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Regular Article |
Receptor Knockout Mice
From the Laboratory for Clinical and Molecular Virology, Department of Veterinary Pathology, University of Edinburgh, Edinburgh, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor knockout mice (IFN-R-/-) results
in splenic fibrosis and excessive loss of splenocytes. In our present
study we found that MHV-68 infection in
IFN-R-/- mice also resulted in fibrosis
and atrophy of the mediastinal lymph nodes, interstitial
pulmonary fibrosis and fibrotic changes in the liver. Atrophy and
cellular depletion of the spleen in
IFN-R-/- was not the result of increased
cell death. The loss of splenocytes in
IFN-R-/- mice, which was most
evident on day 23 after infection, correlated with an increase
in the number of leukocytes in peripheral blood. At the peak of
leukocytosis, on day 23 after infection, peripheral
blood cells from infected IFN-R-/- mice
were unable to traffic through the fibrosed spleens of
IFN-R-/- mice but were able to enter the
spleens of wild-type mice. This indicates that leukocytosis was in part
the result of emigration of cells from the spleen and their subsequent
exclusion of re-entry at the height of fibrosis. Significant cytokine
and chemokine changes were observed in spleens of
IFN-R-/- mice. IFN-, tumor
necrosis factor-
(TNF- ), TNF-ß,
interleukin-1ß (IL-1ß), transforming growth factor-ß1
(TGF-ß1), lymphotactin, and MIP-1ß were elevated on
day 14 after infection whereas chemokines IP-10 and MIG were
significantly reduced. These changes suggest a role for dysregulated
cytokines and chemokines in severe organ-specific fibrosis with
implications for immune-mediated fibrotic disorders.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interferon- is a key Th1 cytokine,7
and it has been
implicated in protective immune responses to several herpes
viruses.8-10
In herpes simplex virus-1 infection,
lack of IFN- diminished the ability to clear the skin
infection, with a delayed-type hypersensitivity
response,11,12
and infection in
IFN-R-/- mice with
murine cytomegalovirus caused chronic aortic inflammation with
prolonged shedding of infectious virus.13
IFN- is produced at high levels during the acute
phase of MHV-68 infection,14
and diminished levels of
IFN- have been associated with recrudescence of lytic MHV-68
infection in lungs.15,16
However, an unexpected outcome of
MHV-68 infection in
IFN-R-/- mice via the
respiratory route was severe splenic atrophy.17
This
observation demonstrated a novel function for this cytokine in
maintaining the splenic architecture during a gammaherpesvirus
infection.
The most striking feature of lymphoid pathology in
IFN-R-/- mice is
splenic atrophy coupled with excessive deposition of collagen. The
entire pathology is linked to the activity of CD8 T cells, because
depletion of CD8 T cells resulted in maintenance of normal lymphoid
architecture during the course of infection.17
Excessive collagen deposition in MHV-68-infected
IFN-R-/- mice would
suggest a dysregulated cytokine response, because overexpression of
cytokines IL-4, IL-1ß, and TGF-ß1, and reduced expression of
interferon- have been shown to promote
fibrosis18,19
in, for example, systemic
sclerosis20,21
and murine models of
fibrosis.22,23
In the present study we have identified hitherto unreported pathologies
in the mediastinal lymph nodes, lungs, and liver and have further
characterized the changes in the spleen of MHV-68-infected
IFN-R-/- mice.
Moreover, we have explored possible mechanisms underlying spleen cell
depletion and the pathological changes observed in these mice. Our data
demonstrate that fibrosis and loss of splenocytes observed in
IFN-R-/- mice are
associated with inhibition of leukocyte trafficking through the
excessively fibrosed spleens of
IFN-R-/- mice,
culminating in leukocytosis. Importantly, a dysregulated pattern of
splenic cytokines and chemokines was observed in
IFN-R-/- mice,
suggesting a possible explanation for the overt pathologies in
IFN-R-/- mice infected
with MHV-68.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult wild-type (129/Sv/Ev) and
IFN-R-/- (129/Sv/Ev)
mice were purchased from Bantin and Kingman (Hull, UK) and bred
in-house.
Virus Infection and Sampling
MHV-68 virus stocks were prepared as previously described.4 Mice (age- and sex-matched) were anesthetized with Halogen (Rhone Merieux Ltd., UK) and were inoculated via the intranasal route with 4 x 105 plaque-forming units of MHV-68 in 40 µl of sterile phosphate-buffered saline. At various time intervals, mice were sacrificed by CO2 asphyxiation. Tissues were fixed in 10% buffered formal saline, paraffin-embedded, and 5-µm thick sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome stain.
Death Assay
Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) was performed on paraffin-embedded sections (in situ TUNEL) as previously described.24
Hematology and Flow Cytometry
Venous blood (inferior vena cava, 500 µl) was collected immediately at postmortem in EDTA tubes, stained with monoclonal antibodies against CD4 (YTS177.9.6.1), CD8 (YTS105.18.10), and CD19 (PharMingen), and analyzed on a FACStar Plus (Becton Dickinson) as previously described.25 Total granulocyte counts were obtained from fluorescence-activated cell sorter profiles.
A Baker System 9100 hematology analyzer was used for hematological analyses. Differential white blood cell staining was carried out on a Bayer Hematek 2000 Stainer (Department of Veterinary Clinical Studies, Clinical Laboratory, Easter Bush Veterinary Center, Edinburgh, UK).
Leukocyte Trafficking
Peripheral blood was collected in EDTA tubes, and leukocytes were obtained by density centrifugation on Histopaque-1077 (Sigma). One milliliter of leukocytes (108/ml in diluent C) was mixed with one ml (10 µmol/L in diluent C) of green fluorescent dye PHK-67 (Sigma) for 6 minutes at room temperature with constant pipetting, followed by 2 ml of fetal calf serum and 4 ml of tissue culture medium Dulbecco’s Modified Essential (DME) to terminate the labeling reaction. Labeled cells were centrifuged at low speed (50 x g, three minutes) and resuspended in PBS. Each mouse was injected with 2 x 107/100 µl via the tail vein. Three mice were injected for each time point. After overnight injections, peripheral blood leukocytes (PBL) and splenocytes were obtained by density centrifugation. The presence of labeled cells was detected by FACS analysis of 200,000 events per each sample. The success of injection was based on the presence of labeled cells in PBL.
Ribonuclease Protection Assay
Ribonuclease protection assay (RPA) was used to quantitatively
measure the expression of splenic cytokines and chemokines. Spleens
were snap-frozen in liquid nitrogen immediately at postmortem and
stored at -70°C before processing. Total RNA was isolated by RNAzolB
solution (Biogenesis). Five micrograms of total RNA was hybridized to
RPA probe sets: ML11 set (tumor necrosis factor-ß, tumor necrosis
factor-, interleukin-4, IL-5, IL-1, IFN-, IL-2, IL-6, IL-1ß,
IL-3, and L32), ML26 set (IL-3, IL-10, GM-CSF, TGF-ß1, IL-13,
IL-12p40, IL-12p35, IL-7, and L32), mCK5 set (LTN, RANTES, EOTAXIN,
MIP-1ß, MIP-1, MIP-2, IP-10, MCP-1, TCA-3, L32, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH); BD PharMingen;
45026P), and CCR7 set (IL-4, IL-12p40, IL-10, IL-1ß, IL-13, CCR7,
L32, and GAPDH; BD PharMingen; 557506). ML11 and ML26 sets were a
kind gift from Dr. M. Hobbs, Scripps Research Institute, USA. The
cloned murine monokine induced by interferon- (MIG) was a kind gift
from Dr. M. Buchmeier (Scripps Research Institute). RPA assays were
performed according to the RiboQuant protocol (BD PharMingen).
Bone Marrow Cellularity Measurement
Single cell suspensions of femoral bone marrow were prepared in RPMI 1640 [10% (v/v) fetal calf serum, 2 mmol/L glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin] and cell numbers determined by hemocytometry.
Cytokine Protein Assays
Splenocytes were prepared by centrifugation over Ficoll-Hypaque
(Histopaque 1077, Sigma), resuspended at 107
cells/ml in RPMI 1640 [10% (v/v) fetal calf serum, 2 mmol/L
glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin] and
incubated at 37°C for 24 hours.26
Supernatants
were then harvested for enzyme-linked immunosorbent assays (ELISA).
IFN- was measured using IFN- antibody pair (BD PharMingen) and
according to the recommended protocol. TNF- was assayed using the
Quantikine M mouse TNF- immunoassay kit (R&D Systems).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R-/- mice at
intervals up to 45 days after infection. Histopathological Changes
The histological changes in spleens of wild-type and
IFN-R-/- mice were
similar up to day 10, which included germinal centers and neutrophil
infiltration of sinusoids. An apparent increase in neutrophils was
noted in IFN-R-/-
mice. Extensive fibrosis was detected in spleens of
IFN-R-/- mice by day
23 after infection (all mice) with fibrosis in sinusoids and adjacent
to germinal centers. Furthermore, spleens of
IFN-R-/- mice were
significantly smaller than spleens of wild-type mice [130 mg ±
26.5 (SD) versus 31 ± 5.7, P <
0.05]. By day 45 after infection, spleens of
IFN-R-/- mice appeared
more cellular compared to day 23, and showed signs of resolving
pathology. In contrast, the spleens of wild-type mice maintained their
normal architecture throughout the course of infection (Figure 1)
.
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R-/- mice. Initial
changes (up to and including day 10) were similar in both wild-type and
IFN-R-/- mice (all
mice), and consisted primarily of increased numbers of blast cells. By
day 14, early fibrosis was observed in
IFN-R-/- mice, and by
day 23, MLN in
IFN-R-/- mice showed
signs of cellular depletion and atrophy. However, by day 45 we observed
an increase in nodal cellularity with a partial resolution of scarring
(Figure 1)
. Importantly, the pathological changes noted above were not
observed in other secondary lymphoid tissues examined. For example,
both the submandibular and the mesenteric lymph nodes in
IFN-R-/- mice appeared
normal (data not shown). The lymphoid tissues examined in the wild-type mice, similar to the spleens of these mice, maintained their normal architecture throughout the course of infection (data not shown).
Pathological changes were also noted in lungs and livers of
IFN-R-/- mice infected
with MHV-68. The initial pulmonary changes were similar in both groups
of mice by day 10 after infection. These changes included interstitial
pneumonia and interstitial and intraalveolar fibrosis, accompanied by
pulmonary phlebitis. The pulmonary changes were most severe by day 14
after infection in
IFN-R-/- mice and
included inflammation and interstitial fibrosis, accompanied by
pulmonary phlebitis. The lungs also showed signs of resolution by day
45 after infection (Figure 1)
.
The hepatic changes in
IFN-R-/- mice were
apparent on day 10 after infection and included multifocal dilation of
sinusoids with fibrin deposits and infiltration by neutrophils and
lymphocytes. Mild changes were observed in livers of wild-type mice,
which included focal dilation of sinusoids with minor fibrin deposition
and mixed inflammatory infiltrates (data not shown).
The mild pathological changes observed in the livers of wild-type mice
were resolved by day 23 after infection (data not shown). In contrast,
the hepatic changes in
IFN-R-/- mice had
progressed from moderate to severe with extensive portal fibrosis. As
with lymphoid tissue changes, most of hepatic changes had resolved by
day 45 after infection (Figure 1)
.
Lymphoid Tissue Cellularity
To support the histological observations, we measured the cell
numbers in lymphoid tissues during the course of infection. We found
that by day 23 after infection, there was a 120-fold reduction in
spleen cell numbers in
IFN-R-/- mice compared
to wild-type mice [9.7 x 107
± 8.1
x 105
(SD) versus 8.1 x
105
± 5.6 x 105,
P < 0.05]. This reduction was followed by an
increased cellularity of spleen in
IFN-R-/- mice evident
by day 45 after infection [8.1 x 106
±
3.6 x 06
on day 45 versus 8.1
x] 105
± 5.6 x 105
on day 23, P < 0.05] (Figure 2)
. Similarly, the MLN of
IFN-R-/- mice were
also depleted by day 23 after infection [3.3 x
106
± 6.4 x 105
(SD)
versus 8 x 104
± 1 x
104, P < 0.05].
|
R-/- mice on day
10 after infection. This was also reflected in an increase in splenic
neutrophils in
IFN-R-/- mice [17%
± 6.5 (SD) versus 13% ± 4.8]. Kinetics of Apoptotic Death in Spleen
The TUNEL assay was used to determine whether loss of splenocytes
in IFN-R-/- was due to
increased apoptosis. The earliest TUNEL positive cells were detected on
day 4 after infection, and were located in the periarterial lymphatic
sheaths. From this point on, there was a gradual increase in numbers of
TUNEL positive cells, which peaked at two weeks before a decline on day
16 after infection (Figure 3)
. At the
peak of splenic apoptosis, the TUNEL positive cells were almost
exclusively found within the follicular structures. However, both the
tempo and the location of these TUNEL positive cells were similar in
wild-type and IFN-R-/-
mice.
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Lack of a substantial splenic apoptosis in
IFN-R-/- mice
suggested the possibility of altered leukocyte trafficking, which could
account for the loss of splenocytes in
IFN-R-/- mice.
Therefore, we analyzed the hematological parameters in venous blood
during the course of infection.
We found significant hematological changes in
IFN-R-/- mice which
were apparent by day 10 after infection, where levels of circulating
neutrophils were significantly elevated in comparison to those in
wild-type mice (Figure 4)
.
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R-/- mice were, on
average, greater than nearly threefold higher than those in the
wild-type mice [3.3 x 107
± 1.2 x
107
(SD)/ml versus 1.3 x
107
± 5 x 106,
P < 0.05] and lymphocytes made as much as 78% of the
rise in total white blood cells (Figure 4)
. The elevated circulating
leukocytes in IFN-R-/-
mice reached a maximum on day 23 after infection [3.5 x
107
± 5.4 x 106
(SD)/ml versus 1.1 x 107
±
5.4 x 106, P < 0.05].
Again, lymphocytes constituted the major population (89%) of
circulating leukocytes (Figure 4)
.
The hematological changes observed at earlier time points in
IFN-R-/- mice were
resolved on day 45 after infection (Figure 4)
. At this late time point,
the levels of circulating leukocytes in
IFN-R-/- mice were
more than halved when compared to day 14, and were in the same range as
circulating leukocytes in wild-type mice [1.6 x
107
± 5.3 x 106
(SD)/ml versus 1.4 x 107
±
5 x 106] (Figure 4)
. These reductions in
total venous blood leukocytes were similar to reductions in total
lymphocyte levels [1.4 x 107
± 5 x
106
(SD)/ml versus 1.2 x
107
± 4.3 x 106].
Notably, the resolution of leukocytosis also coincided with significant
histological changes in spleens of
IFN-R-/- mice (Figure 1)
.
We found no preferential expansion of any lymphocyte subset during
leukocytosis. Moreover, the percentages of venous blood T (CD4 and CD8)
and B (CD19) lymphocytes were similar between both wild-type and
IFN-R-/- mice (data
not shown). We also analyzed the femoral bone marrow cellularity in
wild-type and IFN-R-/-
mice. Bone marrow cellularity was comparable between wild-type and
IFN-R-/- mice during
the entire course of MHV-68 infection (data not shown).
Leukocyte Trafficking during MHV-68 Infection
To address the cause of leukocytosis, PBL from wild-type and
IFN-R-/- mice were
labeled with the green fluorescent dye, PKH-67, and used in cross
transfer studies. PBL from
IFN-R-/- mice (day 23
after infection) were able to traffic through the spleen of wild-type
mice (day 23 after infection), but not through the spleens of
IFN-R-/- mice on day
23 after infection (but did traffic through the spleens of these mice
on day 10 after infection) (Figure 5)
.
These data clearly show that an inhibition in cell trafficking between
spleen and blood is a major cause of leukocytosis in
IFN-R-/- mice.
|
Multiprobe RPA sets and ELISA assays were used to analyze the
levels of cytokines and chemokines, which may play a role in the
pathologies observed in spleens of
IFN-R-/- mice.
The splenic mRNA levels of IFN- were significantly higher in
IFN-R-/- mice than in
wild-type mice, reaching a peak on day 14 after infection [0.397
OD ± 0.018 (SD) versus 0.075 ± 0.018,
P < 0.05]. Similarly, levels of TNF- [0.683
OD ± 0.05 (SD) 0.3596 ± 0.06, P < 0.05],
TNFß [0.71 OD ± 0.17 (SD) versus 0.42 ± 0.12,
P < 0.05], IL-1ß [0.38 ± 0.03 OD (SD)
versus 0.062 ± 0.01, P < 0.05], and
TGF-ß1 [3.17 ± 0.28 OD (SD) versus 2.44 ±
0.23, P < 0.05] were significantly elevated on day 14
after infection in spleens of
IFN-R-/- mice compared
to the wild-type mice (Figure 6)
.
|
protein concentrations in splenocyte cultures from infected
mice complemented the RPA data for this cytokine [32.075 ± 4.822
(SD) ng/ml versus 0.345 ± 0.466, P <
0.05]. Similarly, a TNF--specific ELISA was used to measure the
protein concentration of this cytokine in splenocyte cultures obtained
from infected mice. The splenic TNF- protein levels were on average
2.8-fold higher in
IFN-R-/- mice on day
14 after infection than in wild-type mice [0.536 ± 0.16 (SD)
ng/ml versus 0.187 ± 0.057, P <
0.05] (Table 1)
.
|
.
Similarly, whereas the mRNA for MIG was readily detected in wild-type
mice, we did not detect the transcript for this chemokine in
IFN-R-/- mice (data
not shown).
RANTES was abundantly expressed in both wild-type and
IFN-R-/- mice.
Interestingly, the elevated levels of this chemokine were sustained
even at late time points when there was a reduction in
other mRNA species studied (Figure 6)
.
MIP1ß levels were on average threefold higher on day 14 after
infection in spleens of
IFN-R-/- mice than in
wild-type mice [0.15 ± 0.08 (SD) versus 0.048 ±
0.02, P < 0.05] (Figure 6)
.
The basal levels of the chemokine lymphotactin were similar in both
wild-type and IFN-R-/-
mice. However, the levels of this chemokine reached a peak on day 14
after infection and were significantly higher in
IFN-R-/- mice than in
wild-type mice (0.12 ± 0.04 (SD) versus 0.07 ±
0.02, P < 0.05), before subsiding to pre-infection
levels by day 45 after infection (Figure 6)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R-/- mice results
in fibrosis and atrophy of the spleen and MLN with severe cellular
depletion of these organs culminating in leukocytosis. The pathologies
in IFN-R-/- mice
include extensive mononuclear cell infiltration of the lung and the
liver, with pulmonary interstitial fibrosis and hepatic portal
fibrosis, respectively. The fibrosis and leukocytosis in
IFN-R-/- mice are
associated with elevated levels of pro-fibrotic cytokines and a
dysregulated chemokine response. These pathologies showed signs of
resolution by day 45 after infection.
A prominent feature of splenic pathology in infected
IFN-R-/- mice is loss
of splenocytes. Despite elevated levels of TNF- and TNF-ß in
spleens of IFN-R-/-
mice, we found no significant increase in splenic apoptosis or changes
in bone marrow cellularity to account for the reduced spleen cell
numbers in these mice.
Our present work shows that the loss of splenic cellularity coincides
with a dramatic increase in the number of T and B lymphocytes in
peripheral blood. This is in agreement with our previous observation,
in which T and B lymphocytes were lost from the spleens of
IFN-R-/-
mice.17
Furthermore, we found no particular increase in
CD8 T cells during leukocytosis as observed in infectious mononucleosis
seen in EBV and MHV-68.5
The increases in PBL in
IFN-R-/- mice were
directly related to splenic atrophy, ie, the most depleted spleens had
the highest levels of circulating leukocytes. This leukocytosis
results, in part, from inhibited trafficking of peripheral blood cells
through the fibrosed spleens of
IFN-R-/- mice. PBL
taken from IFN-R-/-
mice at the height of leukocytosis (day 23) did not traffic through the
spleens of IFN-R-/-
mice on day 23 after infection (ie, at the peak of leukocytosis), but
were able to traffic through the spleens of wild-type mice
synchronously infected with MHV-68 and also entered the spleens of
IFN-R-/- mice on day
10 after infection, ie, before extensive deposition of extracellular
matrix.
We found that levels of IL-1ß, TNF-, TNF-ß, TGF-ß1, and
IFN- were significantly elevated in spleens of
IFN-R-/- mice. Similar
cytokine imbalances have also been associated with several human
diseases with fibrosis and in murine models of
fibrosis.20-23
Of these cytokines, TNF-,
TGF-ß1, and IL-1ß are potent inducers of
fibrosis,27-28
whereas IFN- is a potent
anti-fibrogenic cytokine and down-regulates the expression of both type
I and type III collagens and fibronectin.29-32
Consequently, lack of IFN--mediated responses in
IFN-R-/- mice together
with elevated levels of fibrogenic cytokines would further support a
dysregulated cytokine response as a cause of fibrosis in
IFN-R-/- mice.
Interestingly, a reduced trend in the expression of these cytokines
coincided with a partial resolution of the pathologies by day 45 after
infection. Moreover, despite the absence of a functional IFN-
system, the cytokines IL-4 and IL-10, associated with Th2 responses
were not upregulated in
IFN-R-/- mice.
The inhibited trafficking of leukocytes through the spleens of
IFN-R-/- mice prompted
an analysis of chemokines, because they play a crucial role in
leukocyte trafficking.33
CXC chemokines IP-10 and MIG were
significantly elevated in wild-type mice and are involved in the
recruitment of T cells via the CXCR3 receptor.34
Elevated
levels of IP-10 and MIG were observed on day 14 after infection in
infected wild-type mice (at the peak of splenomegaly), but were
significantly reduced in the spleen of infected
IFN-R-/- mice (which
lacked splenomegaly), further supporting previous observations on
requirement of a functional IFN- for induction of these
chemokines.35
IP-10 has been shown to inhibit fibroplasia
and deposition of extracellular matrix proteins,36
and so
a reduction in the expression of this chemokine and MIG during
infection must affect splenic pathology.
MIP-1ß and LTN are potent chemoattractants.37
Paradoxically, spleens of
IFN-R-/- mice showed
elevated levels of MIP-1ß and LTN compared to wild-type mice.
However, elevated levels of cytokines, for example TNF, can inhibit
cell migration by down-regulating chemokine
receptors.38
Therefore, a similar inhibitory
mechanism may provide an explanation for the elevated levels of
MIP-1ß and LTN despite cellular depletion in
IFN-R-/- mice.
The pathologies associated with MHV-68 infection of
IFN--/- mice may have
important implications for human fibrotic conditions. Of significance
here is the idiopathic pulmonary fibrosis (or cryptogenic fibrosing
alveolitis), which is an immune-mediated disorder and has been
associated with EBV infection.39
Furthermore, several
familial genetic defects associated with disruption of the IFN-R
have been reported in man.40
Although no clinical viral
disease has yet been identified in these patients, it is conceivable
that a similar fate may ensue after infection with EBV or Kaposi’s
sarcoma-associated herpesvirus.
In conclusion, we demonstrated that MHV-68 initiates a cascade of
pathological events in
IFN-R-/- mice. The
expulsion of cells from the spleen leads to a rise in leukocytes in the
peripheral blood. These cells are unable to re-enter the spleen
despite, or because of, the elevated levels of inflammatory cytokines
in the spleen. Tissue remodeling occurs, and there is a repopulation of
the affected lymphoid tissues. Whereas data in this report may explain
the likely molecular basis for fibrosis, further work is needed to
determine how MHV-68 gene expression alters the pattern of cytokines
and chemokines in
IFN-R-/- mice and to
understand the role of CD8 T cells in mediating this process.
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Acknowledgements |
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Footnotes |
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Supported by grant 15/S009323 from the Biotechnology and Biological Sciences Research Council (UK).
Accepted for publication March 13, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Virology 1994, 202:76-88[Medline]
knockout mice. J Immunol 1995, 155:3964-3971[Abstract]
. Proc Natl Acad Sci USA 1999, 96:5135-5140This article has been cited by other articles:
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 B. M. Dutia, D. J. Roy, B. Ebrahimi, B. Gangadharan, S. Efstathiou, J. P. Stewart, and A. A. Nash Identification of a region of the virus genome involved in murine gammaherpesvirus 68-induced splenic pathology J. Gen. Virol., June 1, 2004; 85(6): 1393 - 1400. [Abstract] [Full Text] [PDF]
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J. B. Weinberg, M. L. Lutzke, S. Efstathiou, S. L. Kunkel, and R. Rochford Elevated Chemokine Responses Are Maintained in Lungs after Clearance of Viral Infection J. Virol., September 11, 2002; 76(20): 10518 - 10523. [Abstract] [Full Text] [PDF]
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C. M. Payne, C. J. Heggie, D. G. Brownstein, J. P. Stewart, and J. P. Quinn Role of Tachykinins in the Host Response to Murine Gammaherpesvirus Infection J. Virol., November 1, 2001; 75(21): 10467 - 10471. [Abstract] [Full Text] [PDF]
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, wild-type mice; 
,
IFN-
