From the Third Department of Internal Medicine, Faculty of Medicine,
University of Tokyo (Y.S., N.T., Y.Y.); Department of Immunology (Y.S., H.Y.,
K.O.), Division of Cardiology, and Department of Internal Medicine (S.K.),
School of Medicine, Juntendo University, Tokyo; Cardiovascular Center,
Department of Cardiology, Toranomon Hospital, Tokyo (S. Ishiwata); Department
of Immunology, National Children's Medical Research Center, Tokyo
(M.A.); Institute for Adult Diseases, Asahi Life Foundation, Tokyo (Y.S.);
Second Department of Pathology, Tokyo Women's Medical College (S.
Ishiyama, T.N., T.K.); Second Department of Internal Medicine, School of
Medicine, Tokyo Medical and Dental University (S. Ishiyama, M.H.); and
Department of Cardiology, Sakakibara Heart Institute, Tokyo (S.S.), 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.
Methods and Results—To investigate the roles of the costimulatory
molecules B7–1, B7–2, and CD40 in the development of acute
myocarditis and DCM, we analyzed the expression of these
antigens in the myocardial tissues of patients with acute myocarditis
and DCM. We also examined the expression of a cytolytic factor,
perforin, in the infiltrating cytotoxic T lymphocytes (CTLs) and
natural killer (NK) cells, because both killer lymphocytes are thought
to damage B7–1–expressing APCs. We found that B7–1, B7–2, and CD40
were moderately to strongly expressed in the cardiac myocytes of
patients with acute myocarditis. Weak to moderate expression of these
antigens was also found in the cardiac myocytes of patients with DCM.
There was infiltration of perforin-expressing CTLs and NK cells in the
myocardial tissues of patients with acute myocarditis and DCM.
Conclusions—Our findings strongly suggest that expression of
B7–1, B7–2, and CD40 antigens on cardiac myocytes may make them APCs
for CTLs and NK cells and that they may play an important role in the
direct myocardial damage by these killer cells in acute myocarditis and
DCM.
To investigate whether costimulatory molecules B7 and CD40 antigens
really play an important role in myocardial damage in humans, we
analyzed the expression of these antigens in the myocardial
tissue of patients with acute myocarditis and DCM. We also examined the
expression of the cytolytic factor perforin in the infiltrating CTLs
and NK cells, because both killer lymphocytes are thought to directly
damage B7–1–expressing APCs.6 7
Monoclonal Antibodies
Immunofluorescence
Immunohistochemistry
Expression of Perforin in Infiltrating Killer Lymphocytes
Recently, we found that the expression of B7–1 and B7–2 was strongly
induced on cardiac myocytes in a murine model of acute myocarditis
caused by Coxsackievirus B3. We also found that in vivo, anti–B7–1
mAb treatment markedly reduced the myocardial inflammation, indicating
the pivotal role of B7–1 in the development of the myocardial damage
involved. Furthermore, the expression of B7–1 and B7–2 was clearly
induced on cultured murine cardiac myocytes by treatment with
interferon-
In conclusion, the expression of costimulatory molecules (especially
B7–1) on cardiac myocytes may play a critical role in myocardial
damage in patients with acute myocarditis and DCM; this raises the
possibility of immunotherapy with anti–B7–1 mAb to prevent
T-cell–mediated as well as NK cell–mediated myocardial damage in
acute myocarditis and DCM.
Received November 14, 1997;
revision received December 11, 1997;
accepted December 17, 1997.
2.
Mueller DL, Jenkins MK, Schwartz RH. Clonal expansion
versus functional clonal inactivation: a costimulatory signaling
pathway determines the outcome of T cell antigen receptor occupancy.
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3.
Freeman GJ, Freedman AS, Segil JM, Lee G, Whitman JF,
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Immunol. 1989;143:2714–2722.[Abstract]
4.
Azuma M, Ito D, Yagita H, Okumura K, Phillips JH,
Lanier LL, Somoza C. B70 antigen is a second ligand for CTLA-4 and
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5.
Ranheim EA, Kipps TJ. Activated T cells induce
expression of B7/BB1 on normal or leukemic B cells through a
CD40-dependent signal. J Exp Med. 1993;177:925–935.
6.
Townsend SE, Allison JP. Tumor rejection after direct
costimulation of CD8+ T cells by B7-transfected melanoma cells.
Science. 1993;259:368–370.
7.
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Azuma M, Cayabyab M, Buck D, Phillips JH, Lanier LL.
Involvement of CD28 in MHC-unrestricted cytotoxicity mediated by a
human NK leukemia cell line. J Immunol. 1992;149:1115–1123.[Abstract]
9.
Kawasaki A, Shinkai Y, Kuwana Y, Furuya A, Iigo Y,
Hanai N, Itoh S, Yagita H, Okumura K. Perforin, a pore forming protein
detectable by monoclonal antibodies, is a functional marker for killer
cells. Int Immunol. 1990;2:677–684.
10.
Seko Y, Minota S, Kawasaki A, Shinkai Y, Maeda K,
Yagita H, Okumura K, Sato O, Takagi A, Tada Y, Yazaki Y.
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arteritis. J Clin Invest. 1994;93:750–758.
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© 1998 American Heart Association, Inc.
Brief Rapid Communications
Expression of Costimulatory Molecules B7–1, B7–2, and CD40 in the Heart of Patients With Acute Myocarditis and Dilated Cardiomyopathy
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—In patients with acute
myocarditis and dilated cardiomyopathy (DCM), we
previously reported that antigen-specific T cells infiltrate the heart
and play an important role in the myocardial damage involved. For
antigen-specific T-cell activation to occur, it is necessary for T
cells to receive a costimulatory signal provided by costimulatory
molecules expressed on antigen-presenting cells (APCs) as well as
the main signal provided by binding of T-cell receptors to the
antigen.
Key Words: myocarditis • cardiomyopathy • immunology • immunohistochemistry
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
We previously
reported that activated and antigen-specific T cells infiltrate
the hearts of patients with acute myocarditis and
DCM.1 For antigen-specific T-cell activation to
occur, it is necessary for the T cell to receive two signals from the
APC. The first signal is provided by T-cell receptor engagement with a
combination of antigen and major histocompatibility complex, and the
second signal, called the costimulatory signal, is provided by
costimulatory molecules on the APC.2 Among these,
the B7 family molecules B7–1 (B7, CD80)3 and
B7–2 (B70, CD86),4 which are the ligands for
CD28 and CTLA-4 on the T cell, are the most extensively characterized
and appear to be the most critical. Another costimulatory molecule,
CD40, expressed on various APCs, is known to induce expression of B7
antigens as well as to initiate T-cell–dependent antibody
responses.5
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Patients
Myocardial tissue samples were obtained at biopsy or autopsy
from 7 patients with acute myocarditis and 10 patients with DCM (11 men
and 6 women; age, 45.7±13.6 years [mean±SD]) in whom clinical
diagnoses of acute myocarditis and DCM had been previously determined
by history, physical examination, blood analyses, and
echocardiography. Myocardial tissue samples from 4
patients with other causes of congestive heart failure without
inflammation, such as doxorubicin-induced myocardial injury,
hypertrophic obstructive cardiomyopathy,
valvular heart disease, and old myocardial infarction, were
used as controls.
Mouse anti-human B7–1 (L307)8 and B7–2
(IT2.2)4 mAbs were described previously. Mouse
anti-human CD40 (5C3), CD8 (Leu-2a), and CD16 (3G8) mAbs were purchased
from PharMingen, Becton Dickinson Immunocytometry Systems, and
Immunotech, respectively. The procedures for preparing a rat anti-mouse
perforin (P1–8) mAb, which was also shown to react with human
perforin, were described previously.9
In this study, to amplify the specific signals of
antigen-antibody reaction, we used tyramide signal amplification
technology for fluorescence (TSA-Direct [Green], NEN Life
Science Products) according to the manufacturer's instructions.
Cryostat sections of freshly dissected myocardial tissue samples
(6 mm thick) were fixed in acetone for 5 minutes at 4°C and
incubated with mouse anti-human B7–1, B7–2, or CD40 mAb,
respectively, for 1 hour at 37°C. The sections were incubated with
biotinylated horse anti-mouse IgG antibody (Vector Laboratories, Inc)
for 1 hour at 37°C, blocked with a buffer containing a blocking
reagent for 30 minutes, then incubated with streptavidin–horseradish
peroxidase for 30 minutes. The sections were reacted with
fluorescein-tyramide for the appropriate time (3 to 10
minutes), then examined and photographed under a fluorescence
microscope.
Double staining was performed for surface markers (CD 8 or CD16)
and perforin by an enzyme antibody method as described
previously.10 Surface markers stained brown by
the horseradish peroxidase reaction, and perforin stained blue by the
alkaline phosphatase reaction.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Expression of B7–1, B7–2, and CD40 in Ventricular Tissue
In normal ventricular tissue, several cells weakly to
moderately expressed B7–1, B7–2, and CD40 antigens (Fig 1A
, 1E, and 1I, respectively). These
cells positive for B7 antigens and CD40 are thought to be dendritic
cells.11 There was no expression of these
antigens in cardiac myocytes, whereas in ventricular tissue
of patients with acute myocarditis, most of the infiltrating cells
strongly expressed B7–1, B7–2, and CD40 and some of the cardiac
myocytes moderately to strongly expressed these antigens (Fig 1B, 1F, and 1J, respectively). In ventricular tissue of patients
with DCM, most of the infiltrating cells strongly expressed B7–1,
B7–2, and CD40, and a few cardiac myocytes near or in contact with the
infiltrating cells moderately to strongly expressed these antigens (Fig 1C, 1G, and 1K, respectively; arrows). The expression of these antigens
was found in all patients with acute myocarditis and DCM that we
studied, with some variations. In contrast, there was no expression of
these antigens in ventricular tissue of control patients
with other causes of congestive heart failure without inflammation (Fig 1D, 1H, and 1L, respectively).
View larger version (98K):
[in a new window]
Figure 1. Immunohistochemical study for B7–1, B7–2, and
CD40 antigens in ventricular tissues of patients with acute
myocarditis and DCM. Ventricular tissues of a normal
subject (A, E, and I) and patients with acute myocarditis (B, F, and
J), DCM (C, G, and K), and congestive heart failure due to doxorubicin
cardiotoxicity (D, H, and L) were stained with anti–B7–1 mAb (A, B,
C, and D), anti–B7–2 mAb (E, F, G, and H), or anti-CD40 mAb (I, J, K,
and L) by immunofluorescence by use of tyramide
signal amplification technology. Bar=20 µm.
To analyze the pathogenic role these infiltrating cells
might play, we examined the expression of perforin in CTLs and NK cells
in ventricular tissues of patients with acute myocarditis
and DCM, because both types of lymphocytes are thought to directly
damage B7–1–expressing APCs. Fig 2
shows representative sections with double staining of
the infiltrating cells for perforin as blue and cell surface markers
(CD8 and CD16) as brown. There was clear expression of perforin in the
peripheral cytoplasmic granules of CTLs (Fig 2A and 2B) and
NK cells (Fig 2C and 2D) in patients with acute myocarditis (Fig 2A and 2C) as well as those with DCM (Fig 2B and 2D). This indicated that both
infiltrating CTLs and NK cells were activated killer cells.
There were almost no infiltrating CTLs or NK cells in
ventricular tissue of control patients with other causes of
congestive heart failure without inflammation. The absence of the
expression of B7–1, B7–2, and CD40 in ventricular tissue
of these control patients supported the absence of infiltrating killer
cells, because the infiltrating killer cells strongly expressed these
antigens (Fig 1D
, 1H, and 1L).
View larger version (122K):
[in a new window]
Figure 2. Expression of perforin in infiltrating CTLs and NK
cells in the hearts of patients with acute myocarditis and DCM.
Myocardial tissues of patients with acute myocarditis (A and C) and DCM
(B and D) were double stained by enzyme antibody method for perforin
(blue, arrows) and surface markers (brown) CD8 (A and B) and CD16 (C
and D), respectively. Bar=10 µm.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
In this study, we clearly demonstrated that expression of the
costimulatory molecules B7–1, B7–2, and CD40 was induced on cardiac
myocytes in acute myocarditis and DCM. Furthermore, infiltration of
perforin-expressing activated CTLs and NK cells was found in
myocardial tissues in acute myocarditis and DCM. Because CTLs and NK
cells are known to directly damage B7–1–expressing APCs, these
findings strongly suggested that the expression of costimulatory
molecules, especially B7–1, may make cardiac myocytes the target cells
for these infiltrating killer lymphocytes and play a critical role in
the development of the myocardial damage involved. The expression of
CD40 on cardiac myocytes was thought to play a role in inducing B7
antigens on cardiac myocytes on binding to CD40 ligand (gp39) expressed
on the infiltrating T cells.5 
in vitro (manuscript submitted). Therefore, the
expression of B7–1, B7–2, and CD40 on cardiac myocytes revealed in
the present study seems to have been induced by cytokines
such as interferon- released from the infiltrating cells.
Selected Abbreviations and Acronyms
APC
=
antigen-presenting cell
CTL
=
cytotoxic T lymphocyte
DCM
=
dilated cardiomyopathy
mAb
=
monoclonal antibody
NK
=
natural killer
Acknowledgments
This work 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 the Ryoichi Naito
Foundation for Medical Research; a grant from Japan Heart
Foundation–Pfizer Pharmaceuticals Grant for Research on
Coronary Artery Disease; and a grant from the Study Group of
Molecular Cardiology. We thank Kaori Takahashi for
excellent technical assistance.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Seko Y, Ishiyama S, Nishikawa T, Kasajima T, Hiroe
M, Kagawa N, Osada K, Suzuki S, Yagita H, Okumura K, Yazaki Y.
Restricted usage of T-cell receptor V
-Vß genes in infiltrating
cells in the hearts of patients with acute myocarditis and dilated
cardiomyopathy. J Clin Invest. 1995;96:1035–1041.
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