Cardiovascular Drugs and Therapy 1992;6:403-407 © Kluwer Academic Publishers, Boston. Printed in U.S.A.
Effects of Lobenzarit on Murine Acute Viral Myocarditis T o m o y u k i Y o k o y a m a , Tsugiyasu K a n d a , Tadashi Suzuki, Kazuhiko Murata Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan
Summary. Lobenzarit (CCA) is a newly developed immunomodulating drug that has been demonstrated to enhance suppressor T-cell number and function. In this study we investigated the effect of CCA on murine myocarditis induced by the encephalomyocarditis (EMC) virus. Mice were first inoculated with the EMC virus. CCA (100 mg/kg) was administered daily for 14 days, starting on the day the mice were inoculated. The concentrations of L y t 2 + (suppressor/cytotoxic T) cells in the peripheral blood and heart were examined by laser flow cytometry and in situ staining. The concentration of Lyt2 + cells increased significantly in the peripheral blood and heart of CCA-treated mice when compared with untreated mice (blood: 23.0 ± 2.6% vs. 18.8 ± 2.8%, p < 0.05; heart: 30.1 ± 4.6% vs. 15.8 ± 4.3%, p < 0.05, respectively). Surprisingly, however, the severity of myocardial inflammation in CCA-treated mice was significantly greater than in untreated mice (area of inflammation: 33.1 ± 10.5% vs. 18.8 ± 19.5%, respectively, p < 0.05). Thus, these findings suggest that CCA may aggravate EMC-induced acute myocarditis by enhancing the number of Lyt2 + cells.
Cardiovasc Drugs Ther 1992;6:403-407 Key Words. Lobenzarit, myocarditis, suppressor/cytotoxic T cell, encephalomyocarditis virus
tion, would attenuate or prevent myocardial damage in an experimental model of acute viral myocarditis. Accordingly, the purpose of this study was to investigate the effects of CCA in t e r m s of its effects on suppressor T-cells and myocardial damage, using a murine model of acute myocarditis induced by the encephalomyocarditis (EMC) virus.
Methods Animals
Eight-week-old female C3H/He mice, purchased from Charles River Co. (Atsugi, Japan), were used in the experiments.
Virus A myocarditic variant of EMC virus was obtained from Y. Seto, Ph.D. (Keio University, Tokyo, Japan). Virus preparations were stored at -70°C in Eagle's minimum essential medium (MEM) supplemented with 0.1% fetal calf serum (FCS) until use. Chemicals
T h e exact pathogenic mechanisms responsible for myocardial damage in myocarditis are not known. Recent clinical reports suggest that there is association between the decline in suppressor T-cell activity and the development of myocarditis [1,2], thus raising the possibility that augmentation of suppressor T-cell function might have a beneficial effect in acute viral myocarditis. Lobenzarit (CCA) is a new immunomodulating drug that has been used for the treatment of rheumatoid arthritis [3]. While the exact mechanisms of actions of CCA are not known, this drug has been shown to inhibit the age-related decline of suppressor T-cell activity and the production of autoantibodies in NZB/ NZW F1 mice [4]. Furthermore, prophylactic administration of CCA has prolonged the survival rate of mice infected with herpes simplex virus [5]. Based on the above reports, we hypothesized that CCA-induced augmentation of suppressor T-cell number, as well as the inhibition of autoantibody produc-
Lobenzarit (CCA; disodium-4-chloro-2'2-iminodibenzoic acid, lot A6E03) was supplied by Chugai Pharmaceutical Co. Ltd. (Tokyo, Japan). It was dissolved in phosphate-buffered saline and used after passing through a 0.45-~m Millipore filter. Infection protocol
Animals were inoculated intraperitoneally (i.p.) with 500 plaque-forming units of EMC virus in 0.1 ml saline.
Address for correspondence: TomoyukiYokoyama,M.D., Cardiology Section, Veterans Affairs MedicalCenter, 2002 HolcombeBlvd., Houston, TX 77030, USA. Address for reprints: Tsugiyasu Kanda, M.D., Second Department of Internal Medicine, Gunma University Schoolof Medicine, 3-39-15 Showamachi, Maebashi, 371, Japan. This study was presented at the 63rd Annual Scientific Session of the American Heart Association in Dallas, Texas, November, 1990. 403
404
Yokoyama, Kanda, Suzuki and Murata
Treatment protocol Sixty mice were randomly assigned to two groups, each consisting of 30 mice. In the treated group (CCA+ EMC group), each mouse were administered 100 mg/kg of CCA i.p. every day for 14 days on the day the mice were inoculated. Control mice were injected i.p. with 0.1 ml of isotonic saline in the same manner. The dose of CCA was based on a previous report [6] and our preliminary experiments, which suggested that the dose of CCA used was optimal. Ten mice in each group were observed for survival rate during 21 days, and the remaining mice were sacrificed on days 3, 7, and 21 postinoculation (p.i.) with the virus for pathological study, assay of viral titer in the heart, and assay of the peripheral and myocardial lymphocyte subset. Additional control groups of uninfected mice treated for 14 days with CCA (CCA group, administered as above, n = 8) or without CCA (normal control group, n = 3) were also prepared. Virus assay Viral titer in the heart was determined in terms of the resultant cytopathic effect and was expressed as tissue-culture mean infectious dose (TCD50). The heart was homogenized in 2 ml of MEM, the homogenate was centrifuged, and the supernatant was inoculated into a 96-well microtiter plate containing human amnion (FL) cells in MEM containing 10% FCS. The microtiter plate was examined daily for 5 days for the appearance of any cytopathic effect. Pathologic examination The heart and other body organs were weighed and then fixed in 10% buffered formalin followed by staining with hematoxylin-eosin. The sections were examined by a pathologist who was blinded as to the results of the study. Myocardial sections were projected onto paper, where the total area of myocardium and the area of inflammation were outlined. The percent area of the myocardium undergoing inflammation and necrosis was then determined using a computer program that calculated the percent of necrotic myocardium as (total area inflamed/total myocardial area) x 100. No distinctions were made between inflammation and necrosis because the two processes generally occurred simultaneously. Assay for peripheral and myocardial lymphocyte subset The heart was rapidly removed into RPMI1640 culture medium containing 2.5% FCS, then minced gently with a sterile slide glass and an injector. Heart sample from five mice were pooled to obtain adequate numbers of lymphocytes. After mincing, the cell suspension was filtered through nylon mesh to eliminate debris and was centrifuged at 1500 rpm for 5 minutes. Peripheral blood was collected in heparin. The cells were finally suspended in 0.1 ml of the same medium.
T cells were stained with monoclonal rat anti-Thyl.2, anti-Lyt2, and anti-L3T4 antibodies (Becton-Dickinson Immunocytometry Systems, San Jose, CA). The preparations were immediately analyzed by laser flow cytometry (FACScan, Becton-Dickinson Immunocytometry Systems). The following abbreviations are used for the identification of cell types: Thyl,2 = pan T cells, Lyt2 = suppressor/cytotoxic T cells, and L3T4 = helper/inducer T cells.
Positively-stained lymphocytes in the heart The hearts were sectioned on day 7 p.i. and immediately frozen at -70°C. Cryostat sections obtained subsequently were stained in situ by the four-step avidin-biotin immunoperoxidase method of Adamson et al. [7] using monoclonal rat anti-Thyl.2, Lyt2, and L3T4 antibodies as primary antibody and mouse antirat immunoglobulin antibody as the secondary antibody. The total number of small, round nucleated cells and the number of lymphocytes stained with a monoclonal antibody were counted by one blinded pathologist; 300-500 lymphocytes were counted for each sample. The percentage of positively stained lymphocytes was then calculated. Statistics The Kaplan-Meier test was used to determine the significance of differences in the survival rate and the Mann-Whitney U test was used to evaluate differences in the histologic grading, viral titer in the heart, and lymphocyte-cell subset.
Results
Survival rate As shown in Figure 1, survival periods for mice in the CCA + EMC group had a tendency to be shortened in comparison with mice in the EMC group, but there was no significant difference in the survival rate over 21 days between the two groups. Viral titers in the heart Viral titer in the hearts were determined on day 3 p.i. The average viral titer was 2.6 - 0.2 logl0TCD~0/mg (n = 3, mean -+ SO) in the EMC group and 2.5 0.2 logmTCDs0/mg (n = 3) in the CCA+ EMC group. There was no significant difference between the two groups.
Heart weight There were no significant differences in the heart weight/body weight ratio on days 7 and 21 after infection between the EMC group and the C C A + E M C group (Table 1). Histologic findings in the heart On day 7 after infection, moderate lesions of myocardial necrosis with calcification and small lesions of
Lobenzarit in Viral Myocarditis
Table 2. Percent myocardial area of inflammation in
%
CCA
%4
•
100
80 o
CCA-treated, encephalomyocarditis virus inoculated mice Days after infection
o
• __~s
7
60
21
.>_
~g 4o
405
Group
No. of mice
% Myocardial area of inflammation
EMC CCA+EMC EMC CCA+EMC
6 7 6 6
18.0 33.1 38.6 40.4
-+ -+ +-+
17.7 10.5 a 13.2 12.8
o----EMC (n=lO) ¢
Values a r e m e a n -+ SD. ap < 0.05 vs. E M C g r o u p .
CCA+F'MC ( n = | O )
20-
I
0
'
'
. . . .
,
,
,
,
7
,
i
I
i
.
.
.
.
.
14 J1 Days After Inoculation
Fig. 1. Effect of CCA on survival rate in murine myocarditis due to encephalomyocarditis virus. The survival periods of mice treated with CCA (CCA + EMC group) had a tendency to be shortened in comparison with untreated mice (EMC), but there was no significant difference in the survival rate over 21 days between the two groups.
mononuclear cell infiltration were seen in the EMC group. The myocardial necrosis with calcification was more evident, and the percent myocardial area of inflammation was significantly greater in the CCA+ EMC group than in the EMC group on day 7. However, on day 21 after infection there was no difference between the two groups (Table 2).
Lymphocyte subsets in the p e r i p h e r a l blood and heart Lymphocyte subsets in the peripheral blood and heart were determined on day 7 p.i. by laser flow cytometry. In the peripheral blood study, the percentage of Lyt2 + cells was significantly greater (p < 0.05) in the C C A + E M C group than in the EMC group. The percentage of Lyt2 + cells was also significantly greater in the CCA group than in the normal control group. Thus, CCA enhances Lyt2 + cells in both peripheral blood as well as infected and uninfected mice. In the examination of the heart, the percentage of Lyt2 + cells in the CCA + EMC group had a tendency
Table 1. Heart weight~body weight ratios in CCA-treated, encephalomyocarditis virus inoculated mice Days after infection 7 21
Group
No. of mice
EMC CCA+EMC EMC CCA+EMC
6 6 6 6
Weight (g) 17.2 15.7 17.4 18.7
HW = heart weight; BW = body weight. Values a r e m e a n -+ SD.
± ± ± ±
1.6 1.0 1.9 1.2
H W / B W × 10 3 6.4 6.5 7.2 6.9
± 1.0 ± 0.6 ± 1.7 -+ 0.8
to be greater than in the EMC group, although the difference was not significant statistically (Table 3).
Positively-stained lymphocytes in the heart Cryostat sections of the heart on day 7 p.i. were stained with monoclonal antibodies. Few Lyt2 + cells were present between myocytes in the EMC group, while many Lyt2 + cells were seen in the CCA + EMC group. The percentage of Lyt2 + cells was significantly greater in the CCA + EMC group than in the EMC group (Table 4). Additional control group In these groups, mice were inoculated with isotonic saline only or CCA only. There were no deaths over a 3-week period or any microscopic evidence of mycardial inflammation. Discussion
Recent clinical reports have suggested that there is an association between suppressor T-cell activity and the development of myocarditis. For example, Koga et al. [2] observed decreased concentrations of CD8 + (suppressor/cytotoxic T) cells in the peripheral blood of patients with acute myocarditis. In the investigations of concanavalin A stimulated suppressor T-cell activity, Eckstein et al. [1] described a significant reduction of suppressor T cell activity in patients with myocarditis. Thus, these results suggest the following: a) depression of suppressor T-cell production is somehow involved in the pathogenesis of myocarditis and b) augmentation of suppressor T-cell function might possibly have a salutary effect in acute viral myocarditis. In the present study, CCA increased the number of Lyt2 + (suppressor/cytotoxic T) cells in the peripheral blood of both uninfected and infected mice, and in the myocardium of infected mice. CCA treatment did not affect the number of L3T4 + (helper/inducer T) cells in this study. As noted, not only was there no beneficial effect in the CCA-treated mice with acute viral myocarditis, but there was actually an increase in the extent of myocardial damage in the treated mice; that is, there were no significant differences be-
406
Yokoyama, Kanda, Suzuki and Murata
Table 3. Lymphocyte subset in the peripheral blood and heart at 7 days after inoculation in CCA-treated, encephalomyocarditis virus inoculated mice
Group Peripheral blood EMC CCA+EMC Cont. CCA Heart a EMC CCA+EMC
n
Thyl, 2 (pan T c e l l )
Lyt2 (suppressor/cytotoxie T cell)
L3T4 (helper/inducer T celt)
7 5 4 3
67.6 - 4.9 72.2 +- 6.2 65.4 +- 4.9 68.0 +- 6.1
18.8 +- 2.8 23.0 - 2.6b 18.3 -+ 1.2 20.8 +- 0.2¢
44.3 +- 4.9 46.4 +- 3.7 42.7 +- 9.5 39.5 +- 7.8
4 4
78.9 +- 18.3 82.7 -+ 17.4
47.5 -+ 23.1 54.4 +. 18.6
32.4 +- 27.0 35.8 +- 30.5
EMC = infected with EMC virus and untreated mice; CCA+EMC = infected and treated mice; Cont. = uninfected and untreated mice; CCA = uninfectedand treated mice. aIn heart study, the percentage of lymphocytesubset was determined in lymphocytespooled from five mice per sample. bp < 0.05 vs. EMC group. Cp < 0.05 vs. Cont. group. Values are mean -+ SD%.
tween CCA-treated and untreated mice with respect to the survival rate, cardiac viral titer, or heart weight, whereas the severity of myocardial inflammation was significantly greater in CCA-treated mice than in untreated mice. Since the levels of L3T4 + cells were not elevated in this model, it is unlikely that these cells played a major role in the pathogenesis of myocardial injury in the EMC-infected C3H/He mice. A previous experimental study in mice [8] showed that there was no significant change in the percent of suppressor/cytotoxic T cells or helper/inducer T cells during the acute stage of EMC myocarditis, despite the fact that there was a significant decrease in the percent of pan T-cell and immature T-cell subsets. In contrast, our study showed that there were no significant decreases in the percentages of Lyt2 + , L3T4 +, or Thyl.2 + (pan T) cells in the peripheral blood of EMC-infected animals. We did not, however, examine the percentage of immature T cells in this study. The discrepancy between the two studies m a y be related to the murine strain that was used. The results of the present experimental study differ from those in humans, wherein a depression of suppressor T cells has been observed. Thus, our original hypothesis that augmentation of suppressor T-cell Table 4. Positively stained lymphocytes in the heart at 7 days after inoculation in CCA-treated, encephalomyocarditis virus inoculated mice
Group
n
Thyl, 2
Lyt2
L3T4
EMC CCA+EMC
4 4
41.7 +- 13.0 36.9 +- 16.8
15.8 +- 4.3 30.1 +- 4.6a
8.5 +- 0.8 5.6 -+ 0.1
The percentageof positivelystained cells w a s of 300-500 lymphocytesper sample. Values are m e a n -+ SD%. ap < 0.05 vs. E M C group.
determined
by a count
levels might be beneficial in a murine model of myocarditis did not prove to be correct. Moreover, it appears as though the augmentation of Lyt2 + cells might play a pathogenic role in the myocardial injury in our experimental myocarditis model. However, the mechanism for this effect is not known. Important to the above discussion is that Ito et al. [5] have shown that CCA augments cytotoxic T-cell activity against herpes simplex virus-infected cells. CCA has also been reported to enhance the number and function of suppressor T cells [4]. Lyt2 + cells have both suppressor and/or cytotoxic functions, thus making it difficult to differentiate suppressor T-cell activity from cytotoxic T-cell activity. Recently, both CD4+ and CD8+ cells have been shown to have helper and cytotoxic functions. It is conceivable, therefore, that the CCA-induced enhancement of Lyt2 + cells in our model may have resulted in increased cytotoxic activity. Moreover, this problem is further compounded by differences in the activities of Lyt2 + cells in different strains of mice. For example, Huber et al. [9-12] demonstrated that BALB/c mice selectively depleted of Lyt2 + cells in vivo will develop minimal myocarditis when infected with coxsackie B3 virus. A similar depletion of L3T4 + cells in vivo was shown to cause a moderate but nonstatistically significant decrease in cardiac injury. In contrast, the development of myocarditis in DBA/2 mice depends entirely on the presence of L3T4+ cells. Thus, these studies indicate that the role of T cells in picornavirus-induced myocarditis depends on the strain of mouse. Furthermore, these studies demonstrate that autoreactive cytotoxic T cells, which belong to the Lyt2 + cell population, will specifically lyse uninfected myocytes. In the present study we have examined the effect of CCA on the development of myocardial injury in a murine model of EMC myocarditis. This study shows
Lobenzarit in Viral Myocarditis
t h a t C C A i n c r e a s e d t h e n u m b e r of L y t 2 + cells in t h e p e r i p h e r a l blood of b o t h uninfected and infected mice, b u t did not affect t h e n u m b e r of L3T4 + cells. This increase in L y t 2 + cells a p p e a r e d to be associated with an i n c r e a s e in t h e e x t e n t of m y o c a r d i a l d a m a g e in the t r e a t e d mice. A l t h o u g h t h e i m m u n o p a t h o g e n e t i c mechanism for m y o c a r d i a l d a m a g e of t h e C 3 H / H e mice infected with E M C v i r u s is not known, we sugg e s t t h a t excessive a u g m e n t a t i o n of L y t 2 + cells b y C C A m a y be t h e cause of t h e enhanced m y o c a r d i a l d a m a g e in o u r s t u d y . Acknowledgment
5.
6.
7.
8.
We are indebted to Dr. D.L. Mann (Houston, Veterans Affairs Medical Center) for critically reviewing the manuscript.
References
9. 1. Eckstein R, Mempel W, Bolte HD. Reduced suppressor cell activity in congestive cardiomyopathy and myocarditis. Circulation 1982;65:i224-1229. 2. Koga Y, Miyazaki Y, Toshima H, et al. Lymphocyte subsets in patients with acute myopericarditis, arrhythmias and dilated cardiomyopathy. Jpn Circ J 1989;53:78-86. 3. Shiokawa Y, Horiuchi Y, Mizushima Y, et al. A multicenter double-blind controlled study of lobenzarit, a novel immunomodulator, in rheumatoid arthritis. J Rheumatol 1984;11: 615-623. 4. Nakano T, Yamashita Y, Ohsugi Y, et al. The effect of CCA (lobenzarit disodium) on suppressor T cell function and the
10.
11.
12.
407
production of autoantibodies in New Zealand Black and New Zealand White F1 mice. Immunopharmacology 1983;5: 293-302. Itoh K, Kurane I, Saito F, et al. Regulatory effect of CCA on immune responses. Clin Immunol 1983;15:242-252 (in Japanese). Ohsugi Y, Nakano T, Hata S, et al. N-(2-carboxyphenyl)-4chloroanthranilic acid disodium salt: A novel anti-arthritic agent without anti-inflammatory and immunosuppressive activities. J Pharm Pharmac 1977;29:636-637. Adamson TC, Frisman DM, Howell FV. Immunohistologic analysis of lymphoid infiltrates in primary Sjogren's syndrome using monoclonal antibodies. J Immunol 1983;130: 203-208. Kishimoto C, Kuribayashi K, Fukuma K, et al. Immunologic identification of lymphocyte subsets in experimental murine myocarditis with encephalomyocarditis virus: Different kinetics of lymphocyte subsets between the heart and the peripheral blood, and significance of Thyl.2+ (pan T) and Lytl + ,23+ (immature T) subsets in the development of myocarditis. Circ Res 1987;61:715-725. Huber SA, Lodge PA. Coxsackievirus B-3 myocarditis in Balb/c mice: Evidence for autoimmunity to myocyte antigens. Am J Pathol 1984;116:21-29. Gutherie M, Lodge PA, Huber SA. Cardiac injury in coxsackievirus group B, type 3 induced myocarditis in Balb/ c mice is mediated by Lyt2 + cytolytic lymphocytes. Cell Immunol 1984;88:558-567. Houten VN, Huber SA. Role of cytotoxic T cells in experimental myocarditis. Springer Semin Immunopathol 1989; 11:61-68. Blay R, Simpson K, Leslie K, et al. Coxsackievirus-induced disease CD4 + cells initiate both myocarditis and pancreatitis in DBA/2 mice. Am J Pathol 1989;135:899-907.
Effects of Lobenzarit on Murine Acute Viral Myocarditis T o m o y u k i Y o k o y a m a , Tsugiyasu K a n d a , Tadashi Suzuki, Kazuhiko Murata Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan
Summary. Lobenzarit (CCA) is a newly developed immunomodulating drug that has been demonstrated to enhance suppressor T-cell number and function. In this study we investigated the effect of CCA on murine myocarditis induced by the encephalomyocarditis (EMC) virus. Mice were first inoculated with the EMC virus. CCA (100 mg/kg) was administered daily for 14 days, starting on the day the mice were inoculated. The concentrations of L y t 2 + (suppressor/cytotoxic T) cells in the peripheral blood and heart were examined by laser flow cytometry and in situ staining. The concentration of Lyt2 + cells increased significantly in the peripheral blood and heart of CCA-treated mice when compared with untreated mice (blood: 23.0 ± 2.6% vs. 18.8 ± 2.8%, p < 0.05; heart: 30.1 ± 4.6% vs. 15.8 ± 4.3%, p < 0.05, respectively). Surprisingly, however, the severity of myocardial inflammation in CCA-treated mice was significantly greater than in untreated mice (area of inflammation: 33.1 ± 10.5% vs. 18.8 ± 19.5%, respectively, p < 0.05). Thus, these findings suggest that CCA may aggravate EMC-induced acute myocarditis by enhancing the number of Lyt2 + cells.
Cardiovasc Drugs Ther 1992;6:403-407 Key Words. Lobenzarit, myocarditis, suppressor/cytotoxic T cell, encephalomyocarditis virus
tion, would attenuate or prevent myocardial damage in an experimental model of acute viral myocarditis. Accordingly, the purpose of this study was to investigate the effects of CCA in t e r m s of its effects on suppressor T-cells and myocardial damage, using a murine model of acute myocarditis induced by the encephalomyocarditis (EMC) virus.
Methods Animals
Eight-week-old female C3H/He mice, purchased from Charles River Co. (Atsugi, Japan), were used in the experiments.
Virus A myocarditic variant of EMC virus was obtained from Y. Seto, Ph.D. (Keio University, Tokyo, Japan). Virus preparations were stored at -70°C in Eagle's minimum essential medium (MEM) supplemented with 0.1% fetal calf serum (FCS) until use. Chemicals
T h e exact pathogenic mechanisms responsible for myocardial damage in myocarditis are not known. Recent clinical reports suggest that there is association between the decline in suppressor T-cell activity and the development of myocarditis [1,2], thus raising the possibility that augmentation of suppressor T-cell function might have a beneficial effect in acute viral myocarditis. Lobenzarit (CCA) is a new immunomodulating drug that has been used for the treatment of rheumatoid arthritis [3]. While the exact mechanisms of actions of CCA are not known, this drug has been shown to inhibit the age-related decline of suppressor T-cell activity and the production of autoantibodies in NZB/ NZW F1 mice [4]. Furthermore, prophylactic administration of CCA has prolonged the survival rate of mice infected with herpes simplex virus [5]. Based on the above reports, we hypothesized that CCA-induced augmentation of suppressor T-cell number, as well as the inhibition of autoantibody produc-
Lobenzarit (CCA; disodium-4-chloro-2'2-iminodibenzoic acid, lot A6E03) was supplied by Chugai Pharmaceutical Co. Ltd. (Tokyo, Japan). It was dissolved in phosphate-buffered saline and used after passing through a 0.45-~m Millipore filter. Infection protocol
Animals were inoculated intraperitoneally (i.p.) with 500 plaque-forming units of EMC virus in 0.1 ml saline.
Address for correspondence: TomoyukiYokoyama,M.D., Cardiology Section, Veterans Affairs MedicalCenter, 2002 HolcombeBlvd., Houston, TX 77030, USA. Address for reprints: Tsugiyasu Kanda, M.D., Second Department of Internal Medicine, Gunma University Schoolof Medicine, 3-39-15 Showamachi, Maebashi, 371, Japan. This study was presented at the 63rd Annual Scientific Session of the American Heart Association in Dallas, Texas, November, 1990. 403
404
Yokoyama, Kanda, Suzuki and Murata
Treatment protocol Sixty mice were randomly assigned to two groups, each consisting of 30 mice. In the treated group (CCA+ EMC group), each mouse were administered 100 mg/kg of CCA i.p. every day for 14 days on the day the mice were inoculated. Control mice were injected i.p. with 0.1 ml of isotonic saline in the same manner. The dose of CCA was based on a previous report [6] and our preliminary experiments, which suggested that the dose of CCA used was optimal. Ten mice in each group were observed for survival rate during 21 days, and the remaining mice were sacrificed on days 3, 7, and 21 postinoculation (p.i.) with the virus for pathological study, assay of viral titer in the heart, and assay of the peripheral and myocardial lymphocyte subset. Additional control groups of uninfected mice treated for 14 days with CCA (CCA group, administered as above, n = 8) or without CCA (normal control group, n = 3) were also prepared. Virus assay Viral titer in the heart was determined in terms of the resultant cytopathic effect and was expressed as tissue-culture mean infectious dose (TCD50). The heart was homogenized in 2 ml of MEM, the homogenate was centrifuged, and the supernatant was inoculated into a 96-well microtiter plate containing human amnion (FL) cells in MEM containing 10% FCS. The microtiter plate was examined daily for 5 days for the appearance of any cytopathic effect. Pathologic examination The heart and other body organs were weighed and then fixed in 10% buffered formalin followed by staining with hematoxylin-eosin. The sections were examined by a pathologist who was blinded as to the results of the study. Myocardial sections were projected onto paper, where the total area of myocardium and the area of inflammation were outlined. The percent area of the myocardium undergoing inflammation and necrosis was then determined using a computer program that calculated the percent of necrotic myocardium as (total area inflamed/total myocardial area) x 100. No distinctions were made between inflammation and necrosis because the two processes generally occurred simultaneously. Assay for peripheral and myocardial lymphocyte subset The heart was rapidly removed into RPMI1640 culture medium containing 2.5% FCS, then minced gently with a sterile slide glass and an injector. Heart sample from five mice were pooled to obtain adequate numbers of lymphocytes. After mincing, the cell suspension was filtered through nylon mesh to eliminate debris and was centrifuged at 1500 rpm for 5 minutes. Peripheral blood was collected in heparin. The cells were finally suspended in 0.1 ml of the same medium.
T cells were stained with monoclonal rat anti-Thyl.2, anti-Lyt2, and anti-L3T4 antibodies (Becton-Dickinson Immunocytometry Systems, San Jose, CA). The preparations were immediately analyzed by laser flow cytometry (FACScan, Becton-Dickinson Immunocytometry Systems). The following abbreviations are used for the identification of cell types: Thyl,2 = pan T cells, Lyt2 = suppressor/cytotoxic T cells, and L3T4 = helper/inducer T cells.
Positively-stained lymphocytes in the heart The hearts were sectioned on day 7 p.i. and immediately frozen at -70°C. Cryostat sections obtained subsequently were stained in situ by the four-step avidin-biotin immunoperoxidase method of Adamson et al. [7] using monoclonal rat anti-Thyl.2, Lyt2, and L3T4 antibodies as primary antibody and mouse antirat immunoglobulin antibody as the secondary antibody. The total number of small, round nucleated cells and the number of lymphocytes stained with a monoclonal antibody were counted by one blinded pathologist; 300-500 lymphocytes were counted for each sample. The percentage of positively stained lymphocytes was then calculated. Statistics The Kaplan-Meier test was used to determine the significance of differences in the survival rate and the Mann-Whitney U test was used to evaluate differences in the histologic grading, viral titer in the heart, and lymphocyte-cell subset.
Results
Survival rate As shown in Figure 1, survival periods for mice in the CCA + EMC group had a tendency to be shortened in comparison with mice in the EMC group, but there was no significant difference in the survival rate over 21 days between the two groups. Viral titers in the heart Viral titer in the hearts were determined on day 3 p.i. The average viral titer was 2.6 - 0.2 logl0TCD~0/mg (n = 3, mean -+ SO) in the EMC group and 2.5 0.2 logmTCDs0/mg (n = 3) in the CCA+ EMC group. There was no significant difference between the two groups.
Heart weight There were no significant differences in the heart weight/body weight ratio on days 7 and 21 after infection between the EMC group and the C C A + E M C group (Table 1). Histologic findings in the heart On day 7 after infection, moderate lesions of myocardial necrosis with calcification and small lesions of
Lobenzarit in Viral Myocarditis
Table 2. Percent myocardial area of inflammation in
%
CCA
%4
•
100
80 o
CCA-treated, encephalomyocarditis virus inoculated mice Days after infection
o
• __~s
7
60
21
.>_
~g 4o
405
Group
No. of mice
% Myocardial area of inflammation
EMC CCA+EMC EMC CCA+EMC
6 7 6 6
18.0 33.1 38.6 40.4
-+ -+ +-+
17.7 10.5 a 13.2 12.8
o----EMC (n=lO) ¢
Values a r e m e a n -+ SD. ap < 0.05 vs. E M C g r o u p .
CCA+F'MC ( n = | O )
20-
I
0
'
'
. . . .
,
,
,
,
7
,
i
I
i
.
.
.
.
.
14 J1 Days After Inoculation
Fig. 1. Effect of CCA on survival rate in murine myocarditis due to encephalomyocarditis virus. The survival periods of mice treated with CCA (CCA + EMC group) had a tendency to be shortened in comparison with untreated mice (EMC), but there was no significant difference in the survival rate over 21 days between the two groups.
mononuclear cell infiltration were seen in the EMC group. The myocardial necrosis with calcification was more evident, and the percent myocardial area of inflammation was significantly greater in the CCA+ EMC group than in the EMC group on day 7. However, on day 21 after infection there was no difference between the two groups (Table 2).
Lymphocyte subsets in the p e r i p h e r a l blood and heart Lymphocyte subsets in the peripheral blood and heart were determined on day 7 p.i. by laser flow cytometry. In the peripheral blood study, the percentage of Lyt2 + cells was significantly greater (p < 0.05) in the C C A + E M C group than in the EMC group. The percentage of Lyt2 + cells was also significantly greater in the CCA group than in the normal control group. Thus, CCA enhances Lyt2 + cells in both peripheral blood as well as infected and uninfected mice. In the examination of the heart, the percentage of Lyt2 + cells in the CCA + EMC group had a tendency
Table 1. Heart weight~body weight ratios in CCA-treated, encephalomyocarditis virus inoculated mice Days after infection 7 21
Group
No. of mice
EMC CCA+EMC EMC CCA+EMC
6 6 6 6
Weight (g) 17.2 15.7 17.4 18.7
HW = heart weight; BW = body weight. Values a r e m e a n -+ SD.
± ± ± ±
1.6 1.0 1.9 1.2
H W / B W × 10 3 6.4 6.5 7.2 6.9
± 1.0 ± 0.6 ± 1.7 -+ 0.8
to be greater than in the EMC group, although the difference was not significant statistically (Table 3).
Positively-stained lymphocytes in the heart Cryostat sections of the heart on day 7 p.i. were stained with monoclonal antibodies. Few Lyt2 + cells were present between myocytes in the EMC group, while many Lyt2 + cells were seen in the CCA + EMC group. The percentage of Lyt2 + cells was significantly greater in the CCA + EMC group than in the EMC group (Table 4). Additional control group In these groups, mice were inoculated with isotonic saline only or CCA only. There were no deaths over a 3-week period or any microscopic evidence of mycardial inflammation. Discussion
Recent clinical reports have suggested that there is an association between suppressor T-cell activity and the development of myocarditis. For example, Koga et al. [2] observed decreased concentrations of CD8 + (suppressor/cytotoxic T) cells in the peripheral blood of patients with acute myocarditis. In the investigations of concanavalin A stimulated suppressor T-cell activity, Eckstein et al. [1] described a significant reduction of suppressor T cell activity in patients with myocarditis. Thus, these results suggest the following: a) depression of suppressor T-cell production is somehow involved in the pathogenesis of myocarditis and b) augmentation of suppressor T-cell function might possibly have a salutary effect in acute viral myocarditis. In the present study, CCA increased the number of Lyt2 + (suppressor/cytotoxic T) cells in the peripheral blood of both uninfected and infected mice, and in the myocardium of infected mice. CCA treatment did not affect the number of L3T4 + (helper/inducer T) cells in this study. As noted, not only was there no beneficial effect in the CCA-treated mice with acute viral myocarditis, but there was actually an increase in the extent of myocardial damage in the treated mice; that is, there were no significant differences be-
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Yokoyama, Kanda, Suzuki and Murata
Table 3. Lymphocyte subset in the peripheral blood and heart at 7 days after inoculation in CCA-treated, encephalomyocarditis virus inoculated mice
Group Peripheral blood EMC CCA+EMC Cont. CCA Heart a EMC CCA+EMC
n
Thyl, 2 (pan T c e l l )
Lyt2 (suppressor/cytotoxie T cell)
L3T4 (helper/inducer T celt)
7 5 4 3
67.6 - 4.9 72.2 +- 6.2 65.4 +- 4.9 68.0 +- 6.1
18.8 +- 2.8 23.0 - 2.6b 18.3 -+ 1.2 20.8 +- 0.2¢
44.3 +- 4.9 46.4 +- 3.7 42.7 +- 9.5 39.5 +- 7.8
4 4
78.9 +- 18.3 82.7 -+ 17.4
47.5 -+ 23.1 54.4 +. 18.6
32.4 +- 27.0 35.8 +- 30.5
EMC = infected with EMC virus and untreated mice; CCA+EMC = infected and treated mice; Cont. = uninfected and untreated mice; CCA = uninfectedand treated mice. aIn heart study, the percentage of lymphocytesubset was determined in lymphocytespooled from five mice per sample. bp < 0.05 vs. EMC group. Cp < 0.05 vs. Cont. group. Values are mean -+ SD%.
tween CCA-treated and untreated mice with respect to the survival rate, cardiac viral titer, or heart weight, whereas the severity of myocardial inflammation was significantly greater in CCA-treated mice than in untreated mice. Since the levels of L3T4 + cells were not elevated in this model, it is unlikely that these cells played a major role in the pathogenesis of myocardial injury in the EMC-infected C3H/He mice. A previous experimental study in mice [8] showed that there was no significant change in the percent of suppressor/cytotoxic T cells or helper/inducer T cells during the acute stage of EMC myocarditis, despite the fact that there was a significant decrease in the percent of pan T-cell and immature T-cell subsets. In contrast, our study showed that there were no significant decreases in the percentages of Lyt2 + , L3T4 +, or Thyl.2 + (pan T) cells in the peripheral blood of EMC-infected animals. We did not, however, examine the percentage of immature T cells in this study. The discrepancy between the two studies m a y be related to the murine strain that was used. The results of the present experimental study differ from those in humans, wherein a depression of suppressor T cells has been observed. Thus, our original hypothesis that augmentation of suppressor T-cell Table 4. Positively stained lymphocytes in the heart at 7 days after inoculation in CCA-treated, encephalomyocarditis virus inoculated mice
Group
n
Thyl, 2
Lyt2
L3T4
EMC CCA+EMC
4 4
41.7 +- 13.0 36.9 +- 16.8
15.8 +- 4.3 30.1 +- 4.6a
8.5 +- 0.8 5.6 -+ 0.1
The percentageof positivelystained cells w a s of 300-500 lymphocytesper sample. Values are m e a n -+ SD%. ap < 0.05 vs. E M C group.
determined
by a count
levels might be beneficial in a murine model of myocarditis did not prove to be correct. Moreover, it appears as though the augmentation of Lyt2 + cells might play a pathogenic role in the myocardial injury in our experimental myocarditis model. However, the mechanism for this effect is not known. Important to the above discussion is that Ito et al. [5] have shown that CCA augments cytotoxic T-cell activity against herpes simplex virus-infected cells. CCA has also been reported to enhance the number and function of suppressor T cells [4]. Lyt2 + cells have both suppressor and/or cytotoxic functions, thus making it difficult to differentiate suppressor T-cell activity from cytotoxic T-cell activity. Recently, both CD4+ and CD8+ cells have been shown to have helper and cytotoxic functions. It is conceivable, therefore, that the CCA-induced enhancement of Lyt2 + cells in our model may have resulted in increased cytotoxic activity. Moreover, this problem is further compounded by differences in the activities of Lyt2 + cells in different strains of mice. For example, Huber et al. [9-12] demonstrated that BALB/c mice selectively depleted of Lyt2 + cells in vivo will develop minimal myocarditis when infected with coxsackie B3 virus. A similar depletion of L3T4 + cells in vivo was shown to cause a moderate but nonstatistically significant decrease in cardiac injury. In contrast, the development of myocarditis in DBA/2 mice depends entirely on the presence of L3T4+ cells. Thus, these studies indicate that the role of T cells in picornavirus-induced myocarditis depends on the strain of mouse. Furthermore, these studies demonstrate that autoreactive cytotoxic T cells, which belong to the Lyt2 + cell population, will specifically lyse uninfected myocytes. In the present study we have examined the effect of CCA on the development of myocardial injury in a murine model of EMC myocarditis. This study shows
Lobenzarit in Viral Myocarditis
t h a t C C A i n c r e a s e d t h e n u m b e r of L y t 2 + cells in t h e p e r i p h e r a l blood of b o t h uninfected and infected mice, b u t did not affect t h e n u m b e r of L3T4 + cells. This increase in L y t 2 + cells a p p e a r e d to be associated with an i n c r e a s e in t h e e x t e n t of m y o c a r d i a l d a m a g e in the t r e a t e d mice. A l t h o u g h t h e i m m u n o p a t h o g e n e t i c mechanism for m y o c a r d i a l d a m a g e of t h e C 3 H / H e mice infected with E M C v i r u s is not known, we sugg e s t t h a t excessive a u g m e n t a t i o n of L y t 2 + cells b y C C A m a y be t h e cause of t h e enhanced m y o c a r d i a l d a m a g e in o u r s t u d y . Acknowledgment
5.
6.
7.
8.
We are indebted to Dr. D.L. Mann (Houston, Veterans Affairs Medical Center) for critically reviewing the manuscript.
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