Biochemical, Lakewood, NJ). After incubation with different dilutions of sera, goat anti-mouse IgG ({gamma} chain specific) conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) was added to the plates. For the determination of anti-DNA antibodies of various Ig isotypes, horseradish peroxidase conjugated to goat anti-mouse {gamma}1, {gamma}2a, {gamma}2b or {gamma}3 chain-specific antibodies (Southern Biotechnology Associates, Birmingham, AL) was used. Plates were then incubated with the substrate, ABTS [2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid); Sigma] and read at 405 nm using an ELISA reader.

Detection of proteinuria
Proteinuria was measured by a standard semi-quantitative test, using an Albustix kit (Bayer Diagnostic, Newbury, UK). Results were graded according to the manufacturer as: negative, + = 0.3g/l, ++ = 1g/l, +++ = 3g/l or ++++ = >=20g/l.

Immunohistology
Mice were sacrificed at the age of 8–9 months, and kidneys were removed and frozen immediately in liquid nitrogen. Frozen cryostat sections (5 µm) were air-dried and fixed in acetone. For the detection of Ig deposits, sections were incubated with FITC-conjugated goat anti-mouse IgG ({gamma} chain specific) (Jackson ImmunoResearch). For the determination of the various Ig isotypes FITC-conjugated goat anti-mouse IgG2a ({gamma}2a chain specific) or IgG3 ({gamma}3 chain specific) antibodies (Southern Biotechnology Associates) were used. Staining was visualized using a fluorescence microscope.

Histopathology
Mice were sacrificed and their kidneys were preserved frozen at –70°C, and then trimmed and routinely processed for light microscopy. Paraffin-embedded, 5-µm thick sections were stained with hematoxylin & eosin. Lesions were scored using semiquantitative grading as follows: 0 = no lesions, 1 = minimal lesions, 2 = mild lesions, 3 = moderate lesions and 4 = severe lesions. Histopathology was evaluated with the pathologist blinded to whether mice belonged to treated or untreated groups.

Cytokine production by splenocytes
Spleen cells (5 x 106 /ml) of the tested mice were incubated with either enriched medium only or concanavalin A (Con A; 2.5 µg/ml).Supernatants were removed after 24 h and analyzed for cytokine content. IL-2, IFN-{gamma}, IL-4 and IL-10 were determined by ELISA using OptEIA sets (PharMingen, San Diego, CA) and according to the manufacturer’s instructions. For detection of TGF-ß, plates were coated with a recombinant human TGF-ß sRII/Fc chimera. Supernatants were added after activation of latent TGF-ß1 to immunoreactive TGF-ß1 according to the manufacturer (R & D Systems) A standard recombinant TGF-ß1 was used as well. Thereafter, a biotinylated anti-human TGF-ß1 antibody was added and the assay was developed according to the manufacturer’s instructions (R & D Systems).

Detection of intracellular cytokines
Single-cell suspensions of spleens were exposed to a Cytoperm kit (Serotec, Oxford, UK) according to the company’s protocol. Thereafter, cells were incubated with the appropriate anti-cytokine–FITC conjugated antibody. Cells were assessed by a FACScan cytometer and the data was analyzed using Lysys software.

Statistical analysis
To evaluate the significance of the difference between untreated and treated groups, the Student’s t-test and the non-parametric Mann–Whitney test were used. Values of P <= 0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effect of treatment with pCDR1 using different routes
We have previously shown that treatment of (NZB x NZW)F1 mice by administration of either pCDR1 or pCDR3 at the age of 2 months either prevented the development of the SLE-like disease or resulted in a milder disease in the treated mice (10). It was of interest to find out whether treatment of (NZB x NZW)F1 mice at the age of 5 months, when SLE clinical symptoms are already detected, will affect the disease course. To this end, (NZB x NZW)F1 mice at the age of 5 months were injected with pCDR1 (100 or 250 µg/mouse) either i.v. or s.c., once a week for 10 weeks. A group of mice was treated similarly with the vehicle only. Animals were monitored for the presence of anti-dsDNA autoantibodies, levels of protein in the urine (proteinuria) and immune complex deposits in their kidneys. Table 1 demonstrates representative results of such an experiment. As shown in Table 1, i.v. treatment with 100 µg/mouse of pCDR1 resulted in a moderate reduction in anti-dsDNA antibody titers. Subcutaneous treatment either with 100 or 250 µg/mouse led to a similar reduction in the anti-DNA autoantibody titers (Table 1). Both administration routes led to a significant reduction in proteinuria and in the immune complex deposits in the kidneys of the pCDR1-treated mice (Table 1). Thus, analysis indicated that 50–55% of the kidneys in the groups of pCDR1-treated mice were completely free of immune complex deposits. Kidneys of the additional mice in the treated groups had immune complexes of lower intensity than in the control groups. Thus, both routes of administration of the pCDR1 are similarly effective in down-regulating the clinical symptoms of the SLE-like disease in the (NZB x NZW)F1 mice. Therefore, in further experiments the s.c. route was used.


View this table:
[in this window]
[in a new window]
 
Table 1. Comparison of the effects of the i.v. and s.c. treatment protocolsa
 
Treatment of mice by s.c. administration of pCDR3 at a dose of either 100 or 250 µg/mouse ameliorated the clinical manifestations similarly to pCDR1. Thus, levels of proteinuria were lower by 75–90% of those in the PBS-treated groups. Furthermore, in comparison to average intensity of immune complex deposits of 2 ± 0.37 in the group of PBS-treated mice, values of 1 ± 0.27 (P = 0.02) and 1 ± 0.42 (P = 0.04) were determined for kidneys in the groups of mice treated with 100 and 250 µg/mouse respectively.

Peptides pCDR1 and pCDR3 ameliorate specifically the SLE-like disease in (NZB x NZW)F1 mice
The specificity of the beneficial effects of pCDR1 and pCDR3 was also tested. Thus, groups (10 mice per group) of 5-month-old (NZB x NZW)F1 mice were treated s.c. with 250 µg/mouse of the CDR-based peptides or with a control peptide (reversed pCDR1) weekly for 10 weeks. As can be seen in Table 2, a decrease in anti-dsDNA antibody titer was determined in the sera of the treated mice as compared to either untreated mice or to mice that were treated with the reversed pCDR1. Table 2 also shows that the treatment with the CDR-based peptides significantly decreased the levels of proteinuria and severity of immune complex deposits in the kidneys (P < 0.05 for both pCDR1 and pCDR3 treatment protocols as compared to the untreated and reversed pCDR1-treated groups).


View this table:
[in this window]
[in a new window]
 
Table 2. Treatment with the CDR-based peptides down-regulates specifically the clinical manifestations in (NZB x NZW)F1 micea
 
Treatment of (NZB x NZW)F1 mice with pCDR1 and pCDR3 down-regulates the pathogenic isotypes of the anti-DNA autoantibodies
It was of interest to find out whether the reduction in anti-DNA autoantibody titers could be attributed to one or more of the IgG isotypes. Therefore, in addition to the total anti-DNA antibody detected in sera of individual mice, the sera were tested for the presence of anti-DNA antibodies of the various isotypes. Representative results are shown in Fig. 1. It can be seen in Fig. 1(A) that treatment with pCDR1 resulted in a slight decrease in anti-DNA antibodies of the IgG2a isotype, whereas the decrease in antibody titers of the IgG2b and IgG3 isotypes was much more pronounced. Figure 1(B) shows that treatment with pCDR3 led to a decrease mainly in the IgG2a and IgG2b antibody isotypes, and a very mild decrease in the IgG3 isotype. Similar results were obtained in three independent experiments.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Reduction of anti-DNA autoantibody titers. (NZB x NZW)F1 mice were treated with either PBS or the CDR-based peptides (A) pCDR1 or (B) pCDR3 starting at the age of 5 months (s.c., 250 µg/mouse). Mice were bled monthly and sera of individual mice were tested for anti-DNA antibody titer [total IgG and the various isotypes (sera dilution 1:40) as described in Methods]. Results are of sera of mice that were bled shortly after treatment and are representatives of three individual experiments.

 
Immunohistology examination of the immune complex deposits in kidneys of mice that were treated with the CDR-based peptide showed that the reduction in total IgG immune complex deposits could be also observed in the immune complexes of IgG2a and IgG3. Thus, it can be seen in Fig. 2 that treatment with pCDR1 (Fig. 2B) and pCDR3 (Fig. 2C) reduced the intensity of immune complex deposits, as compared to treatment with PBS (Fig. 2A), when the latter were assessed with antibodies to total IgG as well as with antibodies to IgG2a and IgG3.



View larger version (51K):
[in this window]
[in a new window]
 
Fig. 2. Immunohistology examination of immune complex deposits of total and IgG isotypes in kidneys of (NZB x NZW)F1 mice that were treated with the CDR-based peptides. Frozen cryostat sections (5 µm) of 8-month-old (NZB x NZW)F1 mice were air dried, fixed in acetone and stained with FITC-conjugated to goat anti-mouse IgG ({gamma} chain specific), goat anti-mouse IgG2a ({gamma}2a chain specific) or goat anti-mouse IgG3 ({gamma}3 chain specific). Representative kidney sections of (A) PBS-treated mice, (B) pCDR1-treated mice and (C) pCDR3-treated mice (x400).

 
Histopathological evaluation of kidneys of untreated (NZB x NZW)F1 mice and of mice treated with the CDR-based peptides
The histopathological findings are presented in Table 3 and Fig. 3. Three components of renal pathological changes were evaluated: glomerulosclerosis, nephropathy and interstitial lymphocytic infiltration. Glomerulosclerosis was diagnosed when the glomerular basement membrane appeared thickened. The process involved hyaline obliteration of the glomeruli, transforming them into acellular eosinophilic masses (crescentic formation). Nephropathy diagnosis was determined when the renal tubules appeared atrophic or regenerative, basophilic, having dilated lumen with presence of proteinaceous casts. The term interstitial lymphocytic infiltration was applied to define the presence of scattered and discrete interstitial lymphocytic and other mononuclear cell aggregations, present at the medullary regions. Increased severity of glomerulosclerosis and nephropathy was accompanied by dissemination of lymphocytic infiltration within cortical and medullary regions. The mean severities of the histopathological findings are presented in Table 3. The incidence and, in particular, the severity of these parameters were significantly reduced in the groups treated with the peptides, as compared to the group treated only with PBS. In particular, the severities of glomerulosclerosis and nephropathy, which are characteristic components of the spontaneous glomerulonephritis in (NZB x NZW)F1 mice, were prominently reduced in the pCDR1- and pCDR3-treated mice (Table 3). Figure 3 demonstrates kidney sections of mice that were given the vehicle, PBS, only (Fig. 3A and B) and of mice treated s.c. with 10 weekly injections of either pCDR1 (Fig. 3C) or pCDR3 (Fig. 3D). Figure 3(B) represents a kidney of a mouse with early stages of glomerulonephritis characterized by mesangial cell proliferation, whereas Fig. 3(A) depicts a kidney section of a mouse with a severe kidney damage: (a) glomerulosclerosis, (b) interstitial nephritis and (c) cast formation. The histological analysis of kidneys of mice treated with the CDR-based peptides (Fig. 3C and D) shows a normal pattern with no morphological changes.


View this table:
[in this window]
[in a new window]
 
Table 3. Histopathological findings in kidneys of (NZB x NZW)F1 mice
 


View larger version (150K):
[in this window]
[in a new window]
 
Fig. 3. Histopathology of kidney sections of (NZB x NZW)F1 mice untreated or treated with the CDR-based peptides. Groups (10 mice per group) of 5-month-old (NZB x NZW)F1 mice were treated s.c. with PBS only (controls) or with 250 µg/mouse of either pCDR1 or pCDR3 in PBS. The mice were treated once a week for 10 weeks. Paraffin-embedded, 5-µm thick sections of kidneys of mice that were sacrificed at the age of 8 months (~2 weeks after the end of treatment) were stained with hematoxylin & eosin. (A) A kidney section of a mouse given the vehicle, PBS, only. Immune glomerulonephritis. (a) Cresentic formation: thickening of Bauman’s capsule and glomerulosclerosis. (b) Interstitial nephritis (lymphocytes and other mononuclear cells). (c) Protein casts in the lumen of the tubuli. Late stage. (B) A kidney section of a mouse given the vehicle, PBS, only. Mesangial cell proliferation (mesangial proliferative glomerulonephritis). Early stage. (C) A kidney section of a mouse that was treated with the CDR1-based peptide. Normal pattern of kidney tissue. (D) A kidney section of a mouse that was treated with the CDR3-based peptide. Normal pattern of kidney tissue. Hematoxylin & eosin (x350).

 
Treatment of 7-month-old (NZB x NZW)F1 mice with the CDR-based peptides ameliorated SLE manifestations at the advanced stage
We have further tested the ability of both CDR-based peptides to mitigate the SLE-like manifestation in aged (NZB x NZW)F1 mice that suffer already from severe clinical manifestations. To this end 7-month-old (NZB x NZW)F1 mice in which high levels of dsDNA autoantibodies and high proteinuria have been detected were treated with the peptides once a week for 10 weeks by the administration of either 250 or 500 µg/mouse s.c. in PBS. Control mice were treated with PBS alone. Table 4 shows that, in addition to the observed reduction in dsDNA-specific antibody levels in all treated groups, lower proteinuria levels were measured as well. Beneficial effects were also observed when immune complex deposits were determined as demonstrated in Table 4. It is also shown in Table 4 that the survival rate was significantly higher in the groups treated with 250 µg of pCDR1 and with 250 and 500 µg of pCDR3 when compared to PBS-treated animals. Thus, >60% of the PBS-treated mice died before the last injection, whereas most of the treated mice survived and either were healthy or had mild disease manifestations. It is noteworthy that in the present experiment mice were treated with the 250 µg dose that was used in most experiments and with a higher dose of 500 µg/mouse. Nevertheless, the results indicate that the 250 µg dose is efficient enough in ameliorating disease manifestations.


View this table:
[in this window]
[in a new window]
 
Table 4. Treatment of 7-month-old (NZB x NZW)F1 mice with the CDR-based peptides reduces the clinical manifestations of their SLE-like disease
 
Treatment of (NZB x NZW)F1 mice with the CDR-based peptides affects their cytokine profile
Imbalance in the cytokine network plays an important role in the induction and development of experimental SLE (1214). It was therefore of interest to find out whether treatment with the CDR-based peptides affects the cytokine production by splenocytes of the treated mice. Thus, 5-month-old (NZB x NZW)F1 mice (10–12 mice per group) were treated weekly for 10 weeks with 250 µg/mouse of either pCDR1 or pCDR3 given s.c. in PBS.

Groups of mice were sacrificed after 5 weeks of treatment and at the end of treatment, and the cytokines secreted by their Con A-stimulated splenocytes were measured. Figure 4 demonstrates the cytokine secretion by splenocytes of untreated mice, and of pCDR1- and pCDR3-treated mice as measured following 5 weeks of treatment. It can be seen in Fig. 4 that the treatment reduced the secreted IL-2 and IFN-{gamma} (Th1-type) as compared to the untreated group. A lower production of IL-4 and IL-10 (reported to play a role in the pathogenesis of SLE) was also observed in the groups of mice treated with the CDR-based peptides. In contrast to the down-regulation of the Th1- and Th2-type cytokines, the secretion of the immunosuppressive cytokine, TGF-ß, was up-regulated. Similar results were obtained when the pattern of secreted cytokines was measured at the end of treatment with the CDR-based peptides. The immunomodulation of cytokine secretion by the CDR-based peptides appears to be specific, because peptides with the reversed sequence of the CDR-based peptides did not affect significantly the secretion of the tested cytokines. A down-regulation of 5–10% in the secretion of IFN-{gamma}, IL-2, IL-4 and IL-10 could be observed in supernatants of splenocytes taken from mice treated with the relevant reversed peptides. Occasionally treatment with the reversed peptides resulted in an increased production (by up to 20%) of the later cytokines as compared to the levels of cytokines in supernatants of splenocytes of untreated (NZB x NZW)F1 mice. Further, no significant increase in active TGF-ß levels could be determined in supernatants of splenocytes of mice that were treated with the reversed peptides. It is noteworthy that in most cases the levels of the Th1- and Th2-type cytokines were immunomodulated by the CDR-based peptides to levels measured in young (2-month-old) mice with no symptoms of the SLE-like disease. Thus levels of 652, 630, 142 and 1124 pg/ml of IL-2, IFN-{gamma}, IL-4 and IL-10 respectively were measured in supernatants of Con A-activated splenocytes of 2-month-old (NZB x NZW)F1 mice. These levels are very similar to those determined in supernatants of splenocytes taken from mice that were treated with the CDR-based peptides (Fig. 4). It should be noted that treatment with the CDR-based peptides up-regulated the secretion of TGF-ß to levels higher than those secreted by splenocytes of young mice. Intracellular staining of splenocytes of untreated and CDR-based peptide-treated mice revealed also a reduction in the number of cells expressing the various cytokines. Hence, a reduction of 40% in intracellular IL-2 was observed in splenocytes of both pCDR1- and pCDR3-treated mice. A 17 and 11% reduction in intracellular IFN-{gamma} was observed for pCDR1- and pCDR3-treated mice respectively, and a reduction of 40 and 30% for intracellular IL-4 and IL-10 respectively could be demonstrated for splenocytes of mice treated with either CDR-based peptide as compared to cells of untreated mice. Thus, treatment of (NZB x NZW)F1 mice with the CDR-based peptides down-regulates the secretion of the Th1- and Th2-type cytokines, and up-regulates the secretion of the immunosuppressive cytokine, TGF-ß. These results were reproducible in two independent experiments.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4. The effect of treatment with the CDR-based peptides on the cytokine profile. Five-month-old (NZB x NZW)F1 mice were treated with either pCDR1 or pCDR3, 250 µg/mouse given s.c. in PBS, once a week for 10 weeks. Mice were sacrificed after 5 weeks and at the end of treatment, and the cytokines secreted by their Con A-stimulated splenocytes were measured by ELISA as described in Methods. Summary of results of two experiments, obtained with supernatants of mice sacrificed after 5 weeks of treatment. *P < 0.05 as compared to untreated groups.

 
Amelioration of the clinical manifestations of (NZB x NZW)F1 mice by splenocytes of young mice that were treated with the CDR-based peptides
It was of interest to find out whether the beneficial effects of the treatment with the CDR-based peptides could be adoptively transferred with splenocytes of peptide-treated mice. To this end we treated 10-week-old (NZB x NZW)F1 mice, s.c. 3 times every other day, with pCDR1, pCDR3 or reversed pCDR1, 300 µg/mouse. At the end of this treatment the animals were sacrificed, and their splenocytes were either activated with Con A and tested for the secretion of the immunosuppressive cytokine TGF-ß or injected i.p. (20 x 106/ mouse) to respective groups of 8-month-old (NZB x NZW)F1 mice. Figure 5 shows that the secretion of TGF-ß is significantly higher in the splenocytes of pCDR1 (P = 0.0243)- or pCDR3 (P = 0.0319)-treated groups when compared with the control of untreated mice. TGF-ß production by spleen cells from mice treated with reversed pCDR1 was not significantly different from that of control mice (P = 0.0987). Table 5 represents the clinical manifestations of the SLE-like disease in the old (NZB x NZW)F1 mice that were transferred with splenocytes from animals treated with the pCDR peptides. As shown in Table 5 the transfer of splenocytes from pCDR1- and pCDR3-treated mice ameliorates significantly the kidney disease as manifested by proteinuria and the deposits of the immune complexes in the kidneys. This adoptive transfer of splenocytes from peptide-treated mice resulted in a moderate reduction in anti-dsDNA antibody titers (data not shown). Because the recipient mice were sacrificed ~3 weeks following cell transfer, the effect of the inoculated cells on survival could not be determined in these experiments. Nevertheless, whereas in each experiment at least one mouse of the untreated group died before the end of the experiment, all the recipients of splenocytes of mice treated with the CDR-based peptides survived. Thus, beneficial effects of treatment with the CDR-based peptides can be transferred by splenocytes of peptide-treated mice.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5. Levels of TGF-ß secreted by spleen cells of young (NZB x NZW)F1 mice treated with the CDR-based peptides, before being adoptively transferred into 8-month-old diseased mice. Two-month-old (NZB x NZW)F1 mice were treated 3 times (every other day) with pCDR1, pCDR3 or reversed pCDR1, 300 µg/mouse given s.c. in PBS. Mice were then sacrificed and the levels of active TGF-ß secreted by their Con A-stimulated splenocytes were measured by ELISA. Summary of results of two experiments. *P = 0.0243, **P = 0.0319, ***P = 0.0987 as compared with the non-treated group.

 

View this table:
[in this window]
[in a new window]
 
Table 5. Down-regulation of SLE clinical manifestation in (NZB x NZW)F1 mice by splenocytes of mice treated with the CDR-based peptides
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The main findings of the present report are that treatment of the lupus-like disease of (NZB x NZW)F1 mice, when clinical symptoms were already developed, ameliorated disease manifestations in all measured parameters. Moreover, some beneficial effects could be adoptively transferred into (NZB x NZW)F1 mice with the full-blown SLE-like disease by splenocytes of young (NZB x NZW)F1 mice that were treated with the CDR-based peptides. The observed benefits were associated with a down-regulation of IL-2, IFN-{gamma}, IL-4 and IL-10, and an up-regulation in the production of the immunosuppressive cytokine, TGF-ß. The elevated production of TGF-ß was determined in splenocytes of mice treated with the CDR-based peptides that could transfer ameliorating effects of the peptides.

Anti-DNA antibodies play a central role in the diagnosis of SLE. Indeed, the most characteristic serological abnormality found in patients with SLE is the presence of anti-dsDNA antibodies (15,16). Anti-DNA antibodies are considered instrumental in the pathogenesis of the immune glomerulonephritis in SLE in mice and in human patients (15). In (NZB x NZW)F1 mice as well as in other SLE models, the pathogenic anti-DNA antibodies were reported to be of the IgG2a and IgG3 isotypes (2,17,18). The latter compose the immune complexes that lead to glomerulonephritis. This kidney disease is the major cause of death in SLE afflicted mice (2). Indeed, our results demonstrate that mice treated with the pCDR1 or pCDR3 had lower titers of anti-DNA antibodies of these pathogenic isotypes in their sera and less immune complex deposits of the latter isotypes in their kidneys, and a better survival rate.

Most of the experimental protocols of the present report were not designed to compare mortality rates between the groups that were treated with the CDR-based peptides and the non-treated mice. Therefore, mice were sacrificed in most experiments at the age of 8 months, before death occurred. Nevertheless, when mice were treated for 10 weeks starting at the age of 7 months, it could be seen that mortality was significantly lower in the peptide-treated groups (Table 4). The higher survival rate could be attributed to the amelioration in the renal disease that has a central role in disease course in animal and human SLE.

The use of peptides for treating the lupus-like disease of (NZB x NZW)F1 mice has been recently reported by other laboratories as well. Most of the studies report the successful prevention of SLE manifestations as we have previously reported (11) and not treatment of an already existing disease. Thus, injection of a peptide based on the CDR3 of an anti-DNA antibody from naive BALB/c mice to (NZB x NZW)F1 mice, at the age of 2 months, resulted in a delay in mortality rate and onset of proteinuria (19). Further, induction of tolerance in young (NZB x NZW)F1 mice by i.v. injections of peptides derived from the heavy chain variable (VH) regions of anti-DNA autoantibodies postponed the development of the SLE-like disease in these mice (20). Peptides based on a nephritogenic (R4A) anti-DNA antibody were shown to protect mice from renal deposition of anti-DNA antibodies (21). In agreement with our present study demonstrating the beneficial effects of treatment with the CDR-based peptides on an already established SLE, Kaliyaperumal et al. (22) reported the effectiveness of treatment with nucleosomal histone peptides not only in delaying the onset of disease, but also in halting the progression of an established renal disease in (SWR x NZB)F1 lupus-prone mice. Further, treatment with a peptide [‘consensus peptide’ (pCONS)] based on an algorithm that defines the T cell stimulatory amino acid sequences from the VH regions of multiple (NZB x NZW)F1 IgG antibodies to DNA was effective when given to diseased mice (23). Hence, the reported, as well as our, findings support the crucial effect of heavy chain variable region derived peptides in the modulation of murine lupus.

Cytokines have been suggested to play an important role in the immune dysregulation observed in lupus-prone mice and in SLE patients (14,24). Treatment with the CDR-based peptides reduced the levels of IL-2 and IFN-{gamma}. While the role of IL-2 in SLE has not been conclusively determined (14), the role of IFN-{gamma} in the pathogenesis of lupus has been consistently reported. Thus, treatment of (NZB x NZW)F1 mice with IFN-{gamma} accelerated disease, whereas treatment with anti-IFN-{gamma} antibody (25) or soluble IFN-{gamma} receptor (26) delayed disease progression. A similar role for IFN-{gamma} was reported for MRL-lpr/lpr mice in which deficiency of the IFN-{gamma} gene (27) or the IFN-{gamma} receptor gene (28) protected from disease development. We demonstrated that treatment with the CDR-based peptides down-regulated IL-10 and IL-4 secretion. Increased IL-10 production has been reported in all SLE-prone mice (14) and treatment with IL-10 accelerated disease manifestations, whereas administration of anti-IL-10 delayed onset of SLE (29). As for IL-4, it has been reported that transfer of IL-4-stimulated splenocytes into syngeneic recipients increased anti-DNA production, and administration of anti-IL-4 inhibited the autoantibody production and prevented glomerulonephritis (14). Hahn et al. (23) demonstrated significantly lower levels of IFN-{gamma} and IL-4 in plasma of (NZB x NZW)F1 treated with pCONS as compared to plasma of saline-treated mice, supporting the results of the present report. In contrast to the reduced production of the above cytokines following treatment with the CDR-based peptides, treatment with either pCDR1 or pCDR3 resulted in the secretion of elevated levels of the immunosuppressive cytokine, TGF-ß. In agreement, it has been reported that injection of a TGF-ß cDNA expression vector into the skeletal muscle of the lupus-prone MRL-lpr/lpr mice decreased autoantibody production (30). Furthermore, infection of (NZB x NZW)F1 mice with Plasmodium chabaudi led to an improvement in the clinical lupus-like symptoms of the mice. The latter was associated with an increased mRNA expression of TGF-ß (31). Both constitutive and stimulated levels of TGF-ß are lower in patients with SLE, and the high IgG production is attributed, in part, to low levels of TGF-ß (32). The ability of splenocytes of young (NZB x NZW)F1 mice that were treated with the CDR-based peptides to adoptively transfer their capacity to down-regulate SLE manifestations (Table 5) suggests that one of the mechanisms by which the peptides exert their ameliorating effects is the induction of an immunosuppresive response mediated by regulatory cells (33) and/or by immunosuppresive cytokines like TGF-ß. As a result of that, the secretion of cytokines supporting SLE-associated autoimmune responses is actively suppressed. Indeed, splenocytes of mice that were treated with the CDR-based peptides were shown to secrete elevated levels of TGF-ß (Fig. 5).

Taking together, the reported results indicate that a relatively short course of treatment with the CDR-based peptides ameliorates the clinical manifestations of an established SLE in (NZB x NZW)F1 mice. The treatment with the peptides was shown to be effective in SLE-prone mice [(11) and this report] as well as in a model of induced experimental SLE (9). Thus, the peptides pCDR1 and pCDR3 might be potential candidates for the treatment of human SLE.


    Acknowledgements
 
This study was supported by TEVA Pharmaceutical Industries Ltd, Israel.


    Abbreviations
 
CDR—complementarity-determining region

Con A—concanavalin A

pCDR—peptide based on CDR

SLE—systemic lupus erythematosus

TGF—transforming growth factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Hahn, B. H. 1993. An overview of the pathogenesis of systemic lupus erythematosus. In Wallace, D. J. and Hahn, B. H., eds, Dubois’ Lupus Erythematosus, p. 69. Williams & Wilkins, Philadelphia, PA.
  2. Theofilopoulos, A. N. 1992. Murine models of lupus. In Lahita, R. G., ed., Systemic Lupus Erythematosus, p. 121. Churchill Livingston, New York.
  3. Mendlovic, S., Brocke, S., Shoenfeld, Y., Ben Bassat, M., Meshorer, A., Bakimer, R. and Mozes, E. 1988. Induction of a systemic lupus erythematosus-like disease in mice by a common human anti-DNA idiotype. Proc. Natl Acad. Sci. USA 85:2260.[Abstract]
  4. Waisman, A., Mendlovic, S., Ruiz, J. P., Zinger, H., Meshorer, A. and Mozes, E. 1993. The role of the 16/6 idiotype network in the induction and manifestation of systemic lupus erythematosus. Int. Immunol. 5:1293[Abstract]
  5. Tillman, D. M., Jou, N. T., Hill, R. J. and Marion, T. N. 1992. Both IgM and IgG anti-DNA antibodies are the products of clonally selective B cell stimulation in (NZB x NZW)F1 mice. J. Exp. Med. 176:361.
  6. Waisman, A. and Mozes, E. Variable region sequences of autoantibodies from mice with experimental systemic lupus erythematosus. Eur. J. Immunol. 23:1566.
  7. Wloch, M. K., Alexander, A. L., Pippen, A. M. N., Pisetsky, D. S. and Gilkson, D. S. 1997. Molecular properties of anti-DNA induced in preautoimmune NZB/W mice by immunization with bacterial DNA. J. Immunol. 158:4500.[Abstract]
  8. Waisman, A., Ruiz, P. J., Israeli, E., Eilat, E., Könen-Waisman, S., Zinger, H., Dayan, M. and Mozes, E. 1997. Modulation of murine systemic lupus erythematosus with peptides based on complementarity determining regions of a pathogenic anti-DNA monoclonal antibody. Proc. Natl Acad. Sci. USA 94:4620.[Abstract/Free Full Text]
  9. Eilat, E., Dayan, M., Zinger, H. and Mozes, E. 2001 The mechanism by which a peptide based on the complementarity determining region-1 of a pathogenic anti- DNA autoantibody ameliorates experimental SLE. Proc. Natl Acad. Sci. USA 98:1148.
  10. Eilat, E., Zinger, H., Nyska, A. and Mozes, E. 2000. Prevention of systemic lupus erythematosus-like disease in (NZB x NZW)F1 mice by treating with CDR1-and CDR3-based peptides of a pathogenic autoantibody. J. Clin. Immunol. 20:268.[CrossRef][ISI][Medline]
  11. Schnolzer, M., Alewood, P. F. and Kent, S. B. H. 1992. In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int. J. Pept. Protein Res. 40:180.[ISI][Medline]
  12. Handwerger, B. S., Rus, V., da Silva, L. and Via, C. S. 1994. The role of cytokines in the immunopathogenesis of lupus. Semin. Immunopathol. 16:153.[ISI][Medline]
  13. Segal, R., Bermas, B. L., Dayan, M., Kalush, F., Shearer, G. M. and Mozes, E. 1997. Kinetics of cytokine production in experimental systemic lupus erythematosus: involvement of T helper cell 1/T helper cell 2-type cytokines in disease. J. Immunol. 158:3009.[Abstract]
  14. Theofilopoulos, A. N., and Lawson, B. R. 1999. Tumour necrosis factor and other cytokines in murine lupus. Ann. Rheum. Dis. 58:149.
  15. Hahn, B. H. 1998. Antibodies to DNA. N. Engl. J. Med. 338:1359.[Free Full Text]
  16. Spronk, P. E., Horst, G., VabDerGun, B. T., Limburg, P. C. and Kallenberg, C. G. 1996. Anti-dsDNA production coincides with concurrent B and T cell activation during development of active disease in systemic lupus erythematosus (SLE). Clin. Exp. Immunol. 104:446.[ISI][Medline]
  17. Izui, S., Iwamoto, M., Fossati, L., Merino, R., Takahashi, S. and Ibanou-Zekri, N. 1995. The Yaa gene model of systemic lupus erythematosus. Immunol. Rev. 144:137.[ISI][Medline]
  18. Dayan, M., Zinger, H., Kalush, F., Mor, G., Amir Zaltzman, Y., Kohen, F., Sthoeger, Z. and Mozes, E. 1997. The beneficial effects of treatment with tamoxifen and anti-oestradiol antibody on experimental systemic lupus erythematosus are associated with cytokine modulations. Immunology 90:101.[ISI][Medline]
  19. Jouanne, C., Avrameas, S. and Payelle-Brogard, B. 1999. A peptide derived from a polyreactive monoclonal anti-DNA natural antibody can modulate lupus development in (NZB x NZW)F1 mice. Immunology 96:333.[CrossRef][ISI][Medline]
  20. Singh, R. R., Ebling, F. M., Sercarz, E. E. and Hahn, B. H. 1995. Immune tolerance to autoantibody-derived peptides delays development of autoimmunity in murine lupus. J. Clin. Invest. 96:2990.[ISI][Medline]
  21. Gaynor, B., Putterman, C., Valadon, P., Spatz, L., Scharff, M. and Diamond, B. 1997. Peptide inhibition of glomerular deposition of an anti-DNA antibody. Proc. Natl Acad. Sci. USA 94:1955.[Abstract/Free Full Text]
  22. Kaliyaperumal, A., Michaels, M. A. and Datta, S. K. 1999. Antigen-specific therapy of murine lupus nephritis using nucleosomal peptides: tolerance spreading impairs pathogenic function of autoimmune T and B cells. J. Immunol. 162:5775.[Abstract/Free Full Text]
  23. Hahn, B. H., Singh, R. S., Wong, W.-K., Tsao, B. P., Bulpitt,K. and Ebling,F. M. 2001. Treatment with a consensus peptide based on amino acid sequences in autoantibodies prevents T cell activation by autoantigens and delays onset in murine lupus. Arthritis Rheum. 44:432.[ISI][Medline]
  24. Dean, G. S., Tirrell-Price, J., Crawley, E. and Isenberg, D. A. 2000. Cytokines and systemic lupus erythematosus. Ann. Rheum. Dis. 59:243.[Free Full Text]
  25. Jacob, C. O., van der Meide, P. H. and McDevitt, H. O. 1987. In vivo treatment of (NZB x NZW)F1 lupus-like nephrithis with monoclonal antibody to gamma interferon. J. Exp. Med. 166:798.[Abstract]
  26. Ozmen, L., Roman, D., Fountoulakis, M., Schmid, G., Ryffel, B. and Garotta, G. 1995. Experimental therapy of systemic lupus erythematosus: the treatment of NZB/W mice with mouse soluble interferon-gamma receptor inhibits the onset of glomerulonephritis. Eur. J. Immunol. 25:6.[ISI][Medline]
  27. Balomenos, D., Rumold, R. and Theofilopoulos, A. N. 1998. Interferon-{gamma} is required for lupus-like disease and lymphoaccumulation in MRL-lpr mice. J. Clin. Invest. 101:364.[Abstract/Free Full Text]
  28. Peng, S. L., Moslehi, J. and Craft, J. 1997. Roles of interferon-{gamma} and interleukin-4 in murine lupus. J. Clin. Invest. 99:1936.[Abstract/Free Full Text]
  29. Ishida, H., Muchamuel, T., Sakaguchi, S., Andrade, S., Menon, S. and Howard, M. 1994. Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice. J. Exp. Med. 179:305.[Abstract]
  30. Raz, E., Watanabe, A., Baird, S. M., Isenberg, R. A., Parr, T. B., Lotz, M., Kipps, T. J. and Carson, D. A. 1993. Systemic immunological effects of cytokine genes injected into skeletal muscle. Proc. Natl Acad. Sci. USA 90:4523.[Abstract]
  31. Sato, M. N., Minoprio, P., Avrameas, S. and Terninck, T. 2000. Changes in the cytokine profile of lupus-prone mice (NZB/NZW)F1 induced by Plasmodium chaubaudi and their implications in the reversal of clinical symptoms. Clin. Exp. Immunol. 119:333.[CrossRef][ISI][Medline]
  32. Ohtsuka, K., Dixon Gray, J., Stimmler, M. M., Toro, B. and Horwitz, D. A. 1998. Decreased production of TGF-ß by lymphocytes from patients with systemic lupus erythematosus. J. Immunol. 160:2539.[Abstract/Free Full Text]
  33. Shevach, EM. 2001. Certified professionals: CD4+CD25+ suppressor T cells. J. Exp. Med. 193:F41.