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Effects of mycophenolate mofetil in the development of systemic lupus erythematosus in (NZBxNZW)F1 mice
Effects of Mycophenolate Mofetil in the Development of Systemic Lupus Erythematosus in (NZBxNZW)F1 Mice Ma A. Ramos, C. Pin˜era, E. Cibria´n, Ma A. Setie´n, L. Buelta, Ma A. de Cos, A.L.M. de Francisco, R. Merino, and M. Arias
S
YSTEMIC LUPUS ERYTHEMATOSUS (SLE) is a chronic and progressive autoimmune syndrome characterized by the production of multiple autoantibodies (autoAb) resulting in the generation of circulating immune complexes (IC). The deposition of these IC leads to the development of tissue lesions such as glomerulonephritis. The study of several strains of mice that spontaneously develop an autoimmune syndrome resembling human SLE have been of considerable value to analyze cellular and molecular mechanisms responsible for the development of the disease as well as to explore the efficiency of different therapies using immunesuppressive drugs. One of these new immunosuppressive agents, Mycophenolate Mofetil (MMF), have been used successfully to prevent SLE in several lupus-prone mice such as MRL.1pr and (NZBxNZW)F1 mice.1– 4 However, the precise mechanism by which MMF modulates the development of SLE in these animals remains controversial. Whereas in some studies the therapeutic effect of MMF is secondary to the inhibition of autoAb production,1–3 in others the administration of MMF blocks the development of IC-mediated glomerulonephritis but not the production of autoAb.4 To gain insight into the mechanisms responsible for the therapeutic effect of MMF, in the present study (NZBxNZW)F1 female mice have been treated with either high or low doses of MMF from 3 months of age, when increased serum levels of anti-nuclear autoAbs are already present.
MATERIAL AND METHODS (NZBxNZW)F1 females (Jackson Laboratories, Bar Harbor, Maine) were treated from 3 months of age with either 100 mg/kg per day or 30 mg/kg per day of MMF IP. Solvent-treated (NZBxNZW)F1 females and untreated BALB/c mice (Jackson Laboratories) were used as positive and negative controls, respectively. Serum levels of total IgG and IgG1 and IgG2a subclasses as well as the production of IgG anti-DNA autoAbs were measured by ELISA.5 Some of the treated and control mice were immunized with 400 g IV of the T-dependent antigen heat aggregated human gammaglobulin (AHGG) and serum levels of total IgG, IgG1, and IgG2a anti-HGG antibodies were measured by ELISA 10 days later.5 The development of IC-mediated glomerulonephritis was evaluated after histologic examination of kidneys obtained after autopsy at the time of animal death. The severity of kidney disease was determined as previously described.6
From Servicio de Nefrologia (M.A.R., C.P., M.A.S., A.L.M.dF., M.A.) and Unidad de Investigacio´n (E.C., R.M.), Hospital Universitario Marque´s de Valdecilla, and Departamento de Ciencias Me´dicas y Quiru´rgicas (L.B.) and Departamento de Fisiologia y Farmacologia (M.A.dC.), Universidad de Cantabria, Santander, Spain. Address reprint requests to Dr Ramo´n Merino, Unidad de Investigacio´n, Hospital Universitario Marque´s de Valdecilla, Escuela de Enfermeria 4a planta, Av Valdecilla s/n, 39008 Santander, Spain.
Table 1. Histological and Serological Parameters in (NZBxNZW)F1 Serum Igs Mice
BALB/c (NZBxNZW)F1 Untreated MMF 30 mg/kg/d MMF 100 mg/kg/d
Glomerular 50% Score§ Mortality†
0 3.5 ⫾ 0.3 1.7 ⫾ 0.5 0.9 ⫾ 0.3
⬎15 9 ⬎15 ⬎15
IgG anti-DNA‡
3.5 ⫾ 2
IgG*
10.1 ⫾ 3.5
Anti-HGG Antibodies
IgG1*
IgG2a*
IgG¶
IgG1¶
IgG2a¶
ND
ND
ND
ND
ND
282.7 ⫾ 120.7 48.1 ⫾ 15.3 5.3 ⫾ 4.3 23.3 ⫾ 9.7 1.769 ⫾ 0.173 1.668 ⫾ 0.426 1.168 ⫾ 0.621 288.2 ⫾ 177.5 41.3 ⫾ 19.7 7.9 ⫾ 5.8 9.1 ⫾ 7.7 1.761 ⫾ 0.153 1.561 ⫾ 0.273 0.563 ⫾ 0.209 58.7 ⫾ 20.1 17.2 ⫾ 5.9 ND ND ND ND ND
§
The severity of glomerular lesions was scored on a 0 (no evidences of glomerular changes) to 4 (severe glomerulonephritis) scale as described previously.6 Values ⱖ3 can be considered as indicative of a lethal glomerulonephritis. † 50% of mortality by gromerulonephritis expressed in months. ‡ Results expressed in titration units/mL in sera from 6-month-old mice. *Results expressed in mg/mL in sera from 6-month-old mice. ¶ Results expressed in absorbance units at 405 nm in sera from mice 10 days after immunization IV with 400 g of AHGG.
0041-1345/01/$–see front matter PII S0041-1345(01)02408-3
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
3316
Transplantation Proceedings, 33, 3316–3317 (2001)
DEVELOPMENT OF SYSTEMIC LUPUS ERYTHEMATOSU
RESULTS AND DISCUSSION
Continuous treatment of (NZBxNZW)F1 females from 3 months of age with either high (100 mg/kg per day) or low (30 mg/kg per day) doses of MMF, inhibited the development of SLE, manifested by the reduction of glomerular lesions and by the prolonged survival (Table 1). The treatment with 100 mg/kg per day of MMF significantly decreased the levels of serum IgG and IgG anti-DNA auto Abs at 6 months of age in comparison to untreated (NZBxNZW)F1 females (P ⬍ .005 and P ⬍ .02, respectively) (Table 1), the treatment with 30 mg/kg per day of MMF had no effect in the development of hypergammaglobulinemia or anti-DNA production (Table 1). These results suggested that the protective effect of low-dose treatment with MMF on the development of SLE of (NZBxNZW)F1 females was probably related with differences in the quality of autoAbs produced in these mice. In fact, the analysis of IgG subclasses revealed an important reduction in the levels of IgG2a antibodies (P ⬍ .001) and a moderate, although not significant, increase in the titres of IgG1 in (NZBxNZW)F1 females treated with 30 mg/kg per day of MMF in comparison to untreated littermates (Table 1). This observation was additionally supported by the analysis of anti-HGG antibody responses in untreated and MMF-low dosetreated (NZBxNZW)F1 female mice. Again, the treatment
3317
with 30 mg/kg per day of MMF had little if any effect in the levels of IgG anti-HGG antibodies but promoted a selective and significant reduction in the levels of IgG2a anti-HGG antibodies (P ⬍ .02) (Table 1). Our results clearly indicate that MMF modulates the development of SLE in (NZBxNZW)F1 mice in a dosedependent manner. At high doses, MMF protects from SLE by inhibiting the production of pathogenic autoAbs. However, at lower doses, MMF promotes qualitative changes in the autoAbs produced in these animals. The selective reduction in the production of IgG2a antibodies suggests that MMF may interfere with the functional maturation of TH1 cells.5 REFERENCES 1. Corna, D, Morigi M, Facchinetti D, et al: Kidney Int 51:1583, 1997 2. McMurray RW, Elbourne K, Lagoo A, et al: J Rheum 25:2364, 1998 3. Jonsson CA, Svensson L, Carlsten H: Clin Exp Immunol 116:534, 1999 4. Van Bruggen MCJ, Walgreen B, Rijke TPM, et al: J Am Soc Nephrol 9:1407, 1998 5. Takahashi S, Fossati L, Iwamoto M, et al: J Clin Invest 97:1597, 1996 6. Morel L, Mohan C, Yu Y, et al: J Immunol 158:6019, 1997
S
YSTEMIC LUPUS ERYTHEMATOSUS (SLE) is a chronic and progressive autoimmune syndrome characterized by the production of multiple autoantibodies (autoAb) resulting in the generation of circulating immune complexes (IC). The deposition of these IC leads to the development of tissue lesions such as glomerulonephritis. The study of several strains of mice that spontaneously develop an autoimmune syndrome resembling human SLE have been of considerable value to analyze cellular and molecular mechanisms responsible for the development of the disease as well as to explore the efficiency of different therapies using immunesuppressive drugs. One of these new immunosuppressive agents, Mycophenolate Mofetil (MMF), have been used successfully to prevent SLE in several lupus-prone mice such as MRL.1pr and (NZBxNZW)F1 mice.1– 4 However, the precise mechanism by which MMF modulates the development of SLE in these animals remains controversial. Whereas in some studies the therapeutic effect of MMF is secondary to the inhibition of autoAb production,1–3 in others the administration of MMF blocks the development of IC-mediated glomerulonephritis but not the production of autoAb.4 To gain insight into the mechanisms responsible for the therapeutic effect of MMF, in the present study (NZBxNZW)F1 female mice have been treated with either high or low doses of MMF from 3 months of age, when increased serum levels of anti-nuclear autoAbs are already present.
MATERIAL AND METHODS (NZBxNZW)F1 females (Jackson Laboratories, Bar Harbor, Maine) were treated from 3 months of age with either 100 mg/kg per day or 30 mg/kg per day of MMF IP. Solvent-treated (NZBxNZW)F1 females and untreated BALB/c mice (Jackson Laboratories) were used as positive and negative controls, respectively. Serum levels of total IgG and IgG1 and IgG2a subclasses as well as the production of IgG anti-DNA autoAbs were measured by ELISA.5 Some of the treated and control mice were immunized with 400 g IV of the T-dependent antigen heat aggregated human gammaglobulin (AHGG) and serum levels of total IgG, IgG1, and IgG2a anti-HGG antibodies were measured by ELISA 10 days later.5 The development of IC-mediated glomerulonephritis was evaluated after histologic examination of kidneys obtained after autopsy at the time of animal death. The severity of kidney disease was determined as previously described.6
From Servicio de Nefrologia (M.A.R., C.P., M.A.S., A.L.M.dF., M.A.) and Unidad de Investigacio´n (E.C., R.M.), Hospital Universitario Marque´s de Valdecilla, and Departamento de Ciencias Me´dicas y Quiru´rgicas (L.B.) and Departamento de Fisiologia y Farmacologia (M.A.dC.), Universidad de Cantabria, Santander, Spain. Address reprint requests to Dr Ramo´n Merino, Unidad de Investigacio´n, Hospital Universitario Marque´s de Valdecilla, Escuela de Enfermeria 4a planta, Av Valdecilla s/n, 39008 Santander, Spain.
Table 1. Histological and Serological Parameters in (NZBxNZW)F1 Serum Igs Mice
BALB/c (NZBxNZW)F1 Untreated MMF 30 mg/kg/d MMF 100 mg/kg/d
Glomerular 50% Score§ Mortality†
0 3.5 ⫾ 0.3 1.7 ⫾ 0.5 0.9 ⫾ 0.3
⬎15 9 ⬎15 ⬎15
IgG anti-DNA‡
3.5 ⫾ 2
IgG*
10.1 ⫾ 3.5
Anti-HGG Antibodies
IgG1*
IgG2a*
IgG¶
IgG1¶
IgG2a¶
ND
ND
ND
ND
ND
282.7 ⫾ 120.7 48.1 ⫾ 15.3 5.3 ⫾ 4.3 23.3 ⫾ 9.7 1.769 ⫾ 0.173 1.668 ⫾ 0.426 1.168 ⫾ 0.621 288.2 ⫾ 177.5 41.3 ⫾ 19.7 7.9 ⫾ 5.8 9.1 ⫾ 7.7 1.761 ⫾ 0.153 1.561 ⫾ 0.273 0.563 ⫾ 0.209 58.7 ⫾ 20.1 17.2 ⫾ 5.9 ND ND ND ND ND
§
The severity of glomerular lesions was scored on a 0 (no evidences of glomerular changes) to 4 (severe glomerulonephritis) scale as described previously.6 Values ⱖ3 can be considered as indicative of a lethal glomerulonephritis. † 50% of mortality by gromerulonephritis expressed in months. ‡ Results expressed in titration units/mL in sera from 6-month-old mice. *Results expressed in mg/mL in sera from 6-month-old mice. ¶ Results expressed in absorbance units at 405 nm in sera from mice 10 days after immunization IV with 400 g of AHGG.
0041-1345/01/$–see front matter PII S0041-1345(01)02408-3
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
3316
Transplantation Proceedings, 33, 3316–3317 (2001)
DEVELOPMENT OF SYSTEMIC LUPUS ERYTHEMATOSU
RESULTS AND DISCUSSION
Continuous treatment of (NZBxNZW)F1 females from 3 months of age with either high (100 mg/kg per day) or low (30 mg/kg per day) doses of MMF, inhibited the development of SLE, manifested by the reduction of glomerular lesions and by the prolonged survival (Table 1). The treatment with 100 mg/kg per day of MMF significantly decreased the levels of serum IgG and IgG anti-DNA auto Abs at 6 months of age in comparison to untreated (NZBxNZW)F1 females (P ⬍ .005 and P ⬍ .02, respectively) (Table 1), the treatment with 30 mg/kg per day of MMF had no effect in the development of hypergammaglobulinemia or anti-DNA production (Table 1). These results suggested that the protective effect of low-dose treatment with MMF on the development of SLE of (NZBxNZW)F1 females was probably related with differences in the quality of autoAbs produced in these mice. In fact, the analysis of IgG subclasses revealed an important reduction in the levels of IgG2a antibodies (P ⬍ .001) and a moderate, although not significant, increase in the titres of IgG1 in (NZBxNZW)F1 females treated with 30 mg/kg per day of MMF in comparison to untreated littermates (Table 1). This observation was additionally supported by the analysis of anti-HGG antibody responses in untreated and MMF-low dosetreated (NZBxNZW)F1 female mice. Again, the treatment
3317
with 30 mg/kg per day of MMF had little if any effect in the levels of IgG anti-HGG antibodies but promoted a selective and significant reduction in the levels of IgG2a anti-HGG antibodies (P ⬍ .02) (Table 1). Our results clearly indicate that MMF modulates the development of SLE in (NZBxNZW)F1 mice in a dosedependent manner. At high doses, MMF protects from SLE by inhibiting the production of pathogenic autoAbs. However, at lower doses, MMF promotes qualitative changes in the autoAbs produced in these animals. The selective reduction in the production of IgG2a antibodies suggests that MMF may interfere with the functional maturation of TH1 cells.5 REFERENCES 1. Corna, D, Morigi M, Facchinetti D, et al: Kidney Int 51:1583, 1997 2. McMurray RW, Elbourne K, Lagoo A, et al: J Rheum 25:2364, 1998 3. Jonsson CA, Svensson L, Carlsten H: Clin Exp Immunol 116:534, 1999 4. Van Bruggen MCJ, Walgreen B, Rijke TPM, et al: J Am Soc Nephrol 9:1407, 1998 5. Takahashi S, Fossati L, Iwamoto M, et al: J Clin Invest 97:1597, 1996 6. Morel L, Mohan C, Yu Y, et al: J Immunol 158:6019, 1997