å¡ ORIGINAL
ARTICLE
å¡
Immunosuppressive Effect of FK506 on Experimental Allergic Neuritis in Lewis Rats: Change of T Cell Subsets Akiko Adachi, Shigeru Araga and Kazuro Takahashi The effect of a new immunosuppressant, FK506, on experimental allergic neuritis (EAN) was examined in Lewis rats. EAN was induced by inoculation with bovine peripheral myelin. The EAN rats were divided into two groups. FK506-treated EAN rats were prophylactically administered FK506 by injection into the peritoneal cavity at a dosage of 5.0 mg/kg/day for 13 days beginning the day after inoculation. The control EAN rats were injected with only saline solution. FK506 prevented the development of EAN, histologically and clinically. In FK506treated EAN rats, flow cytometric analysis of T cell subsets of the lymph nodes showed a significant decrease in W3/13 positive T cells on the 14th and 21st day and a decrease in W3/25 positive T cells on the 21st day after inoculation when compared with the control EAN rats. The percentage of OX-8 positive T cells were not significantly different between the two groups. Our results suggest that FK506 prevented the development of EAN by decreasing W3/13 and W3/25 positive T cells. (Internal Medicine 31: 6-10, 1992) Key words: flow cytometry, W3/13 positive T cells, W3/25 positive T cells
Introduction
subsets were analyzed.
Experimental allergic neuritis (EAN) , which resembles Materials and Methods human Guillain-Barre syndrome, is an autoimmune disease induced Animals and thein antigen. laboratory Female animals Lewis byrats inoculation (9 with peripheral 10wk old; 180-200g) nerve were myelin obtained emulsified frominSeiwa complete Freund's Experimental adjuvant Animals (1). Several Ltd. (Fukuoka, new agents Japan). haveBovine been used to treat rats with EAN for prophylactic and theraperipheral nerve myelin was prepared from bovine peutic effects, such as glucocorticoid (2, 3), cyclosporin dorsal roots according to the method of Cammer (13) (4, and monoclonal antibody (6). is a and5)stored at a concentration ofART18 10mg/ml inFK506 phosphate new immunosuppressive agent isolated from the fermenbuffered saline (PBS, pH 7.4) at -80°C until use. tation broth Induction ofof EAN. Streptomyces The rats were tsukubaensis. inoculatedItinto belongs to the macrolides groups bypoints chemical structure each footpad and in two of back skin and withthe a total molecular weight is 822 kD. FK506 has similar effects volume of 0.4ml of an emulsion of bovine peripheral and more potent immunosuppressive activity than cyclosporin , nerve myelin solutionisand andistinct equal volume of complete although its structure quite from cyclosporin, Freund's adjuvant. Each rat received 2mg of (7, 8). FK506 has been reported to extend thebovine survival peripheral nerve myelin and 400 pig of Mycobacterium time of cardiac and skin allografts in rats (9, 10), and to tuberculosis. prevent Treatment thewith development FK506. FK506 of typewas II collagen-induced generously arthritis 1) and experimental allergic encephalomyelitis donated(1 from Fujisawa Pharmaceutical Co. , Ltd. (Osaka, in rats (12). Thewas suppressive effects of FK506 EAN Japan). FK506 suspended in sterile salineon solution have neverfor been reported. Here the suppression EAN and used treatment of EAN. EAN rats were of divided by FK506 was examined and the changes theanimals) T cell into two groups. The FK506-treated groupof(30
From Division of Neurology, Institute of Neurological Science, Tottori University School of Medicine, Yonago Received for publication September 17, 1990; Accepted for publication March 25, 1991 Reprint requests should be addressed to Akiko Adachi, MD , Division of Neurology, Institute of Neurological Sciences, Tottori Univers of Medicine, 86 Nishimachi, Yonago 683, Japan 6 Internal Medicine Vol. 31, No. 1 (January 1992)
Immunosuppressive Effect of FK506 on EAN was injected with FK506 into the peritoneal cavity at a dosage of 5mg/kg/day for 13 days beginning the day after inoculation. The control EAN group (30 animals) was injected with 0.5 ml of saline in the same manner as described above. Clinical observation ofEAN, Rats were daily weighed and assessed for clinical signs. The severity of EAN was graded as follows: 0, no abnormality; 1, limp tail; 2, monoparesis or paraparesis; 3, monoplegia or paraplegia; 4, tetraparesis or tetraplegia; 5, moribund or death. Histological examination. To assess the infiltration of mononuclear cells, six parts of the sciatic nerve were removed from each rat and stained with hematoxylin and eosin. The severity of cell infiltration was graded as follows: 0, no abnormality; 1 , cellular infiltration adjacent to a vessel; 2, cellular infiltration in immediate proximity to a vessel; 3, cellular infiltration around a vessel and in more distant Analysis of Tsites. cell subsets. The T cell subsets were analyzed with flow cytometry using mouse anti-rat monoclonal antibodies purchased from Serotec (U.K.). Rats were sacrificed at 10, 14 and 21 days after inoculation under diethylether anesthesia. Peripheral blood samples were drawn by cardiac puncture. The spleen and the inguinal lymph nodes were passed through 200 /im stain less steel mesh. The lymphocyte of the spleen and per ipheral blood were obtained by Ficoll-Paque gradient centrifugation at room temperature at 800x g for 20min (14). The lymphocytes obtained in peripheral blood, spleen and inguinal lymph nodes were suspended at 1.0 x 106 cells per 0.1 ml in dilution buffer [PBS, contain ing 2% (v/v) fetal calfwas serum and 0.1% (w/v) Each cell suspension incubated with 0.25NaN3]. pig of either monoclonal antibody of W3/13, W3/25, or OX-8 at 4°C for 30 min. After three washings with dilution buffer, cell suspension solutions were incubated with 0.25 jug of FITC labeled goat anti-mouse Ig (Tago Inc., Burlingame, U.S.A.) for 30min at 4°C. After three washings, cells were resuspended in 2ml of saline containing 1% (w/v)
paraformaldehyde and kept at 4°C until analyses. Analysis of T cell subsets was performed with a laser fluorescence activated cell sorter, model FCA-1A (JASCO Ltd., Tokyo, Japan). An argon laser with an excitation fre quency of 488 nm was used in combination with a 550 nm "long-pass" filter. Results were reported as the percent ages of cells labeled with a specific monoclonal antibody Statistical out of the analysis. total lymphocytes Statisticalcounted. analysis was carried out using Student's t-test. A significant difference was considered to be p values of 0.01. Results Clinical and histological examination of EAN The FK506-treated rats showed suppression of the development of EAN, whereas the control EAN rats developed EAN (Fig. 1). The mean onset of EAN in the control EAN rats was 13.0 ± 0.8 days. The histological examination showed no mononuclear cell infiltration in the sciatic nerves of the FK-506 treated rats. At the 21st day after inoculation, the FK506-treated rats showed a slight degree of cell infiltration, although EAN was clinically suppressed (Table 1 and Fig. 2). Analysis of T cell subsets T cell subsets in the spleen (Fig. 3). There were no significant differences in the percentages of either W3/ Table 1. Histological Score D ays after ino cu lation G roup
10
14
A B
0.75 ｱ 1.19 0 .54 ｱ 0 .68
2.39 ｱ 0.72
83
'o2
lh
1 2 101112131415161718192021222324252627282930 Days after inoculation Fig. 1. Mean clinical grades of rats. The FK506-treated EAN rats were injected with FK506 in the peritoneal cavity at a dosage of 5.0mg/kg/day for 13 days beginning the day after inoculation (à"-à"). The control EAN rats were injected with a saline solution in the same way as mentioned above (o-o). Medicine
Vol. 31, No. 1 (January
1992)
2 .58 ｱ 0 .93
Mean ± S.D. Group A: FK506 prophylaxis (5.0mg/kg/day) (each n =4), Group B: control (each n=4). à"-« FK 506 prophylaxis (5.0mg/kg/days) (n=10) Ch-O Control (n= ll)
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Fig. 2. Histopathology of FK506-treated EAN. Longitudinal section of sciatic nerve 21 days after inoculation. FK506-treated rats showed a slight degree of cell infiltration around a vessel (A). Control EAN rats demonstrated a heavy mononuclear cell infiltration (B). H-E stain. X200.
21
inoculation
Fig. 3. Percentages of W3/13, W3/25 and OX-8 positive T cells in the spleen. Numbers of rats are shown in the parentheses. Open circle, control EAN rats; closed circle, FK506-treated EAN rats; bar, standard deviation.
13, W3/25 or OX-8 positive T cells, between the two groups. T cell subsets in the inguinal lymph nodes (Fig. 4). The FK506-treated group showed significantly lower percentages of W3/13 positive T cells than the control group on the 14th day (p
ARTICLE
å¡
Immunosuppressive Effect of FK506 on Experimental Allergic Neuritis in Lewis Rats: Change of T Cell Subsets Akiko Adachi, Shigeru Araga and Kazuro Takahashi The effect of a new immunosuppressant, FK506, on experimental allergic neuritis (EAN) was examined in Lewis rats. EAN was induced by inoculation with bovine peripheral myelin. The EAN rats were divided into two groups. FK506-treated EAN rats were prophylactically administered FK506 by injection into the peritoneal cavity at a dosage of 5.0 mg/kg/day for 13 days beginning the day after inoculation. The control EAN rats were injected with only saline solution. FK506 prevented the development of EAN, histologically and clinically. In FK506treated EAN rats, flow cytometric analysis of T cell subsets of the lymph nodes showed a significant decrease in W3/13 positive T cells on the 14th and 21st day and a decrease in W3/25 positive T cells on the 21st day after inoculation when compared with the control EAN rats. The percentage of OX-8 positive T cells were not significantly different between the two groups. Our results suggest that FK506 prevented the development of EAN by decreasing W3/13 and W3/25 positive T cells. (Internal Medicine 31: 6-10, 1992) Key words: flow cytometry, W3/13 positive T cells, W3/25 positive T cells
Introduction
subsets were analyzed.
Experimental allergic neuritis (EAN) , which resembles Materials and Methods human Guillain-Barre syndrome, is an autoimmune disease induced Animals and thein antigen. laboratory Female animals Lewis byrats inoculation (9 with peripheral 10wk old; 180-200g) nerve were myelin obtained emulsified frominSeiwa complete Freund's Experimental adjuvant Animals (1). Several Ltd. (Fukuoka, new agents Japan). haveBovine been used to treat rats with EAN for prophylactic and theraperipheral nerve myelin was prepared from bovine peutic effects, such as glucocorticoid (2, 3), cyclosporin dorsal roots according to the method of Cammer (13) (4, and monoclonal antibody (6). is a and5)stored at a concentration ofART18 10mg/ml inFK506 phosphate new immunosuppressive agent isolated from the fermenbuffered saline (PBS, pH 7.4) at -80°C until use. tation broth Induction ofof EAN. Streptomyces The rats were tsukubaensis. inoculatedItinto belongs to the macrolides groups bypoints chemical structure each footpad and in two of back skin and withthe a total molecular weight is 822 kD. FK506 has similar effects volume of 0.4ml of an emulsion of bovine peripheral and more potent immunosuppressive activity than cyclosporin , nerve myelin solutionisand andistinct equal volume of complete although its structure quite from cyclosporin, Freund's adjuvant. Each rat received 2mg of (7, 8). FK506 has been reported to extend thebovine survival peripheral nerve myelin and 400 pig of Mycobacterium time of cardiac and skin allografts in rats (9, 10), and to tuberculosis. prevent Treatment thewith development FK506. FK506 of typewas II collagen-induced generously arthritis 1) and experimental allergic encephalomyelitis donated(1 from Fujisawa Pharmaceutical Co. , Ltd. (Osaka, in rats (12). Thewas suppressive effects of FK506 EAN Japan). FK506 suspended in sterile salineon solution have neverfor been reported. Here the suppression EAN and used treatment of EAN. EAN rats were of divided by FK506 was examined and the changes theanimals) T cell into two groups. The FK506-treated groupof(30
From Division of Neurology, Institute of Neurological Science, Tottori University School of Medicine, Yonago Received for publication September 17, 1990; Accepted for publication March 25, 1991 Reprint requests should be addressed to Akiko Adachi, MD , Division of Neurology, Institute of Neurological Sciences, Tottori Univers of Medicine, 86 Nishimachi, Yonago 683, Japan 6 Internal Medicine Vol. 31, No. 1 (January 1992)
Immunosuppressive Effect of FK506 on EAN was injected with FK506 into the peritoneal cavity at a dosage of 5mg/kg/day for 13 days beginning the day after inoculation. The control EAN group (30 animals) was injected with 0.5 ml of saline in the same manner as described above. Clinical observation ofEAN, Rats were daily weighed and assessed for clinical signs. The severity of EAN was graded as follows: 0, no abnormality; 1, limp tail; 2, monoparesis or paraparesis; 3, monoplegia or paraplegia; 4, tetraparesis or tetraplegia; 5, moribund or death. Histological examination. To assess the infiltration of mononuclear cells, six parts of the sciatic nerve were removed from each rat and stained with hematoxylin and eosin. The severity of cell infiltration was graded as follows: 0, no abnormality; 1 , cellular infiltration adjacent to a vessel; 2, cellular infiltration in immediate proximity to a vessel; 3, cellular infiltration around a vessel and in more distant Analysis of Tsites. cell subsets. The T cell subsets were analyzed with flow cytometry using mouse anti-rat monoclonal antibodies purchased from Serotec (U.K.). Rats were sacrificed at 10, 14 and 21 days after inoculation under diethylether anesthesia. Peripheral blood samples were drawn by cardiac puncture. The spleen and the inguinal lymph nodes were passed through 200 /im stain less steel mesh. The lymphocyte of the spleen and per ipheral blood were obtained by Ficoll-Paque gradient centrifugation at room temperature at 800x g for 20min (14). The lymphocytes obtained in peripheral blood, spleen and inguinal lymph nodes were suspended at 1.0 x 106 cells per 0.1 ml in dilution buffer [PBS, contain ing 2% (v/v) fetal calfwas serum and 0.1% (w/v) Each cell suspension incubated with 0.25NaN3]. pig of either monoclonal antibody of W3/13, W3/25, or OX-8 at 4°C for 30 min. After three washings with dilution buffer, cell suspension solutions were incubated with 0.25 jug of FITC labeled goat anti-mouse Ig (Tago Inc., Burlingame, U.S.A.) for 30min at 4°C. After three washings, cells were resuspended in 2ml of saline containing 1% (w/v)
paraformaldehyde and kept at 4°C until analyses. Analysis of T cell subsets was performed with a laser fluorescence activated cell sorter, model FCA-1A (JASCO Ltd., Tokyo, Japan). An argon laser with an excitation fre quency of 488 nm was used in combination with a 550 nm "long-pass" filter. Results were reported as the percent ages of cells labeled with a specific monoclonal antibody Statistical out of the analysis. total lymphocytes Statisticalcounted. analysis was carried out using Student's t-test. A significant difference was considered to be p values of 0.01. Results Clinical and histological examination of EAN The FK506-treated rats showed suppression of the development of EAN, whereas the control EAN rats developed EAN (Fig. 1). The mean onset of EAN in the control EAN rats was 13.0 ± 0.8 days. The histological examination showed no mononuclear cell infiltration in the sciatic nerves of the FK-506 treated rats. At the 21st day after inoculation, the FK506-treated rats showed a slight degree of cell infiltration, although EAN was clinically suppressed (Table 1 and Fig. 2). Analysis of T cell subsets T cell subsets in the spleen (Fig. 3). There were no significant differences in the percentages of either W3/ Table 1. Histological Score D ays after ino cu lation G roup
10
14
A B
0.75 ｱ 1.19 0 .54 ｱ 0 .68
2.39 ｱ 0.72
83
'o2
lh
1 2 101112131415161718192021222324252627282930 Days after inoculation Fig. 1. Mean clinical grades of rats. The FK506-treated EAN rats were injected with FK506 in the peritoneal cavity at a dosage of 5.0mg/kg/day for 13 days beginning the day after inoculation (à"-à"). The control EAN rats were injected with a saline solution in the same way as mentioned above (o-o). Medicine
Vol. 31, No. 1 (January
1992)
2 .58 ｱ 0 .93
Mean ± S.D. Group A: FK506 prophylaxis (5.0mg/kg/day) (each n =4), Group B: control (each n=4). à"-« FK 506 prophylaxis (5.0mg/kg/days) (n=10) Ch-O Control (n= ll)
Internal
21
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Fig. 2. Histopathology of FK506-treated EAN. Longitudinal section of sciatic nerve 21 days after inoculation. FK506-treated rats showed a slight degree of cell infiltration around a vessel (A). Control EAN rats demonstrated a heavy mononuclear cell infiltration (B). H-E stain. X200.
21
inoculation
Fig. 3. Percentages of W3/13, W3/25 and OX-8 positive T cells in the spleen. Numbers of rats are shown in the parentheses. Open circle, control EAN rats; closed circle, FK506-treated EAN rats; bar, standard deviation.
13, W3/25 or OX-8 positive T cells, between the two groups. T cell subsets in the inguinal lymph nodes (Fig. 4). The FK506-treated group showed significantly lower percentages of W3/13 positive T cells than the control group on the 14th day (p
