JACC
Vol.
September
14. No. 3
799
1989:7Y!GUO2
Characteristics of Lymphocytes Cultured From Murine Viral
Myocarditis Specimens: A Preliminary and Technical Report
CHIHARU
KISHIMOTO,
JOHN 1‘. FALLON,
MD, PHD, JAMES
T. KURNICK,
MD,
MD, PHD, FACC, CLYDE S. CRUMPACKER,
WALTESR H. ABELMANN,
MD,
MD, FACC
Boston, hlassachusetts
Long-term culture of lymphocytes from myocardial biopsy
specimens has been reported. To test the usefulness of this
technique in experimental models of murine myocarditis,
the culture and characterization of lymphocytes from mice
infected with encephalomyocarditisvirus or coxsackievirus
B3 were studied. Lymphocytes were successfully cultured
from three hearts of encephalomyocarditis virus-infected
mice in interleukin 2.containing culture.
After approximately 10 days, lymphocytes migrated out
of myocardium in the chronic stage of myocarditis and
The current hypothesis that best fits what is known about the
natural history of viral myocarditis indicates that the initial
cardiac damage and manifestations are due to viral replication in heart muscle cells, and that the extent of damage is
determined not only by the cardiotropism and virulence of
the virus, but also by the genetic and immunologic characteristics of the host (1,2). The phase of viral replication in the
heart, however, is short (1 to 2 weeks at most), and subacute
and chronic active phases of myocarditis are thought to be a
function of autoimmune or immune processes (3). To address some of these unresolved questions, experimental
models of viral myocarditis in mice (4), namely, encephalomyocarditis virus disease and coxsackievirus B3 myocarditis, may be of value.
Attention has been focused on the role of lymphocytes in
the diseased myocardium and in the extracardiac systems in
the pathogenesis of myocarditis (5). Recently, long-term
From the Charles A. Dana Research Institute and the Harvard-Thorndike
Laboratory of Beth Israel Hospital. Department of Medicine, Beth Israel
Hospital; Drpartment of Pathology. Massachusetts General Hospital and the
Harvard Medical School, Boston. Massachusetts. This work was performed
in part during the tenure of a research fellowship from the American Heart
Association, Massachusetts .r\ffiliate. Inc. (Dr. Kishimoto), Needham. Massachusetts.
Manuscript received August 4, 1988; revised manuscript received February 21, 1989, accepted March 29. 1989.
Address for remin&: Walter H. Abelmann. MD, Cardiovascular Division,
Beth Israel Hospital, 330 Brookline Avenue. Boston, Massachusetts 02215.
Cl989 by the Amrrlcan
College of Cardiology
gradually increased in numbers. More than half of the
exuding cells were Thy l.tpositive. None of the myocardial
samples from coxsackievirus B3Gfected mice grew out
lymphocytes. Pathologic examination of aB specimens
showed myocardial necrosis with lymphoid infiltration.
Although further studies are needed, this preliminary
study suggests that the technique of lymphocyte culture
may promote new insights into the pathogenesis of experimental myocarditis.
(J Am Coil Cardiol1989:14:799-802)
culture of lymphocytes has been reported from human
biopsy specimens (69). To test the potential usefulness of
this technique in mouse models of myocarditis, we attempted the culture and characterization of lymphocytes
from hearts of mice infected with encephalomyocarditis
virus and coxsackievirus B3.
Methods
Viruses. The myocardiotropic variant of encephalomyocarditis virus and coxsackievirus B3 (Nancy strain) were
used; the viral stock was prepared in cultures of the kidney
of African green monkey cells in Eagle’s minimum essential
medium. Cell-conditioned medium containing suspensions
of the virus was centrifuged after the cytopathic effect had
developed. Viral stocks were stored at -70°C until use.
Mice. Three to 8 week old male BALBlc, DBAI2 and
C,H/He mice (Jackson Laboratory) were used.
Experimental infections. The precise methodology of experimental infection has been described previously (5). In
brief, inbred mice (BALBlc, n = 48; DBA/Z, n = 52;
C,H/He, n = 30) were inoculated intraperitoneally with 0. I
ml of an encephalomyocarditis virus or coxsackievirus B3
suspension containing 100 to 300 plaque-forming units. Mice
were killed periodically. After confirmation of myocarditis
from the gross appearance of the heart (yellowish-white
0735-109718963.50
800
KISHIMOTO
LYMPHOCYTE
ET AL.
CULTURE
IN EXPERIMENTAL
JACC Vol. 14. No. 3
September 1989:799-802
MYOCARDITIS
Table 1. Results of Lymphocyte Cultures from Murine Mvocardium
Sample No.
Mouse
Virus
Days after
inoculation
Duration of
culture (days)
Thy 1.2+ (%)
Lyt 1+ (%)
L3T4+ (%)
Lyt 2+ (%)
Approximate
cell number
1
2
3
DBA/Z
EMCV
13
DBAR
DBA/2
EMCV
?I
20
10
EMCV
19
120
150
49.3
61.2
14.4
50.3
34.8
57.7
11.4
47.7
4 x IO’ I x IOX
-
4
5
6
7
8
9
B.4LB/c
EMU’
28
BALBlc
EMCV
28
BALB/c
EMCV
30
C,H/He
CB3V
14
DBAl2
CB3V
27
&H/He
CB3V
28
90
90
32
10
I5
15
56.0
2.4
14.8
None
_*
60.9
10.4
22.3
None
_*
6X104
-
4 x 10’
1 x lo4
CB3V = coxsackievirus 83: EMCV = encephalomyocarditis virus.
*Cell number was not examined. Many macrophages appeared in the wells of samples
patches on the surface), the heart was removed aseptically
and cut into about 20 pieces (2 to 3 mm in length): 1 piece
was processed for histologic study (hematoxylin-eosin
stain), the others for lymphocyte culture.
Lymphocyte culture. Culture of specimens in the acute
stage was not attempted in order to avoid contamination
with virus. Lymphocytes were maintained in a 24 well,
flat-bottomed tissue culture plate by the replenishment of
medium containing 10% of interleukin 2 with RPM1 1640,
supplemented with 5% to 10% fetal calf serum, at 37°C in a
humidified atmosphere with 5% carbon dioxide. One piece
was maintained in medium free of interleukin 2 as control.
Tissues were observed daily on an inverted microscope to
monitor cells that exuded from the tissue. The medium was
replenished at least every 3rd day. Proliferating lymphocytes
from positive wells were removed, pooled and restimulated
with phytohemagglutinin. Cells were divided into additional
wells when they had reached a concentration of 106/ml.
When growth within a particular well was slow, medium was
replenished by aspirating two thirds of the medium and
adding fresh medium to fill the well.
Phenotypic analysis. Cell phenotypes were analyzed by
laser flow cytometry; the exudate cells were stained with
monoclonal antibodies, as described previously (10). The
antibodies used were Thy 1.2 (pan T, Becton Dickinson);
Lyt 1 (helper/suppressor T, Becton Dickinson); Lyt 2 (suppressorlcytotoxic T, Becton Dickinson) and L3T4 (activated
helper T, Becton Dickinson). Thus, the lymphocytes may
represent co-expression of these surface markers. In each
case, 2 to 5 x IO5cells were stained with each antibody, and
1,000 to 2,000 cells were analyzed per stain.
Results
Table I summarizes
the results. After 1 week, macro-
phages and fibroblasts first appeared in wells with interleukin
I and 7.
2. Thereafter, macrophages gradually diminished. In each
case. lymphocytes migrated out of the tissue after approximately 10 days and increased in number, especially adjacent
to the tissue (Fig. I). The continued presence of the tissue
enhanced the viability and growth of the lymphocytes.
Approximately 50% to 60% of the exuding cells were Thy
1.2-positive in the three successfully cultured cases; the
L3T4-positive cells predominated over the Lyt l-positive or
the Lyt 2-positive cells. In none of the cases of CB3V
myocarditis did the exuding cells proliferate.
Pathologic study of all specimens revealed moderate to
severe myocardial necrosis with lymphocytic infiltration
(Fig. 2).
Discussion
Pathogenesis of myocarditis. Acute myocarditis is characterized by lymphoid infiltration of the heart and myocardial cell necrosis (l-3,1 1). Abnormal behavior of the immune
system has been postulated as a pathogenic mechanism of
subacute and chronic myocardial dysfunction or dilated
cardiomyopathy (5). recognized as sequelae of acute myocarditis (12). However, evaluations of in situ cellular immune reactions in humans have been hampered by limited
access to relevant tissues. Most studies have used peripheral
mononuclear cells as surrogates for cells at the actual sites of
injury; indeed, cells present at the site of inflammation may
be quite different from those in the peripheral blood (5).
More recently, histologic sections of tissues containing
lymphoid infiltrates have been studied with immunocytochemical methods (10,13). However. it remains to be shown
whether these cells possess functional capacities. Until
recently. such study has been impossible because of the
small number of cells recovered from endomyocardial biopsy specimen of patients with idiopathic myocarditis (9).
JACC Vol. 14, No. 3
Seutember 3989:799-802
LYMPHOCYTE
CULTURE
KISHIMOTO
ET AL.
IN EXPERIMENTAL
MYOCARDITIS
801
Figure 1. Appearance of lymphocytes in culture
with myocarditis tissue (encephalomyocarditis
virus; sample 3). A, Photomicrograph taken
through an inverted microscope 2 months after
the tissue was placed into interleukin 2containing medium. The dark shadow (left) is
the tissue. B, Photomicrograph of the lymphocytes adjacent to the tissue. A, original~magnification X300, reduced by 10%; B, original
magnification x 100. reduced by 10%.
The aim of this brief report is to describe a technique that
allows the in vitro expansion of the lymphoid cell population
contained in experimentally induced myocarditis samples in
long-term cultures and thus the simultaneous analysis of
their phenotypes.
Lymphocyte culture in experimental myocarditis. The culture of lymphocytes from specimens of coxsackievirus B3
myocarditis was unsuccessful. Although the reason for this
is unknown, we may speculate that there are few interleukin
2-sensitive T cells in the myocardium of coxsackievirus B3
myocarditis or that there are fewer lymphoid infiltrates in the
myocardium of coxsackievirus B3 myocarditis as compared
with encephalomyocarditis virus.
Most of the cells exuding from encephalomyocarditis
virus-infected myocardium were T cells and consisted of Lyt
1’ and L3T4’ (Table 1). In two samples (no. 4 and 5), Lyt
7’
_ lymphocytes were absent. With regard to the precise
assigned functions of each murine lymphocyte subset, the
Lyt I+ and Lyt 2’ subset is the precursor of other T cell
subsets and has been demonstrated to be an alloreactive and
syngeneic cytotoxic lymphocyte subset in addition to the
Lyt I- and Lyt 2’ subset. The Lyt 1’ and Lyt 2- subset is
not only a helper/inducer in antibody and cytotoxic lymphocyte inductions, but also an effector of delayed type hypersensitivity in which many lymphokines and monokines are
involved (14). The L3T4+ subset is the activated helper T
cell (15). So-called null cells (non-T and non-B cells) are also
found.
802
KISHIMOTOETAL.
LYMPHOCYTECULTUREIN
EXPERIMENTALMYOCARDITLS
JACC Vol.14,No. 3
September 1989~799402
Figure 2. Histopathology of myocarditis tissue
caused by encephalomyocarditis virus (sample
3). Myocardial necrosis and fibrosis are predominant, but infiltrating cells are still present
(hematoxylin-eosin stain). Original magnification x380, reduced by 10%
Conclusions. This report presents rest&s that clearly
indicate that lymphocyte culture technology may be useful
and may promote new insights into the pathogenesis of
myocarditis.
References
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