Effects of In Vivo Administration of Anti–B7-1/B7-2 Monoclonal Antibodies on Murine Acute Myocarditis Caused by Coxsackievirus B3

From the Third Department of Internal Medicine (Y.S., N.T., Y.Y.), Faculty of Medicine, University of Tokyo; the Department of Immunology (Y.S., M.A., H.Y., K.O.), School of Medicine, Juntendo University, Tokyo, Japan; and the Institute for Adult Diseases (Y.S.), Asahi Life Foundation, Tokyo, Japan.

Correspondence to Yoshinori Seko, MD, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7–3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.

	Abstract

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Abstract—In viral myocarditis, we previously reported that antigen-specific T cells infiltrate the heart and play an important role in the pathogenesis of myocardial damage. For antigen-specific T-cell activation to occur, it is necessary for T cells to receive costimulatory signals provided by costimulatory molecules expressed on antigen-presenting cells as well as main signals provided by binding of T-cell receptors to antigens. To investigate the roles of costimulatory molecules B7-1 and B7-2 in the development of acute viral myocarditis, we first analyzed the expression of B7-1/B7-2 in the hearts of mice with acute viral myocarditis induced by coxsackievirus B3 (CVB3). Second, we evaluated the induction of B7-1/B7-2 in cultured cardiac myocytes treated with interferon gamma (IFN-). Third, we examined the effects of in vivo administration of anti–B7-1/B7-2 monoclonal antibodies (mAbs) on the development of acute viral myocarditis. We found that CVB3-induced murine acute myocarditis resulted in enhanced expression of B7-1/B7-2 in cardiac myocytes. The expression of B7-1/B7-2 in cardiac myocytes could be induced in vitro by IFN-. We found that in vivo anti–B7-1 mAb treatment markedly decreased myocardial inflammation, whereas anti–B7-2 mAb treatment abrogated the protective effect of anti–B7-1. Our findings indicate that distinct roles for B7-1 and B7-2 antigens are involved in the development of acute viral myocarditis and raise the possibility of immunotherapy with anti–B7-1 mAb to prevent T-cell–mediated myocardial damage in viral myocarditis.

Key Words: immunology • T lymphocyte • infection • killer cell • cardiomyopathy

	Introduction

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Evidence has accumulated that a cell-mediated autoimmune mechanism plays an important role in the pathogenesis of myocardial cell damage involved in viral myocarditis.^{1 2 3 4} Furthermore, prolonged myocardial cell damage initiated by acute myocarditis is thought to be one of the main causes of dilated cardiomyopathy.^{5 6 7} We previously reported that MHC class I antigen was strongly induced on cardiac myocytes in murine acute myocarditis caused by CVB3.² We also demonstrated that NK cells expressing a cytolytic factor, perforin, infiltrate the heart first, and then infiltration by antigen-specific T cells subsequently occurs,^{3 8 9} suggesting that the expression of MHC class I antigens on cardiac myocytes facilitates the interaction between cardiac myocytes and T cells, especially CTLs, and leads to further myocardial cell damage. This is supported by the finding that activated and antigen-specific T cells infiltrate the hearts of patients with dilated cardiomyopathy as well as acute myocarditis.⁷

It is necessary for T cells to receive two signals from the antigen-presenting cell for antigen-specific T-cell activation to occur. The first signal is provided by T-cell receptor engagement with the antigen/MHC complex, and the second signal, termed the costimulatory signal, is provided by costimulatory molecules on the antigen-presenting cell.^{10 11} Among these molecules, B7-1 (B7, CD80)^{12 13} and B7-2 (B70, CD86),^{14 15 16} the B7 family molecules that are the ligands for CD28 and CTLA-4 on T cells, are the most extensively characterized and appear to be the most critical.

The purpose of the present study was to investigate in more detail the T cell–mediated autoimmune mechanism, especially the roles for B7 family costimulatory molecules in the development of acute viral myocarditis. For this purpose, first, we analyzed the expression of B7-1/B7-2 in the heart tissue of mice with acute viral myocarditis induced by CVB3. Second, to confirm the expression of B7-1/B7-2 in myocardial cells in vitro, we induced the expression of B7-1/B7-2 in cultured murine myocardial cells with IFN- and evaluated the expression of B7-1/B7-2 in these cells by immunofluorescence. Third, we examined the effects of in vivo administration of anti–B7-1/B7-2 mAbs on the inflammation associated with acute viral myocarditis.

	Materials and Methods

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Virus and Animals
CVB3 (Nancy strain) was a kind gift from Dr Y. Kitaura (Osaka Medical College, Osaka, Japan). It was grown in cultures of FL cells (human amnion), which were supplied by the Japanese Cancer Research Bank–Cell Bank (National Institute of Hygienic Sciences, Tokyo). The virus preparation had a titer of 1x10¹¹ PFU/L and was stored at -80°C. Six-week-old C3H/He male mice and 14- to 16-day-old fetal C3H/He mice were purchased from the Shizuoka Laboratory Animal Center (Shizuoka, Japan). They were inoculated intraperitoneally with 1x10⁶ PFU of CVB3 in 0.2 mL PBS.

Monoclonal Antibodies
Anti-mouse B7-1 mAb (hybridoma 1G10, rat IgG2a) and anti-mouse B7-2 mAb (GL1, rat IgG2a) were purchased from PharMingen and were used for immunohistochemical study. Another anti-mouse B7-1 mAb (RM80, rat IgG2a) and another anti-mouse B7-2 mAb (PO3, rat IgG2b) were generated by immunizing SD rats with a mouse B-cell line, BCL1, and fusing immune splenocytes with P3U1 myeloma cells.¹⁷ They were used for in vivo mAb treatment study. The preparation of a mouse CMA19 was previously described.¹⁸ The reactivity of CMA19 for C3H/He mouse ventricular myosin heavy chain was confirmed by immunoblot analysis (data not shown).

Preparation of Cultured Cardiac Myocytes
Cultured cardiac myocytes were prepared from 14- to 16-day-old fetal C3H/He mice as described previously.^{2 4} The isolated cardiac myocytes were dispersed into LAB-TEK II Chamber Slide (Nalge Nunc Intl). They were cultured overnight at 37°C in a humidified 5% CO₂/95% air incubator and were then divided into two groups, designated A and B. After replacement with fresh culture medium, recombinant murine IFN- (10⁵ U/L) (Shionogi & Co, Ltd) was added to group B. After 48 hours under these conditions, the cardiac myocytes were subjected to immunocytochemical study.

Immunohistochemistry
In this study, to amplify the specific signals of antigen-antibody reaction, we used TSA technology for fluorescence (TSA-Direct [Green], NEN Life Science Products, according to the manufacturer's instructions). Mice were killed on day 7 after virus inoculation. Cryostat sections (6 µm thick) of spleen and heart ventricles were prepared, air-dried, and fixed in acetone for 5 minutes at 4°C. After they were washed in PBS, the sections were incubated with rat anti-mouse B7-1 or B7-2 mAb, respectively, for 1 hour at 37°C. After they were washed again in PBS, the sections were incubated with biotinylated rabbit anti-rat IgG antibody (Vector Laboratories, Inc), which was preabsorbed with mouse serum, for 1 hour at 37°C. They were then washed in TNT buffer (0.1 mol/L Tris-HCl, pH 7.5, 0.15 mol/L NaCl, and 0.05% Tween 20), blocked with TNB buffer containing a blocking reagent for 30 minutes, and then incubated with streptavidin–horseradish peroxidase for 30 minutes. After the sections were washed again in TNT buffer, they were incubated with fluorescein tyramide for the appropriate time (3 to 10 minutes), washed in TNT buffer, examined, and photographed under a MICROPHOT-FX fluorescence microscope (Nikon).

Immunocytochemistry
For immunocytochemical analysis, to distinguish cardiac myocytes from nonmuscle cells (mainly consisted of fibroblasts), we performed double staining for cardiac myosin heavy chain and B7-1/B7-2. The cultured cells on the slides were washed in PBS and fixed in acetone for 5 minutes. They were then incubated with CMA19 for 1 hour at 37°C, washed in PBS, incubated with TRITC-conjugated anti-mouse IgG antibody for 1 hour at 37°C, and washed again in PBS. The subsequent procedure for the staining of B7-1 or B7-2 was the same as that for the tissue samples.

In Vivo Treatment of Mice With Anti–B7-1/B7-2 mAbs
Six-week-old C3H/He mice were divided into four groups, designated A, B, C, and D (8 mice were used for each group). Mice in group B received the anti–B7-1 mAb (5 mg/kg IP) on the day of virus inoculation (day 0) and on day 3. Mice in group C received the anti–B7-2 mAb (5 mg/kg), mice in group D received anti–B7-1 plus anti–B7-2 mAbs (5 mg/kg each), and mice in group A (control group) received saline; the method of administration for these groups was the same as for group B.

The hearts were removed and laterally sectioned approximately midway between the apex and the atria, which resulted in cross sections of both ventricles. Half of each heart was fixed in 10% buffered formalin and used for histological study. The other half of each heart was frozen in liquid nitrogen and used for polymerase chain reaction.

Histology
The cross sections of formalin-fixed heart tissue from mice in each group were stained with hematoxylin and eosin, then photographed, and printed onto color copy papers. The total area of the myocardium and the areas of inflammation (consisting of cell infiltration and necrosis) were accurately outlined on the color copy papers by microscopically examining the original hematoxylin/eosin-stained cross sections and scanned. The percent area of the myocardium undergoing inflammation was determined by analysis performed on a Macintosh computer using the public domain NIH Image program (written by Wayne Rasband at the US National Institutes of Health and available on the Internet by anonymous FTP, zippy.nimh.nih.gov, or on floppy disk from NTIS, 5285 Port Royal Rd, Springfield, VA 22161, part No. PB93–504868).

Statistical Analysis
One-way ANOVA (with significance at P<.05, corrected by Bonferroni for multiple comparison) was used to evaluate differences between the groups.

	Results

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Expression of B7-1 and B7-2 in Ventricular Tissue
In ventricular tissue of normal mice, B7-1 was only weakly expressed by several cells, and B7-2 was strongly expressed by several cells almost uniformly distributed over the myocardium (Fig 1A and 1C, respectively). The cells positive for B7 antigens are thought to be dendritic cells.¹⁹ There was almost no expression of B7-1 and B7-2 antigens in the cardiac myocytes of normal mice. On day 5 after virus inoculation, just after massive cell infiltrations appeared, expression of B7-1 and B7-2 (Fig 1B and 1D, respectively) was clearly induced on the sarcolemma of cardiac myocytes and the infiltrating cells and then reached a maximum level on about day 7. The expression level of B7-2 on these cells seemed to be stronger than that of B7-1. The expression of B7-1 and B7-2 continued for >4 weeks after virus inoculation with a gradual decrease and was seen nonuniformly over the myocardium and around the areas of cell infiltration or adjacent to them in serial sections.

View larger version (123K): [in this window] [in a new window]   Figure 1. Immunohistochemical study for B7-1 and B7-2 antigens in ventricular tissue. Normal ventricular myocardium (A and C) and ventricular myocardium of mice killed on day 7 of CVB3 infection (B and D) were stained with anti–B7-1 mAb (A and B) and anti–B7-2 mAb (C and D). In normal ventricular myocardium, only a slight or undetectable level of expression of B7-1 (A) and B7-2 (C) was observed on the sarcolemma of cardiac myocytes. Strong expression of B7-2 was observed on many dendritic cells (C). There was strong expression of B7-1 (B), and B7-2 (D) was observed on the sarcolemma of many cardiac myocytes and the infiltrating cells in ventricular myocardium infected with CVB3 (day 7). Bar=20 µm.

Expression of B7-1 and B7-2 in Cultured Ventricular Myocytes
Fig 2 shows double-stained ventricular myocytes cultured in a medium with or without IFN- for 48 hours. Figs 2A and 2B show the staining pattern specific for B7-1, and Figs 2E and 2F show the staining pattern specific for B7-2. Figs 2C and 2D, which correspond to Figs 2A and 2B, respectively, and Figs 2G and 2H, which correspond to Figs 2E and 2F, respectively, show the staining pattern specific for cardiac myosin heavy chain and indicate that most of the cells are cardiac myocytes. There was very slight or no expression of B7-1 and B7-2 in the ventricular myocytes of the control group (Figs 2A and 2E). After treatment with IFN-, most of the ventricular myocytes moderately to strongly expressed B7-1 and B7-2 on their surfaces (Figs 2B and 2F, respectively). No significant level of expression of B7-1 or B7-2 was induced in most of the nonmuscle cells, which mainly consisted of fibroblasts, by treatment with IFN-.

View larger version (118K): [in this window] [in a new window]   Figure 2. Immunocytochemical study of cultured ventricular myocytes for B7-1 (A to D) and B7-2 (E to H). A and B, Myocytes in control group (A) and IFN-–treated group (B), stained with anti–B7-1 mAb and labeled with FITC by the TSA method. C and D, Myocytes stained with CMA19 and labeled with TRITC, corresponding to panels A and B, respectively. Most of the cells reacted in both groups, indicating that they were cardiac myocytes. Slight or no reactions to anti–B7-1 mAb were seen in the control group (A), whereas strong reactions were seen in cardiac myocytes in the IFN-–treated group (B). E and F, Myocytes in the control group (panel E) and IFN-–treated group (panel F), stained with anti–B7-2 mAb and labeled with FITC by the TSA method. G and H, Myocytes stained with CMA19 and labeled with TRITC, corresponding to panels E and F, respectively. Most of the cells reacted in both groups, indicating that they were cardiac myocytes. Slight or no reactions to anti–B7-2 mAb were seen in the control group (E), whereas strong reactions were seen in cardiac myocytes in the IFN-–treated group (F). Bar=20 µm.

In Vivo Anti–B7-1/B7-2 mAb Treatment Study
The incidence of myocarditis was 100% in all of the groups. Panels A, B, C, and D of Fig 3 show representative sections from the hearts of mice from groups A, B, C, and D, respectively. Extensive cell infiltration and necrosis were seen in the mice from groups A, C, and D, whereas both cell infiltration and necrosis were much less severe in the mouse from group B. The results of the histological study are summarized in Fig 4. The mean±SE percent area of myocardium undergoing inflammation was significantly decreased in group B compared with group A (P<.005), group C (P<.005), and group D (P<.001). There were no significant differences among groups A, C, and D. Thus, anti–B7-1 mAb treatment markedly decreased the myocardial inflammation, whereas anti–B7-2 mAb treatment had no significant effect. Anti–B7-1 plus anti–B7-2 mAb treatment also had no significant effect, indicating that anti–B7-2 mAb treatment abrogated the protective effect of anti–B7-1 mAb treatment.

View larger version (165K): [in this window] [in a new window]   Figure 3. Histological analysis of the effects of in vivo anti–B7-1/B7-2 mAb treatment. Cross sections of the heart of a mouse (day 7) in the saline-treated group (A), anti–B7-1 mAb–treated group (B), anti–B7-2 mAb–treated group (C), and anti–B7-1 plus anti–B7-2 mAb–treated group (D) were stained with hematoxylin and eosin. Note the extensive cell infiltration and necrosis in the myocardium of the saline-treated, anti–B7-2 mAb–treated, and anti–B7-1 plus anti–B7-2 mAb–treated groups (A, C, and D, respectively). Both cell infiltration and necrosis were much less in the myocardium of the anti–B7-1 mAb–treated group (panel B). Bar=1 mm.

View larger version (38K): [in this window] [in a new window]   Figure 4. Mean±SE (n=8) percent area of myocardium undergoing inflammation in each group. Mean myocardial inflammation was significantly decreased in the anti–B7-1 mAb–treated group compared with other groups.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrated that CVB3-induced murine acute myocarditis resulted in the enhanced expression of B7-1 and B7-2 on the surface of myocardial cells, especially on cardiac myocytes. In vitro study showed that the enhanced expression of B7-1 and B7-2 was induced in cardiac myocytes by IFN-, which was shown to be mainly synthesized by the infiltrating cells in vivo.⁴ This strongly suggested that the expression of B7-1 and B7-2 in cardiac myocytes from mice with acute myocarditis was induced by the cytokines (such as IFN-) mainly released from the infiltrating cells. This is supported by the fact that the expression of these antigens was clearly induced on cardiac myocytes on day 5 after virus inoculation, just after massive cell infiltrations appeared. There have been no previous studies reporting the expression of B7 antigens in myocardial tissue, especially cardiac myocytes. The expression of B7 antigens in cardiac myocytes may costimulate T cells antigen-specifically and induce immune responses.

In the present study, we also demonstrated that in vivo anti–B7-1 mAb treatment markedly decreased the myocardial inflammation in murine acute myocarditis, whereas anti–B7-2 mAb treatment abrogated the protective effect of anti–B7-1 mAb treatment. This indicates that distinct roles for B7-1 and B7-2 are involved in the development of acute viral myocarditis and raises the possibility of immunotherapy with anti–B7-1 mAb to prevent T cell–mediated myocardial damage in viral myocarditis. In the present study, we did not analyze the effects of anti–B7-1 treatment on the cardiac function of mice with viral myocarditis, and it is known that there is not always good correlation between cardiac function and the severity of inflammation in mice with myocarditis.²⁰ Therefore, we analyzed the effects of anti–B7-1 treatment on the survival of A/J mice with CVB3 myocarditis, because C3H/He mice with CVB3 myocarditis rarely die throughout the course of the disease. And, we found that anti–B7-1 mAb treatment clearly improved the survival of A/J mice with myocarditis caused by CVB3 (authors' unpublished data, 1997).

It is known that CD4⁺ Th cells differentiate into two subsets capable of secreting distinct patterns of cytokines on antigenic stimulation. Th1 cells secrete IL-2, IFN-, and TNF-ß, which activate macrophages and are critical for inducing cell-mediated immune responses. Th2 cells secrete IL-4, IL-5, and IL-10, which are critical for IgG1 and IgE antibody production and also suppress cell-mediated immune responses.^{21 22} Up to now, roles for costimulatory molecules B7-1/B7-2 and CD28/CTLA-4 have been studied in several autoimmune diseases, and one of the best characterized models is murine EAE, which is an animal model of human multiple sclerosis. Kuchroo et al²³ have reported that administration of anti–B7-1 mAb results in predominant generation of Th2 clones and significantly suppresses the induction of this autoimmune disease, whereas administration of anti–B7-2 mAb facilitates Th1 development and substantially increases disease severity. This suggests that treatment with anti-B7 mAbs can alter the course of autoimmune diseases by differentially influencing the development of Th1/Th2 cells from Th precursor cells. From the analysis of the cytokine profile of the infiltrating cells, we found that this model of acute viral myocarditis is mainly mediated by Th1 cells like EAE.²⁴ To investigate the effects of anti–B7-1 mAb treatment on these immune mediators, we also examined the expression of Th cell–related cytokines and inducible NO synthase as well as CVB3 genomes in the heart tissues of mice by a semiquantitative polymerase chain reaction method. However, there was no significant difference in the expression of IFN-, IL-2, and IL-4 transcripts in the ventricular tissues between the anti–B7-1 mAb–treated group and the saline control group, suggesting that no significant change in the ratio of infiltrating Th1/Th2 cells occurred (data not shown). There was also no significant difference in the expression of CVB3 genomes and inducible NO synthase transcript, which are thought to aggravate the inflammation (data not shown).²⁵

It was shown that the expression of B7-1 on tumor cells induces an immune response mediated by CD8⁺ CTLs and leads to tumor regression.^{26 27} Therefore, it is thought that the expression of B7-1 on cardiac myocytes in acute viral myocarditis may activate infiltrating CTLs and induce direct cytotoxicity against the cardiac myocytes. This may be one of the mechanisms of the in vivo effect of anti–B7-1 mAb treatment. Recently, it has been reported that the expression of B7-1 on tumor cells triggers NK cell–mediated cytotoxicity, which leads to tumor regression, and that this response occurs even in the absence of CD28 and cannot be inhibited by the expression of MHC class I antigen.^{28 29} This would be one of the most likely explanations for the in vivo effect of anti–B7-1 mAb treatment revealed in the present study, because NK cells consist of the dominant population of the infiltrating cells and strong expression of MHC class I antigen is induced on cardiac myocytes in the early stage of this murine viral myocarditis.² In addition, no significant expression of CD28 was detected on the infiltrating NK cells (data not shown).

Thus, at least two main phenotypes of the infiltrating cells, which are CTLs and NK cells, may be involved in the mechanism of the in vivo effect of anti–B7-1 mAb treatment. Although the results of in vivo anti–B7-1/B7-2 mAb treatment in the present study were similar to those of EAE, the precise mechanism by which anti–B7-1 mAb treatment decreases the myocardial inflammation and by which cells serve as antigen-presenting cells for T cells are still unknown and remain to be clarified.

	Selected Abbreviations and Acronyms

 

CMA19 = anti–cardiac myosin heavy chain mAb CTL = cytotoxic T lymphocyte CVB3 = coxsackievirus B3 EAE = experimental autoimmune encephalomyelitis IFN = interferon IL = interleukin mAb = monoclonal antibody MHC = major histocompatibility complex NK cell = natural killer cell PFU = plaque forming unit(s) Th cell = T-helper cell TNF = tumor necrosis factor TSA = tyramide signal amplification

	Acknowledgments

 
This study was supported by a grant for cardiomyopathy from the Ministry of Health and Welfare, Japan; a grant for scientific research from the Ministry of Education, Science and Culture, Japan; a grant from Sankyo Foundation of Life Science; a grant from the Ryoichi Naito Foundation for Medical Research; and a Japan Heart Foundation/Pfizer Pharmaceuticals grant for research on coronary artery disease. We thank Kaori Takahashi for excellent technical assistance.

	Footnotes

 
This manuscript was sent to Laurence H. Kedes, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received September 8, 1997; accepted January 9, 1998.

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