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Journal of Virology, February 1999, p. 1665-1667, Vol. 73, No. 2
Division of Immunology and Cell Biology, John
Curtin School of Medical Research, Australian National University,
Canberra, Australia,1 and
Max-Planck-Institut für Immunbiologie, Freiburg,
Germany2
Received 27 July 1998/Accepted 15 October 1998
Lack of perforin renders the relatively resistant mouse strain
C57BL/6 highly susceptible to the natural mouse pathogen ectromelia virus, a cytopathic orthopoxvirus. This is indicated by increased mortality, elevated virus titers and pathology in liver and spleen, and
increased levels of liver enzymes in blood. Cowpox virus on the other
hand is more virulent in the presence of perforin than in its absence.
An additional lack of granzyme A which together with perforin is a
constituent of cytoplasmic granules from cytotoxic T cells increases
the virulence of cowpox virus.
Cytototoxic T (Tc) cells are of
primary importance in the recovery of mice from infection with mousepox
ectromelia virus (ECT) (1, 2). Tc cells exert their effector
function by two very different mechanisms, with one being mediated by
cytokines such as gamma interferon and interleukins (11) and
the other being mediated by cytotoxic molecules. To date two major
pathways of target cell killing by cytolytic leukocytes (mainly natural
killer [NK] and Tc cells) have been described. Firstly, the granule
exocytosis pathway mediated by perforin or cytolysin and serine
proteases or granzymes (Gzm) (5, 13). This is generally
believed to be the dominant mechanism by which Tc and NK cells
eliminate virus-infected cells (7). The second mechanism,
called the Fas-mediated pathway, requires the interaction of the
Fas receptor on the target cell with the Fas ligand on the
killer cell (14) and is supposedly involved in
immunregulation and tolerance (12).
From the three main constituents of the granules involved in the
exocytosis pathway Thus, it was of interest to us to have a fresh look at the role of
perforin in the survival of mice to two additional poxvirus infections,
one being the natural pathogen ECT and the other being cowpox virus
(CPV). The virulent Moscow strain of ECT was grown in mice and prepared
from infected spleens and titrated as described previously
(10). CPV was grown on CV-1 cell monolayers and titrated as
was ECT. B6 animals are relatively resistant to ECT administered via
the hind footpad. Doses of >106 PFU of virulent Moscow
strain are required to cause disease ending in mortality. We used
three strains of mice, wild-type B6, the perforin-defective KO
strain (6) (perf
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Perforin Is Essential for Control of Ectromelia
Virus but Not Related Poxviruses in Mice
ABSTRACT
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perforin and the two Gzm, GzmA and GzmBonly GzmA
has been thoroughly investigated so far for its role in recovery of
mice from ECT infection. By comparing gzmA
/
knockout (KO) mice with wild-type C57BL/6 (B6) mice (10) it was found that the lack of GzmA does not affect the cytolytic potential
of ECT-immune Tc cells but leads to increased mortality and morbidity,
as well as higher virus titers and tissue damage in liver and spleen.
The actual role of GzmA in recovery from ECT infection is still elusive
but possibly involves reduction of progeny virus infectivity by its own
proteolyic activity or via secondary mediators (10). As for
the role of perforin, conclusions have been reached only in regard to
the non-mouse pathogen vaccinia virus (VV). In their study, concerning
a comparison of the role of perforin on protection against cytopathic
and noncytopathic viruses, Kägi et al. (8) came to the
conclusion that cytopathic viruses are not controlled by perforin, as
mice survived infection with this virus in the absence of perforin.
However, it was already known that mice survive even high doses of VV
in the absence of CD8+ T cells (16).
/), and the
double-KO mouse lacking both the perforin and gzmA genes
(gzmA/ × perf/). The latter
strain was obtained by crossing the perf/
mice with the gzmA/ mice (4) and
breeding to homozygosity. All animals were monitored for the correct
genotype by PCR analysis as has been described (see reference
15 and the legend to Fig.
1). In Fig. 1 the results of a
dose-response experiment using ECT, ranging from 101 to
106 PFU/mouse (administered via the footpad), are
illustrated. B6 mice were only infected with the two highest doses, and
although morbidity was noticed no mortality occurred. On the other
hand, mice of the two KO strains started to die at the highest dose 6 to 7 days postinfection and all had died at day 8. Even at the lowest
inoculum of only 10 PFU, the mutant mice started to die at day 9 and
all had succumbed by day 10. These results indicate that the animals
are as susceptible to ECT as the least-resistant strains known
(3) and clearly point to perforin's being of paramount
importance in the recovery from primary ECT infection. No statistically
significant differences were found between the single
perf/ KO mice and the double-KO mice also
defective in GzmA, again suggesting perforin is the overriding
prerequisite for survival. However as shown before, the presence of
GzmA contributes to control of ECT infection by a mechanism(s) other
than cytolytic activity (10). To obtain a more detailed
analysis, a kinetic study was undertaken using the three mouse strains
and an infectious dose of 102 PFU of ECT. Three individual
mice of each strain were sacrificed 2, 4, 6, and 8 days after
infection. Livers and spleens were analyzed for virus titers (Table
1) and histology and blood samples were assayed for liver enzyme (Fig. 2). Virus
was not detectable 2 days postinfection in any of the mouse strains. In
B6 mice virus titers in liver and spleen reached a maximum by day 6 and
then declined in liver to undetectable levels by day 8. In the
perf/ and gzmA/ × perf/ mice virus titers were at least 1 log higher in both organs on day 6. By day 8 the difference was at
least 3 logs in liver and up to 2 logs in spleen. One animal of the
perf/ genotype had died by day 8 (Table 1).
Histological examinations of liver and spleen mirror virus load, with
increased necrosis and cellular infiltration in the KO mice from day 6 on compared to B6 wild-type mice (data not shown). Using an objective
assay of liver damage, namely levels in the blood of the liver-derived enzyme aspartate aminotransferase (AST) (10), and using the same animals for which virus titer determinations (Table 1) and histology examinations were undertaken, we found that at day 8 postinfection, despite the generally observed high variability in this
assay (10, 17) the perf/ and
gzmA/ × perf/ mice had
significantly higher liver enzyme levels than B6 mice (Fig. 2).
View larger version (14K):
[in a new window]
FIG. 1.
Dose-response curves for ECT infection via the footpad
of C57BL/6 ( 
), perforin-deficient (
), and GzmA-plus
perforin-deficient (
) mice. Surviving mice were monitored for 21 days. For detection of the respective mutations, DNA of all individual
mice was analyzed by PCR, as described previously (15),
using the following primers: for gzmA/ mice,
5'-AGG AGC AAT ATA TAC CAA TGG-3' and 5'-AGG TAG GTG AAG GAT AGC CAC-3'
(neo-primer 5'-CGG AGA ACC TGC GTG CAA TC-3'); for
perf/ mice, 5'-CCA CTC CAC CTT GAC TTC AAA
AAG GCG-3' and 5'-TGG GCA GCA GTC CTG GTT GGT GAC CTT-3'.
TABLE 1.
Kinetics of virus titers in liver and spleen of
individual micea infected with ECT
View larger version (22K):
[in a new window]
FIG. 2.
Liver enzyme levels in serum of ECT-infected mice. Shown
are mean AST levels in units per liter of serum (± standard deviations
[error bars]) from three individual animals B6 (),
perf/ (
), and gzmA/ × perf/ ()immunized via the footpad with
102 PFU of ECT (Moscow strain). Values are enzyme levels
from a single surviving animal.
In contrast to ECT, CPV is much less virulent in mice, and mortality is
obtained most consistently after intraperitoneal infections. The same
three strains of mice were infected with 105, 1 × 106, and 2 × 106 PFU of CPV (Fig.
3). None of the animals died as a result
of receiving the lowest concentration. At the two higher doses it was
found that the lack of both GzmA and perforin made the double-KO mice
more susceptible than wild-type B6 mice. This was especially significant at 106 PFU. Comparing
perf/ mice with wild-type B6 mice, the
absence of perforin provided a significant protective effect, with 0 versus 50% mortality at 106 PFU of CPV and only 30 versus
100% mortality at the highest dose, respectively. This striking
contrast in virulence between these two closely related cytopathic
orthopoxviruses, ECT and CPV, in the presence or absence of perforin
must reflect totally different pathogenic mechanisms which control
these two viruses. Thus, for a meaningful interpretation of
host-parasite relationships only natural pathogens will uncover
strategies of either host or virus which are of evolutionary
significance.
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The finding that perforin is essential in the recovery of mice from ECT infection questions the proposed role of poxvirus-encoded serpins, one of interfering in the death pathway, in the evasion of poxviruses from the Tc cell response (9). Thus, perforin is an essential element in the survival strategy of mice to recover from cytopathic and noncytopathic viruses.
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ACKNOWLEDGMENTS |
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This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Si 214/7-1).
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia. Phone: 61 26 249-4392. Fax: 61 26 248-6271. E-mail: arno.mullbacher{at}anu.edu.au.
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Journal of Virology, February 1999, p. 1665-1667, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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