Download Captopril Prevents Experimental Autoimmune

The Journal of Immunology
Captopril Prevents Experimental Autoimmune Myocarditis1
Lisa M. Godsel,2* Juan S. Leon,2* Kegiang Wang,* Jamie L. Fornek,* Agostino Molteni,† and
David M. Engman3*
Captopril, an angiotensin-converting enzyme inhibitor, is widely used in the treatment of a variety of cardiomyopathies, but its
effect on autoimmune myocarditis has not been addressed experimentally. We investigated the effect of captopril on myosininduced experimental autoimmune myocarditis. A/J mice, immunized with syngeneic cardiac myosin, were given 75 mg/L of
captopril in their drinking water. Captopril dramatically reduced the incidence and severity of myocarditis, which was accompanied by a reduction in heart weight to body weight ratio and heart weight. Captopril specifically interfered with cell-mediated
immunity as myosin delayed-type hypersensitivity (DTH) was reduced, while anti-myosin Ab production was not affected. Captopril-treated, OVA-immunized mice also exhibited a decrease in OVA DTH. In myosin-immunized, untreated mice, injection of
captopril directly into the test site also suppressed myosin DTH. Interestingly, captopril did not directly affect Ag-specific T cell
responsiveness because neither in vivo nor in vitro captopril treatment affected the proliferation, IFN-␥ secretion, or IL-2 secretion
by Ag-stimulated cultured splenocytes. These results indicate that captopril ameliorates experimental autoimmune myocarditis
and may act, at least in part, by interfering with the recruitment of cells to sites of inflammation and the local inflammatory
environment. The Journal of Immunology, 2003, 171: 346 –352.
M
yocarditis, inflammation of the heart, is characterized
by myocyte necrosis and degeneration with mononuclear cell infiltration in the presence or absence of fibrosis (1). In the U.S., ⬃2500 people develop myocarditis each
year (2, 3), although the prevalence of this disease is probably
underestimated (4, 5). Myocarditis may be caused by infections
with bacteria, viruses, parasites, and fungi, as well as by drugs and
toxins. Virally induced myocarditis is commonly caused by coxsackievirus infection in North America and Europe, and parasiteinduced myocarditis is commonly caused by Trypanosoma cruzi
infection in Latin America (1, 3, 5–7).
Treatments for myocarditis are directed toward reducing or
eliminating the infectious agent and associated disease complications, such as congestive heart failure, cardiogenic shock, conduction abnormalities, dysrhythmias, and thromboembolism (8).
These complications are typically treated with diuretics, digitalis,
␤ blockers, and vasodilators, such as angiotensin II receptor antagonists and angiotensin-converting enzyme (ACE)4 inhibitors
(9). One commonly prescribed ACE inhibitor is captopril, which
binds to ACE via its peptide-binding pocket and inhibits the functions of ACE, particularly the formation of angiotensin II from
angiotensin I and the breakdown of bradykinin (10). Among the
*Departments of Microbiology-Immunology and Pathology and The Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL 60611; and †Department of Pathology, University of Missouri,
Kansas City, MO 64110
Received for publication January 3, 2003. Accepted for publication April 17, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was conducted with support from the National Institutes of Health and
American Heart Association.
2
L.M.G. and J.S.L. contributed equally to this study.
3
Address correspondence and reprint requests to Dr. David M. Engman, Northwestern University Feinberg School of Medicine, Department of Pathology, 303 East
Chicago Avenue, Ward 6-175, Chicago, IL 60611. E-mail address: d-engman@
northwestern.edu
4
Abbreviations used in this paper: ACE, angiotensin-converting enzyme; DTH, delayed-type hypersensitivity; EAM, experimental autoimmune myocarditis.
Copyright © 2003 by The American Association of Immunologists, Inc.
many ACE inhibitors and angiotensin II receptor antagonists, captopril has been shown to modulate chemotaxis, motility, adhesion,
differentiation, activation, and cytokine and chemokine production
of immune cells (reviewed in Ref. 11). Captopril is effective at
ameliorating many human cardiomyopathies (12, 13), although its
direct effect on human myocarditis has not been addressed. The
drug decreases inflammation, calcification, and fibrosis in several
models of infectious myocarditis, including encephalomyocarditis
virus (14 –16), coxsackievirus B3 (17–19), and T. cruzi (20). However, the effect of captopril on experimental models of Ag-induced
autoimmune myocarditis has not been addressed.
An established model of experimental autoimmune myocarditis
(EAM) is induced in susceptible strains of mice upon immunization with the ␣ H chain of cardiac myosin (21). EAM is histologically similar to human myocarditis, with myocyte swelling and
necrosis accompanied by mononuclear cell infiltration and fibrosis.
Studies have shown that EAM is a T cell-mediated disease, requiring both CD4⫹ and CD8⫹ subsets (22–26). B cells are not
vital for Ag presentation in EAM, and autoantibodies are not necessary for the progression of myocarditis (22, 27, 28).
To study the effect of ACE inhibition on the development of
autoimmune myocarditis, we administered captopril to A/J mice
immunized with cardiac myosin. We found that captopril ameliorates myocarditis and decreases cell-mediated inflammatory responses found in EAM. These results demonstrate that captopril
treatment is indeed an effective method for inhibiting autoimmune
myocarditis, and although it decreases cell-mediated immune responses, it does not directly affect T cell function.
Materials and Methods
Experimental animals
Male A/J mice (The Jackson Laboratory, Bar Harbor, ME) were 6 – 8 wk
of age at initiation of the experiments. DO11.10 BALB/c mice were a gift
from S. Miller (Northwestern University, Chicago, IL). Mice were anesthetized by a single i.p. injection of 60 mg/kg sodium pentobarbital for
each experimental manipulation. The use and care of mice were conducted
in accordance with the guidelines of the Center for Comparative Medicine
at Northwestern University.
0022-1767/03/$02.00
The Journal of Immunology
Preparation of myosin
Cardiac myosin H chains were purified according to the method of Shiverick et al. (29), with modifications as described (30).
Induction of autoimmune myocarditis
Mice were immunized with myosin (300 ␮g) in an emulsion of CFA
(Difco, Detroit, MI) in a total volume of 0.1 ml. Mice received s.c. injections in three sites in the dorsal flank. Seven days later, mice were boosted
in an identical manner.
OVA immunization
Mice were immunized with an emulsion of OVA (100, 75, 50, or 25 ␮g;
Sigma-Aldrich, St. Louis, MO) in CFA in a total volume of 0.1 ml. Mice
received s.c. injections in three sites in the dorsal flank. Seven days later,
mice were boosted in an identical manner.
Captopril treatment regimen
Mice were given drinking water containing or lacking 75–100 ␮g/ml captopril from the day of immunization through the day of sacrifice. We tested
a variety of doses of captopril and chose for the actual experiment the
highest dose that gave a decrease in myosin-induced myocarditis without
mortality. Administration of captopril at concentrations greater than 120
␮g/ml in the water led to excessive mortality. The range of doses tested
was chosen based on other reports (15, 31), with 2 mg/ml as the upper end
dose (32). The amount of water consumed and the weights of the mice were
monitored and the amount of captopril was adjusted so that the mice received ⬃25 mg captopril/kg body weight/day. Captopril stimulated water
consumption in our mice, as reported (33), to 5–7 ml per mouse per day
throughout the course of treatment.
Serum ACE activity
Serum ACE activity was determined by the spectrophotometric method of
Cushman and Cheung (34) using the synthetic substrate hippuryl-L-histidyl-L-leucine. Serum was treated with HCl and ethyl acetate and dehydrated. Hippuryl-L-histidyl-L-leucine content was then determined using
the biuret method utilizing BSA as a standard. ACE levels were expressed
as milliunits per ml of serum. The increase in serum ACE correlates with
the presence of captopril due to the deregulation of a negative feedback
loop in the renin-angiotensin system.
Histologic evaluation of disease
Hearts were excised, fixed in 10% buffered Formalin, and embedded in
paraffin. Four sections per heart were stained with H&E or Masson’s
trichrome and examined by light microscopy. Masson’s trichrome was
used to determine the extent of collagen deposition as an indicator of fibrosis. Each section was examined blinded for evidence of mononuclear
and polynuclear cellular inflammation, necrosis and mineralization, and
fibrosis, and was assigned a histologic score between 0 (no involvement
noted) and 4 (100% involvement), with 1, 2, and 3 representing 25, 50, and
75% involvement of the histologic section. Independent blinded observers
obtained substantial (0.60 – 0.79) to almost perfect (0.80 –1.00) agreement
on their scoring by weighted ␬ statistic (35), as measured on a representative sample of 20 heart sections: overall agreement (0.82), inflammation
(0.80), necrosis (0.96), and fibrosis (0.92).
347
lysed by hypotonic shock in Tris-NH4Cl (pH 7.3), and the cells were
washed and resuspended in HBSS (Life Technologies, Grand Island, NY).
Cells were cultured in 96-well microtiter plates (Corning-Costar, Acton,
MA) at 5 ⫻ 105 cells/well in DMEM containing 5 ⫻ 10⫺5 M 2-ME, 2 mM
L-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 0.1 M nonessential amino acids, and 5% FCS (HyClone, Logan, UT) in the presence of
10 ␮M OVA, 10 ␮M myosin, or medium alone as a negative control, and
anti-murine CD3 (500 ng/ml, 2C11 clone; a gift from W. Karpus, Northwestern University) as a positive control. Cells were incubated at 37°C in
a humidified atmosphere containing 5% CO2. The cells were pulsed with
1 ␮Ci of [3H]TdR (ICN Radiochemicals, Irvine, CA) after 72 h and harvested after 96 h, and [3H]TdR uptake was detected using a Topcount
microplate scintillation counter (Packard Instruments, Meriden, CT). Results are presented as the mean ⫾ SEM of triplicate wells. Assessment of
cytokine production was determined from lymph node culture supernatants
harvested following 48-h stimulation with 10 ␮M OVA, 10 ␮M myosin, or
medium alone as a negative control, and anti-CD3 as a positive control.
The supernatants were tested for the presence of IL-2, IL-4, and IFN-␥ by
commercial ELISA kits (Endogen, Cambridge, MA). The detection limit of
the cytokine ELISA kits is as follows: IL-2, 15.6 pg/ml; IL-4, 31.3 pg/ml;
and IFN-␥, 48.8 pg/ml.
Statistical evaluation
All values are expressed as mean ⫾ SEM. The statistical significance of
DTH, T cell proliferations, cytokine levels, and Ab isotypes was analyzed
by one-way ANOVA, followed by a two-tailed t test and post hoc Bonferroni analysis. The statistical significance of disease incidence and comparison of histologic scores were analyzed by Pearson’s ␹2. Agreement of
blinded observer histopathology scores was analyzed by weighted ␬. Mean
histologic scores ⫾ SD, although statistically invalid, will be provided to
aid the reader and to continue the convention of other autoimmune myocarditis papers. Values of p ⬍ 0.05 were considered significant.
Results
Captopril increases serum ACE levels
To study the effect of captopril on autoimmune-mediated inflammation and fibrosis, we added captopril to the drinking water of
myosin-immunized and saline-immunized A/J mice. Each captopril-treated mouse received 25 mg drug/kg body weight per day.
We confirmed that the captopril treatment was effective by assaying for increased levels of ACE in treated mice at day 21 postimmunization. Increased ACE levels result from the captopril-induced negative feedback loop of the renin-angiotensin system in
which a decrease of angiotensin II stimulates the production of
ACE (34). Captopril-treated myosin and saline-immunized mice
had significantly ( p ⬍ 0.001) higher levels of ACE than did untreated controls (Fig. 1). These results indicate that captopril was
Measurement of Ag-specific delayed-type hypersensitivity
Myosin- and OVA-specific delayed-type hypersensitivity (DTH) was
quantitated using a standard ear-swelling assay (30). Ag-induced ear swelling was the result of mononuclear cell infiltration and exhibited typical
DTH kinetics (i.e., minimal swelling at 4 h, maximal swelling at 24 – 48 h
postinjection).
Serologic analysis
Myosin- and OVA-specific ELISAs were used to analyze the levels and
isotype specificities of the Ag-specific Abs, as described (36). Each serum
sample was analyzed separately, and the data were compiled for each
group. Each plate was also tested with sera from one particular mouse
immunized with myosin/CFA, a second immunized with OVA/CFA, and a
third immunized with saline/CFA as controls.
In vitro T cell proliferation and cytokine assays
Spleens were forced through 100-mesh stainless steel screens to yield single cell splenocyte suspensions. RBCs in the spleen preparations were
FIGURE 1. Captopril up-regulates serum ACE levels in myosin-immunized mice. At 21 days postimmunization, sera were collected and analyzed for serum ACE levels from four groups of mice: myosin immunized
and captopril treated (Myosin/CFA ⫹ Captopril), myosin immunized (Myosin/CFA), saline immunized and captopril treated (PBS/CFA ⫹ Captopril), and saline immunized (PBS/CFA). ⴱ, p ⬍ 0.001 compared with the
respective untreated group.
348
CAPTOPRIL PREVENTS AUTOIMMUNE MYOCARDITIS
Table II. Captopril treatment decreases inflammation, necrosis, and
fibrosis in mice with experimental autoimmune myocarditis
Scoresa
All Mice
Inflammation
Necrosis
Fibrosis
Affected Mice
Inflammation
Necrosis
FIGURE 2. Captopril prevents cardiac pathology in myosin-immunized
mice. At 21 days postimmunization, hearts from the four groups described
in Fig. 1 were analyzed grossly (Gross) and histopathologically by staining
with H&E or Masson’s trichrome (Trichrome). Representative hearts and
tissue sections are shown. Hearts from saline-immunized mice were indistinguishable from hearts from saline-immunized, captopril-treated mice
(PBS/CFA ⫹ Captopril).
effective in blocking the production of angiotensin II by inhibiting
ACE in treated mice. Increasing the captopril dose or prolonging
captopril treatment resulted in elevated serum ACE levels (37–39).
Captopril reduces cardiac hypertrophy in myosin-immunized mice
Twenty-one days postimmunization, gross analysis of the hearts of
captopril-treated mice revealed a significant decrease in the typical
signs of myocarditis: hypertrophy, induration, and pallor. Captopril-treated, myosin-immunized mice resembled saline-immunized
mice (Fig. 2). Hypertrophy was reduced in treated mice, as evidenced by body weight and heart weight measurements (Table I).
Captopril significantly reduced the heart weight, body weight, and
heart weight to body weight ratios in myosin-immunized mice.
Interestingly, both the heart weight and the heart weight to body
weight ratios of myosin-immunized, captopril-treated mice were
equivalent to those of saline-immunized mice. Captopril did not
significantly affect the heart weight or heart weight to body weight
ratio in saline-immunized mice, suggesting that the reduction of
these parameters in myosin-immunized, captopril-treated mice was
due to reduction in hypertrophy.
Captopril ameliorates myocarditis in myosin-immunized mice
To investigate whether the reduction of hypertrophy reflected a
lack of tissue inflammation, we performed histopathologic analysis
of hearts excised from myosin- and saline-immunized mice 21
days postimmunization. Captopril significantly reduced the incidence (Table I) and severity (Fig. 2, Table II) of myocarditis in
Fibrosis
Captopril
0
1
2
3
4
Total
Severity
⫹*
⫺
⫹**
⫺
⫹**
⫺
22
12
25
14
24
11
5
8
3
10
3
11
1
7
0
5
1
7
0
2
0
0
0
0
0
0
0
0
0
0
28
29
28
29
28
29
0.3 ⫾ 0.5
1.0 ⫾ 1.0
0.1 ⫾ 0.3
0.7 ⫾ 0.8
0.2 ⫾ 0.5
0.9 ⫾ 0.8
0
2
3
4
2
1
5
8
3
10
3
11
1
7
0
5
1
7
0
2
0
0
0
0
0
0
0
0
0
0
6
19
6
19
6
19
1.2 ⫾ 0.4
1.5 ⫾ 0.8
0.5 ⫾ 0.5
1.1 ⫾ 0.7
0.8 ⫾ 0.8
1.3 ⫾ 0.6
⫹
⫺
⫹
⫺
⫹
⫺
a
Histologic scores were assigned by blinded observers and ranged from 0 (no
involvement noted) to 4 (100% involvement), with 1, 2, and 3 representing 25, 50, and
75% involvement of the histologic section. The number of mice and the percentage
of mice in the group receiving that score are given. Mice injected with saline (n ⫽ 8)
or mice injected with saline and treated with captopril (n ⫽ 10) received scores of 0
for all parameters. The severity score is presented as the average ⫾ SD.
ⴱ, p ⬍ 0.05 compared with untreated myosin-immunized mice.
ⴱⴱ, p ⬍ 0.01 compared with untreated myosin-immunized mice.
myosin-immunized mice. Specifically, captopril reduced inflammation, fibrosis, and necrosis in these animals. Restricting the
analysis to only diseased hearts showed that disease severity in
affected mice was reduced, although not to a statistically significant level (Table II).
Captopril reduces Ag-specific DTH
We hypothesized that the reduction in cardiac inflammation in captopril-treated, myosin-immunized mice was due to an effect on
myosin-specific immunity, which mediates cardiac inflammation
in these mice (36). We assayed myosin-specific cellular immunity
and found that captopril significantly reduced myosin DTH in myosin-immunized mice (Fig. 3a). These results suggest that captopril affects T cell-mediated inflammation in vivo, explaining the
reduction of inflammation and damage observed in hearts of the
captopril-treated, myosin-immunized mice. To determine whether
the reduction of DTH by captopril was due to a suppression of T
cell responses and not due to a reflection of captopril’s cardioprotective role (i.e., less myocardial damage results in decreased myosin autoimmune response (21)), we assayed OVA DTH in OVAimmunized mice treated with captopril. Captopril significantly
reduced OVA DTH in OVA-immunized mice (Fig. 3c). These results suggest that captopril can reduce inflammatory responses to
both a self and foreign Ag, irrespective of myocardial damage.
Captopril has been shown to reduce local inflammatory processes
Table I. Captopril treatment decreases disease incidence, heart weight, and heart weight to body weight ratio in mice with experimental autoimmune
myocarditis
Group
Myosin/CFA ⫹ Captopril
Myosin/CFA
PBS/CFA ⫹ Captopril
PBS/CFA
ⴱ, p ⫽ 0.001 compared with myosin/CFA.
ⴱⴱ, p ⬍ 0.05 compared with myosin/CFA.
ⴱⴱⴱ, p ⬍ 0.05 compared with PBS/CFA.
Disease
Incidence
6/30 (20%)*
21/33 (64%)
0/11
0/10
Heart Weight (g)
Body Weight (g)
Heart Weight to Body Weight Ratio
0.087 ⫾ 0.016**
0.110 ⫾ 0.023***
0.084 ⫾ 0.008
0.095 ⫾ 0.011
21.1 ⫾ 1.76**
22.8 ⫾ 2.49
21.0 ⫾ 2.22
22.8 ⫾ 1.88
3.97 ⫻ 10⫺3 ⫾ 1.03 ⫻ 10⫺4**
4.83 ⫻ 10⫺3 ⫾ 1.65 ⫻ 10⫺4***
4.03 ⫻ 10⫺3 ⫾ 2.95 ⫻ 10⫺4
4.15 ⫻ 10⫺3 ⫾ 2.86 ⫻ 10⫺4
The Journal of Immunology
349
icantly affected by captopril treatment (Fig. 4), suggesting that
captopril does not affect humoral immunity in our model system.
Ag-specific T cell proliferation and cytokine secretion are not
affected in vivo or in vitro by captopril administration
To determine whether the in vivo effects of captopril or Ag-specific cellular immunity are due to direct effects on T cell function,
we measured the effect of captopril treatment in vitro on the Agspecific stimulation of splenocytes derived from DO11.10 TCR
transgenic mice in which the majority of T lymphocytes are specific for OVA. Splenocytes from untreated DO11.10 cultured with
OVA exhibited strong proliferation and cytokine secretion in the
presence or absence of captopril in the culture medium (Fig. 5).
Splenocytes did not respond to myosin, a negative control. This
prompted us to test splenocytes from captopril-treated OVA-immunized BALB/c mice. Splenocytes from mice receiving captopril
for 21 days showed normal in vitro proliferation and cytokine secretion when stimulated by OVA or anti-CD3 (Fig. 6). These results suggest that captopril does not directly affect Ag-specific T
cell responses.
Discussion
FIGURE 3. Captopril treatment inhibits DTH responses. A, Twenty-one
days postimmunization, myosin-specific DTH was measured in 10 mice
from the four groups described in Fig. 1 by a 24-h ear-swelling assay, as
described in Materials and Methods. ⴱ, p ⬍ 0.001 compared with the
myosin/CFA group. B, Twenty days postimmunization, untreated, myosinimmunized mice received ear injections of myosin plus 1 ⫻ 10⫺4 M captopril (Myosin/CFA ⫹ Captopril) or myosin alone (Myosin/CFA). Salineimmunized mice received ear injections of myosin plus 1 ⫻ 10⫺4 M
captopril (PBS/CFA ⫹ Captopril) or myosin alone (PBS/CFA). Myosinspecific DTH was measured the following day, and bars represent the mean
swelling of five mice per group. ⴱ, p ⬍ 0.01 compared with the myosin/
CFA group. C, Eight mice per group were immunized and boosted with the
indicated amounts of OVA (OVA/CFA) or saline (PBS/CFA). Four mice in
each group received captopril (Captopril) or nothing (No Treatment) in their
water. Twenty-one days postimmunization, OVA-specific DTH was measured
in four mice from each group. ⴱ, p ⬍ 0.005. Error bars represent SEM.
Captopril ameliorated autoimmune myocarditis, as evidenced by a
reduction in cardiac hypertrophy and the incidence and severity of
inflammation, necrosis, and fibrosis. Captopril also reduced in vivo
cell-mediated inflammatory responses, as measured by a reduction
of myosin- and OVA-specific DTH in Ag-immunized mice. This
reduction in cell-mediated immunity by captopril was not due to a
direct effect on T cells because these cells proliferated normally
and secreted normal amounts of proinflammatory cytokines when
necessary for the induction of DTH responses, including: deposition of extravasated fibrin (17), production of proinflammatory cytokines (40 – 42), recruitment of leukocytes (43, 44), and regulation of the renin-angiotensin system in dendritic cell function (45).
Therefore, we tested whether captopril could suppress DTH locally
or required systemic administration. In myosin-immunized, untreated mice, local injection of captopril into the DTH site also
significantly decreased myosin DTH compared with controls (Fig.
3b). These results indicate that captopril can act locally to mediate
its effects and that its reduction of DTH may be due to a direct
effect on T cell function, trafficking of T cells, or a generalized
suppression of the inflammatory environment.
Captopril does not affect Ag-specific Ab production
Prevention of myocarditis in myosin-immunized mice by restoring
peripheral T cell tolerance to myosin is associated with a decrease
in myosin-specific Ab production (36). Moreover, other studies
suggest that captopril decreases bulk Ab levels (46, 47). We investigated whether captopril treatment affected humoral responses
to myosin and OVA at day 21 postimmunization, and found that
myosin-specific and OVA-specific Ab production was not signif-
FIGURE 4. Captopril does not affect myosin-specific or OVA-specific
Ab production. A, At 21 days postimmunization, sera from mice in the four
groups described in Fig. 1 were analyzed by myosin-specific ELISA using
isotype-specific secondary Abs. Bars represent the averages of 10 mice. B,
At 21 days postimmunization, serum was collected and analyzed by OVAspecific ELISA using isotype-specific detecting Abs from four groups of
mice: OVA immunized and captopril treated (OVA/CFA ⫹ Captopril),
OVA immunized (OVA/CFA), saline immunized and captopril treated
(PBS/CFA ⫹ Captopril), and saline immunized (PBS/CFA). Bars represent
the mean OD450 of 10 samples. Error bars represent SEM.
350
FIGURE 5. In vitro captopril administration does not affect Ag-specific
T cell proliferation or cytokine secretion. Splenocytes from untreated
DO11.10 mice were cultured with 10 ␮M OVA or 10 ␮M myosin (Myosin), as a negative control, in the presence of varying concentrations of
captopril. A, Proliferative responses were measured by [3H]TdR incorporation and represented as the mean of triplicate wells. B, Culture supernatants were assayed for the presence of IL-2 or IFN-␥ secreted by splenocytes cultured with 10 ␮M OVA or 10 ␮M myosin. Splenocytes were
pooled from at least five mice. Symbols represent the mean of triplicate
wells. Error bars represent SEM.
removed from the animal and tested in vitro even when captopril
was present in the culture medium. Interestingly, myosin- and
OVA-specific Ab responses were not affected by captopril.
Our finding that captopril inhibits the development of experimental autoimmune myocarditis is supported by several studies
showing the beneficial effects of captopril on human cardiomyopathies (12, 13), infection-induced experimental myocarditides
(14 –20), and experimental autoimmune disease models (48, 49).
In addition, another ACE inhibitor, temocapril, reduces myosininduced myocarditis in rats via redox regulation mechanisms involving thioredoxin, although the effect of this agent on the immune system is not known (50). Our data on the reduction of DTH
responses by captopril are novel and are supportive of the drug’s
role in suppressing inflammatory processes (reviewed in Ref. 11).
Captopril reduced both heart weight and heart weight to body
weight ratio almost to normal, as is true of other disease models
(14 –19, 51). This reduction is due to a decreased inflammation,
myocyte necrosis, and consequent reparative fibrosis (Table II).
Our data also indicate that captopril completely prevents myocarditis in some mice and reduces disease severity in those mice that
CAPTOPRIL PREVENTS AUTOIMMUNE MYOCARDITIS
FIGURE 6. In vivo captopril administration does not affect Ag-specific
T cell proliferation or cytokine secretion by splenocytes of OVA-immunized mice. At 21 days postimmunization, splenocytes were prepared and
cultured with 10 ␮M OVA, 10 ␮M myosin (Myosin) as a negative control,
or anti-CD3 (anti-CD3) as a positive control, from four groups of mice:
OVA immunized and captopril treated (OVA/CFA ⫹ Captopril), OVA
immunized (OVA/CFA), saline immunized and captopril treated (PBS/
CFA ⫹ Captopril), and saline immunized (PBS/CFA). Captopril was administered to mice from day of immunization to day of sacrifice. Splenocytes were pooled from at least five mice in each group and did not receive
captopril in the culture medium. A, Proliferative responses were measured
by [3H]TdR incorporation and represented as the mean of triplicate wells.
B, Culture supernatants were assayed for the presence of IFN-␥ and IL-2
and represented as the mean of triplicate wells. Error bars represent SEM.
did develop some disease (Table II). Although the reduction in
disease severity in affected mice does not reach statistical significance, it may still be of clinical significance. We are currently
addressing whether treatment of captopril at later times postdisease
induction also reduces myocarditis, as is true of mice infected with
coxsackievirus B3 (17, 18).
The precise mechanism by which captopril reduces autoimmune
myocarditis remains to be determined. We initially hypothesized
that captopril functions by inhibiting myosin-specific T cell responses. Although we found that captopril decreased myosin DTH
in myosin-immunized mice (Fig. 3), we also found that captopril
could work locally by reducing myosin DTH when injected into
the DTH test site. In addition, the reduction in myosin DTH is not
due to the cardioprotective role of captopril (less damage leading
to decreased autoimmune response (21)), because captopril also
reduced OVA DTH in OVA-immunized mice. These results suggest that captopril may suppress cell-mediated immunity against a
self or foreign Ag irrespective of the presence of myocardial damage. We wondered whether the reduction in DTH induced by captopril was due to suppression of T cell function, as reported by
other groups (42, 49). Instead, we found that captopril, whether
administered in vitro or in vivo, had no effect on the proliferative
capacity or cytokine secretion of splenocytes stimulated by Ag or
by anti-CD3 (Fig. 5). Our results are in accordance with those
showing that proinflammatory and anti-inflammatory cytokines secreted by human PBMCs are not affected by captopril administration (52). These results are also counter to those of other groups
The Journal of Immunology
reporting that captopril enhances T cell function in their model
systems (53–56). The main difference between our results and
those of the other groups is that we tested the effect of captopril on
Ag-specific T cell proliferation and cytokine secretion, while other
groups addressed this question in an Ag-nonspecific manner (e.g.,
by Con A, LPS, and PHA responses, among others). We do not yet
know the mechanism by which captopril affects T cell responsiveness in vivo. Some groups have suggested that differences in these
effects could be due to duration of captopril exposure (57), dosage
(52–54, 58), in vitro vs in vivo administration (54), and plasma
levels of PGs and bradykinin (56, 59), among other possibilities.
We are currently addressing these hypotheses in our model system.
If captopril does not directly affect T cell function, captopril
may reduce inflammation and DTH by reducing recruitment of T
cells to the site of antigenic stimulation or by altering the local
inflammatory environment. These hypotheses are supported by our
result that local injection of captopril into the DTH site reduced
DTH. We are currently testing both hypotheses. Captopril has been
shown to inhibit lymphocyte recruitment by decreasing chemotaxis in capillary endothelial cells (43) and neutrophils (44). The
effects of captopril on chemokines have not been addressed, but
there is mounting evidence that angiotensin II affects the activities
of a number of chemokines, including RANTES (CCL-5), monocyte chemoattractant protein-1 (CCL-2), and macrophage-inflammatory protein 1-␣ (CCL-3) (reviewed in Ref. 11). Captopril also
reduces local inflammatory processes necessary for the induction
of DTH responses, including: deposition of extravasated fibrin
(17); production of proinflammatory cytokines such as TNF-␣ (40,
41), IFN-␥ (42), and IL-12 (42); and dendritic cell function (45).
Taken together, these results show that captopril significantly
reduces experimental autoimmune myocarditis. Additional mechanisms of action of captopril could involve suppression of angiotensin II levels, enhancement of bradykinin levels, or a pharmacologic effect of captopril’s thiol group, among other mechanisms.
Antagonists of angiotensin II receptors reduced encephalomyocarditis virus-induced myocarditis (16, 51, 60). Increased bradykinin
levels and activation of NO and PGs by ACE inhibitors have been
implicated in providing cardiac protection via reduction of infarct
size (61), reduction of hypertrophy (62), and reduction of collagen
gene expression (63). The cardioprotective effect may also be due
to up-regulation of bradykinin, leading to NO synthesis (63, 64),
which is most likely an important molecule in autoimmune myocarditis (65). Finally, the thiol group of captopril is thought to
ameliorate encephalomyocarditis virus-induced myocarditis by
elimination of oxygen radicals (14).
In conclusion, captopril ameliorates autoimmune myocarditis,
as shown by its reduction of cardiac hypertrophy, inflammation,
necrosis, and fibrosis. Our data suggest that captopril reduces inflammation by decreasing cell-mediated immunity, without a direct effect on T cells, but not by affecting humoral immunity. These
results have direct clinical import because patients suffering from
autoimmune myocarditis would benefit from captopril. Future
work from our laboratory will address the mechanisms of action of
captopril in decreasing inflammation, so as to increase the specificity of treatment.
Acknowledgments
We thank Dr. William Ward for the kind gift of captopril, and Joann Taylor-Hinz for technical expertise in performing the ACE activity assay. We
thank Dr. Alfred Rademaker (Center for Preventative Medicine, Northwestern University) for advice on statistical analysis, and Drs. Stephen
Miller, William Karpus, and Carl Waltenbaugh for reagents and advice.
351
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