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From the Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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expression is barely detectable
at any time point in PKO mice, but it is readily identified in
perforin+ animals, peaking just before the time of
maximal myocarditis; this is particularly interesting, given
that MIP-1 knockout mice are resistant to CVB myocarditis,
but remain able to control viral infection. Thus, the chemokine
pathway offers a second route of intervention to diminish myocarditis
and its sequelae, while permitting the host to eradicate the
virus.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many viruses can cause myocarditis, but enteroviruses in general, and coxsackieviruses in particular, are important human pathogens. Coxsackieviruses are members of the Picornaviridae family and fall into groups A and B, defined by their pathogenicity in newborn mice.6 The proportion of cases of acute myocarditis, and of dilated cardiomyopathy, with suspected coxsackievirus etiology varies among studies, but is usually 25 to 50% and most commonly involves B group viruses (CVB). CVB has been isolated from stool or pharyngeal specimens of many patients with acute myocarditis.7,8 Isolation of infectious virus particles directly from the human myocardium is less common, because myocardial biopsy remains unusual, but specimens obtained at necropsy after a lethal infection have yielded infectious CVB,7,9,10 which causes myocarditis in mice.9 Although five of the six CVB types can cause myocarditis, the majority of animal studies have focused on CVB3 and have shown unequivocally that this virus can cause cardiac disease. Studies have been carried out in many species, including primates,11 but the most popular animal model is the mouse. The murine model systems show early death, acute myocarditis, and longer-term disease, thus paralleling the human situation. CVB3 infection of mice can result in death at around 4 days postinfection (p.i.), at which time histologically apparent myocarditis is rarely detectable.12 In surviving mice of several strains, a biphasic myocarditis occurs; an acute stage, at which infiltration is first seen 4 to 7 days p.i., resolving by day 14 or so, and a chronic stage, in which low-grade inflammation is maintained. CVB can be isolated from myocardium up to day 9 to 10 p.i., but no infectious virus is detected beyond day 14 (at least in immunocompetent mice).
Although there is no doubt that CVB3 can cause myocarditis and myocyte destruction in mice, the precise mechanisms underlying these processes remain uncertain. The role of cytotoxic T lymphocytes (CTLs) in CVB-induced myocarditis was first shown more than 20 years ago13 and has been confirmed in subsequent studies (see, eg, Refs. 12 and 14-16 ). Acute myocarditis becomes histologically evident 4 to 7 days p.i., and cellular infiltrate comprises CD8+ T cells, natural killer cells, and macrophages.12,17-19 This infiltration usually resolves after viral clearance, leading to host recovery (sometimes with residual myocardial fibrosis). We have shown that CTLs play a major role in myocarditis; functional paralysis of this T-cell population (either by monoclonal antibody depletion, or by the use of mice deficient in class I recognition) greatly diminished the acute inflammatory response and prevented subsequent myocardial scarring.12 However CD8+ T-cell depletion is a double-edged sword: in the absence of functional CTLs, viral titers were significantly increased, indicating that CTLs are both protective (diminishing virus titers) and immunopathological (causing extensive myocarditis).
The mechanisms by which CTLs exert their biological effects are varied. Major histocompatibility complex class I expression, required for CTL-mediated recognition and lysis, is low to undetectable on normal myocardiocytes in vivo, but can be readily detected in vivo after CVB infection,20 perhaps being induced by interferon. CTLs have at least three methods whereby they may affect virus-infected target cells: 1) perforin-mediated lysis, 2) Fas-dependent lysis, and 3) cytokine release. Perforin is critical to the control of some virus infections, given that perforin knockout (PKO) mice are unable to clear infection by lymphocytic choriomeningitis virus.21,22
In this article, we evaluate the role of perforin both in the etiology
of CVB3 myocarditis and in CVB3 infection and clearance.
Perforin-mediated lysis of CVB-infected cells is a plausible foundation
for myocarditis and for control of myocardial virus titers, both of
which we found to be CTL dependent.12
Perforin-containing
cells enter the myocardium soon after CVB infection,18
and
circumstantial evidence indicates that perforin may be required for
CVB-induced myocarditis;23
in addition, splenocytes from
CVB-infected mice can lyse CVB-infected myocytes in
vitro.24
In this communication, we show that the
absence of perforin is, broadly, beneficial to CVB-infected mice. PKO
mice survive a CVB3 challenge that is lethal to 90% of perforin +/+
littermates, and although myocarditis occurs in the absence of
perforin, it is much less extensive than in perforin+ mice,
resolves more rapidly, and does not result in severe myocardial
fibrosis. Despite the diminished myocardial infiltration in PKO mice,
viral titers, myocardial distribution, and growth kinetics are
indistinguishable in PKO and perforin+ mice, and virus is
undetectable by 21 days p.i. in both mouse phenotypes. Therefore, in
CVB myocarditis, the antiviral and immunopathological functions of CTLs
can be uncoupled. We also evaluate apoptosis and chemokine expression
in PKO and perforin+ mice. In both mouse genotypes,
chemokine expression approximately parallels myocarditis. Expression of
MIP-1, a chemokine whose disruption renders mice resistant to
myocarditis,25
is barely detectable in PKO mice.
These findings have important therapeutic implications; the development of a specific inhibitor of perforin or of the chemokine pathways might permit us to prevent immunopathology without diminishing the antiviral efficacy of the immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cardiopathogenic H3 strain of CVB3 (Nancy) was utilized in all experiments. The titer of the virus was determined by plaque assay in HeLa cells according to previously published procedures.
Mice
PKO mice were generated by Drs. C. M. Walsh and W. R. Clark (University of California, Los Angeles, Los Angeles, CA), using AB-1 embryonal stem cells, which were introduced into the C57 background and bred on this background.22 Heterozygous PKO mice were sent to Scripps Research Institute, where they were bred to generate homozygous knockout mice, as well as heterozygous and homozygous positive littermates (as controls). We determined the perforin gene status by polymerase chain reaction on tail DNA, for every mouse bred. All experiments utilized adult (at least 8 weeks old) male PKO mice and their transgenic wild-type (homozygous or heterozygous) littermates.
Infection Protocol and Organ Processing
Mice were infected (0.2 ml intraperitoneally) with CVB3 diluted in saline. For evaluation of susceptibility to early death, mice were given 100 plaque-forming units (pfu) CVB3 on day 0 and observed for 21 days. For evaluation of myocarditis, mice were given a sublethal dose (35 pfu) to ensure survival, and at the indicated time points, the organs were removed, dissected in equal parts, washed, and either snap frozen in liquid nitrogen or fixed in 10% zinc normal buffered formalin for histological analysis. Quantitation of virus from heart tissue was performed by homogenization of the tissue and centrifugal clarification of the supernatant, followed by virus plaque assay in HeLa cells as described.
Histology and Immunohistochemistry
For histological analysis, heart tissue was fixed in 10% neutral buffered formalin, sectioned into 5-µm slices, and stained either with hematoxylin and eosin (H&E) or with Masson's trichrome as specified in the text. Inflammation was scored on a scale of 0 to 4 based on the number of lesions present in a section. A score of 1 represents 1 to 10 lesions; 2, 11 to 20; 3, 21 to 40; and 4, >40.
Multiprobe RNase Protection Assay
The RNase protection assay was performed as previously
described.26
Probes used were C10 (a murine member of the
C-C chemokine family); MIP-1 and MIP-1ß (macrophage inflammatory
proteins 1 and 1ß); MIP-2 (macrophage inflammatory protein 2);
MCP-1 and MCP-3 (monocyte chemoattractant proteins 1 and 3); crg-2
(cytokine-regulated gene-2); and RANTES (regulated upon activation
normal T cell expressed and secreted).
In Situ Hybridization
A previously published protocol27 was followed on paraffin sections (5 µm) of heart tissue previously fixed in 10% normal buffered formalin. After preparation of the slides and prehybridization at 42°C, 1 x 106 cpm of a radiolabeled antisense RNA probe was applied and allowed to hybridize at 42°C overnight. After washing, slides were dipped in photographic emulsion and were held at 4°C for 3 to 14 days, when they were developed and fixed. To visualize the cells, the developed slides were stained in H&E for 2 to 3 minutes.
Identification of Apoptotic Cells
The Apoptag kit (Oncor, Gaithersburg, MD) was used according to the manufacturer's instructions.
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Results |
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Three groups of mice (10 mice per group) were infected with 100
pfu CVB3 and were observed for 21 days. As shown in Figure 1
, 90% of homozygous positive
littermates (wild type, +/+) died, whereas 100% of PKO mice survived.
Thus, CTL-mediated lysis appears to contribute to a lethal outcome of
CVB infection. This is consistent with our findings of diminished
susceptibility in ß2mKO mice, which lack CD8+ T cells,
and of protection against lethal outcome after CD8 depletion of CD4KO
mice.12
There may be a gene dosage effect, because 50% of
heterozygous mice survived, compared with only 10% of homozygous
positive mice. No animals died after day 10 p.i. Thus, perforin
appears important to the immunopathological function of
CD8+ T cells during lethal acute infection.
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Results for +/+ and +/- mice were similar, and henceforth both
genotypes will be referred to as "perforin+" mice.
Figure 2
shows the development of
myocarditis lesions over time in PKO and perforin+ mice. At
each time point, in each group, a minimum of four mice was used (thus,
perforin+ groups include at least eight mice, four +/+ and
four +/-). At 4 days p.i., no infiltration was seen in PKO mice, and
only very occasional small foci were detected in perforin+
mice. By 7 days p.i., mild myocarditis could be seen in both
perforin+ and PKO mice. Myocarditis was much more marked by
day 10 in perforin+ mice, but declined in PKO littermates.
By day 14, myocarditis had almost resolved in PKO mice (four out of
four mice), but remained severe in perforin+ animals. Note
that this scoring system reflects only the number of individual lesions
and not the total area of myocardium involved. In a particularly
extensive myocarditis, the lesion score may even be reduced as adjacent
lesions "fuse" to produce a single large lesion. Consequently,
lesion scores must be complemented by representative histology (Figure 3)
. The lesions in PKO mice were very
much smaller, and more circumscribed, than those in
perforin+ animals; the appearance in the PKO mice was
similar to that seen after CD8 depletion of CD4KO mice and to that seen
in ß2mKO mice.12
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No infiltration was detectable in either genotype by day 21
p.i., but by >30 days p.i., marked myocardial fibrosis was present in
perforin+ mice, but not in PKO animals (Figure 4)
. Thus, perforin exacerbates
myocarditis, and its ablation prevents not only acute disease, but also
the subsequent scarring.
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CTLs are critical to the control of many virus infections, but, in
combating picornavirus infections, antibodies appear to play a vital
role. Nevertheless, we have shown that CD8+ T cells play
some role in limiting CVB replication, because depletion of
CD8+ T cells leads to increased CVB load in the
heart.12
To determine whether this antiviral effect was
mediated by perforin, a time course of CVB3 replication in the heart
was carried out, comparing PKO mice with perforin+
littermates. As shown in Figure 5
, there
was no statistically significant difference between the mouse strains.
Regardless of the perforin status, the growth curve peaked on day
4 p.i., falling rapidly thereafter, and no virus was detectable by
21 days p.i. (not shown). Furthermore, the absolute titers at each time
point were not significantly different. Thus, perforin is not required
for clearance of CVB3, and it appears to play little if any role in the
limitation of viral replication in the heart.
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Although we show above that myocardial virus titers are similar
regardless of the perforin status of the host, it remained possible
that the infection would be more widely distributed in the myocardia of
mice lacking perforin. Therefore, at the peak of virus titer, 4 days
p.i., myocardial sections were evaluated by in situ
hybridization with antisense probes for CVB3. For this positive-strand
virus, the antisense probe detects the abundant viral genome and the
almost identical single mRNA. Although the virus titer is very high
(~108 pfu/g), virus was detectable only in well separated
and tightly circumscribed foci in both PKO and perforin+
hearts (shown by dark-field microscopy in Figure 6
); the lesion numbers were similar in
all mice, regardless of perforin status. Bright-field examination of
the infected foci revealed no inflammation, consistent with our finding
that myocarditis is absent on day 4 p.i., as shown in Figure 3
.
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Apoptosis, or programmed cell death, is an important facet of the
host defense against virus infection and also plays a critical part in
several aspects of host development, including regulation of the immune
response. Apoptosis can be induced by several mechanisms. We looked for
apoptosis in +/+ and +/- littermates and found signal in the
infiltrate, but not in noninflamed muscle (Figure 7)
; this is consistent with our previous
observation in the CD4KO mouse strain (also
perforin+).12
The picture differed in PKO mice;
little or no signal could be detected in the small inflammatory
lesions, whereas many myocyte nuclei were positive. Therefore, in
perforin+ mice, there was a marked inflammation, with
apoptosis restricted to these areas whereas in PKO mice the myocarditis
was reduced, but apoptosis was detected in myocyte nuclei.
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CVB-induced myocarditis does not occur in MIP-1 transgenic
knockout mice.25
This chemokine is secreted by macrophages,
lymphocytes, and neutrophils and is chemoattractant for T cells. We
considered it possible that perforin-mediated lysis of infected cells
might increase neutrophil and macrophage infiltration, which would,
through release of MIP-1 and other chemokines, increase the influx
of CTLs. If so, the limited myocardial infiltration in PKO mice might
result, in part, from lower chemokine levels in the infected
myocardium, which in turn resulted from the reduced cytolysis. Normal
and PKO mice were infected with a sublethal dose of CVB3 and chemokine
mRNA expression in the hearts was examined at the indicated time
points, using an RNase protection assay. As demonstrated in Figure 8
, many of the proinflammatory chemokines
were up-regulated during the course of infection. Both the onset and
decay of chemokine expression seemed advanced in PKO mice; crg-2 and
MCP-1 were detectable at day 2 p.i. and peaked at day 4 p.i.
In contrast, chemokine expression in perforin+ mice was
undetectable on day 2, and barely detectable on day 4 p.i.; in
these mice, expression peaked around day 7, when myocarditis developed.
In both PKO and perforin+ mice, signal reached background
levels by day 14. When considering specific chemokines, MIP1- was
barely detectable in PKO animals but was readily detectable in +/+ mice
at 7 and 10 days p.i., whereas MCP-1, MCP-3, crg-2, and RANTES mRNA
expression increased in response to infection in both PKO and wild-type
mice. Thus, CVB3 infection resulted in an increase of proinflammatory
chemokines that correlated with the development of myocarditis, and the
expression kinetics differed between PKO and perforin+
mice.
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To confirm the above, and to localize the expression of the
chemokines, in situ hybridization studies were carried out
on the hearts of PKO and perforin+ mice, 7 days p.i.
Antisense probes for MIP-1, RANTES, and crg-2 were used, and
specificity controls included sense probes (Figure 9
, B and D). In all cases, chemokine RNA
expression was noted in infiltrating cells. Representative results for
crg-2 are shown in Figure 9
for perforin+ mice (Figure 9
, A
and B) and for PKO mice (Figure 9
, C and D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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How does CVB cause myocarditis and long-term disease? Some proposed pathogenic mechanisms include 1) direct destruction of cardiac muscle cells by CVB3, 2) immunopathological destruction mediated by immune effector mechanisms directed against viral antigens, 3) immune-mediated destruction by effector mechanisms targeting novel host-encoded antigens expressed on the surface of cardiac cells after CVB3 infection, 4) immune-mediated destruction targeted against host antigens sharing determinants with CVB3 antigens ("molecular mimicry"), and 5) immune-mediated destruction targeted against host antigens normally expressed but made more immunogenic as a consequence of CVB3 infection. This array of possible mechanisms is further complicated by variability in outcome depending on virus genotype33,34 and the strain,35,36 sex,37,38 age,39 and immune status8,40 of the murine host.
Our laboratory has chosen to focus on the first two mechanisms (1 and
2) described above, because they offer the most direct explanation for
the pathology observed, and because there already is evidence in
support of both. First, the virus itself can cause cell lysis. In the
acute phase of infection, many organs have high viral titers (heart,
pancreas, liver, spleen, lung, and kidney), and viral dissemination is
largely hematogenous, as reflected by a high viremia. In most mouse
strains, the virus titer in the heart peaks at 4 days p.i. and
diminishes thereafter, with infectious virus being undetectable after
14 days. The virus appears to enter the heart by first infecting
endothelial cells and then spreading to the
myocardiocytes.41
Myocardiocytes can be infected in
vitro,42,43
and infected cells are rapidly
lysed.44
The in vitro findings are corroborated
by in vivo ultrastructural studies of myocardial tissue that
show clear evidence of virus infection of cardiac muscle cells and cell
death45-47
and by studies showing that some pathology can
occur even in immunocompromised mice unable to mount a normal T-cell
response.48,49
Thus, virus-mediated lysis of
myocardiocytes in vivo almost certainly occurs and most
probably contributes to myocarditis. Many laboratories have provided
convincing evidence for the second mechanism, implicating T cells
in myocarditis.12,13,18,38,50-54
The relative
contributions of CD4+ and CD8+ T cells appear
to vary somewhat between different mouse strains,38,54
and
a role for 
-
T cells has been suggested.55,56
We previously showed that CD8+ T cells play two roles
during CVB3 infection, one beneficial to the host, and the other
harmful; CTLs limit viral titers in the myocardium, but also cause
extensive myocarditis.12
Here we establish the part played
by perforin in these events and find that perforin function is,
broadly, damaging to the host. Perforin expression results in a
dramatically increased mortality upon CVB3 infection (Figure 1)
; some
90% of perforin+/+ mice succumb, in contrast to 100%
survival in the PKO littermates, and there appears to be a gene dosage
effect, as approximately half of the heterozygous littermates died.
This parallels our previous finding that depletion of CD8+
T cells enhanced survival.12
Furthermore, again consistent
with our earlier study, we show that PKO mice have a much less
extensive myocarditis than heterozygous or +/+ littermates. The lesions
in PKO mice are less frequent, smaller, and better circumscribed, than
those in perforin+ littermates (Figures 2 and 3)
. Thus,
perforin appears to play a major role in the CD8+ T
cell-mediated immunopathology that accompanies CVB3 infection.
We have shown that CD8+ T cells limit viral titers in the
heart, as depletion of these cells resulted in a 17-fold increase in
CVB3 titer.12
Here we demonstrate that this antiviral
effect does not require perforin; virus growth kinetics, and viral
titers, are indistinguishable in PKO and perforin+ mice
(Figure 5)
. It remained possible that, despite these findings, perforin
played a role in restricting the myocardial distribution of CVB;
however, in situ studies show that the number of infected
foci at the peak of viral infection are similar in
perforin+ and PKO mice (Figure 6)
. Our demonstration that
PKO mice clear virus in a manner indistinguishable from +/+ mice
strengthens the argument that, although perforin plays a vital role in
control of relatively "nonlytic" viruses (such as lymphocytic
choriomeningitis virus), it may play less of a role in control of
highly lytic viruses such as vaccinia, Semliki Forest, and vesicular
stomatitis viruses57
and CVB (our data). Thus, we show that
the antiviral and immunopathological results of CTL activity can be
uncoupled by removing perforin; perforin blockade therefore represents
a promising therapeutic target in the treatment of CVB myocarditis.
How might CD8+ CTLs contribute to CVB clearance, given that
our studies show that perforin is not required? CTLs have a second
method of delivering a contact-mediated lethal signal, the fas pathway.
Fas is a membrane protein, a member of the tumor necrosis factor
receptor family,58
which is commonly expressed on
hematopoietic cells and whose activation leads to apoptosis of the
carrier cell. The in vivo ligand for the fas protein, FasL,
is expressed on CTLs and activated splenocytes; these cells can
interact with, and induce apoptosis of, Fas+ target
cells.59,60
Whereas FasL expression is very restricted, Fas
is abundantly expressed on a few nonhematopoietic tissues, including
heart and liver.61
We therefore consider it possible that
CD8+ CTLs might control CVB replication by inducing
apoptosis in infected myocardiocytes. We show here (Figure 7)
that
apoptosis is readily detectable in the hearts of both PKO and
perforin+ mice. In the perforin+ mice, in which
myocarditis is extensive, apoptosis is seen primarily within the
infiltrates; in contrast, in PKO mice, we note clusters of
myocardiocytes with nuclear staining consistent with apoptosis. This
apoptosis may be T cell mediated (given that perforin is not required
for the function of the Fas pathway), or it may be virus induced.
Although these results are consistent with the hypothesis that the fas
pathway is important in regulating the outcome of this infection,
further studies, perhaps using mice defective in the fas
pathway,62
will be required to investigate this question.
Alternatively, CTLs may exert their effects not through lysis but
rather, as argued by Ramshaw et al63
and
others,64
by showering infected cells and the surrounding
area with a variety of cytokines. Others have recently analyzed
cytokine expression in the CVB3-infected myocardium of normal mice and
have found high levels of interleukin-2, interferon-, and tumor
necrosis factor-ß.65
In addition, we have evaluated the expression of chemokines in
CVB-induced myocarditis. Cook et al25
found that MIP-1KO
mice were resistant to myocarditis, and that CVB titers were
indistinguishable from those in normal animals.25
As shown
in Figure 8
, the mRNA encoding MIP-1, a chemokine whose ablation
prevents CVB-induced myocarditis,25
is detectable in
perforin+ mice, but barely detectable in the PKO
littermates. Furthermore, the kinetics of chemokine mRNA expression
vary somewhat between PKO and perforin+ mice. In general,
responses are accelerated in PKO mice, in which mRNA expression is
detectable by day 2 p.i., has lower levels of maximum expression,
and recede sooner. It is tempting to suggest that this accelerated
chemokine production may reflect enhanced replication of the CVB
inoculum in the periphery of PKO mice and thus the earlier delivery of
CVB to the PKO myocardium. However, our data (Figure 5)
show high viral
titers at 2 days p.i. in both PKO and perforin+ mice, and
the similarity in growth kinetics renders this explanation less likely.
The levels of chemokine mRNA expression approximate the severity of
myocarditis. We have previously shown that macrophages are the largest
cellular subset in the infiltrate, and that their recruitment is
diminished by depletion of CD8+ T cells.12
We
suggest that, in the normal host, perforin-mediated CTL lysis of
infected myocytes results in necrosis and a corresponding influx of
macrophages and neutrophils; the chemokines expressed by these
infiltrating cells (Figure 9)
induce further lymphocytic infiltration,
which in turn causes further tissue damage. In contrast, in PKO mice,
antiviral CD8+ T cells invade the myocardium, limiting CVB
replication, but cell death is largely apoptotic, and therefore the
major trigger for myocarditis is absent. Consequently, phagocytic
infiltration is minimized, and expression of the relevant chemokines is
reduced, as we show here. This may offer a second avenue for
therapeutic intervention; blockade of chemokine gene expression, or of
their effector functions, at an early stage p.i. might greatly limit
myocarditis without interrupting virus clearance.
The interaction between CVB and its host is complex; some immune components protect against myocarditis (CD4+ cells in C57BL/6 mice12 ), whereas others greatly exacerbate disease (perforin+ CD8+ cells, shown here). In our initial work, we showed that depletion of CD8+ T cells was effective in diminishing myocarditis, but this therapeutic benefit was achieved at a cost, as viral titers were significantly elevated.12 It is, therefore, perhaps not surprising that the outcome of wholesale immunosuppressive treatment is unpredictable, varying among different mouse strains.66 Inhibition of T cells using lobenzarit disodium exacerbates disease,67 and treatment with nonsteroidal anti-inflammatory agents is deleterious,68,69 whereas FK-506 treatment reduces myocarditis.70 A recent human clinical trial concluded that immunosuppressive drugs should not be routinely used.71 Here we present evidence that the costs and benefits of anti-CVB T cell responses can be dissociated. This has important therapeutic implications, suggesting that blockade of the perforin pathway, or of chemokine responses, might ameliorate disease without diminishing the host's capacity to eradicate the virus.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants AI 42314 (to JLW), and MH 50426 and MH 47680 (to ILC). IM was supported by a fellowship from the Fundacion Ramon Areces.
John R. Gebhard's present address is Pharmadigm, Inc., Salt Lake City, UT.
Accepted for publication May 11, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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