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-Deficient Mice Infected with Lymphocytic Choriomeningitis Virus1
*
Institute of Medical Microbiology and Immunology and
Medical Anatomy, University of Copenhagen, Copenhagen, Denmark
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in the control of acute infection
with a noncytopathogenic virus, mice with targeted defects of the genes
encoding IFN-, perforin, or both were infected i.v. with two strains
of lymphocytic choriomeningitis virus differing markedly in their
capacity to spread in wild-type mice. Our results reveal that IFN-
is pivotal to T cell-mediated control of a rapidly invasive stain,
whereas it is less important in the acute elimination of a slowly
invasive strain. Moreover, the majority of mice infected with the
rapidly invasive strain succumb to a wasting syndrome mediated by
CD8+ effector cells. The primary effector mechanism
underlying this disease is perforin-dependent lysis, but other
mechanisms are also involved. Wasting disease can be prevented if naive
CD8+ cells from mice transgenic for an MHC class
I-restricted lymphocytic choriomeningitis virus-specific TCR are
adoptively transferred before virus challenge, indicating that the
disease is the result of an unfortunate balance between virus
replication in internal organs, e.g., liver and spleen, and the host
response; resetting this balance by increasing host responsiveness will
again lead to a rapidly controlled infection and limited tissue damage.
Thus, the presence or absence of IFN- determines whether CTLs will
clear infection with this noncytopathogenic virus or induce severe
immunopathology. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which may interfere with viral replication in
various ways. While the role of cytotoxicity seems firmly established
(1, 2, 3), the importance of cytokines is less clear,
especially in the case of noncytolytic viruses (4). The
present study focuses on the importance of IFN- as a critical
modifier of the efficiency of CTLs in antiviral immunity against the
latter type of viruses.
Although originally named for its antiviral activity, the role of
IFN- as an essential mediator in the context of virus clearance has
been elusive. Thus, experiments conducted in mice made deficient for
IFN- by either Ab treatment or gene knockout have revealed that many
viral infections may be controlled in absence of IFN-
(5, 6, 7, 8, 9, 10), although in other cases a significant effect may
be observed (5, 6, 11, 12, 13, 14). With regard to one of the most
studied viral model systems, the murine lymphocytic choriomeningitis
virus (LCMV)4
infection, the majority of studies support the view that IFN- plays
only a marginal role in clearance of the acute infection (10, 11, 15, 16, 17), whereas this cytokine seems to be important for
adoptive immunotherapy of persistent virus carriers (mice infected at
birth) (15, 18). As opposed to this, one group studying
infection with high doses of a highly invasive strain of LCMV has
reported a complete inhibition of virus clearance following treatment
with anti-IFN- (19, 20). However, since also the
generation of CTLs, known to be the primary mediators of LCMV clearance
in the acute phase of the infection (1), was found to be
greatly reduced in anti-IFN--treated mice, the authors concluded
that IFN- was essential for the generation of virus-specific CTLs,
and that the antiviral effects of the cytokine therefore eluded
evaluation in this system. Contradicting this
interpretation, more recent studies have clearly established that
clonal expansion and differentiation of virus-specific (including
LCMV-specific) CTLs can proceed normally in the absence of IFN-
(9, 11, 15, 21, 22). Therefore, a more likely explanation
for their results is that the importance of IFN- varies with the
biological properties of the virus strain, and that the observed
suppression of the CTL response is secondary to an extensive
uninhibited virus spreading. In this context, it is pertinent to note
that similar abortive CTL responses can be found both in normal mice
infected with very high doses of virus (23, 24) and in
mice made deficient for IFN-
/ß (16, 25)).
Consequently, it is possible that the importance of IFN- is stongly
influenced by the invasiveness of the virus stain studied; several
studies have recently emphasized this parameter as a critical factor in
determining the spectrum of relevant host defense mechanisms (18, 26, 27, 28).
The present study was therefore undertaken to clarify the role of
IFN- as a modifier of CTL-dependent clearance/control of acute viral
infection with strains varying in their capacity to rapidly replicate
and spread in the host. Furthermore, with the purpose of reevaluating
the relative importance of perforin and IFN- in virus clearance and
immunopathology, mice were generated with targeted defects of both
perforin and IFN- genes. Our results reveal that not only is IFN-
pivotal in controlling infection with the rapidly invasive LCMV strain,
but in its absence most infected mice succumb from the extensive
CTL-mediated, primarily perforin-dependent, immunopathology induced
during the futile attempt to clear the infection.These findings are
pertinent to the understanding of the mechanisms underlying T
cell-mediated control of systemic infections with noncytopathogenic
viruses such as hepatitis B, and perhaps HIV, in which an unbalanced T
cell response may significantly contribute to the induced
pathology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IFN--deficient mice (IFN--/-) were
bred locally from breeding pairs obtained from The Jackson Laboratory
(Bar Harbor, ME). In most experiments, these mice were on a C57BL/6
background, but in a few experiments IFN--/-
mice on a BALB/c background were used. Perforin-deficient
(Pfp-/-) mice were obtained from Taconic
(Germantown, NY), and transgenic mice expressing a TCR specific for
LCMV (gp aa 33–41, immundominant epitope in H-2b
mice) were the progeny of breeding pairs kindly provided by H. Pircher
and R. M. Zinkernagel, University of Zürich, Switzerland
(29).
Mice deficient in both IFN- and perforin production were produced in
the following way: Pfp-/- mice were mated with
IFN--/- mice to generate an
F1 generation heterozygous at both loci. These
mice were then backcrossed to IFN--/- mice,
and offspring heterozygous at the perforin locus (+/-) and homozygous
for the disrupted Ifg gene (-/-) were then selected and
interbred. Assessment of genotypes was performed by PCR, and functional
analysis was used to verify both defects: these mice did not produce
alloreactive CTLs in a MLR, and in response to polyclonal stimulation
with Con A extensive cell proliferation, but no IFN- production was
observed. The capacity of these mice to produce T cell-dependent Abs
was intact, as evidenced by the ability to produce neutralizing IgG Abs
in response to vesicular stomatitis virus (50). Because
these mice are on a mixed B6,129 background, mice heterozygous for the
pfp gene, but homozygous for the disrupted Ifg
gene, were used as controls in some experiments studying the effect of
perforin deficiency.
Wild-type C57BL/6 (B6) and BALB/c mice were purchased from Bomholtgaard (Ry, Denmark). Mice from outside sources were always allowed to acclimatize for at least 1 wk before use; at that time, the animals were 7–10 wk old. Animals were housed under controlled conditions (specific pathogen free (SPF)) that included testing of sentinels for unwanted infections according to FELASA standards; no such infections were revealed. All experiments were conducted in accordance with the guidelines of the Danish Committee on Laboratory Animal Care.
Virus
LCMV of the Traub strain was produced and stored as described previously (30). LCMV of the Armstrong (ARM) strain (clone 53b) was kindly provided by M. B. A. Oldstone, Scripps Clinic and Research Foundation (La Jolla, CA) (31). Titrations of organ virus levels were conducted by intracerebral (i.c.) inoculation of 10-fold dilutions of 10% w/v organ homogenates into young adult NMRI mice. Titration endpoints were calculated by the Kärber method and expressed as mean lethal dose (LD50).
Infection
Unless otherwise specified, mice to be infected received a virus
dose of 200 PFU i.v. (1 PFU is 
5 LD50 by i.c.
titration) in a volume of 0.3 ml. Intravenous injection of LCMV
normally results in a transient, immunizing infection, whereas i.c.
inoculation induces a fatal T cell-mediated meningitis from which the
animals succumb days 6 to 8 postinfection (p.i.). The two virus strains
used in this study differ markedly in the speed with which they
replicate in the host, particularly in the liver and lungs. Thus, when
organ virus levels are compared on day 4 p.i. (i.e., immediately
before the beginning of the adaptive phase of the host response),
spleen virus titers differ only slightly between LCMV ARM- and
Traub-infected mice, whereas much higher virus levels (2–3 logs)
are found in livers and lungs of Traub-infected mice (data not shown).
Eventually, generally high organ virus levels can be attained by both
viruses in immune-deficient mice.
Clinical disease
Weight loss and mortality were used to evaluate the clinical severity of the LCMV infection. Mice were monitored daily for a period of 28 days after i.v. inoculation.
Histopathology
Livers from representative animals were fixed in 4% Formalin, embedded in paraffin, and used for histological examination after hematoxylin-eosin staining of 5-µm sections.
Cell preparations
Spleens were removed from mice killed by ether-anesthetization. Single cell suspensions were obtained by pressing the organs through a fine steel mesh. In some experiments, splenocytes were depleted of CD8+ T cells by treatment with anti-CD8 (Cedarlane, Hornby, Ontario, Canada), followed by incubation with rabbit complement (Cedarlane), as previously described (27); incubation with complement not preceded by Ab treatment served as control.
In vivo depletion of CD8+ T cells
Depletion of CD8+ T cells was obtained by i.v. injection on day -1 and +1 relative to virus infection with the Ig fraction of ascites from mice carrying the clone YTS169.4 (Sera-lab, Crawley Down, Sussex, U.K.) or ascitic fluid from mice carrying the 2.43 hybridoma. Cell depletion was verified by flow cytometry on day 10 p.i.; <1% CD8+ cells were found in the spleen.
Cytotoxicity assays
Cytotoxic T cell activity was evaluated in 51Cr release assays. MC57G fibrosarcoma cells infected with LCMV 48 h earlier were used to assay virus-specific cytotoxicity; uninfected cells served as control targets. Assay time was 5–6 h, and percent specific release was calculated as described previously (30).
Flow-cytometric analysis
Cells were stained with relevant rat anti-mouse Abs obtained from PharMingen (San Diego, CA), washed, and fixed with 1% paraformaldehyde. Samples were analyzed using a Becton Dickinson FACSCalibur (San Jose, CA), and 1–5 x 104 viable mononuclear cells were gated using a combination of forward and side scatter to exclude dead cells and debris (32). Data analysis was conducted using the PC-LYSYS program. For analysis of proliferating cells, mice were given 5-bromo-2'-deoxyuridine (BrdU) in their drinking water (0.8 mg/ml) for a 3-day period. Single cell suspensions of splenocytes were prepared, and cells were surface labeled with anti-CD8, fixed with ethanol, permeabilized, and treated with DNase. Cells were then washed and stained with anti-BrdU (Becton Dickinson).
IFN- ELISA
Serum or cell culture supernatants were assayed using a sandwich ELISA (Endogen, Cambridge, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is critical for control of acute infection with a rapidly
invasive LCMV strain
To determine the role of IFN- in the control of acute infection
with a rapidly invasive strain of LCMV,
IFN--/- and wild-type B6 mice were infected
i.v. with 200 PFU of LCMV Traub, and the time course of spleen and
liver virus titers was studied. As can be seen in Fig. 1
, lack of IFN- leads to a marked
impairment of virus clearance: no substantial decrease in virus levels
is observed in IFN--deficient mice between day 4 and 10 p.i.,
during which period virus is almost cleared in wild-type mice. Even on
day 28 p.i., substantial levels of virus were found in all tested
IFN--/- mice.
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-/- and wild-type mice were compared
with those in similarly infected T cell-deficient nude mice 4 and 10
days after infection, i.e., before and after the predominant phase of T
cell-dependent virus clearance. No differences in virus loads were
observed between the groups on day 4 p.i. (not shown), and on day
10 p.i. similar high virus levels were observed in
IFN--/- mice and nude mice, whereas
significantly lower (p < 0.05 Mann-Whitney
rank sum test) virus titers were detected in wild-type mice (Fig. 2). This pattern strongly indicates that
IFN- plays little or no role in controlling the infection before the
development of the antiviral T cell response, and that T cell-mediated
clearance of LCMV Traub is markedly dependent on the presence of this
cytokine.
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, virus levels in perforin
and perforin/IFN- knockout mice were studied and compared with those
obtained in IFN--/- mice. In addition, virus
levels in CD8+-depleted
IFN--/- mice were analyzed. As can be seen
in Fig. 2
, identical spleen virus titers were obtained on day 10
p.i. in all test groups, and a similar pattern was observed in the
liver (data not shown). Thus, CTLs contributed little to virus
elimination in the absence of IFN-.
To evaluate the long-term importance of either effector mechanism for
virus control, virus levels were also analyzed after 4 wk of infection.
On day 28 p.i., a clear hierachy of virus levels was noted. Thus,
the highest virus titers were observed in nude mice lacking all T
cells. Lower virus titers were seen in
perforin/IFN--/- mice. Compared with the
latter, significantly lower (p < 0.05,
Mann-Whitney rank sum test) spleen virus levels were found in mice
lacking perforin only, and this finding was repeated in the liver (data
not shown). Together this pattern indicates that not only may IFN-
contribute significantly to virus control in this late phase of
infection, but also that T cell-dependent effector mechanisms in
addition to both perforin-mediated lysis and IFN- exist and play
some role in controlling virus replication.
Because the above results are in contrast to previous findings obtained
in experiments involving primarily the ARM strain of LCMV
(15), we also studied virus levels in mice infected 10 and
28 days earlier with a matching dose of this less invasive LCMV strain.
As can be seen in Fig. 3, a slight
negative impact of IFN- deficiency on virus clearance was observed
also in LCMV ARM-infected mice, but in this case the T cell response
clearly resulted in substantial reduction of spleen virus titers in
IFN--/- mice. Notably, significantly lower
(p < 0.05, Mann-Whitney rank sum test) virus
levels were found on day 10 p.i. in Pfp-/-
mice compared with nude mice, suggesting that IFN- may have some
CTL-independent antiviral activity. In keeping with this assumption,
very high levels of IFN- were detected in the circulation of
Pfp-/- mice (50–100 ng/ml vs <0.25 ng/ml
in wild types) and, more important, the virus levels in double knockout
mice matched those in similarly infected nude mice. On day 28
p.i., virus levels in LCMV-infected perforin-deficient mice equalled
those in nude mice, a pattern contrasting with our findings in LCMV
Traub-infected mice (see Fig. 2).
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-deficient mice
One further observation that was made in the course of the above
experiments was that most IFN--/- mice
succumbed to i.v. infection with 200 PFU of LCMV Traub (overall
mortality, 82%; median time to death, 14 days, range 9–27 days), and
similar results were obtained in knockouts on a BALB/c background. This
mortality was associated with marked weight loss in IFN--deficient
mice starting at about day 7 p.i. (Fig. 4, A and D),
coinciding with the appearance of high numbers of activated T cells in
the spleen (32, 33). Only limited disease was observed in
similarly infected wild-type mice or in
IFN--/- mice infected with LCMV ARM, and
none died (Fig. 4, A and B). LCMV Traub did not
induce any symptoms in T cell-deficient nude mice, and
CD8+ T cell depletion completely protected
IFN--/- mice (Fig. 4D). Based on
these observations, the most likely explanation to the observed disease
pattern appeared to be that wasting disease was the result of augmented
CD8+ T cell-dependent immunopathology induced in
the attempt to control the more disseminated virus infection in
Traub-infected IFN--deficient mice. To test this hypothesis, we
compared liver virus levels in day 10 infected mice (Fig. 5) with LCMV-induced weight loss (Fig. 4)
and observed a clear correlation. Thus, no weight loss was found in
LCMV ARM-infected wild-type mice in which virus is also rapidly
cleared. Moderate/transient wasting was observed in wild-type mice
infected with LCMV Traub as well as in
IFN--/- mice infected with LCMV ARM, and in
both of these groups intermediate virus levels were noted. Finally,
severe wasting is induced in LCMV Traub-infected
IFN--/- mice in which virus infection is not
controlled. To further test the correlation between delayed virus
clearance and disease severity, wild-type B6 were infected with a
100-fold higher dose of LCMV Traub. Under these conditions, even
wild-type mice were unable to rapidly reduce virus levels in internal
organs (Fig. 5), and the increase in inoculum resulted in severe,
albeit transient wasting (Fig. 4C).
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-deficient and wild-type mice. Histological
analysis of liver sections revealed moderate hepatitis in both
wild-type mice and IFN--/- mice on day 8
after infection with LCMV Traub (Fig. 6A–D). However, the extent
and localization of infiltrating cells were substantially different. In
wild-type mice, some mononuclear infiltration of the portal areas was
seen together with small, clearly defined granulomatous lesions
scattered throughout the lobuli, many of which contained eosinophilic
bodies, probably necrotic liver cells. In contrast, portal mononuclear
inflammation was much more pronounced in
IFN--/- mice, together with a diffuse
panlobular infiltration with predominantly mononuclear cells. On day
10 p.i., wild-type mice had a mild portal infiltration, and fewer,
well-demarcated mononuclear lesions were found in the lobuli. In
IFN--/- mice, the lesions had progressed
further, and there was marked portal infiltration containing many
granulocytes. Many necrotic liver cells were noted, and few mononuclear
cells were associated with these lesions; instead, granulocytes often
dominated.
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To determine the effector mechanism through which
CD8+ T cells were causing disease,
IFN--/- mice, perforin-deficient mice, and
double knockout mice were infected i.v. with 200 PFU of LCMV Traub and
compared with regard to severity of weight loss on day 9 p.i. as
well as mortality over a 4-wk observation period (Fig. 7). It was found that wasting disease was
significantly milder in mice lacking perforin in addition to IFN-
(despite similar high virus levels and high numbers of activated
CD8+ T cells (see below)), and only few of these
mice died. To test whether the residual disease seen in infected double
knockout mice was caused by a CD8+ T
cell-dependent mechanism, some of these mice were depleted of
CD8+ cells using mAb. Following this treatment,
all mice appeared healthy and no weight loss was observed,
demonstrating that CD8+ cells contribute to
development of wasting also through perforin-independent mechanisms.
Interestingly, despite a lesser initial weight loss, more
perforin-deficient mice succumbed from the infection than in the case
of double knockouts (Fig. 7). This supports the recent contention that
IFN- contributes to the pathogenesis of LCMV-induced disease in
Pfp-/- mice (which seems to represent a
different disease entity, a T cell-dependent form of aplastic anemia
(34)). Notably, for the experimental groups compared in
this section, the differences in severity of disease did not relate to
the extent of viral replication, as virus levels were found to be
identical in all three knockout strains (Fig. 5).
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and
double knockout mice
Besides hepatitis, marked involution of the spleen was observed in
LCMV Traub-infected IFN--/- mice: at about
day 8 p.i., spleen cell numbers were approx. 10–20% of those in
matched wild-type mice. Furthermore, LCMV-specific cytotoxicity was
about 8-fold lower (data not shown). No reduction in spleen cell
numbers was found in IFN--deficient mice also lacking perforin,
indicating that spleen cell depletion was also a result of CTL-mediated
immunopathology. To study this further, wild-type mice, mice lacking
IFN-, and mice deficient in both perforin and IFN- were infected
with LCMV Traub, and on day 6 p.i., the number of proliferating
CD8+ T cells was evaluated flow cytometrically
based on in vivo BrdU incorporation (mice were given BrdU in their
drinking water from day 3 p.i.). On days 6 and 8 p.i., the
percentage of phenotypically activated (VLA-4high
(33)) CD8+ T cells was also
analyzed. As can be seen in Fig. 8, the
initial expansion of the CD8+ T cell population
was found to be of similar magnitude in all three mouse strains.
However, the subsequent expansion in the number of activated
CD8+ T cells was aborted in
IFN--/- mice, whereas this was not the case
if the mice were also deficient in perforin; even on day 28 p.i.,
high numbers of actvated T cells were present in the latter mice (not
shown). Thus, IFN- is not essential for T cell activation and
proliferation, and the splenic involution and reduction in
CD8+ T cell number therefore are integrated
components of the symptomatology of the disease, probably resulting
from perforin-mediated cell damage to the Ag-presenting environment
and/or to the CTLs themselves (35, 36).
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-deficient mice
If the wasting disease in LCMV Traub-infected
IFN--/- mice resulted from an impaired
capacity of IFN--deficient T cells to control virus replication,
increasing host T cell responsiveness should prevent disease. To test
this prediction, groups of IFN--deficient mice were given either
3 x 106 cells from naive mice transgenic
for an LCMV-specific class I-restricted TCR or the same number of cells
from naive wild-type controls. Three days after cell transfer, the mice
were infected with LCMV Traub. As can be seen in Fig. 9, cells from transgenic mice completely
prevented the wasting syndrome normally observed in IFN- mice, and
this protection correlated with rapid virus control, essentially
matching that in wild-type animals (day 10 organ virus titers are
presented in Fig. 9, and nearly identical titers were obtained on day
8 p.i.). Normal splenocytes only slightly affected the initial
weight loss, but although these mice did not fully regain their initial
body weight during the observation period, mortality was low. The
protective effect of adoptive cell transfer was eliminated by prior
depletion of CD8+ T cells from the donor cell
population, demonstrating that wasting disease could be prevented as
well as induced by effector cells belonging to this subset.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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may play a decisive role in the T
cell-mediated control of acute infection with a noncytopathogenic
virus. However, the importance of IFN- varies markedly with
biological properties of the infecting virus. Thus, following infection
with an LCMV strain that rapidly attains high titers in the major
internal organs, failure to produce IFN- not only results in
markedly impaired capacity to control virus replication, but also in
severe, mostly fatal CTL-mediated immunopathology. A small influence on
virus clearance is also observed in mice infected with the less
invasive LCMV strain, but immunopathology is minimal. A schematic
summary of our findings is presented in Table I.
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has been implicated as a critical mediator
in adoptive immunotherapy of chronically infected virus carriers
(15, 18), whereas its role in the control of acute
infection with LCMV has generally been played down compared with that
of perforin-dependent lysis (37). The reason for this view
appears to be the use in most of the previous studies of either i.v.
infection with the slowly invasive LCMV ARM strain or peripheral
infection with very low doses of invasive isolates (10, 11, 15). Under those conditions, in contrast to the situation in
mice infected systemically with moderate to high doses of more
viscerotropic strains such as WE (19, 20) and Traub
(present study), the potential for extensive viral dissemination in the
internal organs is limited, and perforin-dependent lysis may suffice
for the effector T cells to accomplish the initial decrease in virus
load, although the infection may not be completely eliminated. Probably
this race between virus on the one hand and immune response on the
other also explains the recent finding that in mice with only low
affinity TCRs for LCMV, due to genetic manipulation, IFN- may be a
decisive factor even following infection with LCMV ARM
(17), and we have preliminary observations indicating that
depletion of CD4+ T cells in
IFN--/- animals may have a similar effect. Thus, the
overall impression is that the antiviral potential of IFN- becomes
critical in situations in which the balance between virus replication
on the one hand and host immune response on the other is somehow
shifted in favor of the virus. This interpretation is also in keeping
with the finding that although IFN- is not pivotal in intact LCMV
ARM-infected mice, the ability of effector T cells to rapidly control
this virus following early adoptive transfer (i.e., while the virus is
maximally replicating and effector cell capacity limited) is virtually
abolished by co-inoculation of anti-IFN- mAb (38).
In this context, it may be relevant to note that we do not know
precisely how IFN- is working to assist in T cell-mediated virus
control. We have recently shown that IFN- is not required for
targeting of effector cells to sites of viral replication
(22). However, it is possible that this cytokine is needed
to up-regulate critical molecules on the target cell surface promoting
effective CTL/target conjugation (39, 40). Although this
possibility cannot be ruled out, we do not find either MHC class I or
ICAM-1 to be expressed at markedly different levels in infected
IFN--/- mice compared with infected
wild-type mice (unpublished observation). IFN- could also function
through induction of NO (41); however, virus clearance is
unimpaired in mice lacking inducible NO synthase (manuscript in
preparation). Therefore, we assume that IFN- works primarily by
reducing the number of cells susceptible to infection, and thereby
reduces the rate with which the virus successfully establishes a
productive infection in new host cells; previous studies have shown
that pretreatment with IFN- will reduce virus take in vivo
(38). That IFN- directly inhibits viral replication is
also supported by the present finding that perforin-deficient mice
transiently have the capacity to reduce spleen virus levels when
infected with a low dose of LCMV ARM, whereas virus titers matching
those in nude mice are found in similarly infected mice lacking both
IFN- and perforin. Because the kinetics requirements for
CTL-mediated control of acute LCMV infection appear to be very
demanding (42), it is easy to envisage that lack of
IFN- may lead to a situation in which the capacity of the virus to
spread exceeds that of CTL-mediated elimination of infected cells,
particularly when the intrinsic invasiveness of the virus strain is
already high.
That a change in the virus/host balance may lead to fatal
immunopathology following i.v. infection is not unprecedented in the
LCMV model. Thus, ß2-microglobulin-deficient
mice may succumb to a severe CD4+ T cell-mediated
chronic wasting disease (43, 44, 45), and perforin-deficient
mice develop a similar disease (1, 2). Perhaps more
pertinent to the present situation, T cell low responder mice infected
with low-moderate doses of virus may also succumb to CTL-mediated
hepatitis (46). Therefore, it would seem that severe
immunopathology is induced when the primary CTL response is incapable
of rapidly controlling the acute infection, but the virus spreads too
slowly to cause early exhaustion of the immune response. Under these
conditions, the ongoing T cell response may gradually cause sufficient
tissue damage to kill the host. The primary mechanism underlying
CD8+ T cell-mediated pathology in
IFN--deficient mice appears to be perforin-dependent cell lysis
because most double-deficient mice survive the infection. However,
significant wasting was observed in these mice, although it was rarely
severe enough to be fatal. As no evidence of wasting disease was
detected following CD8+ T cell depletion of
double-deficient mice, it may be concluded that
CD8+ T cells contribute to immunopathology
through effector mechanisms in addition to IFN- and perforin.
Analysis of mRNA levels for cytokines in spleen and liver has failed to
disclose any major differences between infected
IFN--/- mice and similarly infected
wild-type mice, and, notably, regarding the type I/type II cytokine
balance, we do not see a different pattern in mice lacking the ability
to produce IFN- (unpublished observation). The latter is consistent
with findings in other viral models in which lack of IFN- does not
generally lead to a type II profile (47, 48), indicating
that other virus-induced mediators suffice to induce a type I cytokine
profile. Because the main clinical symptoms of LCMV Traub-infected
IFN--/- mice are those of a wasting
syndrome, we extensively searched for evidence of augmented TNF-
production. However, little or no TNF- could be demonstrated in
serum or in culture supernatants from stimulated T cells, and TNF-
production from stimulated adherent cells is reduced compared with
wild-type mice (unpublished observation). Consequently, it is tempting
to suggest that the Fas/Fas ligand-dependent pathway of T cell-mediated
lysis may constitute the underlying mechanism. Studies addressing this
issue are presently underway.
Also, in the later phase of infection, differences in the relative
importance of antiviral effector mechanisms appear to exist between the
two viral strains studied in this work. Thus, virus titers in LCMV
ARM-infected perforin-deficient mice are identical to those in nude
mice 4 wk after virus inoculation. This is in contrast to the pattern
in LCMV Traub-infected mice in which neither deficiency of perforin
alone nor of both perforin and IFN- appears to have the same effect.
This finding bears a striking resemblance to the outcome of studies on
adoptive immunotherapy of chronically infected virus carriers, in which
it has been found that CD8+ T cells suffice to
clear infection with LCMV ARM, whereas also CD4+
T cells and B cells play a role in mice infected with the viscerotropic
WE stain (18). We have ourselves previously found that
some CD4+ T cell-dependent effector capacity
reduces virus replication in LCMV Traub-infected
ß2-microglobulin-deficient mice
(49), and the present observation that residual antiviral
capacity exists in mice lacking perforin is consistent with this
finding. Taken together, the presented data serve to underscore the
clear impact the biological properties of the infecting virus may have
in determining the relative importance of various host defense
mechanisms.
In conclusion, our study demonstrates that IFN- plays a critical
role as a modifier of CD8+ effector capacity with
regard to both protective immunity and immunopathology. Thus, our
findings indicate that not only contact-dependent, perforin-mediated
lysis (1, 2), but also T cell-derived cytokine is
important for the control of an acute infection with a noncytolytic
virus. In addition, our results clearly illustrate that deficient
cytokine production may transform an otherwise harmless infection into
a highly lethal disease. Thus, the presence or absence of IFN-
determines whether the antiviral CTL response will be protective or
cause fatal immunopathology.
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Footnotes |
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2 Current address: Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105.
3 Address correspondence and reprint requests to Dr. Allan Randrup Thomsen, Institute of Medical Microbiology and Immunology, the Panum Institute, 3C Blegdamsvej, Copenhagen, DK-2200 N, Denmark. E-mail address:
4 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; ARM, Armstrong strain; BrdU, 5-bromo-2'-deoxyuridine; i.c., intracerebral; p.i., postinfection.
Received for publication May 10, 1999. Accepted for publication September 14, 1999.
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, Wild-type B6 mice infected with 2
x 104 PFU of LCMV Traub; all other groups were given 200
PFU i.v.
