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Circulation. 1995;91:1116-1122

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(Circulation. 1995;91:1116-1122.)
© 1995 American Heart Association, Inc.

Articles

Effects of 15-Deoxyspergualin on Experimental Autoimmune Giant Cell Myocarditis of the Rat

Makoto Kodama, MD; Shaosong Zhang, MD; Haruo Hanawa, MD; Makihiko Saeki, MD; Takayuki Inomata, MD; Keisuke Suzuki, MD; Sen Koyama, MD; Akira Shibata, MD

From the First Department of Internal Medicine (M.K., H.H., M.S., T.I., K.S., A.S.), Niigata (Japan) University School of Medicine; the Department of Medical Technology (M.K.), The College of Biomedical Technology, Niigata University, Niigata, Japan; the Division of Cardiology (S.K.), Tachikawa General Hospital, Nagaoka, Japan; and the Department of Medicine (S.Z.), University of Wisconsin Medical School, Milwaukee, Wis.


*    Abstract
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 *Abstract
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Background The benefits of immunosuppressive therapy for human myocarditis are controversial. The effects of a new immunosuppressant agent, 15-deoxyspergualin (DSG), on rats with experimental autoimmune myocarditis (EAM), an animal model of human giant cell myocarditis, were examined.

Methods and Results Lewis rats were immunized with cardiac myosin in Freund's complete adjuvant on day 0. In the first experiment, the effective doses of DSG required to prevent EAM were investigated. Rats were placed into one of five groups: the control group (A) was administered saline from days 1 to 10; group B, 0.3 mg/kg per day of DSG; group C, 1.0 mg/kg per day of DSG; group D, 3.0 mg/kg per day of DSG, and group E, 10.0 mg/kg per day of DSG. Rats were killed on day 28. The heart weight/body weight ratios of the rats of groups D and E were significantly lower than that of the control group. Macroscopic and microscopic scores for myocarditis decreased in groups D and E. In the next experiment, the effects of delayed administration of DSG in preventing autoimmune myocarditis were studied. Two groups of rats received 3.0 and 10.0 mg/kg per day of DSG from days 6 to 15, respectively. Two other groups of rats received the same doses of DSG from days 11 to 20. No preventive effect of delayed DSG treatment was observed. The effects of long-term, delayed initiation therapy then were evaluated. Rats were administered 10.0 mg/kg per day of DSG from days 6 to 25. The heart weight/body weight ratio and macroscopic and microscopic scores of the rats so treated significantly decreased compared with the controls.

Conclusions It was demonstrated that DSG can prevent the development of cardiac myosin–induced autoimmune myocarditis.


Key Words: myocarditis • cells • immune system


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowDiscussion
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Effective strategies for treating myocarditis have not yet been established. While many studies concerning immunosuppressive therapy for human myocarditis have been reported, their efficacy remains controversial.1 2 3 4 5 6 7 8 9 10 11 12 This controversy depends on the possibility of spontaneous remission of myocarditis and the lack of large-scale randomized clinical trials. Another possibility is that the etiology of myocarditis in humans varies. Therapy should be based on etiology; thus, treating myocarditis without reference to its etiology could lead to confusion. If the cause of the myocarditis can be verified during the acute phase of the disease, some patients may be effectively treated with immunosuppressant agents. Clinical and experimental studies indicate that immunosuppressant agents should not be used in treating viral myocarditis.13 14 15 16 17 18 The applicability of such agents in treating giant cell myocarditis, hypersensitivity myocarditis, and other forms of this disease are not known.

Unique models for autoimmune myocarditis in the mouse and rat recently have been established by immunization with cardiac myosin.19 20 Experimental autoimmune myocarditis (EAM) in the rat follows a severe course that is characterized by congestive heart failure and the appearance of multinucleated giant cells. Macroscopic findings of EAM resemble the fulminant form of human myocarditis.21 22 The onset of EAM is about day 15 after immunization. The period of active inflammation of EAM in rats continues over 2 weeks, and it exceeds that of murine viral myocarditis.22 Most infiltrating mononuclear cells in EAM are composed of macrophages and CD4+ T cells. CD8+ T cells are scarce, and B cells are rare in the lesions.23 It also has been demonstrated that EAM is transferable into syngeneic rats not by antibodies but by activated T cells.24 EAM is close to the pathogenesis of some forms of human myocarditis, such as giant cell myocarditis, hypersensitivity myocarditis, and idiopathic myocarditis with a chronic or recurrent course.22 25

The immunosuppressant agent 15-deoxyspergualin (DSG) was isolated from Bacillus laterosporus.26 It has two biological actions, an antitumor effect in murine leukemia and an immunosuppressive effect.27 28 Studies on DSG recently have focused on its intracellular interaction with a family of heat shock proteins.29 The side effects of various immunosuppressant agents occasionally can be serious. Cyclosporine and FK506, recently introduced into clinical medicine, are nephrotoxic. Otherwise, DSG suppresses bone marrow function, but nephrotoxicity has not been described.

In this study, we investigated the effects of DSG on EAM in rats, an animal model of human giant cell myocarditis.


*    Methods
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 *Methods
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Animals
Male Lewis rats were purchased from Charles River Japan Inc. They were bred and maintained at the Facility for Comparative Medicine and Animal Experimentation, Niigata University School of Medicine.

Antigen
Purified cardiac myosin was used as the antigen. Cardiac myosin was prepared and purified from human hearts according to methods described previously.20

Immunization
Cardiac myosin was dissolved in a solution of potassium chloride, 0.3 mol/L, and phosphate-buffered saline (PBS), 0.2 mol/L, at a concentration of 10.0 mg/mL. Rats were immunized with 1.0 mg of cardiac myosin in an equal volume of Freund's complete adjuvant containing 6.0 mg/mL of heat-killed Mycobacterium tuberculosis on day 0.20 22

Agent
Rats with EAM were treated with DSG, which was kindly provided by Nippon Kayaku Inc. The agent was dissolved in saline for intraperitoneal injection.

Treatment
In the first experiment, we determined the dose of DSG required to prevent EAM. Rats were placed in one of five groups, according to the dose of DSG (Fig 1Down, treatment of early phase). Group A (control, n=10) was intraperitoneally injected with 0.5 mL of saline from days 1 to 10; group B (n=9), 0.3 mg/kg per day of DSG; group C (n=8), 1.0 mg/kg per day of DSG; group D (n=8), 3.0 mg/kg per day of DSG; and group E (n=8), 10.0 mg/kg per day of DSG during the same period.



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  		Figure 1. Chart of experimental protocols. 1, Clinical course of the disease: schematic representation of the clinical course of experimental autoimmune myocarditis; 2, treatment of early phase: experimental groups and treatment protocols during the early phase; 3, delayed therapy: experimental groups of the delayed therapy.

In the second experiment, we investigated the effects of the delayed administration of DSG in preventing EAM (Fig 1Up, delayed therapy). Rats of groups F (n=8) and G (n=7) were treated with 3.0 and 10.0 mg/kg per day of DSG from days 6 to 15, respectively. Rats in groups H (n=8) and I (n=8) were injected with 3.0 and 10.0 mg/kg per day of DSG from days 11 to 20, respectively. The effects of delayed initiation and long-term therapy then were evaluated. Group K (n=8) received 10.0 mg/kg per day of DSG from days 6 to 25. Group J (n=8) was injected with 0.5 mL of saline during the same period.

Sampling
Rats were killed under ether anesthesia on day 28. Macroscopic findings of the hearts then were assessed. Findings were classified as 0, normal; 1, focal discolored area present; and 2, multiple or diffuse discolored areas present on the cardiac surface.20 22 Macroscopic scores were judged by two investigators, one of whom had no knowledge of the experimental protocol.

Hearts were weighed immediately after the animals were killed. Because the lesions of EAM spread diffusely, precise calculation of the proportion of diseased muscle volume against total muscle mass was difficult. Our observations confirmed that the ratio of heart weight/body weight best represented the severity of EAM during the acute phase.20 22

Histopathology
Hearts were removed and fixed in 10% formalin, then embedded in paraffin. Several transverse sections were cut from the paraffin-embedded samples and stained with hematoxylin and eosin. The extent of the lesions were graded as 0, normal; 1, few small lesions not exceeding 0.25 mm2 present; 2, multiple small or a few moderately sized lesions not exceeding 6.25 mm2 present; and 3, multiple moderately sized lesions or larger lesions exceeding 6.25 mm2 present. We measured the areas of the lesions using a square lattice scale placed in the eye lens of the microscope. The scale covers 6.25 mm2 under magnification x40; this area occupies about 20% of a transverse section of the heart of a normal Lewis rat. The scale also covers 0.25 mm2 under magnification x200. This area occupies about 1% of a transverse section of the normal rat heart. Microscopic findings were examined by three of the authors, two of whom had no knowledge of the protocol.

Anti–Cardiac Myosin Antibodies
Production of antibodies against cardiac myosin was measured by an enzyme-linked immunosorbent assay according to the previously described procedure, with some modifications.20 The optical density (OD) values of pooled sera of normal Lewis rats at dilution x100 served as the negative control. Each serum sample was diluted with PBS at twofold increments starting at x100. Titers of anti–cardiac myosin antibodies of the samples were determined as the dilution values until the OD of sample sera exceeded 0.1 over the OD of the negative control.

Hematologic and Biochemical Variables
In order to elucidate the mechanisms of protective actions of DSG on EAM and to investigate the side effects of DSG, another four groups of rats were examined. Group A2 (n=5) and group E2 (n=5) were immunized and treated according to the same protocol as groups A and E, respectively, and were killed on day 13. Group J2 (n=5) and group K2 (n=5) were also immunized and treated in the same way as groups J and K, respectively, and were killed on day 26. Hematologic and biochemical variables of the blood obtained at the time the animals were killed were measured. Blood of the normal syngeneic rats (n=8) served as the control.

Immunohistochemical Analysis
Expression of major histocompatibility complex (MHC) antigens in the heart, subsets of infiltrating mononuclear cells in the lesions, and the presence of bound immunoglobulins and complement were immunohistochemically investigated in the rats that were treated with DSG or saline. Hearts from rats of groups A2, E2, J2, and K2 were frozen in OCT compound (Miles Inc.). Frozen sections were cut in a cryostat and fixed in ether for 10 minutes. Immunohistochemical studies were carried out using various monoclonal antibodies: OX18, a marker for rat MHC class I antigens; OX6, a marker for MHC class II antigens; W3/25, a marker for CD4+ T cells; OX8, a marker for CD8+ T cells; and OX42, a marker for macrophages (Serotec Inc). Bound immunoglobulins and complements were detected using peroxidase conjugated goat anti-rat IgG, anti-rat IgM, and anti-rat complement 3 (C3) (Cappel). The staining methods were described previously.22 23

Statistical Analysis
Heart weight/body weight ratio was expressed as mean±1 SD. Both the one-way ANOVA and Duncan's test were used to calculate statistical differences. The Student's t test was used for the comparison of lung weight/body weight, liver weight/body weight, and spleen weight/body weight between the two groups. Macroscopic and microscopic scores were expressed as mean values. The titer of anti–cardiac myosin antibodies was expressed as the dilution titer. Differences were considered significant at P<.05.


*    Results
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*Results
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Treatment in the Early Phase
No rat from the first five groups died spontaneously. The heart weight/body weight ratio of group A was 6.84±1.40; group B, 6.07±1.32; group C, 6.46±1.39; group D, 4.96±1.68; and group E, 4.21±0.99 (Fig 2Down). The heart weight/body weight ratios of groups D and E were significantly lower than that of the control. The ratio of group E was also significantly lower than those of groups B and C.



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  		Figure 2. Graphs show results of the groups with early therapy. 1), Heart weight/body weight ratios. Solid lines indicate a statistically significant difference (P<.05); dotted lines indicate that the difference is not significant. 2), Dilution titers of anti–cardiac myosin antibodies in the serum. 3), Macroscopic scores of myocarditis. 4), Microscopic scores of myocarditis.

The titers of anti–cardiac myosin antibodies of the rats injected with high doses of DSG were slightly suppressed (Fig 2Up). The suppressive effect for the production of anti–cardiac myosin antibodies was dose dependent.

All rats in groups A, B, and C showed discoloration of the cardiac surface. The majority of these animals also had pericardial effusions. Nine rats, 4 from group D and 5 from group E, showed no abnormalities on macroscopic examination of the hearts. Macroscopic scores of groups D and E were low (Fig 2Up).

Extensive myocardial necrosis and mononuclear cell infiltration were observed in the hearts of all animals from groups A, B, and C (Fig 3Down). The center of the myocarditis lesions was composed of myocardial necrosis, degenerated myocardial fibers, large mononuclear cells, and multinucleated giant cells. No calcifications were present. Interstitial cell infiltration consisted of mononuclear cells and polymorphonuclear neutrophils spread around the margin of the lesion. Myocarditis was also detected in the hearts of 7 out of 8 rats in group D. The hearts of 3 rats that appeared macroscopically normal showed microscopically mild inflammation. Five rats in group E that appeared macroscopically normal had no inflammatory lesion in their hearts (Fig 3Down). The microscopic score of group E was markedly low (Fig 2Up). Therefore, EAM was partially suppressed by 3.0 mg/kg per day of DSG and significantly suppressed by 10.0 mg/kg per day of DSG.



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  		Figure 3. A, Photomicrograph of rat heart from group A. Severe myocarditis is observed with multinucleated giant cells. B, Heart of a rat from group E. Bars indicate 60 µm.

Delayed Therapy
One rat in group J died, and severe myocarditis was observed in the heart of this rat. The heart weight/body weight ratio of group F was 5.34±0.81; group G, 6.27±2.13; group H, 5.63±2.42; group I, 5.10±1.86; group J, 6.36±1.73; and group K, 3.87±0.17 (Fig 4Down). The heart weight/body weight ratio of group K was significantly lower than those of groups A, G, and J. There were no other significant differences among the groups.



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  		Figure 4. Graphs show results of the groups with delayed therapy. 1), Heart weight/body weight ratios. Solid lines indicate a statistically significant difference (P<.05); dotted lines indicate that the difference is not significant. 2), Titers of anti–cardiac myosin antibodies. 3), Macroscopic scores of myocarditis. 4), Microscopic scores of myocarditis.

Anti–cardiac myosin antibodies of groups H and I were slightly suppressed compared with those of group J. Those of group K were markedly suppressed (Fig 4Up).

All rats in groups F, G, H, I, and J showed macroscopic evidence of severe myocarditis. Seven of the 8 rats in group K were macroscopically normal. The macroscopic score of group K was low (Fig 4Up).

Severe myocarditis was microscopically observed in the hearts of all rats of groups F, G, H, I, and J. No myocarditis was detected in the hearts of 6 rats in group K. The microscopic score of group K was very low (Fig 4Up).

Weights of other organs were measured in groups J and K. The lung weight/body weight ratios of groups J and K were 6.10±1.26 and 4.68±0.33, respectively (P<.01). The liver weight/body weight ratios of groups J and K were 42.7±3.16 and 39.7±2.99, respectively (P<.1). The spleen weight/body weight ratios of groups J and K were 4.74±0.58 and 3.05±0.86, respectively (P<.001).

Immunohistochemical Analysis
MHC class I antigens were weakly detected on myocardial fibers of rat hearts of the groups A2, E2, J2, and K2, but there were no differences in the findings among the four groups. MHC class II antigens were not expressed on the cardiomyocytes in either the saline-treated or the DSG-treated rats. Otherwise, vascular endothelial cells in the hearts of saline-treated rats were clearly stained with OX6, but the staining of these cells of DSG-treated rats was weak (Fig 5Down). Four out of 5 rats of group A2 and all 5 rats of group E2 showed no inflammatory lesions. All 5 rats of group J2 revealed severe myocarditis, and none of group K2 showed myocarditis. Infiltrating cells in the rat hearts of group J2 were predominantly composed of macrophages and CD4+ T cells. CD8+ T cells were scarce. Bound IgG was detected on the vascular wall and myocardial fibers around the vessels (Fig 5Down). Anti-rat IgG staining of DSG-treated rats was weaker than that of saline-treated rats. The areas of necrosis and degenerated myocardial fibers were intensely stained with anti-rat IgG. There was no difference in the staining by anti-rat IgM among the four groups. Bound C3 was detected in the lesions of rats treated with saline but was not detected in the hearts of DSG-treated groups (Fig 5Down).



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  		Figure 5. Immunohistochemical analysis of saline-treated and 15-deoxyspergualin–treated rats. A and B are stainings using OX6 (see text) of rat hearts from groups J2 and K2, respectively. Vascular endothelial cells of the hearts from group J2 intensely expressed major histocompatibility complex class II antigens. C and D show bound IgG of hearts from groups A2 and E2, respectively. E and F indicate the findings of staining with anti-rat C3 of the rat hearts from groups J2 and K2, respectively. Bar indicates 30 µm; all photographs are in the same magnification.

Side Effects of DSG
Hematologic and biochemical variables were investigated soon after therapy periods. In saline-treated rats, leukocytosis and thrombocytosis developed from day 13, and mild anemia appeared on day 26. On the other hand, marked leukocytopenia and anemia appeared from day 13 in the rats treated with DSG. No significant renal toxicity was detected in either the saline-treated or the DSG-treated rats.


*    Discussion
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*Discussion
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The entire mechanisms of actions of DSG on producing immunosuppression are not known. This agent was initially evaluated for its antitumor effects in murine leukemia, L-1210.26 Its immunosuppressive actions were subsequently described. DSG inhibits the antibody responses to both thymus-dependent and thymus-independent antigens.27 It also suppresses the mixed lymphocyte reaction and the induction of cytotoxic T lymphocytes.28 DSG suppresses the expression of interleukin-2 receptor but has little effect on the production of interleukin-2. An intracellular DSG binding protein was recently identified as a cognate member of the heat shock protein 70 family.29 The beneficial effects of DSG have been demonstrated in animal models, producing the prevention of rejection of cardiac allograft,30 treatment of streptozotocin-induced diabetes mellitus,31 spontaneous murine lupus nephritis,32 33 experimental autoimmune uveoretinitis,34 35 experimental allergic encephalomyelitis, and murine graft-versus-host disease.36 37 This is the first study to evaluate its effects in myocarditis.

Giant cell myocarditis is thought to be a rare and lethal inflammatory heart disease of unknown etiology. However, a recent report demonstrated that it is more common than had been previously thought.38 It is not known whether giant cell myocarditis is an actual homogeneous disease entity. We have reported that EAM could express both forms of myocarditis: giant cell myocarditis and non–giant cell myocarditis, according to the phase and severity of the disease.22 This implies that some patients with non–giant cell myocarditis may have the same etiology as that of giant cell myocarditis. Moreover, the absence of multinucleated giant cells in a few biopsy specimens does not exclude the etiologic similarity to giant cell myocarditis. While the EAM model resembles human giant cell myocarditis in its morphological features, its similarity to other clinical diseases is also noted.23 It resembles human hypersensitivity myocarditis because of the predominance of macrophages in the lesions.39 Therefore, results of this study also may be applied to those diseases.

The preventive effects of DSG on EAM were demonstrated in this study. We previously reported that progression of EAM could be prevented by cyclosporine and FK-506.40 41 However, prednisolone and aspirin were ineffective in preventing EAM.40 The production of anti–cardiac myosin antibodies also was suppressed in parallel with the suppression of myocarditis in this study. The effects of DSG on antibody production resembled that of cyclosporine but differed from that of FK-506, which had little effect on antibody production.41 Immunohistochemical analysis revealed that the intensity of bound IgG and C3 was weaker in the hearts of the rats treated with DSG than those of rats treated with saline. This may imply partly why DSG has revealed therapeutic efficacy on EAM. We have reported previously that anti–cardiac myosin antibodies play a lesser role in the initiation of EAM,24 but complement-mediated target cell injuries by autoantibodies may play a role in the progression of this disease. In the study on delayed therapy, the lung weight/body weight ratio of the effectively treated group was significantly lower than that of the control group, which may indicate the prevention of lung congestion due to heart failure. Similar effects also have been observed after treatment using FK-506 and cyclosporine.40 41

The same dose of DSG produced differing effects on EAM according to the treatment phase, as shown in groups E, G, and I. Therefore, DSG appeared to effectively suppress the initial response of EAM, the afferent limb of the immune response, but its short-term use failed to inhibit the later phase response, the efferent limb. The schema for the clinical course of EAM appears in Fig 1Up (clinical course of the disease). We have previously shown that EAM is a T cell–mediated autoimmune disease.24 The initial immune response of EAM probably involves the activation and proliferation of myocarditogenic T cell clones. In this study, expression of MHC class II antigens was suppressed in the hearts of the rats treated with DSG. Our previous reports imply that CD4+ T cells with {alpha}/ß T cell receptors play critical roles in the initiation of EAM.42 Because CD4+ T cells recognize antigens in the complex form with MHC class II molecules, suppression of the expression of MHC class II antigens will lead to prevention of the disease. Recruitment of myocarditogenic T cells to the target organ is the second stage. Effector-target interaction is probably the last stage, which may involve anti–cardiac myosin antibodies, macrophages, polymorphonuclear leukocytes, and various inflammatory mediators. DSG proved to be most effective on the initial antigen-priming process in EAM, from the findings of this study. Similar effects of DSG on lymphocyte proliferation also were demonstrated in an in vitro study of mixed lymphocyte reaction.28 Long-term therapy is necessary to suppress the efferent limb of the immune response of EAM, as shown in group K.

Immunosuppressant agents exhibit a variety of adverse effects due to their biological actions. Cyclosporine and FK506 are strong immunosuppressant agents, especially against T cell–mediated immune responses, and nephrotoxicity is their well-known side effect. No nephrotoxicity was observed with DSG in this study. Bone marrow suppression is the most important side effect of DSG. Critical dosages for these adverse effects of immunosuppressant agents differ among species and strains. Therefore, the clinical study concerning DSG should be performed cautiously. Clinically, immunosuppressant agents should be chosen according to the valance of their beneficial and harmful actions. Because biological actions and side effects of DSG differ from cyclosporine and FK506, it may be useful in treating patients in whom these agents are ineffective or toxic.

The treatment of myocarditis has been extensively investigated using murine viral myocarditis models. Immunosuppressant agents such as prednisolone,13 cyclosporine,14 15 16 17 cyclophosphamide,18 antibodies against T cells, and FK50643 44 45 have been used in treating murine myocarditis caused by coxsackievirus B3 or encephalomyocarditis virus. Those studies revealed that such immunosuppressive therapy has neither preventive nor therapeutic efficacy. Our studies demonstrated that immunosuppressive therapy is quite effective in treating autoimmune myocarditis. It is suggested that myocarditis should be treated according to its etiology. Autoimmune myocarditis can be treated effectively with immunosuppressant agents, whereas viral myocarditis can be treated effectively with antiviral agents.46 47 DSG eventually may be used in treating myocarditis in humans who have an autoimmune etiology.


*    Acknowledgments
 
This study was supported in part by a Japan Heart Foundation Research Grant, research grants from the Niigata Medical Association, and grants for scientific research from the Ministry of Education, Science, and Culture of Japan (05770454, 06770486). We thank Nippon Kayaku Inc, Tokyo, Japan, for providing us with 15-deoxyspergualin. We also gratefully acknowledge Miss T. Watanabe and Mr Yoshiyuki Komatsu, Tashiro Shyokakika Hospital, Niigata, Japan, for their excellent technical assistance.


*    Footnotes
 
Reprint requests to Makoto Kodama, MD, First Department of Internal Medicine, Niigata University School of Medicine, Asahimachi 1-754, Niigata, 951 Japan.

Received May 31, 1994; revision received August 31, 1994; accepted September 23, 1994.


*    References
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 up arrowIntroduction
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up arrowResults
up arrowDiscussion
*References
 

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