Successful treatment of autoimmunity in (NZB X NZW) F1 mice with ...

Clin. exp. Immunol. (1986) 64, 234-242

Successful treatment of autoimmunity in (NZB x NZW)F1 mice with cyclosporin and (Nva2)-cyclosporin: II. Reduction of glomerulonephritis H. C. GUNN & B. RYFFEL Preclinical Research, Sandoz, Switzerland

(Acceptedfor publication 9 December 1985)

SUMMARY

Autoimmune (NZB x NZW)F1 mice were treated with the immunosuppressive agent, cyclosporin, and its new derivative (Nva2)-cyclosporin. Both compounds prevented the deposition of immunocomplexes in the kidneys, and the subsequent development of glomerulonephritis and proteinuria in young mice. They also reduced established proteinuria in old mice. Therefore, we feel that both cyclosporin and (Nva2)-cyclosporin may be useful in the treatment of human glomerulonephritis where there is an autoimmune component. Keywords glomerulonephritis proteinuria NZB/W cyclosporin (Nva2)-cyclosporin INTRODUCTION

Female (NZB x NZW)F1 hybrid (NZB/W) mice spontaneously develop an autoimmune disease characterized by the appearance of circulating antinuclear antibodies and a fatal immune complex (IC) glomerulonephritis (GN) (Howie & Helyer, 1968). These features are similar to those seen in human systemic lupus erythematosus (SLE). Immunological abnormalities including spontaneous B cell activation and age-related T cell suppressor defects have been implicated in its pathogenesis (Moutosopoulous et al., 1977; Krakauer, Waldmann & Stober, 1976). Therefore, these F. mice offer an excellent model in which to study therapeutic regimes possibly applicable to the human disease. Various immunosuppressive and anti-inflammatory treatments (e.g. cyclophosphamide, azathioprine and corticosteriods) have been shown to be effective in preventing the development of NZB/W nephritis (Gelfand et al., 1976; Morris et al., 1976; Cavallo, Graves & Granholm, 1984). Three other studies have started treatment of the disease after the onset of proteinuria; Kotzin & Strober (1979) using total lymphoid irradiation and Bowles, Holley & Handwerger (1983) with cyclosporine, both managed to reverse the disease. The fungal metabolite cyclosporin (CS) has potent immunosuppressive effects with low myelocytotoxicity (Borel et al., 1977) and is used with considerable success in suppressing allograft rejection in experimental animals and man (Kahan, 1984). (Nva2)-cyclosporin ((Nva2)-CS) is a derivative of CS which is at present thought to possess similar properties to CS except for reduced nephrotoxicity in rats (Hiestand et al., 1985). In our previous report (Gunn, 1986) we showed both a preventive and a therapeutic effect of CS and (Nva2)-CS on autoantibody production in female NZB/W mice. In this report, we present data on the effect of these compounds on the kidney pathology of the female NZB/W mice. Correspondence: Dr H. C. Gunn, University Hospital, Rm 8-L25, 339 Windermere Rd., London, Ontario, Canada, N6A 5A5.

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Autoimmunity in (NZB x NZW) F1 mice

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MATERIALS AND METHODS Mice. Female NZB/W mice were purchased from TND Central Institute (Zeist, The Netherlands). The mice were randomly sorted into control and treatment groups each containing 10 mice. Mice were ear-marked to permit individual identification. Experiments were started when the mice were aged 12, 24 or 36 weeks old and they received drug treatment for 12 weeks. Drug dosage. Both CS and (Nva2)-CS were dissolved in a small amount of ethanol. Olive oil was added to this solution until a concentration had been obtained where a dose of 100 mg/kg could be given in a volume of 0 3 ml. The final percentage of ethanol was less than 6% in all cases. The mean weight of the group of mice was used to calculate the dose, which was given by gavage once daily five times a week, using a blunt ended curved needle and syringe. The serum levels of the cyclosporins were assayed by radioimmunoassay. These tests were kindly performed by Dr P. Donatsch. Evaluation ofproteinuria. The degree of proteinuria was estimated by staining 10 yl urine spots on filter paper with bromophenol blue (Knight, Adams & Purves, 1977). A thin-layerchromatography scanner connected to an integrating computer was then used to quantify the intensity of the stained urine spots compared to a serial dilution of bovine serum albumin (Serva, Heidelberg, FRG). Levels of protein above 100 mg% were considered abnormal. Histology. At the end of each experiment the kidneys were fixed in a 10% neutral formalin solution and processed for histological examination. Staining of the sections included haematoxylin and eosin, and chromotrope aniline blue. Twenty-five glomeruli per mouse were analysed for protein deposits and the percentage of glomeruli with protein deposits recorded. Direct immunofluorescent studies were performed on paraffin sections using fluorescein-labelled antisera directed against mouse Ig and C3 (Nordic, The Netherlands). A working dilution of 1:10 was used for an incubation period of 30 min. Membrano-proliferative glomerulonephritis was scored with an arbitrary scale from 0 (absence of lesions) to 4 (the most severe form of the lesions). Immunofluorescent scoring was as follows: 0, no visible deposits; 1, weak deposits; and 2, moderate deposits). Results are presented in terms of incidence (any figure > 0) and mean score. The statistical test used for comparing incidences was Fisher's exact probability and the Student's t-test was used to compare the mean scores.

RESULTS

(Nva2) -Cyclosporin prevents the development of proteinuria Figure 1 shows the pooled data from two experiments: (Nva2)-CS prevents the occurrence of proteinuria in NZB/W mice when the treatment started at 24 weeks of age. At the age of 36 weeks, 50% of the control mice became proteinuria positive (> 100 mg%) while none of the mice that received (Nva2)-CS became positive. This difference is highly significant (P< 0-001) when analysed by Fisher's exact probability test. Identical experiments were also performed with CS and, likewise, none of the mice treated with the drug developed proteinuria. However, very few of the control mice in these CS experiments became proteinuric and hence no statistical difference could be shown (data not shown). Effect of drug treatment on established proteinuria in old NZB/ W mice Thirty-six-week-old NZB/W mice were randomly sorted into control groups given olive oil or groups given the compounds. Proteinurea tests were performed a few days before first administration of the drug, and 6, and 12 weeks after the commencement of treatment. The results, pooled data from three experiments with CS and two experiments with (Nva2)-CS, are analysed in two ways. Table 1 shows the cumulative incidences (mice dying of proteinuria are included in the calculation of the incidence at the next time point) of proteinuria. The results show that the

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Table 1. Cumulative percentages of proteinuria positive mice at various ages

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cumulative incidence of proteinuria increased from 36 to 48 weeks of age in the control mice, but decreased when mice were treated with either CS or (Nva2)-CS during this time period. Fisher's exact probability test performed between control and drug-treated groups at 48 weeks shows the results to be highly significant: P=O OO5 for CS and P=O OO1 for (Nva2)-CS. Figure 2 shows the individual levels of proteinuria of the drug-treated mice that were positive at the beginning of the experiment. There was a reduction of proteinuria in all but one of the CS treated mice. Some mice, however, died soon after the start of the experiment, presumably because the disease was too far advanced.

Effect of drug treatment on glomerulonephritis in the NZB/ W mice NZB/W mice were given CS or (Nva2)-CS for 12 weeks starting at various ages, i.e. 12, 24 and 36 weeks of age. At the end of each experimental period the mice were killed and the kidneys were examined histologically. Membrano-proliferative glomerulonephritis, protein, Ig and C3 deposits were reduced by treatment with both drugs (Tables 2 & 3). The drugs prevented the glomerular pathology in younger mice. The results also suggest that there may be a reduction of glomerular lesions when older mice are treated. Effect of drug treatment on survival of old NZB/ W mice When both CS and (Nva2)-CS were given to 24-week-old NZB/W mice, they do not develop proteinuria (Fig. 1) or die (data not shown) as long as the drug is given. When these drugs were given to the mice starting at 36 weeks of age, the survival rates were increased compared to the control group (Fig. 3). However, the differences are not significantly different from controls which may be due to the fact that a few drug-treated mice died within a few weeks of the start of treatment. The mice that died already had very high levels of proteinuria and their disease states were presumably too advanced to be treated. It may be possible that the drug-treated animals died due to cyclosporin nephrotoxicity. However, this is not likely because after 4 weeks of treatment, no mice died. Also, none of the drug-treated animals which were proteinuria negative at the start of treatment died.

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DISCUSSION In our previous report (Gunn, 1986) we demonstrated the inhibitory effect of CS and (Nva2)-CS on autoantibody titres in NZB/W mice. The present report indicates that treatment of young mice with these compounds prevents the onset of proteinuria and development of kidney pathology. This is probably a result of suppression of autoantibody production, and subsequent immune complex formation and deposition in the glomeruli. The precise mechanism of this suppression is not clear. Current evidence suggests that the B cells in the NZB/W mice are hyper-responsive to T-cell-derived soluble factors (Theofilopoulos et al., 1983). CS is known to interfere with the release of these lymphokines (Granelli-Piperno, Inaba & Steinman, 1984) and it is perhaps by this mechanism that autoantibodies, and hence the whole disease course in these mice, is suppressed. However, CS has been reported to induce a slight fall of plasma protein levels (Thompson et al., 1981). We cannot exclude this reduction as a possible exploration for the reduction of proteinurea in the NZB/W mice. At present, it seems unlikely to be the case since none of the NZB/W mice (young, 24-week-old or old) lost weight during the 12 week treatment periods (data not shown). Similar results with CS were obtained by Bowles et al. (1983). However, Jones & Harris (1985) reported that there was no difference in proteinuria and kidney pathology between CS-treated and untreated groups. One possible reason for this descrepancy is that the ethanol/olive oil mixture we used may be a better galenical form than olive oil alone. We obtained trough serum levels of


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231 + 52 ng/ml ofCS and 593 + 168 ng/ml of (Nva2)-CS after 12 weeks of treatment. However, Jones and Harris did not check the level of CS obtained in the serum of their mice. The most pertinent question to us was whether these immunosuppressive compounds could stop glomerular damage in mice with moderate or advanced disease when the autoimmune component of the disease was arrested. Two lines of evidence suggest that individual mice do respond to therapy. Firstly, CS and (Nva2)-CS therapy resulted in decreased proteinuria. Secondly, those animals which initially had high levels of proteinuria (and assumed to have had extensive protein deposition in the glomeruli) had minimal protein deposition when examined histologically after several weeks of therapy. Bowles et al. (1983) obtained similar results using CS. Kotzin & Stober (1979) also managed to reverse proteinuria after total lymphoid irradiation, but they did not perform any histology. Israel-Biet et al. (1983) observed no alteration of proteinuria, in male NZB/ W mice treated for only 30 days. However, they did observe diminished Ig deposits in mesangial areas even after this short treatment period. The mechanism for the inhibition of GN and improvement of renal function has not been fully elucidated. Reduced autoantibody production probably resulted in the decreased production of IC, thus allowing more effective clearance of the IC by the reticuloendothelial system. Decreased deposition of IC in the glomeruli could allow the on-going inflammation to be suppressed, for example, by the inhibition of lymphokine production by CS and (Nva2)-CS. A reduction in the GN could then have a beneficial effect on glomerular filtration and hence reduce proteinuria. The question of the effect of CS on IC-mediated renal injury and proteinuria has also been investigated by several workers using various other models. Israel-Biet et al. (1983) showed a reduction of IC deposition in the glomeruli without a reduction in proteinuria. However, they treated old male NZB/W mice with CS for only 2 weeks. Baran et al. (1985) using a mercury-induced autoimmune GN model in rats, and Cattran and Moller (1985) in the active Heymann's nephritis model in rats, obtained essentially the same results as ours, i.e. a suppression of both IC deposition and proteinuria by CS. However, Jones and Harris (1985) using the NZB/W model, Neild et al. (1983) using a model ofacute serum sickness nephritis in rabbits, and Thaiss et al. (1985) using an in situ model of IC-GN in rats, all showed a reduction in proteinuria without a reduction of IC deposition in the glomeruli. Since there was a reduction of proteinuria in six of the seven reports, we may assume that CS really does affect on this parameter. The interpretation of the available data on IC deposition in the glomeruli is more difficult. The negative data of Jones and Harris may be due to their lack of effect on autoantibody levels. Without further experiments, one can only speculate that the nature, and perhaps amount of IC generated in the models of Neild et al. (1983) and Thaiss et al. (1986) were different to those where CS was shown to have decreased glomerular deposition. One possibility for the reduction of proteinuria in the absence of IC deposition is that CS may be affecting glomerular filtration rate (Tonnesen, Hamner & Weinmann 1983). The lack of statistical differences in the survivl rates between drug-treated and control groups may be due to the fact that it takes at least 4 weeks for any appreciable drop in serum autoantibodies to be seen (Gunn, 1986). We feel that the absence of any deaths from weeks 40 to 48 of our experiments may be biologically significant. We have presented results that suggest that CS and (Nva2)-CS may prove to be of clinical benefit in the treatment of autoimmune glomerulonephritis. The data available on the effect of CS in other on-going autoimmune response is not clear. The results range from having clear-cut beneficial effects, e.g. experimental autoimmune uveitis (Nussenblatt et al., 1981) and experimental myasthenia gravis (Drachman et al., 1985), to having no effect at all, e.g. obese strain chicken (Wick, Muller & Schwarz, 1982), experimental autoimmune thyroiditis in mice (Vladutiu, 1983) and the non-obese diabetic BB rat (unpublished observations). Due to the wide range of autoimmune disorders, each disease would probably give a different answer. Our present opinion is that CS can suppress an on-going autoimmune response but by itself does not 'cure' the disease. Some other form of immune manipulation must also be used to re-regulate the immune system whilst CS immunosuppression gives the body 'breathing space' from pathological damage. In this sense, CS may be superior to the other forms of immunosuppression currently used because of its wider therapeutic index, and especially its lack of myelotoxicity, if we expect the immune system to

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recover. If (Nva2)-CS does prove to be non-nephrotoxic in man, it would then be a true alternative to conventional immunotherapy currently used in the treatment of autoimmune diseases. The authors would like to thank Mr P. C. Hiestand and Professor J. F. Borel for constant advice and encouragement during the course of the work, Dr B. P. Richardson and Dr M. J. Mihatsch for reading the manuscript, and Mr A. Vizeli and Mr W. Etter for expert technical assistance.

REFERENCES BARAN, D., VENDEVILLE, B., VIAL, M.C., COSSON, C., KRAKAUER, R.S., WALDMANN, T.A. & STOBER, W. (1976) Loss of suppressor T cells in adult NZB/ BASCON, C., TEYDENNE, P. & DRUET, P. (1986) NZW mice. J. exp. Med. 129, 123. Effect of Cyclosporin A on mercury induced autoimmune glomerulonephritis in the Brown Nor- KNIGHT, J.G., ADAMS, D.D. & PURVES, H.D. (1977) The genetic contribution of the NZB mouse to the way rat. Clin. Nephrology. In press. renal disease of the NZB x NZW hybrid. Clin. exp. BOREL, J.F., FEURER, C., MAGNEE, C. & STAHELIN, H. Immunol. 28, 362. (1977) Effects of the new anti-lymphocytic peptide KOTZIN, B.Z. & STROBER, S. (1979) Reversal of NZB/ Cyclosporin-A in animals. Immunology, 32, 1017. NZW disease with total lymphoid irradiation. J. BOWLES, C.A., HOLLEY, K.E. & HANDWERGER, B.S. exp. Med. 150, 371. (1983) Cyclosporin A therapy in (NZB x NZW)F1 mice. Abstract presented at Am. Rheumatism Asso. MORRIS, A.D., ESTERLY, J., CHASE, G. & SHARP, G. (1976) Cyclophosphamide protection in NZB/W June 1-4. Disease. Mechanism and therapeutic regimens. CATTRAN, C. & MOLLER, G. (1985) Effects of CyclosArthritis Rheum. 19, 49. porin (CS) on the early and late phase of active Heymann's nephritis (AHN). Kidney Int. 27, 207. MOUTOSOPOULOS, H.M., BOEHM-TRuITr, M., KASSAN, S.S. & CHUSED, T.M. (1977) Demonstration of CAVALLO, T., GRAVEs, K. & GRANHOLM, N.A. (1984) activation of B lymphocytes in New Zealand Black Murine lupus nephritis: Effects of Glucocorticoids mice at birth by an immunoradiometric assay for on circulating and tissue-bound immunoreactants. murine IgM. J. Immunol. 119,1639. Lab. Invest. 49, 476. DRACHMANN, D.B., ADAMS, R.N., MCINTOSH, K. & NEILD, G.H., IVORY, K., HIRAMATSU, M. & GWYN WILLIAMS, D. (1983) Cyclosporin A inhibits acute PESTRONK, A. (1985) Treatment of Myasthenia serum sickness nephritis in rabbits. Clin. exp. Gravis with Cyclosporin A. Clin. Immunol. ImmuImmunol. 52, 586. nopathol. 34, 174. GELFAND, M.C., SCHUR, P.H., ASOFSKY, R. & STEIN- NUSSENBLATT, R.B., RODRIGUES, M.R., WACKER, W.B., CEVARIO, S.J., SALINAS-CARMODA, M.C. & BERG, A.D. (1976) Therapeutic Studies in NZB/W GERY, I. (1981) Cyclosporin A: Inhibition of experistudies. IV. Effect of combinational drug therapy mental autoimmune uveitis in Lewis rats. J. clin. on immune complex deposition. Arthritis Rheum. Invest. 67, 1228. 19,43. GRANELLI-PEPERNO, A., INABA, K. & STEINMAN, R.M. THAISS, F., MIHATSCH, M.J., BATSFORD, S., VOGT, A. & SCHOLLMEYER, P. (1986) Cyclosporin A in experi(1984) Stimulation of lymphokine release from T mental in situ immune complex glomerulo-nephrilymphoblasts. Requirement for mRNA synthesis tis. Clin. Nephrology. In press. and inhibition by cyclosporin A. J. exp. Med. 160, THEOFILOPOULOS, A.N., PRUD'HOMME, G.J., FIESER, 1792. T.M. & DIXON, F.J. (1983) B-cell hyperactivity in GUNN, H.C. (1986) Successful treatment of autoimmurine lupus. II. Defects in response to and munity in (NZB x NZW)F, mice with cyclosporin production of accessory signals in lupus prone and (Nva2)-cyclosporin: I. Reduction of autoantimice. Immunol. Today, 4, 317. bodies. Clin. exp. Immunol. 64, 225. HIESTAND, P.C., GUNN, H.C., GALE, J.M., RYFFEL, B. THOMSON, A.W., WHITING, P.H., BLAIR, J.T., DAVIDSON, R.J.L. & SIMPSON, J.G. (1981) Pathological & BOREL, J.F. (1985) Comparison of the pharmacoChanges Developing in the Rat during a 3-week logical profiles of ciclosporin, (Nva2)-ciclosporin, course of high dosage Cyclosporin A and their and (Val2)-Dihydrociclosporin. Immunology, 55, reversal following drug withdrawal. Transplan249. tation, 32, 271. HOWIE, J.B. & HELYER, B.J. (1968) The immunology and pathology of NZB mice. Adv. Immunol. 9, 215. TONNESEN, A.S., HAMNER, R.W. & WEINMANN, E.J. (1983) Cyclosporin and Sodium and Potassium ISRAEL-BIET, D., NOEL, L-H., BACH, M.-A., DARExcretion in the Rat. Trans. Proc. XV (Suppl. 1), DEANE, M. & BACH, J.F. (1983) Marked reduction 514. of DNA antibody production and glomerulopathy in thymulin (FTS-Zn) or cyclosporin A treated VLADUTIU, A.G. (1983) Effect of Cyclosporine on Experimental Autoimmune Thyroiditis in Mice. (NZB x NZW)F, mice. Clin. exp. Immunol. 54,359. Transplantation, 35, 518. JONES, M.G. & HARRIS, G. (1985) Prolongation of life in female NZB x NZW(F,) hybrid mice by cyclos- WICK, G., MULLER, P.-U. & SCHWARZ, S. (1982) Effect of cyclosporin A on spontaneous autoimporin A. Clin. exp. Immunol. 59, 1. mune thyroiditis of Obese strain (OS) chickens. KAHAN, B.D., editor (1984) Cyclosporin, Grune & Eur. J. Immunol. 12, 877. Stratton, Orlando.