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The effects of a new immunosuppressive agent, FK506, on the glomerular injury in rats with accelerated nephrotoxic serum glomerulonephritis
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
57, 351-362 (l%(b)
The Effects of a New Immunosuppressive Agent, FK506, on the Glomerular injury in Rats with Accelerated Nephrotoxic Serum Glomerulonephritis SOUSHUN
HARA, ATSUSHI
The Third
Department
FUKATSU, NORIHIKO SUZUKI, AND SEIICHI MATSUO
of Internal
Medicine, Nagoya,
Nagoya Japan
University
NOBUO School
SAKAMOTO,
of Medicine,
The aim of the present work was to study the effects of a new immunosuppressive drug, FK506, in accelerated nephrotoxic serum glomerulonephritis. Glomerulonephritis was induced in female Wistar rats by the preimmunization with normal rabbit IgG (Day -4) and the subsequent intravenous injection of rabbit anti-GBM serum (Day 0). Without treatment with FK506, rats developed proteinuria at Day 6 and onward. Rat anti-rabbit IgG was strongly detected at Day 6 and the titer was maintained through Day 20. Moderate hypercellularity and focal crescent formation were observed at Day 20. Rats injected intramuscularly with 0.3 or 1 mgikg of FK506 did not develop proteinuria and the anti-rabbit IgG titer was much less or was undetectable throughout the experiments. These data suggest that FK506 is effective in the present model of glomerulonephritis. Q 1990 Academic
Press. Inc.
INTRODUCTION Recently a new immunosuppressive drug, FK506, was isolated from a strain of soil fungus, Streptomyces tsukubaensis (1) and is now thought to be much more potent against allograft rejection and for the inhibition of experimental autoimmune diseases than cyclosporine A (CyA) (2-5). The mechanisms of the immunosuppressive action of FK506 have been extensively studied, mainly from the aspect of cellular immunity (6), i.e., suppression of reactivity in mixed lymphocyte culture, suppression of production of cytokines (IL-2, IL-3, and interferon-y), and inhibition of the expression of the IL-2 receptor on lymphocytes. So far there has been no evidence that FK506 directly affects B cells in vitro (7). In vivo effects of FK506 have also been studied and most of the studies (2-5) showed that the beneficial effects of this drug were through its suppressive effects on cellular immunity. To date, many drugs have been introduced clinically to prevent or treat glomerulonephritis. Although corticosteroids and a number of immunosuppressants are now known to be effective in certain forms of glomerulonephritis, their effects are limited and sometimes have serious adverse effects (8, 9). In an attempt to explore the possibility of introducing this new drug, FK506, in the treatment of glomerulonephritis, we studied the effects of FK506 in an established model of experimental glomerulonephritis, accelerated nephrotoxic serum glomerulonephritis (10). Glomerular injury in this model is mediated by the autologous antibodies against the glomerular-fixed heterologous immunoglobulins and several reports suggest that the cell-mediated immune system (T cell and/or macrophage) 351 0090-1229/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ET AL.
participates in glomerular pathology (11-14). This report describes the beneficial effects of FK506 in this model of glomerulonephritis and discusses the mechanisms of action. MATERIALS
AND METHODS
Animals. Female Wistar rats weighing about 170 g and female Japanese white rabbits weighing about 2 kg were obtained from the Chubu Kagaku Shizai Co. (Nagoya, Japan). They were allowed free access to food and water. Preparation of rabbit anti-rat glomerular basement membrane (GBM) antiserum. Rat GBM was obtained from female Wistar rats by a modified method of Meezan et al. (15). Briefly, rat kidneys were perfused with ice-cold T&-buffered
saline (150 miU NaCl, 10 r&f Tris-HCl, 1 t&ml pepstatin A, 1 &ml Antipain, 1 mM benzamidine, 1 mM diisopropyl fluorophosphate). Cortex was removed and glomeruli were isolated by a mesh-sieving technique at 4°C using the same buffer. Tubular fragment contamination was less than 5%. Glomeruli were sonicated for 30 min at 4°C. After centrifugation at 15,000 t-pm for 30 min the sediment was incubated in 1% deoxycholate in phosphate-buffered saline (PBS), pH 7.4, containing protease inhibitors for 1 hr at room temperature. The fraction was washed three times with cold PBS and was then incubated in I M NaCl containing 2000 Kuniz units of deoxyribonuclease 1 (DNAase, Sigma, St. Louis, MO) for 2 hr at 37°C. After centrifugation at 10,000 t-pm for 30 min the sedimented material was obtained as purified GBM and was used as immunogen. Japanese white rabbits were immunized subcutaneously with GBM in complete Freund’s adjuvant every 2 weeks and were bled 32 days after the first immunization. Complement was inactivated by incubating antisera at 56°C for 30 min. The reactivity of antisera to rat GBM (RbAGBM) in vitro was tested by the indirect immunofluorescence technique (IF) using frozen sections of normal rat kidneys as substrate. Typical linear binding of RbAGBM was seen in GBM and tubular basement membrane (TBM) up to the dilution of 16400. In a preliminary experiment, it was confirmed that an intravenous injection of 1.0 ml of RbAGBM induced very mild cellular infiltration in glomeruli but did not induce significant proteinuria during the heterologous phase (Day 0 to Day 10) of nephrotoxic serum glomerulonephritis. Histology and immunohistology. For light microscopy study, kidney tissues were fixed in buffered formalin and embedded in paraffin. Sections (2 pm) were stained with hematoxylin and eosin and periodic acid-Schiff reagent. The number of nuclei was counted in 20 equatorially cut glomeruli in each section and the average number of nuclei per glomerulus was calculated (16). The number of the glomenrli with crescents was also calculated. For the immunofluorescence study, small fragments of kidney tissue were snapfrozen in liquid nitrogen and were cut at 2 p.m by a cryostat. Frozen sections were fixed for 10 min in 100% acetone at 4°C and were stained with goat anti-rabbit IgG labeled with fluorescein isothiocyanate (FITC), FITC-labeled rabbit anti-rat IgG, FITC-labeled goat anti-rat C3, or FITC-labeled goat anti-rat fibrinogen (Cappel, West Chester, PA). In order to avoid the cross-reactivity, FITC-labeled goat anti-rabbit IgG was preabsorbed with normal rat serum.
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FKSW. FK506 was supplied by Fujisawa Pharmaceutical Co. (Osaka, Japan) and was suspended in saline at the concentration of 1 mg/ml. Experimental design. Rats were divided into six groups and each group contained five rats. Accelerated nephrotoxic serum glomerulonephritis was induced according to the modified method of Schreiner (17). Rats were preimmunized with 1 mg of normal rabbit IgG in complete Freund’s adjuvant at Day -4. Four days later (Day 0), 1 ml of RbAGBM was intravenously injected into rats. These were used as control rats (Group IV). Rats of Group I were intramuscularly injected with 0.3 mg/kg/day of FK506 from Day -4 to Day 9. Rats of Group II were intramuscularly injected with 1 mg/kg/day of FK506 from Day 2 to Day 15. Rats of Group III were treated in the same way as Group I except for the dose of FK506 (1 mg/kg/day instead of 0.3). Rats of Group V were injected with 1 mg/kg/day of FK506 from Day -4 to Day 9 without administration of RbAGBM. Rats of Group VI were the age- and sex-matched controls. The composition and treatment in each group of rats are summarized in Table 1. Rats were sacrificed at Day 20 and kidneys were obtained for histological and immunohistological studies. Measurement of urinary protein excretion. Rats were housed in metabolic cages at Day -4, Day 6, Day 13, and Day 20. Urine was collected overnight (16 hr) and the amount of urinary protein was measured by the quantitative sulfosalicylic acid method (18) using bovine serum albumin as a standard. Food was removed during urine collection in order to avoid the contamination of food protein into urine. Serum creatinine. Rats were bled at Days -4, 2, 6, 13, and 20 by retroorbital puncture under ether anesthesia. At each puncture, 0.5 to 1 .O ml of whole blood was obtained and rats were never blinded by this procedure. Serum creatinine was measured by a creatinine measuring kit, CRE(E) KAINOS DR-2300 (KAINOS Co., Tokyo, Japan), which required only 50 pJ of serum. Measurement of rat anti-rabbit IgG in the circulation. Rat anti-rabbit IgG was measured by the ELISA assay using polystylene multiwell plates (Immulon 2, Dynatech Laboratories Inc., Chantilly, VA). Each well was coated with 10 kg/ml of normal rabbit IgG overnight at 4°C. Wells were incubated with rat sera and then with peroxidase-labeled goat anti-rat IgG absorbed with normal rabbit serum. A 0.04% o-phenylenediamine containing 0.005% H,O, was added to the wells. The reactivity of rat serum with rabbit IgG was then measured by the optical density at 492 nm using a SLT-210 photoreader (SLT-Labinstruments, Austria). TABLE
1
EXPERIMENTALPROTOCOLS FORSIXGROUPSOFRATS
Group
Number of rats
Preimmunization with rabbit IgG (at Day -4)
Injection with RbAGBM (at Day 0)
Amount of FKSW bg/kg/day)
FK506 administration
I II III IV V VI
5 5 5 5 5 5
Yes Yes Yes Yes No No
Yes Yes Yes Yes No No
0.3 1.0 1.0 Not injected 1.0 Not injected
Day Day Day Not Day Not
-4-Day 2-Day -4-Day injected -4-Day injected
9 15 9 9
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ET AL.
RESULTS Histological study. Glomeruli from rats of Group IV exhibited diffuse proliferative glomerulonephritis (Fig. Id). Cellular crescents were seen in 12.5% of the glomeruli in this group but were not seen in other groups. In the glomeruli from Group I, III, V, and VI, there was no proliferation (Figs. la and lc). In rats of Group II, the glomeruli showed mild hypercellularity (Fig. lb). The quantitative data are shown in Fig. 2. The number of nuclei per glomerulus is significantly increased in rats of Group IV in comparison with rats of other groups. There was no significant tubulointerstitial alteration in any group. In rats of Group V and VI there were no morphological changes in the kidney. Zmmunofluorescence study. In all groups, except for Groups V and VI, rabbit IgG bound strongly along the GBM (Fig. 3). In rats of Groups II and IV, binding of rat IgG was clearly seen along GBM (Figs. 4b and 4d), while only faint or no binding of rat IgG along GBM was seen in rats of Groups I and III (Figs. 4a and 4~). Deposition of rat C3 along GBM was seen in rats of Group IV (Fig. 5b), but was not observed in rats of Groups I, II, and III (Fig. 5a). Deposition of
FIG. 1. Light microscopic appearance of a representative glomerulus from each experimental group. A glomerulus from a rat of Group I (a) or Group III (c) showed normal histology, while a glomerulus from a rat of Group IV (d) showed diise proliferative glomerulonephritis. A glomerulus from a rat of Group II showed mild hypercellularity (b). PAS stain. a. b, c, d: X200.
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Number of Nuclei / Glomerulus I
IED:
a0 70 60
r-" I---“’
T
1
I
13 Group m Group 0 Group
II III IV
SO 40 30 20 10 0
2. The number of nuclei in a glomerular cross section at Day 20. Each column and bar are expressed as mean k SD. *The significant difference (P < 0.01) between two groups by Student’s t test. FIG.
fibrinogen was seen in the crescents in rats of Group IV (Fig. 5d). In Groups V and VI, rat IgG, rat C3, and fibrinogen were not detected. These data are summarized in Table 2. Circulating rut anti-rabbit IgG. In rats of Group IV, circulating rat antibodies to rabbit IgG were detected at Day 6 and reached plateau at Day 13 and Day 20. In rats of Group II, anti-rabbit IgG was detected at Day 6 and Day 13 but was not detected at Day 20. Antibody titer was not significantly different between Group II and Group IV at Day 6 and at Day 13. Circulating rat anti-rabbit IgG titer was slightly elevated at Day 6 but it returned to the baseline level by Day 20 in rats of Group I. In rats of Group III, rat anti-rabbit IgG war not detected in the circulation throughout the experiment. These data are summarized in Fig. 6.
FIG. 3. Binding of RbAGBM along glomerular capillary wall in a rat of Group I shown by the direct IF technique. x200.
356
HARA
ET AL.
FIG. 4. Binding of rat IgG to a rat glomerulus shown by the direct IF technique. There is strong binding of rat IgG in a rat of Group IV (d) or moderate binding in a rat of Group II (b). There is faint or no binding of rat IgG in a rat of Group I (a) or Group III (c). a. b. c. d: x200.
Proteinuria. The amount of protein excreted in the urine for 16 hr in rats of Groups I, II, and III was less than 3 mg throughout the experiment. This amount is normal in our laboratory for female Wistar rats. In contrast, most rats of Group IV showed mild to moderate proteinuria at Day 6 and onward (Fig. 7). Serum creatinine. The serum creatinine concentration of rats of Groups I through V was compared with that of Group VI (untreated age- and sex-matched controls). There was no significant difference in the serum creatinine concentration. The data are given in Fig. 8. DISCUSSION
In the present model of accelerated nephrotoxic serum glomerulonephritis, RbAGBM, which when bound to rat GBM cannot induce a significant glomerular lesion by itself, served as a planted antigen during the autologous phase of the disease (10). The severity of glomerular damage is dependent on the autologous response to heterologous (rabbit) immunoglobulins and the lesions are mediated by cellular components such as polymorphonuclear leukocytes, macro&ages,
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357
FIG. 5. Binding of rat C, (a,b) and fibrin/fibrinogen (cd) in a glomerulus of a rat of Group III (a,c) and Group IV (b,d). There is strong binding of C, along the glomerular capillary wall in a rat of Group IV (b), while no binding is seen in a rat of Group III (a). Fibrin deposition is not seen in a glomerulus of a rat of Group III, while fibrin is strongly positive in the area of crescent in a glomerulus of a rat of Group IV (d). a, b, c, d: x200.
and sensitized T lymphocytes (11-14). In rats of Group IV the severity of proteinuria and histological changes were somewhat variable in individual rats and this was possibly due to the difference in the immune response against the heterologous (rabbit) immunoglobulins. Despite this variability, our study showed that FK506 effectively suppressed the autologous response to rabbit IgG and minimized the glomerular damage as a consequence. Mechanisms of immunosuppression of FK506 have recently been studied and this drug affects T lymphocytes. There is no evidence that FK506 directly affects B lymphocytes. Sawada et al. (19) showed that FK506 does not inhibit the recognition of antigens by Tim, receptor but it does inhibit the intracellular process downward from the Ti/T, receptor activated by antigens. It is also reported by the same group that FK506 does not inhibit the proliferation of T lymphocytes which are already activated by IL-2 (19). These data suggest that FK506 suppresses T lymphocytes in the early course of the immune response. This could lead to
358
HARAETAL. TABLE 2 SUMMARYOFIFSTUDYATDAY~O Rabbit IgG
Rat IgG
Rat C,
I II III IV
+++ +++ +++ +++
f ++ 2 ++++
++
Group V Group VI
-
-
-
Group Group Group Group
Fibrinogen ++ (mainly in crescents) -
Note. The intensity of staining was semiquantitated from (-) to (+ + + +).
suppression of subsequent antibody production. The fact that the autologous antibody response in rats of Groups I and III is effectively suppressed is consistent with this concept. The alternative explanation concerning the suppression of the autologous antibody response is the induction of suppressor T lymphocytes. Although there is no direct evidence for this, Kawashima et al. (20) reported that, in experimental autoimmune uveitis, there is antigen-specific suppression of antibody response 2 weeks after the initial immunization of autologous antigen together with FK506 and, therefore, it is reasonable to consider the induction of suppressor T lymphocytes. This second mechanism might explain the decrease of anti-rabbit IgG antibodies on Day 20 in rats of Group II which already had an antibody response against rabbit IgG when FK506 was started. It should, however, be noted that FK506 inhibits the expression of the IL-2 receptor (Tat antigen) on human CD8 + cells more effectively than on CD4 + cells (21), suggesting that this drug suppress the activation of suppressor T lymphocytes. In addition to the inhibition of antibody production, it is quite possible that FK506 mitigates the glomerular lesion through the suppression of expansion of sensitized T lymphocytes by heterologous immunoglobulins. Bhan et al. (11) dem0 De92 Group Group Group 0.15- Group
I II III IV
G---% *------a A--.* H
1 10 15 20 Days O -4 0 5 FIG. 6. Circulating rat anti-rabbit IgG titer detected by ELISA. The data are given by means k SE.
FK.506
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X X
50
10
X
fi
X
x
X
-
x I
X X
::
3
A 0.01
I
Day-4
A
q
Day6
Day1
3
A
ci
G-l
0
G-II
0
G-III
x
G-IV
A
G-V
0
G-VI
_
Day20
FIG. 7. Urinary protein excretion for 16 hr. The data are shown in a semilogarithmic Legends for six groups of rats are given on the right side (Group I through Group VI).
manner.
onstrated that passive transfer of T lymphocytes sensitized by heterologous immunoglobulins into the same strain of rats injected with a subnephritogenic dose of nephrotoxic serum induced proliferative glomerulonephritis. Bolton et al. (22) confirmed the role of cellular immunity in the bursectomized chicken system using a subnephritogenic dose of heterologous nephrotoxic serum. These reports
6 Day
FIG. 8. Serum creatinine concentration at Day -4, Day 6, and Day 20. The number (I through VI) at the bottom of each column indicates the group number.
360
HARA ET AL.
suggest that sensitized T cells are involved in the establishment of glomerular lesions during the autologous phase of nephrotoxic serum glomerulonephritis. Macrophages/monocytes and polymorphonuclear leukocytes, rather than complement, are also important effector cells in this phase (22, 23). Although there is no evidence so far that FK506 directly affects macrophages/monocytes or polymorphonuclear leukocytes, it is probable that macrophages/monocytes are influenced by FK506 indirectly through its suppressive effect on T lymphocytes or through a decreased number of Fc portions of host immunoglobulins (24). CyA is now widely used against transplant rejection and it is widely accepted that this is very useful in preventing allograft rejection (25, 26). CyA is also effective in uveitis of Bechet’s disease (27, 28). Although CyA is thought to be effective in patients with glomerulonephritis (29, 30), the effect is limited (31) and there are often adverse effects such as intravascular coagulation and endothelial damage (8, 9). There have been several conflicting reports concerning the effects of CyA in a similar model of experimental glomerulonephritis. Tipping et al. (32) reported that CyA suppressed the accumulation of T lymphocytes and subsequently inhibited the macrophage-induced glomerular injury. In contrast, Wood et al. (33) claimed that CyA worsened the glomerular injury through the increased infiltration of polymorphonuclear leukocytes and the formation of glomerular thrombosis. In the present study we report for the first time the favorable effects of FK506 in accelerated nephrotoxic serum glomerulonephritis in rats. It was demonstrated that the autologous response to rabbit IgG was effectively suppressed and that the glomerular injury was much less severe with the use of FK506. The dose of FK506 employed in this study was about l/20 to 935 of that of CyA reported in the literature (32). Although precise functional studies were not performed in the present study, FK506 did not influence the serum creatinine level, urinary protein excretion, or renal histology in control rats (Group V). According to a recent report the side effect of FK506 was minimal if it was orally administered in patients who received organ transplantation (34). These data suggest the possibility that FK506 can be used for human glomerulonephritis. ACKNOWLEDGMENTS This work was partly supported by The Monbusho Scientific Research Grants of the Japanese Government (No. 63480189 and No. 63944665) and by the research grants from The Nagoya Kyoritsu Host&al and The Masuko Institute for Medical Research. Authors thank the Fujisawa Pharmaceutical Co. for providing FK506. Authors also thank Nobuko Kamei, Minako Miyawaki, and Naoko Kuno for their excellent technical assistance.
REFERENCES 1. Kino, T., Hatanaka, H., Hashimoto, M., Goto, T., Okuhara, M., Kohara, M., Aoki, H., and Imanaka, H., Fk506, a novel immunosuppressant isolated from a streptomyces. I. Fermentation, isolation, and biological characteristics. J. Antibiot. 40, 1249-1255, 1987. 2. Ochiai, T., Nagata, M., Nakajima, K., Suzuki, T., Sakamoto, K., Enomoto, K., Gunji, Y., Uematsu, T.. Goto, T., Hori, S., Kenmochi, T., Nakagouri, T., Asano, T., Isono, K., Hamaguchi, K., Tsuchida, H., Nakahara, K., Inamura, N., and Goto, T., Studies of the effects of IX506 on renal allografting in the beagle dog. Z’runq~lanruiion 44, 729-733, 1987. 3. Inamura, N., Hashimoto, M., Nakahara, K., Aoki, H., Yamaguchi, I., and Kohsaka, M., Immu-
FK506
4. 5. 6. 7. 8.
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nosuppressive effect of FK506 on collagen-induced arthritis in rats. Clin. Immunol. Immunoparho/. 46, 82-90, 1988. Inamura, N., Hashimoto, M., Nakahara, K., Nakajima, Y., Nishio, M., Aoki, H., Yamaguchi, I., and Kohsaka, M., Effect of FK506 on experimental allergic encephalomyelitis in rats. Znt. J. Zmmunopharmacol. 10, 991-995, 1988. Takabayashi, K., Koike, T., Kurasawa, K., Matsumura, R., Sato, T., Tomioka, H., Ito, I., Yoshiki, T., and Yoshida, S., Effect of FK506, a novel immunosuppressive drug on murine systemic lupus erythematosus. Clin. Zmmunol. Zmmunopatho/. 51, 110-l 17, 1989. Kino, T., Hatanaka, H., Miyata, S., Inamura, N., Nishiyama, M., Yajima, T., Goto, T., Okuhara, M., Kohsaka, M., Aoki, H., and Ochiai, T., FK506, a novel immunosuppressant isolated from a streptomyces. II. Immunosuppressive effect of FK506 in vitro. J. Antibiot. 40, 1256-1265, 1987. Yoshimura, N., Matsui, S., Hamashima, T., and Oka, T.. Effect of a new immunosuppressive agent, FK506, on human lymphocyte responses in vitro. II. Inhibition of the production of IL-2 and y-IFN, but not B-cell-stimulating factor 2. Transplanration 47, 351-356, 1989. Meyrier, A., Cyclosporine A nephrotoxicity in patients with Behcet’s disease. Dev. Toxicol. Environ.
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14, 129-133,
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9. Svenson, K., Bohman, S. O., and Hallgren, R., Renal interstitial fibrosis and vascular changes: Occurence in patients with autoimmune diseases treated with cyclosporine. Arch. Intern. Med. 146, 2007-2010,
1986.
10. Unanue, E. R., and Dixon, F. J., Experimental glomerulonephritis, VI. The autologous phase of nephrotoxic serum nephritis. J. Exp. Med. 121, 715-725, 1965. 11. Bhan, A. K., Schneeberger, E. E., Collins, A. B., and McCluskey, R. T., Evidence for a pathogenic role of a cell-mediated immune mechanism in experimental glomerulonephritis. .Z.Exp. Med. 148, 246-260,
1978.
12. Moorthy, A. V., and Abreo, K., Potentiation of nephrotoxic serum nephritis in Lewis rats by Freund’s complete adjuvant-Possible role for cellular immune mechanisms. Clin. Zmmunol. Zmmunopathol. 28, 383-394, 1983. 13. Holdsworth, S. R., and Neale, J. T., Macrophage-induced glomerular injury: Cell transfer studies in passive autologous antiglomerular basement membrane antibody-initiated experimental glomerulonephritis. Lab. Invest. 51, 172-180, 1984. 14. Holdsworth, S. T., Neale, J. T., and Wilson, C. B., Abrogation of macrophage-dependent injury in experimental glomerulonephritis in the rabbit: Use of an antimacrophage serum. J. Clin. Invest. 68, 686-698, 1981. 15. Meezan, E., Hjelle, T. J., and Brendel, K., A simple, nondisruptive method for the isolation of morphologically and chemically pure basement membranes from several tissues. Life Sci. 17, 1721-1732, 1975. 16. Abrass. C. K., and Cohen, A. H., Accelerated glomerulosclerosis in diabetic rats with immune complex injury. Diabetes 36, 1246-1253, 1987. 17. Schreiner, G. F., Cotran, R. S., Pardo, V., Unanue. E. R., A mononuclear cell component in experimental immunological glomerulonephritis. J. Exp. Med. 147, 369384, 1978. 18. Davidsohn, I., and Henry, J. B., “Clinical Diagnosis by Laboratory Methods,” p. 48, Saunders, Philadelphia, 1969. 19. Sawada, S., Suzuki, G., Kawase, Y., and Takaku, F., Novel immunosuppressive agent, FK506: In vitro effects on the cloned T cell activation. J. Zmmunol. 139, 1797-1803, 1987. 20. Kawashima, H., Fujino, Y., and Mochizuki, M., Effects of a new immunosuppressive agent, FK506, on experimental autoimmune uveoretinitis in rats. Invest. Ophthalmol. Vis. Sci. 29, 12651271, 1988. 21. Yoshimura, N., Matsui, S., Hamashima, T., and Oka, T., Effect of a new immunosuppressive agent, FK506, on human lymphocyte responses in vitro. I. Inhibition of expression of alloantigenactivated suppressor cells, as well as induction of alloreactivity. Transplantation 47, 351-356, 1989. 22. Bolton, W. K., Tucker, L. F., and Sturgill, B. C., New avian model of experimental glomerulonephritis consistent with mediation by cellular immunity: Non humorally mediated glomerulonephritis in chickens. J. C/in. Invest. 73, 1263-1276, 1984.
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23. Naish, P. F., Thomson, N. M., Simpson, I. J., and Peters, K., The role of polymorphonuclear leukocytes in the autologous phase of nephrotoxic nephritis. C/in. Exp. Immtcnol. 22, 2102-l 11. 1975. 24. Holdsworth, S. R., Fc dependence of macrophage accumulation and subsequent injury in experimental glomerulonephritis. /. Immunol. 130, 735-739, 1983. 25. Abbud-Filho, M., Ramalho, H. J., Barberato, J. B., Corrente, J. E., Zerati-Filho, M., Verona, C. B., Martucci, R. C., and Bezas, A. G., Inhibition of chronic kidney allograft rejection by cyclosporine. Transplant. Proc. 21, 1793-1795, 1989. 26. Merion, R. M., White, D. J., and Caine, R. Y., Early renal allograft rejection episodes are less aggressive with cyclosporine-A immunosuppression. Transplant. Proc. 15, 2172-2177, 1983. 27. Caspers-Velu, L. E.. Decaux, G., and Liver& J., Cyclosporine in Behcet’s disease resistant to conventional therapy. Ann. Ophthalmol. 21, 11l-l 16, 1989. 28. Binder, A. I.. Graham, E. M., Sanders, M. D., Dinning, W., James, D. G., and Denman, A. M., Cyclosporin A in the treatment of severe Behcet’s uveitis. Brit. J. Rheumatol. 26, 285-291, 1987. 29. Rostoker, G.. Toro, L., Maadi, A., Ryckelynk, J., Sadreux, T., Rivalan, J., Chevet, D., Janin. C.. Lang, P., and Weil, B., Cyclosporin in idiopathic steroid-resistant membranous glomerulonephritis. Lancet 2, 975-976, 1989. 30. Meyrier, A., Ciclosporin in the treatment of nephrosis: Minimal change disease and focalsegmental glomerulosclerosis. Amer. J. Nephrol. 9, 65-71, 1989. 31. Morales, J. M., Andres, A., Prieto, C., Praga, M., Gutierrez Millet, V.. and Rodicio, J. L., Ciclosporin-induced partial and transient improvement of nephrotic syndrome in recurrent focal segmental glomerulosclerosis. Nephron 53, 283-284, 1989. 32. Tipping, P. G., Neale, J. T., and Holdsworth, S. R., T lymphocyte participation in antibodyinduced experimental glomerulonephritis. Kidney Inr. 27, 530-537. 1985. 33. Wood, A.. Adu, D., Birtwistle, R. J., Brewer, D. B., and Michael, J., Cyclosporine A and antiglomerular basement membrane antibody glomerulonephritis in rats. &it. J. Exp. Pathol. 69, 189-195, 1988. 34. Shapiro, R., Fung, J. J., Jain, A. B., Parks, P., and Starzl, T. E.. The side effects of FK506 in humans. Transplanr. Proc. 22, Suppl I, 35-36, 1990. Received April 10, 1990: accepted with revision July 12. 1990
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
57, 351-362 (l%(b)
The Effects of a New Immunosuppressive Agent, FK506, on the Glomerular injury in Rats with Accelerated Nephrotoxic Serum Glomerulonephritis SOUSHUN
HARA, ATSUSHI
The Third
Department
FUKATSU, NORIHIKO SUZUKI, AND SEIICHI MATSUO
of Internal
Medicine, Nagoya,
Nagoya Japan
University
NOBUO School
SAKAMOTO,
of Medicine,
The aim of the present work was to study the effects of a new immunosuppressive drug, FK506, in accelerated nephrotoxic serum glomerulonephritis. Glomerulonephritis was induced in female Wistar rats by the preimmunization with normal rabbit IgG (Day -4) and the subsequent intravenous injection of rabbit anti-GBM serum (Day 0). Without treatment with FK506, rats developed proteinuria at Day 6 and onward. Rat anti-rabbit IgG was strongly detected at Day 6 and the titer was maintained through Day 20. Moderate hypercellularity and focal crescent formation were observed at Day 20. Rats injected intramuscularly with 0.3 or 1 mgikg of FK506 did not develop proteinuria and the anti-rabbit IgG titer was much less or was undetectable throughout the experiments. These data suggest that FK506 is effective in the present model of glomerulonephritis. Q 1990 Academic
Press. Inc.
INTRODUCTION Recently a new immunosuppressive drug, FK506, was isolated from a strain of soil fungus, Streptomyces tsukubaensis (1) and is now thought to be much more potent against allograft rejection and for the inhibition of experimental autoimmune diseases than cyclosporine A (CyA) (2-5). The mechanisms of the immunosuppressive action of FK506 have been extensively studied, mainly from the aspect of cellular immunity (6), i.e., suppression of reactivity in mixed lymphocyte culture, suppression of production of cytokines (IL-2, IL-3, and interferon-y), and inhibition of the expression of the IL-2 receptor on lymphocytes. So far there has been no evidence that FK506 directly affects B cells in vitro (7). In vivo effects of FK506 have also been studied and most of the studies (2-5) showed that the beneficial effects of this drug were through its suppressive effects on cellular immunity. To date, many drugs have been introduced clinically to prevent or treat glomerulonephritis. Although corticosteroids and a number of immunosuppressants are now known to be effective in certain forms of glomerulonephritis, their effects are limited and sometimes have serious adverse effects (8, 9). In an attempt to explore the possibility of introducing this new drug, FK506, in the treatment of glomerulonephritis, we studied the effects of FK506 in an established model of experimental glomerulonephritis, accelerated nephrotoxic serum glomerulonephritis (10). Glomerular injury in this model is mediated by the autologous antibodies against the glomerular-fixed heterologous immunoglobulins and several reports suggest that the cell-mediated immune system (T cell and/or macrophage) 351 0090-1229/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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participates in glomerular pathology (11-14). This report describes the beneficial effects of FK506 in this model of glomerulonephritis and discusses the mechanisms of action. MATERIALS
AND METHODS
Animals. Female Wistar rats weighing about 170 g and female Japanese white rabbits weighing about 2 kg were obtained from the Chubu Kagaku Shizai Co. (Nagoya, Japan). They were allowed free access to food and water. Preparation of rabbit anti-rat glomerular basement membrane (GBM) antiserum. Rat GBM was obtained from female Wistar rats by a modified method of Meezan et al. (15). Briefly, rat kidneys were perfused with ice-cold T&-buffered
saline (150 miU NaCl, 10 r&f Tris-HCl, 1 t&ml pepstatin A, 1 &ml Antipain, 1 mM benzamidine, 1 mM diisopropyl fluorophosphate). Cortex was removed and glomeruli were isolated by a mesh-sieving technique at 4°C using the same buffer. Tubular fragment contamination was less than 5%. Glomeruli were sonicated for 30 min at 4°C. After centrifugation at 15,000 t-pm for 30 min the sediment was incubated in 1% deoxycholate in phosphate-buffered saline (PBS), pH 7.4, containing protease inhibitors for 1 hr at room temperature. The fraction was washed three times with cold PBS and was then incubated in I M NaCl containing 2000 Kuniz units of deoxyribonuclease 1 (DNAase, Sigma, St. Louis, MO) for 2 hr at 37°C. After centrifugation at 10,000 t-pm for 30 min the sedimented material was obtained as purified GBM and was used as immunogen. Japanese white rabbits were immunized subcutaneously with GBM in complete Freund’s adjuvant every 2 weeks and were bled 32 days after the first immunization. Complement was inactivated by incubating antisera at 56°C for 30 min. The reactivity of antisera to rat GBM (RbAGBM) in vitro was tested by the indirect immunofluorescence technique (IF) using frozen sections of normal rat kidneys as substrate. Typical linear binding of RbAGBM was seen in GBM and tubular basement membrane (TBM) up to the dilution of 16400. In a preliminary experiment, it was confirmed that an intravenous injection of 1.0 ml of RbAGBM induced very mild cellular infiltration in glomeruli but did not induce significant proteinuria during the heterologous phase (Day 0 to Day 10) of nephrotoxic serum glomerulonephritis. Histology and immunohistology. For light microscopy study, kidney tissues were fixed in buffered formalin and embedded in paraffin. Sections (2 pm) were stained with hematoxylin and eosin and periodic acid-Schiff reagent. The number of nuclei was counted in 20 equatorially cut glomeruli in each section and the average number of nuclei per glomerulus was calculated (16). The number of the glomenrli with crescents was also calculated. For the immunofluorescence study, small fragments of kidney tissue were snapfrozen in liquid nitrogen and were cut at 2 p.m by a cryostat. Frozen sections were fixed for 10 min in 100% acetone at 4°C and were stained with goat anti-rabbit IgG labeled with fluorescein isothiocyanate (FITC), FITC-labeled rabbit anti-rat IgG, FITC-labeled goat anti-rat C3, or FITC-labeled goat anti-rat fibrinogen (Cappel, West Chester, PA). In order to avoid the cross-reactivity, FITC-labeled goat anti-rabbit IgG was preabsorbed with normal rat serum.
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FKSW. FK506 was supplied by Fujisawa Pharmaceutical Co. (Osaka, Japan) and was suspended in saline at the concentration of 1 mg/ml. Experimental design. Rats were divided into six groups and each group contained five rats. Accelerated nephrotoxic serum glomerulonephritis was induced according to the modified method of Schreiner (17). Rats were preimmunized with 1 mg of normal rabbit IgG in complete Freund’s adjuvant at Day -4. Four days later (Day 0), 1 ml of RbAGBM was intravenously injected into rats. These were used as control rats (Group IV). Rats of Group I were intramuscularly injected with 0.3 mg/kg/day of FK506 from Day -4 to Day 9. Rats of Group II were intramuscularly injected with 1 mg/kg/day of FK506 from Day 2 to Day 15. Rats of Group III were treated in the same way as Group I except for the dose of FK506 (1 mg/kg/day instead of 0.3). Rats of Group V were injected with 1 mg/kg/day of FK506 from Day -4 to Day 9 without administration of RbAGBM. Rats of Group VI were the age- and sex-matched controls. The composition and treatment in each group of rats are summarized in Table 1. Rats were sacrificed at Day 20 and kidneys were obtained for histological and immunohistological studies. Measurement of urinary protein excretion. Rats were housed in metabolic cages at Day -4, Day 6, Day 13, and Day 20. Urine was collected overnight (16 hr) and the amount of urinary protein was measured by the quantitative sulfosalicylic acid method (18) using bovine serum albumin as a standard. Food was removed during urine collection in order to avoid the contamination of food protein into urine. Serum creatinine. Rats were bled at Days -4, 2, 6, 13, and 20 by retroorbital puncture under ether anesthesia. At each puncture, 0.5 to 1 .O ml of whole blood was obtained and rats were never blinded by this procedure. Serum creatinine was measured by a creatinine measuring kit, CRE(E) KAINOS DR-2300 (KAINOS Co., Tokyo, Japan), which required only 50 pJ of serum. Measurement of rat anti-rabbit IgG in the circulation. Rat anti-rabbit IgG was measured by the ELISA assay using polystylene multiwell plates (Immulon 2, Dynatech Laboratories Inc., Chantilly, VA). Each well was coated with 10 kg/ml of normal rabbit IgG overnight at 4°C. Wells were incubated with rat sera and then with peroxidase-labeled goat anti-rat IgG absorbed with normal rabbit serum. A 0.04% o-phenylenediamine containing 0.005% H,O, was added to the wells. The reactivity of rat serum with rabbit IgG was then measured by the optical density at 492 nm using a SLT-210 photoreader (SLT-Labinstruments, Austria). TABLE
1
EXPERIMENTALPROTOCOLS FORSIXGROUPSOFRATS
Group
Number of rats
Preimmunization with rabbit IgG (at Day -4)
Injection with RbAGBM (at Day 0)
Amount of FKSW bg/kg/day)
FK506 administration
I II III IV V VI
5 5 5 5 5 5
Yes Yes Yes Yes No No
Yes Yes Yes Yes No No
0.3 1.0 1.0 Not injected 1.0 Not injected
Day Day Day Not Day Not
-4-Day 2-Day -4-Day injected -4-Day injected
9 15 9 9
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RESULTS Histological study. Glomeruli from rats of Group IV exhibited diffuse proliferative glomerulonephritis (Fig. Id). Cellular crescents were seen in 12.5% of the glomeruli in this group but were not seen in other groups. In the glomeruli from Group I, III, V, and VI, there was no proliferation (Figs. la and lc). In rats of Group II, the glomeruli showed mild hypercellularity (Fig. lb). The quantitative data are shown in Fig. 2. The number of nuclei per glomerulus is significantly increased in rats of Group IV in comparison with rats of other groups. There was no significant tubulointerstitial alteration in any group. In rats of Group V and VI there were no morphological changes in the kidney. Zmmunofluorescence study. In all groups, except for Groups V and VI, rabbit IgG bound strongly along the GBM (Fig. 3). In rats of Groups II and IV, binding of rat IgG was clearly seen along GBM (Figs. 4b and 4d), while only faint or no binding of rat IgG along GBM was seen in rats of Groups I and III (Figs. 4a and 4~). Deposition of rat C3 along GBM was seen in rats of Group IV (Fig. 5b), but was not observed in rats of Groups I, II, and III (Fig. 5a). Deposition of
FIG. 1. Light microscopic appearance of a representative glomerulus from each experimental group. A glomerulus from a rat of Group I (a) or Group III (c) showed normal histology, while a glomerulus from a rat of Group IV (d) showed diise proliferative glomerulonephritis. A glomerulus from a rat of Group II showed mild hypercellularity (b). PAS stain. a. b, c, d: X200.
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Number of Nuclei / Glomerulus I
IED:
a0 70 60
r-" I---“’
T
1
I
13 Group m Group 0 Group
II III IV
SO 40 30 20 10 0
2. The number of nuclei in a glomerular cross section at Day 20. Each column and bar are expressed as mean k SD. *The significant difference (P < 0.01) between two groups by Student’s t test. FIG.
fibrinogen was seen in the crescents in rats of Group IV (Fig. 5d). In Groups V and VI, rat IgG, rat C3, and fibrinogen were not detected. These data are summarized in Table 2. Circulating rut anti-rabbit IgG. In rats of Group IV, circulating rat antibodies to rabbit IgG were detected at Day 6 and reached plateau at Day 13 and Day 20. In rats of Group II, anti-rabbit IgG was detected at Day 6 and Day 13 but was not detected at Day 20. Antibody titer was not significantly different between Group II and Group IV at Day 6 and at Day 13. Circulating rat anti-rabbit IgG titer was slightly elevated at Day 6 but it returned to the baseline level by Day 20 in rats of Group I. In rats of Group III, rat anti-rabbit IgG war not detected in the circulation throughout the experiment. These data are summarized in Fig. 6.
FIG. 3. Binding of RbAGBM along glomerular capillary wall in a rat of Group I shown by the direct IF technique. x200.
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FIG. 4. Binding of rat IgG to a rat glomerulus shown by the direct IF technique. There is strong binding of rat IgG in a rat of Group IV (d) or moderate binding in a rat of Group II (b). There is faint or no binding of rat IgG in a rat of Group I (a) or Group III (c). a. b. c. d: x200.
Proteinuria. The amount of protein excreted in the urine for 16 hr in rats of Groups I, II, and III was less than 3 mg throughout the experiment. This amount is normal in our laboratory for female Wistar rats. In contrast, most rats of Group IV showed mild to moderate proteinuria at Day 6 and onward (Fig. 7). Serum creatinine. The serum creatinine concentration of rats of Groups I through V was compared with that of Group VI (untreated age- and sex-matched controls). There was no significant difference in the serum creatinine concentration. The data are given in Fig. 8. DISCUSSION
In the present model of accelerated nephrotoxic serum glomerulonephritis, RbAGBM, which when bound to rat GBM cannot induce a significant glomerular lesion by itself, served as a planted antigen during the autologous phase of the disease (10). The severity of glomerular damage is dependent on the autologous response to heterologous (rabbit) immunoglobulins and the lesions are mediated by cellular components such as polymorphonuclear leukocytes, macro&ages,
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FIG. 5. Binding of rat C, (a,b) and fibrin/fibrinogen (cd) in a glomerulus of a rat of Group III (a,c) and Group IV (b,d). There is strong binding of C, along the glomerular capillary wall in a rat of Group IV (b), while no binding is seen in a rat of Group III (a). Fibrin deposition is not seen in a glomerulus of a rat of Group III, while fibrin is strongly positive in the area of crescent in a glomerulus of a rat of Group IV (d). a, b, c, d: x200.
and sensitized T lymphocytes (11-14). In rats of Group IV the severity of proteinuria and histological changes were somewhat variable in individual rats and this was possibly due to the difference in the immune response against the heterologous (rabbit) immunoglobulins. Despite this variability, our study showed that FK506 effectively suppressed the autologous response to rabbit IgG and minimized the glomerular damage as a consequence. Mechanisms of immunosuppression of FK506 have recently been studied and this drug affects T lymphocytes. There is no evidence that FK506 directly affects B lymphocytes. Sawada et al. (19) showed that FK506 does not inhibit the recognition of antigens by Tim, receptor but it does inhibit the intracellular process downward from the Ti/T, receptor activated by antigens. It is also reported by the same group that FK506 does not inhibit the proliferation of T lymphocytes which are already activated by IL-2 (19). These data suggest that FK506 suppresses T lymphocytes in the early course of the immune response. This could lead to
358
HARAETAL. TABLE 2 SUMMARYOFIFSTUDYATDAY~O Rabbit IgG
Rat IgG
Rat C,
I II III IV
+++ +++ +++ +++
f ++ 2 ++++
++
Group V Group VI
-
-
-
Group Group Group Group
Fibrinogen ++ (mainly in crescents) -
Note. The intensity of staining was semiquantitated from (-) to (+ + + +).
suppression of subsequent antibody production. The fact that the autologous antibody response in rats of Groups I and III is effectively suppressed is consistent with this concept. The alternative explanation concerning the suppression of the autologous antibody response is the induction of suppressor T lymphocytes. Although there is no direct evidence for this, Kawashima et al. (20) reported that, in experimental autoimmune uveitis, there is antigen-specific suppression of antibody response 2 weeks after the initial immunization of autologous antigen together with FK506 and, therefore, it is reasonable to consider the induction of suppressor T lymphocytes. This second mechanism might explain the decrease of anti-rabbit IgG antibodies on Day 20 in rats of Group II which already had an antibody response against rabbit IgG when FK506 was started. It should, however, be noted that FK506 inhibits the expression of the IL-2 receptor (Tat antigen) on human CD8 + cells more effectively than on CD4 + cells (21), suggesting that this drug suppress the activation of suppressor T lymphocytes. In addition to the inhibition of antibody production, it is quite possible that FK506 mitigates the glomerular lesion through the suppression of expansion of sensitized T lymphocytes by heterologous immunoglobulins. Bhan et al. (11) dem0 De92 Group Group Group 0.15- Group
I II III IV
G---% *------a A--.* H
1 10 15 20 Days O -4 0 5 FIG. 6. Circulating rat anti-rabbit IgG titer detected by ELISA. The data are given by means k SE.
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X X
50
10
X
fi
X
x
X
-
x I
X X
::
3
A 0.01
I
Day-4
A
q
Day6
Day1
3
A
ci
G-l
0
G-II
0
G-III
x
G-IV
A
G-V
0
G-VI
_
Day20
FIG. 7. Urinary protein excretion for 16 hr. The data are shown in a semilogarithmic Legends for six groups of rats are given on the right side (Group I through Group VI).
manner.
onstrated that passive transfer of T lymphocytes sensitized by heterologous immunoglobulins into the same strain of rats injected with a subnephritogenic dose of nephrotoxic serum induced proliferative glomerulonephritis. Bolton et al. (22) confirmed the role of cellular immunity in the bursectomized chicken system using a subnephritogenic dose of heterologous nephrotoxic serum. These reports
6 Day
FIG. 8. Serum creatinine concentration at Day -4, Day 6, and Day 20. The number (I through VI) at the bottom of each column indicates the group number.
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suggest that sensitized T cells are involved in the establishment of glomerular lesions during the autologous phase of nephrotoxic serum glomerulonephritis. Macrophages/monocytes and polymorphonuclear leukocytes, rather than complement, are also important effector cells in this phase (22, 23). Although there is no evidence so far that FK506 directly affects macrophages/monocytes or polymorphonuclear leukocytes, it is probable that macrophages/monocytes are influenced by FK506 indirectly through its suppressive effect on T lymphocytes or through a decreased number of Fc portions of host immunoglobulins (24). CyA is now widely used against transplant rejection and it is widely accepted that this is very useful in preventing allograft rejection (25, 26). CyA is also effective in uveitis of Bechet’s disease (27, 28). Although CyA is thought to be effective in patients with glomerulonephritis (29, 30), the effect is limited (31) and there are often adverse effects such as intravascular coagulation and endothelial damage (8, 9). There have been several conflicting reports concerning the effects of CyA in a similar model of experimental glomerulonephritis. Tipping et al. (32) reported that CyA suppressed the accumulation of T lymphocytes and subsequently inhibited the macrophage-induced glomerular injury. In contrast, Wood et al. (33) claimed that CyA worsened the glomerular injury through the increased infiltration of polymorphonuclear leukocytes and the formation of glomerular thrombosis. In the present study we report for the first time the favorable effects of FK506 in accelerated nephrotoxic serum glomerulonephritis in rats. It was demonstrated that the autologous response to rabbit IgG was effectively suppressed and that the glomerular injury was much less severe with the use of FK506. The dose of FK506 employed in this study was about l/20 to 935 of that of CyA reported in the literature (32). Although precise functional studies were not performed in the present study, FK506 did not influence the serum creatinine level, urinary protein excretion, or renal histology in control rats (Group V). According to a recent report the side effect of FK506 was minimal if it was orally administered in patients who received organ transplantation (34). These data suggest the possibility that FK506 can be used for human glomerulonephritis. ACKNOWLEDGMENTS This work was partly supported by The Monbusho Scientific Research Grants of the Japanese Government (No. 63480189 and No. 63944665) and by the research grants from The Nagoya Kyoritsu Host&al and The Masuko Institute for Medical Research. Authors thank the Fujisawa Pharmaceutical Co. for providing FK506. Authors also thank Nobuko Kamei, Minako Miyawaki, and Naoko Kuno for their excellent technical assistance.
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12. Moorthy, A. V., and Abreo, K., Potentiation of nephrotoxic serum nephritis in Lewis rats by Freund’s complete adjuvant-Possible role for cellular immune mechanisms. Clin. Zmmunol. Zmmunopathol. 28, 383-394, 1983. 13. Holdsworth, S. R., and Neale, J. T., Macrophage-induced glomerular injury: Cell transfer studies in passive autologous antiglomerular basement membrane antibody-initiated experimental glomerulonephritis. Lab. Invest. 51, 172-180, 1984. 14. Holdsworth, S. T., Neale, J. T., and Wilson, C. B., Abrogation of macrophage-dependent injury in experimental glomerulonephritis in the rabbit: Use of an antimacrophage serum. J. Clin. Invest. 68, 686-698, 1981. 15. Meezan, E., Hjelle, T. J., and Brendel, K., A simple, nondisruptive method for the isolation of morphologically and chemically pure basement membranes from several tissues. Life Sci. 17, 1721-1732, 1975. 16. Abrass. C. K., and Cohen, A. H., Accelerated glomerulosclerosis in diabetic rats with immune complex injury. Diabetes 36, 1246-1253, 1987. 17. Schreiner, G. F., Cotran, R. S., Pardo, V., Unanue. E. R., A mononuclear cell component in experimental immunological glomerulonephritis. J. Exp. Med. 147, 369384, 1978. 18. Davidsohn, I., and Henry, J. B., “Clinical Diagnosis by Laboratory Methods,” p. 48, Saunders, Philadelphia, 1969. 19. Sawada, S., Suzuki, G., Kawase, Y., and Takaku, F., Novel immunosuppressive agent, FK506: In vitro effects on the cloned T cell activation. J. Zmmunol. 139, 1797-1803, 1987. 20. Kawashima, H., Fujino, Y., and Mochizuki, M., Effects of a new immunosuppressive agent, FK506, on experimental autoimmune uveoretinitis in rats. Invest. Ophthalmol. Vis. Sci. 29, 12651271, 1988. 21. Yoshimura, N., Matsui, S., Hamashima, T., and Oka, T., Effect of a new immunosuppressive agent, FK506, on human lymphocyte responses in vitro. I. Inhibition of expression of alloantigenactivated suppressor cells, as well as induction of alloreactivity. Transplantation 47, 351-356, 1989. 22. Bolton, W. K., Tucker, L. F., and Sturgill, B. C., New avian model of experimental glomerulonephritis consistent with mediation by cellular immunity: Non humorally mediated glomerulonephritis in chickens. J. C/in. Invest. 73, 1263-1276, 1984.
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23. Naish, P. F., Thomson, N. M., Simpson, I. J., and Peters, K., The role of polymorphonuclear leukocytes in the autologous phase of nephrotoxic nephritis. C/in. Exp. Immtcnol. 22, 2102-l 11. 1975. 24. Holdsworth, S. R., Fc dependence of macrophage accumulation and subsequent injury in experimental glomerulonephritis. /. Immunol. 130, 735-739, 1983. 25. Abbud-Filho, M., Ramalho, H. J., Barberato, J. B., Corrente, J. E., Zerati-Filho, M., Verona, C. B., Martucci, R. C., and Bezas, A. G., Inhibition of chronic kidney allograft rejection by cyclosporine. Transplant. Proc. 21, 1793-1795, 1989. 26. Merion, R. M., White, D. J., and Caine, R. Y., Early renal allograft rejection episodes are less aggressive with cyclosporine-A immunosuppression. Transplant. Proc. 15, 2172-2177, 1983. 27. Caspers-Velu, L. E.. Decaux, G., and Liver& J., Cyclosporine in Behcet’s disease resistant to conventional therapy. Ann. Ophthalmol. 21, 11l-l 16, 1989. 28. Binder, A. I.. Graham, E. M., Sanders, M. D., Dinning, W., James, D. G., and Denman, A. M., Cyclosporin A in the treatment of severe Behcet’s uveitis. Brit. J. Rheumatol. 26, 285-291, 1987. 29. Rostoker, G.. Toro, L., Maadi, A., Ryckelynk, J., Sadreux, T., Rivalan, J., Chevet, D., Janin. C.. Lang, P., and Weil, B., Cyclosporin in idiopathic steroid-resistant membranous glomerulonephritis. Lancet 2, 975-976, 1989. 30. Meyrier, A., Ciclosporin in the treatment of nephrosis: Minimal change disease and focalsegmental glomerulosclerosis. Amer. J. Nephrol. 9, 65-71, 1989. 31. Morales, J. M., Andres, A., Prieto, C., Praga, M., Gutierrez Millet, V.. and Rodicio, J. L., Ciclosporin-induced partial and transient improvement of nephrotic syndrome in recurrent focal segmental glomerulosclerosis. Nephron 53, 283-284, 1989. 32. Tipping, P. G., Neale, J. T., and Holdsworth, S. R., T lymphocyte participation in antibodyinduced experimental glomerulonephritis. Kidney Inr. 27, 530-537. 1985. 33. Wood, A.. Adu, D., Birtwistle, R. J., Brewer, D. B., and Michael, J., Cyclosporine A and antiglomerular basement membrane antibody glomerulonephritis in rats. &it. J. Exp. Pathol. 69, 189-195, 1988. 34. Shapiro, R., Fung, J. J., Jain, A. B., Parks, P., and Starzl, T. E.. The side effects of FK506 in humans. Transplanr. Proc. 22, Suppl I, 35-36, 1990. Received April 10, 1990: accepted with revision July 12. 1990