From the Center for Immunology, Departments of Pathology and Molecular Microbiology, Washington
University School of Medicine, St. Louis, Missouri 63110
To define immune mechanisms that regulate chronic and latent herpesvirus infection, we analyzed the role of interferon 
(IFN-) during murine cytomegalovirus (MCMV) infection. Lethality studies demonstrated a net protective role for IFN-, independent of IFN-
/
, during
acute MCMV infection. Mice lacking the IFN- receptor (IFN-R
/) developed and maintained striking chronic aortic inflammation. Arteritis was associated with inclusion bodies and
MCMV antigen in the aortic media. To understand how lack of IFN- responses could lead to
chronic vascular disease, we evaluated the role of IFN- in MCMV latency. MCMV-infected
IFN-R/ mice shed preformed infectious MCMV in spleen, peritoneal exudate cells, and
salivary gland for up to 6 mo after infection, whereas the majority of congenic control animals
cleared chronic productive infection. However, the IFN-R was not required for establishment of latency. Using an in vitro explant reactivation model, we showed that IFN- reversibly inhibited MCMV reactivation from latency. This was at least partly explained by IFN--
mediated blockade of growth of low levels of MCMV in tissue explants. These in vivo and in
vitro data suggest that IFN- regulation of reactivation from latency contributes to control of
chronic vascular disease caused by MCMV. These studies are the first to demonstrate that a
component of the immune system (IFN-) is necessary to regulate MCMV-associated elastic
arteritis and latency in vivo and reactivation of a herpesvirus from latency in vitro. This provides a new model for analysis of the interrelationships among herpesvirus latency, the immune
system, and chronic disease of the great vessels.
Key words:
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Introduction |
Human cytomegalovirus (HCMV)1 establishes a chronic
infection in normal hosts, characterized by latency and
intermittent shedding of infectious virus. HCMV causes
severe reactivation disease in situations of immunocompromise, and is a frequent cause of morbidity and mortality in
transplant and AIDS patients. The specific components of the
immune system that are responsible for preventing reactivation disease have not been identified. Reactivation disease in
humans involves multiple tissues, including the retina, liver,
lung, and gastrointestinal tract. In apparently immunocompetent hosts, CMV has been implicated in the genesis of atherosclerosis (1), rapidly progressive coronary artery disease and
endothelialitis in cardiac transplant patients (4), coronary
restenosis after angioplasty (8, 9), and inflammatory aortic diseases (10, 11). Although these studies suggest a role for
HCMV in human vascular disease, this is an area of considerable controversy (12). Since MCMV can cause aortic inflammation in weanling mice (16), we focused on the great
vessels as possible targets for chronic MCMV disease.
The role of the innate and adaptive immune systems in
controlling acute MCMV infection has been well characterized. CD8 T cells are primary effector cells in clearance
of MCMV during acute infection (17, 18). However,
MCMV replication can be controlled by CD4 T cells
when CD8 T cells are depleted (19), and CD4 T cells are
required to clear virus from the salivary gland (18). NK
cells play a significant role in the control of acute infection with MCMV, and at least one mechanism for NK cell protection is secretion of IFN- (20). IFN- has a number
of functions that probably play a role in controlling acute
MCMV infection. These include activation of macrophages
during MCMV infection (21), enhancement of MHC class
I-dependent antigen presentation by infected cells to CD8
T cells (23), and inhibition of lytic MCMV replication and
gene expression (21, 24, 25). Administration of recombinant IFN- can protect against lethal infection (26), and administration of anti-IFN- can both decrease the effectiveness of anti-MCMV CD8 and CD4 T cells (23, 27) and
increase viral titers in visceral organs (21, 27). CD4 T cells
specific for the HCMV immediate early 1 protein produce
cytokines including IFN- and TNF- on antigenic stimulation, which inhibit viral replication in MRC5 cells (28).
Although these studies all argue for a protective role for
IFN-, other studies in both rat CMV and HCMV have argued that IFN- is required to generate productive infection of macrophages and efficient replication in vivo (29,
30). These results raise the question of whether the net effect of endogenous IFN- is protective.
After a period of persistent productive infection in the salivary gland, MCMV establishes a predominantly latent infection in immunocompetent mice (31, 32). Latency is characterized by the presence of viral genome in the absence of
preformed infectious virus combined with the capacity to
reactivate either in vivo or in vitro from tissue explants. The
extent to which the immune system regulates and controls
latency is not fully understood. It is thought that the immune
system regulates latency because immunosuppression results
in reactivation of lytic MCMV (33). Reactivation is a
multistep process that may be divided into two phases: cellular reactivation and subsequent growth of virus. Cellular reactivation probably results from a shift from latent to lytic
gene expression; nothing is known about whether or how the immune system alters this process. Cellular reactivation is followed by growth of reactivated virus to levels that cause
disease or are experimentally detectable. The mechanisms
responsible for controlling viral replication after cellular reactivation are also incompletely defined, although the immune
system is likely to play a role at this step in reactivation. For
example, antibody has been shown to limit dissemination of
MCMV after reactivation (36). IFN- is a good candidate in
regulating growth of reactivated MCMV given its importance for clearing persistent productive infection of the
salivary gland (27).
In this study we first demonstrate that the net effect of
endogenous IFN- is protective during acute infection in
vivo. We then show that IFN- plays an important role
during chronic MCMV infection in vivo and during reactivation in vitro. In the course of these studies we have defined a novel role for IFN- in regulating chronic vascular
pathology due to MCMV.
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Materials and Methods |
Viruses and Viral Assays.
MCMV Smith strain was obtained
from the American Type Culture Collection (no. VR-194, Lot
10; Rockville, MD). Tissue culture-passaged MCMV (tcMCMV) and salivary gland MCMV (sgMCMV) were generated as described previously (21, 31). All media were prepared as described previously (37). Murine embryonic fibroblasts (MEFs) were prepared and used during initial passaging or thawed from frozen stocks (31). All MEFs were used before passage 4. To detect and quantitate MCMV genome, nested PCR detecting sequences of the MCMV immediate early 1 gene were performed
on DNA from bone marrow cells as described previously (31).
This nested PCR assay reproducibly detects one copy of target
plasmid (31, 37). MCMV sequences are quantitatively recovered
in tissue DNA preparations (31). Herpes simplex virus (HSV) was
grown and titered as described previously (21). The myocarditic
reovirus serotype 3 8B was grown and titered as described previously (38).
Mice, Mouse Infections, and Tissue Harvests.
129/Sv mice and
mice with null mutations in the IFN- receptor (IFN-R/),
the IFN-/ receptor (IFN-/R/), and both the IFN- and
IFN-/ receptors (IFN-//R/), were obtained from Dr.
Michel Aguet (39) and bred at Washington University (St. Louis,
MO). These mice were infected intraperitoneally with doses of
sgMCMV described in the text. BALB/c (National Cancer Institute, Frederick, MD) and C57BL/6 (The Jackson Laboratory, Bar
Harbor, ME) mice were infected with 2 × 104 or 1 × 105 PFU
sgMCMV intraperitoneally and rested for 4-10 mo to establish a
latent infection. To assess mice for the presence of preformed infectious MCMV, spleens and salivary glands were harvested aseptically and handled individually. Bone marrow cells and peritoneal exudate cells (PECs) were harvested as described previously
(37). Low levels of preformed infectious MCMV were detected
using coculture of sonicated cells or tissues with MEFs in an assay
that detects 1-10 PFU per organ as described (31, 37). In brief,
solid organs were minced, then sonicated twice, and three T75
flasks (Sarstedt, Newton, NC) of confluent MEFs were inoculated with the sonicated tissue. Dilution of tissue sonicates into
large volume cultures (total vol 30 ml/T75 flask) is essential to
generate the high sensitivity of this assay (31). One T75 flask of
confluent MEFs was inoculated per sample of sonicated PECs or
bone marrow cells (generally between 2 and 3 × 106 PECs or 2 and 3 × 107 bone marrow cells). Flasks were observed for 3 wk
to detect MCMV-associated cytopathic effect. When necessary,
results were confirmed by transfer of supernatant onto fresh MEF
monolayers. Positive controls consisted of adding 5 PFU of
MCMV to flasks and were consistently positive.
Pathology and Immunohistochemistry.
To analyze aortic pathology, the heart, lungs and great vessels were resected en bloc after
death and processed as described elsewhere (40). In brief, serial
sections from the posterior of the heart through the thymus were
prepared and stained with hematoxylin and eosin and then read
blindly for aortic pathology by either H.W. Virgin or A.J. Dal
Canto. Samples were scored as positive if evidence of arteritis in
the aorta or pulmonary artery was observed. Since arteritis most
commonly involved the base of the aorta, samples were called
negative only if a clear view of the base of the aorta was available;
samples were excluded if the base of the aorta was not visible.
Murine polyclonal antibody to MCMV was prepared as follows.
BALB/c mice were infected with 105 PFU tcMCMV. Salivary
glands were harvested at 20 d after infection, homogenized in
medium lacking FCS, and titered by plaque assay. BALB/c mice
were infected with 104 PFU of this stock. Mice were bled
through the retroorbital plexus at 1, 2, and 3 mo after additional
boosts with 1.2 × 105 PFU of sgMCMV. This approach was
taken to minimize immunization with FCS in tissue culture preparations of virus. Immunization with murine cellular antigens was
avoided by selection of BALB background mice for both preparation of virus and serum generation. Immunohistochemical staining was performed as described previously (40) except that the
murine polyclonal antibody to MCMV was diluted 1:1000. Preimmune serum was also used at a 1:1000 dilution as a negative
control. Horseradish peroxidase (HRP)-conjugated donkey anti-
mouse secondary antibody (Jackson Immunoresearch Labs., West
Grove, PA) was used at 1:1000. Tyramide signal amplification (NEN Life Science Products, Boson, MA) was performed and
HRP activity was localized as previously described (40).
MCMV Reactivation from Explanted Organs and Bone Marrow.
Spleens or lungs from 8-13 latently infected BALB/c or C57BL/
6 mice were pooled, minced, homogenized using a Tenbroeck
Tissue Homogenizer (Bellco, Vineland, NJ), and plated evenly
into 12 six-well plates (Falcon, Franklin Lakes, NJ). An aliquot
(0.3-0.5 spleen or lung equivalents) was diluted, sonicated, and
cocultured with MEFs to rule out the presence of preformed infectious virus in pooled spleen and lung explants (see above). Explants were cultured in the presence of mock IFN (prepared by
Lee Biomolecular, San Diego, CA, in the same manner as their
IFN preparations, except that it is mock induced), 100 U/ml
IFN- (Lee Biomolecular), IFN- (Lee Biomolecular), or rIFN-
(gift of Genentech, San Francisco, CA, or purchased from Genzyme Corp., Cambridge, MA). Explants were maintained with
media changes with every 3-4 d for 64-116 d. IFN was replaced
with these media changes for the number of days indicated in
Figs. 5 and 6. Reactivation was detected by transfer of 100 µl
culture medium to fresh MEF monolayers which were observed
for cytopathic effect for 12 d (31). For determination of the effects of IFNs on growth of low levels of MCMV in explant cultures, spleen and lung explants from naive mice were prepared as
described above. 12 d after explantation, MCMV was added to the cultures at doses described in the text and the plates were incubated as described above. To determine if IFN or other factors from explant wells could prevent detection of MCMV in secondary MEF cultures, supernatant was harvested from wells before
reactivation and cultured with MEFs to which 5 PFU of MCMV
were added. To assess reactivation of MCMV from bone marrow, bone marrow was harvested, treated with a hypotonic solution to lyse red blood cells, and cultured with L cell-conditioned
medium as described previously (41). After 19 d, media was
changed to DMEM with 10% FCS (31) and MEFs were added as
an indicator monolayer for MCMV-induced cytopathic effect.
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Fig. 5.
Effects of IFN- on reactivation in vitro. (A) Lungs from 8-10 latently infected BALB/c mice were pooled, minced, homogenized, and
divided evenly for culture in six 6-well plates (12 wells per condition in each of two experiments) in the presence of media alone (no IFN ), mock IFN,
or 100 U/ml IFN-, IFN-, or IFN-. Supernatant was tested for the presence of infectious virus, and media was changed every 3-4 d. On day 35 cytokine treatments were removed from all cultures except for two 6-well plates per experiment, which contained 100 U/ml IFN-, until day 116. Data
shown is the average of two independent experiments. (B) Spleen explants prepared from 8-13 latently infected BALB/c or C57BL/6 mice treated in the
same manner as the lung explants (two independent experiments, results from BALB/c and C57BL/6 mice showed similar trends).
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Fig. 6.
Effects of IFN- on growth of 10 or 50 PFU MCMV in tissue explants. Naive lung (A and B) or spleen (C and D) explants were generated
from BALB/c mice and cultured as described for latent explants. On day 12 after explant, cultures were infected with either 10 (A and C ) or 50 (B and
D) PFU of tcMCMV. Infection was carried out in 500 µl total volume per well. Standard infections were 2 h at 37°C with rocking every 15 min. For A
and C, results shown are pooled from four independent experiments with six wells plated per condition. Data shown begins with day 0 after infection,
which was 12 d after explant. For B and D, results shown are pooled from two independent experiments with six wells plated per condition.
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Statistical Methods.
Statistical analysis was carried out through
the Division of Biostatistics at Washington University School of
Medicine. The data were analyzed using the SAS procedure
PHREG, which performs regression analysis of survival data
based on the Cox proportional hazards model. This model was
particularly relevant to these data as it takes into account the censoring of the day of death or killing. The dependent variables
used to analyze lethality data (Fig. 1) were day of death censored
by whether or not the animal died on its own. Cox proportional
hazards models were developed to first test all of the strains
against the control (129 or B6) with dose and sex as covariates.
Then models were developed to pairwise compare each experimental strain against the others, still using dose and sex as covariates. Mouse strain was a significant predictor of outcome independent of viral dose or sex (see text). The arteritis data (Table 1)
were also analyzed using a Cox proportional hazards model, comparing the IFN-R/ strain to the 129 control with dose and sex
as covariates. The dependent variables were day of sacrifice censored by whether or not the animal displayed evidence of arteritis. For the persistence data (Table 2), stepwise proportional hazards regression techniques were used to determine the best fitting
variables among the different experiments. The final analyses
compared IFN-R/ to the 129 controls using as dependent
variables day of killing censored by whether or not the animal
displayed evidence of shedding virus in any organ, or in the salivary gland, spleen, or PECs. The bone marrow results were not
statistically analyzed as there were no mice that displayed evidence of the shedding virus in their bone marrow.
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Fig. 1.
Lethality studies in IFN unresponsive mice. Mice with targeted mutations in IFN responsiveness were infected with between 10 and 5 × 106 PFU of sgMCMV intraperitoneally and followed for mortality for 21 d or for 5 d after the last death observed in an experimental
group. Data is shown as percentage of mortality. Data was derived from
the following numbers of mice and experiments: 129: 84 mice in 7 experiments; C57BL/6: 69 mice in 3 experiments; IFN-R/: 85 mice in 6 experiments; IFN-/R/: 93 mice in 5 experiments; IFN-//R/
mice: 44 mice in 4 experiments; IFN-/: 41 mice in 3 experiments.
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Results |
Protective Role of IFN- in Acute Infection.
Control congenic mice and mice with various null mutations influencing IFN responsiveness were challenged with varying doses of MCMV and followed for mortality (Fig. 1). Sharp
changes in mortality observed with small changes in viral
dose have been previously reported and necessitated the use
of large numbers of age-matched mice across many experiments to obtain clear data (42, 43). We have evaluated 416 mice in the course of these experiments. We aged mice at
least 2 mo before challenge to minimize the impact of large
changes in susceptibility to lethal MCMV infection that
occur over the first weeks of life (our unpublished data). The LD50's for IFN-R/ and IFN-/ mice were approximately five- and fourfold lower, respectively, than for
congenic (129 and C57BL/6) control mice (P <0.02; P < 0.005). IFN-/ might compensate for some effects of
IFN- since both can inhibit MCMV replication in vivo
and in vitro (21, 24, 25). We therefore evaluated IFN-/
R/ mice and found that the LD50 for IFN-/R/
mice was 800-fold lower than for control 129 mice (P
<0.0002). To determine the importance of IFN- in the
absence of IFN-/ responses, we compared IFN-/R/
and IFN-//R/ mice. As few as 10 PFU of MCMV
killed 100% of IFN-//R/ mice, and thus an LD50
could not be determined. However, the LD50 for IFN-//
R/ mice was at least 40-fold lower than for IFN-/
R/ mice (P < 0.0001). These data demonstrate that
IFN- plays a net protective role in MCMV infection, and
that IFN- effects are not completely redundant with those
of IFN-/.
MCMV-infected IFN-R/ Mice Develop Chronic Aortitis.
Given the importance of IFN- to regulating acute
MCMV infection, we examined the course of chronic
MCMV infection in IFN-R/ mice given a dose that
most would survive for at least 21 d of infection (Fig. 1; 1-5 × 104 PFU). Because MCMV can cause aortic disease in
newborn immunocompetent mice (16), we examined the
aorta for signs of pathology over the first 154 d of infection
(Table 1). Early after infection (days 28-56), lesions of the
aorta were seen in 4 out of 10 normal 129 and 5 out of 6 IFN-R/ mice. Interestingly, after day 84 aortic lesions
were not seen in 129 mice (0 out of 24 mice; Table 1). In
contrast, most IFN-R/ mice had significant lesions as
late as 154 d after infection (15 out of 27 mice; Table 1
and Fig. 2). This difference is statistically significant, with P
<0.0033. These lesions involved all layers (intima, media,
and adventitia) of the vessels (Figs. 2, C and D, and 3 A). A
prominent inflammatory infiltrate consisting primarily of
mononuclear cells was observed (Figs. 2 D and 3 A). Cytomegalic inclusion bodies were seen within cells in the
aortic media (Fig. 3 B). Immunohistochemistry revealed
MCMV antigens in the media of affected aortas (Fig. 3, C
and D). Cells in the aortic media containing inclusions and
MCMV antigen were morphologically consistent with
smooth muscle cells. We have previously published that arteritis is not seen in mock infected IFN-R/ mice observed for 6 to 15 wk (40). Furthermore mice infected with
either a myocarditic reovirus reassortant (8B) or HSV failed to show lesions 84 d after infection (Table 1). At this time IFN-R/ mice infected with MCMV showed aortic pathology. These experiments demonstrate that MCMV
causes chronic vascular pathology in IFN-R/ mice, and
illustrate a tropism for cells of the aortic media during
chronic infection.
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Fig. 2.
MCMV induced vascular pathology in IFN-R/ mice. Mice were killed 154 d after infection with 104 PFU MCMV. All sections were
stained with hematoxylin and eosin. L, lumen; I, intima; M, media; Adv, adventitia; V, aortic valves. (A) Normal aorta in an MCMV-infected 129 mouse.
(B) High power view from the boxed region shown in A. Uninfected IFN-R/ mice show the same histology. (C) Aorta from an MCMV-infected
IFN-R/ mouse. (D) High power view of the boxed region shown in C.
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Fig. 3.
MCMV is present in arteritic lesions. L, lumen; I, intima; M, media; Adv, adventitia. (A) Aortic lesion from an MCMV-infected IFN-R/
mouse killed 166 d after infection with 103 PFU MCMV. (B) Higher power view of the media from the aorta shown in Fig. 2 C. Note cytomegalic inclusion body (arrow). (C) Parallel section to A. Immunohistochemical staining with immune mouse sera demonstrating MCMV antigen (dark brown), primarily in the media. (D) Parallel section to C. Absence of immunohistochemical staining with preimmune mouse sera demonstrating specificity of the
signal in C for MCMV antigen.
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IFN-R/ Mice Maintain Chronic Productive MCMV Infection for 180 d after Infection.
Aortic pathology in IFN-R/ mice seemed likely to be due to an important role
of IFN- in controlling chronic MCMV infection. 29 IFN-R/ mice and 28 wild-type 129 mice were therefore evaluated for the presence of preformed infectious virus at times during which MCMV transitions from a
chronic productive salivary gland infection to a latent infection in a majority of immunocompetent mice (Table 2).
Spleen, salivary gland, PECs, and bone marrow cells were sonicated and cultured on MEFs to detect low levels of
preformed infectious virus. IFN-R/ mice were productively infected in at least one of the tissues tested out to 180 d
after infection. Only one of four IFN-R/ mice survived
to the 180 d time point. Although preformed infectious virus was detected in some congenic control 129 mice, the
majority established a latent infection in the absence of persistent productive infection as has been shown for BALB/c
mice (31, 32). Differences between IFN-R/ and 129 mice are statistically significant for total mice shedding (P < 0.009), mice shedding in the spleen (P <0.01) and salivary gland (P <0.009). The presence of low levels of preformed MCMV in IFN-R/ mice in spleen and PECs
demonstrates that IFN- regulates chronic infection with
MCMV in several locations in addition to salivary gland (27).
Establishment of Latent Infection in IFN-R/ Mice.
Since we detected chronic productive infection in several
tissues in IFN-R/ mice, we were interested in assessing
whether a latent infection could be established in the absence of IFN- responsiveness. Of the tissues tested for
chronic productive MCMV infection in IFN-R/ mice,
only bone marrow consistently lacked detectable preformed infectious MCMV. Bone marrow cells (between
10 and 25 × 106 per mouse) were tested from 22 IFN-R/ mice and contained no preformed infectious virus as
detected by coculture with MEFs (six independent experiments). Since coculture with MEFs regularly detects 5 PFU
of MCMV, these experiments showed that there was <1
PFU of MCMV per 2-5 × 106 bone marrow cells from
IFN-R/ mice. To determine if the lack of infectious
MCMV in bone marrow cells reflected a lack of MCMV
infection in the bone marrow of IFN-R/ mice, DNA
was isolated from bone marrow cells and PCR was used to
quantitate the level of viral genome (31). Dilutions of
DNA from 129 and IFN-R/ mice revealed that the
amount of viral genome in DNA samples was similar (Fig.
4). The presence of viral genome in the absence of productive infection suggests that MCMV establishes either a latent or abortive infection in the bone marrow of IFN-R/
mice. To demonstrate that the viral genome that we detected was due to latent infection, bone marrow cells were
cultured in vitro to induce reactivation. 8 out of 19 cultures of IFN-R/ bone marrow reactivated MCMV. 2 out of 18 cultures of wild-type 129 bone marrow reactivated MCMV. The presence of viral genome in bone marrow cells, and the capacity to reactive MCMV from bone marrow cultures demonstrates that IFN- is not required
to establish latency in the bone marrow.
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Fig. 4.
IFN-R/ mice are capable of establishing latency in the
bone marrow. DNA was prepared from bone marrow from IFN-R/
and 129 mice between 72 and 154 d after infection. DNA was adjusted to
0.1 µg/µl in TE and then serially diluted in tRNA (0.1 µg/µl). Each
sample was tested in a nested PCR assay for the presence of MCMV immediate early 1 DNA (sensitivity 1 copy). Data is expressed as the percentage of PCR reactions for a given sample which were positive. 14-26
reactions were performed for each dilution.
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IFN- Directly Inhibits MCMV Reactivation from Latency in
Spleen and Lung Explants.
Since latency can be established
in the absence of IFN- responsiveness, we examined the
hypothesis that IFN- controls reactivation from MCMV
latency. We took advantage of an in vitro assay in which latently infected spleen (31, 33, 44, 45) or lung explants reactivate at high frequency upon culture in vitro. Explants
were cultured in the presence of media alone, media plus
mock IFN, or media with 100 U/ml IFN-, IFN-, or
IFN-. Explant supernatant was sampled every 3-4 d for
64-116 d and tested for infectious virus by culture on
MEFs (Fig. 5, A and B). Explants were tested for chronic
productive MCMV infection present at the time of explantation by culturing a sonicated aliquot with MEFs (see Materials and Methods). These control cultures were consistently negative. Although 100% of lung explants that were
treated with media alone or with mock IFN reactivated by
day 35, only 12.5-15% of explant cultures treated with
IFN- reactivated (Fig. 5 A). Explants treated with IFN-
or IFN- showed an intermediate block to reactivation.
Similar results were seen with spleen explants, although the
frequency of reactivation is lower (Fig. 5 B). We considered several explanations for the lack of detection of reactivated MCMV in explants treated with IFN-. IFN treatment might prevent reactivation of MCMV by killing
latently infected cells. To assess this possibility, IFN treatments were withdrawn on day 35 for lung explants and day 38 for spleen explants. In some explants, IFN- treatment
was continued. After cytokine removal, an additional 9 out
of 21 lung explant cultures previously treated with IFN-
reactivated, suggesting that the blockade of reactivation induced by IFN- is at least partially reversible (Fig. 5 A).
Cultures that continued to receive IFN- during this time
did not reactivate. Similar results were seen in spleen explant cultures (Fig. 5 B). This reversibility of IFN- effect
argues against IFN--induced killing of cells containing latent virus as a mechanism of reactivation blockade. Another explanation for our lack of detection of MCMV reactivation in these treated explants may be that the IFNs
carried over from explant cultures decrease our ability to
detect infectious MCMV in MEF cultures. However, 5 PFU
of MCMV added to media from IFN--treated explants
was detected on MEF monolayers in 18 out of 18 times. These data were consistent with a direct blockade of MCMV
reactivation by IFN- treatment.
Mechanism of Action of IFN- in Explant Reactivation.
IFN- could inhibit reactivation by blocking either cellular
reactivation or growth of reactivated virus. Since the molecular nature of MCMV latency is unknown, we could
not examine an effect of IFN- on cellular reactivation directly. However, we hypothesized that IFN- could block
reactivation in explant cultures by blocking the growth of
small amounts of virus released during cellular reactivation.
We therefore made lung and spleen explant cultures using
organs from uninfected mice and treated them with IFNs
as for explants from latently infected organs. To mimic a
reactivation event, either 10 or 50 PFU of MCMV was
added on day 12, and the explants were observed for production of MCMV for the ensuing 39 d (Fig. 6). The
number of wells productively infected with MCMV was
decreased by IFN- treatment in both spleen and lung explants given either 10 or 50 PFU (Fig. 6). This shows that
IFN- can block productive infection with low levels of
MCMV. IFN- had a similar effect when an inoculum of
10 PFU was used, but was less effective than IFN- when
an inoculum of 50 PFU was used. These data demonstrate
that under certain conditions of infection, IFN- can effectively prevent growth of low levels of MCMV in explant
cultures. This blockade could account for the inhibition of
reactivation we observed in explant cultures from latently infected mice (Fig. 5). The lack of IFN--mediated inhibition of outgrowth of reactivated virus could contribute to
the presence of preformed infectious MCMV in IFN-R/
mice detected during chronic infection (Table 2).
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Discussion |
The experiments reported here demonstrate the following
important points: (a) IFN- regulates chronic infection with
MCMV in multiple tissues in addition to the salivary gland
(27); (b) IFN- is not required for establishment of MCMV
latency; (c) IFN- can reversibly block reactivation from
latency in an in vitro model; (d) one mechanism by which
IFN- might regulate chronic MCMV infection is blockade
of the growth of low levels of infectious virus; and (e) one
consequence of the absence of IFN- responsiveness is
chronic inflammatory disease in the large elastic arteries. Previous work has shown that IFN- is present for prolonged
times in trigeminal ganglia latently infected with the -herpesvirus HSV (46, 47), suggesting IFN- as a candidate for regulating chronic HSV infection. IFN- plays a role in chronic
infection with -herpesviruses, as is illustrated by the severe
chronic elastic arteritis seen in IFN- nonresponsive mice infected by the murine -herpesvirus 68 (HV68; reference
40). Experiments presented here provide the first evidence
that IFN- regulates chronic infection with a -herpesvirus,
MCMV, potentially by controlling reactivation from latency.
In addition, lack of IFN- regulation of MCMV infection
has significant pathologic consequences for the host. The fact
that lack of response to a single cytokine results in severe
MCMV-associated pathology suggests that penetrance and
severity of disease during chronic herpesvirus infection is regulated by relatively subtle changes in host defense.
IFN- and Chronic MCMV Infection.
We found that IFN-
is important for controlling chronic infection with MCMV,
but was not essential for the establishment of latency in bone
marrow cells. It was possible that events during early stages
of infection set the stage for chronic productive infection
(e.g., by altering the cells infected or the viral load in specific
cells or tissues). However, IFN- was effective at preventing
the reactivation of latent MCMV from tissue explants, suggesting that IFN- has significant effects during chronic infection in addition to any effects of IFN- during acute infection. Notably, the effect of IFN- lasted for up to 116 d,
and was reversible after 30-40 d of culture. These results argue that IFN- is very effective in this role, and that IFN-
does not act by killing all latent cells. IFN- and IFN-
were less effective. This, combined with the fact that IFN-
has a role during acute infection that is not redundant to the
effects of IFN- and IFN- (Fig. 1), argues strongly that
IFN- is a key regulator of all phases of MCMV infection.
The effect of IFN- on reactivation from tissue explants
could be due to effects on either cellular reactivation or
growth of virus in explant cultures after cellular reactivation has occurred. We found that IFN- can completely
block the growth of low levels of MCMV in explant cultures. This effect was significant enough that it could account entirely for the effects of IFN- on reactivation from
latent tissue explants. This being the case, we cannot address the possibility that IFN- might have a role in regulating cellular reactivation. The mechanism by which IFN-
works to prevent MCMV growth is controversial. Several
studies have shown that IFN- can decrease viral yields
(21, 24, 25), but studies demonstrating an effective block
on replication of low levels of infectious MCMV have not
previously been reported. Some studies have suggested a role
for IFN- in regulating transcription of the MCMV immediate early genes, whereas others have suggested a role for
IFN- later in the lytic replication cycle (24, 25). Further
studies will be required to define these mechanism(s) at the
molecular level, and to understand how they might contribute to prolonged blockade of viral growth.
It is interesting that MCMV and HCMV both have
mechanisms to block the signaling of IFN- in infected
cells. MCMV inhibits IFN- induction of MHC gene expression at a step downstream from STAT (signal transducer and activator of transcription)-1 phosphorylation and
nuclear translocation (48). HCMV inhibits IFN- signaling in infected cells by altering the turnover of Janus (JAK)
family kinases required for IFN- action (49). Thus, both
the viruses have developed countermeasures that combat
the effects of IFN- in infected cells. This creates an interesting balance between cytokine secretion and effectiveness
in vivo, and virus-encoded anticytokine actions. It is likely
that this critical balance defines the outcome of chronic
CMV infection. The capacity of HCMV to inhibit IFN-
signaling may explain the lack of effectiveness of IFN- in
macrophages treated with IFN- after infection (30).
The source of IFN- during chronic infection has not
been determined. Since IFN- is important in both the
adaptive (both CD4- and CD8-mediated) and the innate
(NK cell-mediated) responses to MCMV infection, future
studies will have to assess the role of all three of these lymphocyte subsets as IFN- producers during chronic infection. Since CD8 T cell-mediated secondary vaccine responses do not require IFN- (42), we favor the hypothesis that CD4 T cells or NK cells are important during chronic
infection. This would be consistent with the proven role
for CD4 T cells and IFN- in controlling persistent productive infection in the salivary gland (18, 27).
MCMV and Vascular Disease.
In this study, we confirmed that MCMV infects the great elastic arteries (16),
and demonstrated for the first time that in the absence of
the IFN- receptor MCMV causes chronic vascular pathology even in adult mice. It was notable that lack of IFN- responsiveness was not a prerequisite for MCMV induction
of vascular pathology. Thus, normal mice showed signs of
disease early after infection. However, IFN-R/ mice
maintained lesions for much longer than normal mice, suggesting that IFN- is critical for controlling infection in the
large elastic arteries. This is remarkably parallel to our studies
in mice infected with HV68. HV68 causes severe elastic
arteritis in IFN-R/ mice (40). The fact that MCMV and
HV68 are quite distinct in primary sequence (50, 51)
but cause disease of the elastic arteries in similar immunodeficient mice argues for the nature of the interaction between the tissue (elastic arteries) and IFN- as the critical
determinant of virus induced vasculitis rather than specific
receptors or other proteins shared between MCMV and
HV68. Alternatively it is possible that both MCMV and
HV68 share specific IFN--sensitive interactions with
smooth muscle cells or other cells of the elastic arteries.
Latent MCMV has been identified in endothelial cells by
PCR in situ hybridization (52). It is tempting to speculate
that the lack of IFN- results in chronic vascular disease
because of a failure to control reactivation in endothelial
cells. An alternative hypothesis is that latently infected macrophages (37, 52) are the source of reactivating MCMV
that causes arteritis. IFN- may also be essential for regulating vascular pathology via effects on the proliferation of
smooth muscle cells (53) or the development of foam cells,
which characterize atherosclerotic lesions (54).
In this report, we provide evidence that IFN- is a critical component in the regulation of chronic MCMV infection, without which severe pathologic consequences develop.
This may have implications for the role of herpesviruses in
vascular pathology. Explant studies suggest that IFN- regulates MCMV latency by inhibiting reactivation from latency. One mechanism for this inhibition is the prevention of outgrowth of low levels of virus. Further studies are
needed to evaluate the mechanism of this block of reactivation, and to define the role of reactivation in the induction
of vascular pathology.
Address correspondence to Herbert W. Virgin IV, Center for Immunology, Departments of Pathology and
Molecular Microbiology, Washington University School of Medicine, Box 8118, 660 South Euclid Ave.,
St. Louis, MO 63110. Phone: 314-362-9223; Fax: 314-362-4096; E-mail: virgin{at}immunology.wustl.edu
We thank Avril Adelman at the Division of Biostatistics, Washington University, for performing statistical
analyses. We also thank Dr. Sam Speck and Dr. David Leib and members of their laboratories for helpful
commentary during the course of these studies.
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Regulates Acute and Latent Murine
Cytomegalovirus Infection and Chronic Disease of the
Great Vessels
Top
Abstract
Introduction
