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Journal of Virology, March 1999, p. 1756-1766, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Protection from Lethal Coxsackievirus-Induced
Pancreatitis by Expression of Gamma Interferon
Marc S.
Horwitz,
Troy
Krahl,
Cody
Fine,
Jae
Lee, and
Nora
Sarvetnick*
Department of Immunology, The Scripps
Research Institute, La Jolla, California 92037
Received 19 October 1998/Accepted 12 November 1998
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ABSTRACT |
Coxsackievirus infection causes severe pancreatitis and myocarditis
in humans, often leading to death in young or immunocompromised individuals. In susceptible strains of mice, coxsackievirus strain CB4
causes lethal hypoglycemia. To investigate the potential of gamma
interferon (IFN-
) in protection and clearance of the viral infection, IFN- knockout mice and transgenic (Tg) mice specifically expressing IFN- in their pancreatic 
cells were infected with CB4. Lack of IFN- in mice normally resistant to CB4-mediated disease
resulted in hypoglycemia and rapid death. However, expression of
IFN- in the cells of Tg mice otherwise susceptible to lethal infection allowed for survival and protected them from developing the
accompanying hypoglycemia. While all the mice had high levels of viral
replication in their pancreata and comparable tissue pathology
following viral infection, the Tg mice had significantly lower levels
of virus at the peak of infection, significantly higher numbers of
activated macrophages before and after infection, and less damage to
their acinar tissue. Additionally, despite having increased levels of
inducible nitric oxide synthetase (iNOS) expression, treatment of Tg
mice with the iNOS inhibitor aminoguanidine did not alter the level of
protection afforded by IFN- expression. In conclusion, IFN-
protects from lethal coxsackievirus infection by activating macrophages
in an iNOS-independent manner.
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INTRODUCTION |
Induced in response to immune
stimulation, tissue damage, and viral replication, gamma interferon
(IFN-) is an important mediator of cellular inflammation. As a
pluripotent inducer of cellular and immune processes, IFN- has been
shown not only to protect tissue from virus-mediated damage
(21) but also to induce tissue damage on its own (16,
24). The ability of this cytokine to both directly protect and
damage tissue is most likely associated with the local host factors
within the target tissue compartment.
Numerous infectious agents have been associated with acute
pancreatitis, and there is well-documented clinical evidence linking coxsackievirus B4 (CB4) to the development of both pancreatitis and
diabetes in humans (4, 32). CB4 was isolated from patients suffering from both pancreatitis and diabetes and, after passage through murine cells, was subsequently demonstrated to infect mice
(14, 31, 32). Following CB4 infection, the virus replicates in a number of tissues, specifically targeting the acinar tissue of the
pancreas for pathology, causing severe pancreatitis similar to that
observed in humans. However, the role of cytokines and inflammatory
mediators in the development of pancreatitis has not been investigated
thoroughly. Since IFN- is an important mediator of immune responses
in vivo, it is a likely participant in the CB4-mediated immunopathogenesis.
To determine whether IFN- has a role in this virus-mediated disease,
mice lacking systemic expression of IFN- (IFN- knockout mice
[GKO]) (7) and transgenic mice overexpressing IFN- in the pancreas (NOD-IFN-) (12) were infected with CB4. The
ability of IFN- to control the infection and to protect mice from
the resulting CB4-mediated pancreatitis was then tested.
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MATERIALS AND METHODS |
Mice.
NOD/SHI and NOD/SCID mice were obtained from the
rodent breeding colony at The Scripps Research Institute (La Jolla,
Calif.). GKO mice of the H-2b haplotype were
provided by D. Dalton (Trudeau Institute, Saranac, N.Y.)
(7). Heterozygous GKO (+/
) mice were crossed with
(129/SvEv × C57BL/6)F1 mice in our animal facility to
generate homozygous (/) GKO (129/SvEv × C57BL/6)F2 mice. (129/SvJ × C57BL/6)F2
mice were obtained from The Jackson Laboratory (Bar Harbor, Maine), bred, and maintained in our colony. In addition, C57BL/6 mice were used
as controls and showed results similar to those of the (129/SvJ × C57BL/6)F2 mice presented herein. Both 129/SvEv and 129/SvJ
mice are derived from the same parental strain. The difference between
the two substrains is that the SvEv was crossed once with C3H and the
F1 was backcrossed 14 times to the Sv parental strain, but
the SvEv substrain is 99.99% similar to SvJ (27).
The NOD-IFN- Tg mice were previously developed in our laboratory
(12) and were bred and maintained there as well. Blood glucose was measured in tail vein or eye bleeds from nonfasting mice at
various times postinfection with a standard glucometer with a range of
20 to 400 mg/dl.
NOD mice were used in this study because the transgene was derived on
the NOD major histocompatibility complex (MHC) background.
Female NOD
mice spontaneously develop diabetes at a rate of 85%
(30% for males)
by 16 to 20 weeks of age in our colony. All mice
were infected between
6 and 8 weeks of age and were tested prior
to infection to ensure that
they were not
diabetic.
Virus.
Virus stocks of coxsackievirus group B type 4 Edwards
strain 2 (CB4 strain E2) were obtained from Charles Gauntt (University of Texas
San Antonio) and were derived from stocks originating from
Roger Loria (Medical College of Virginia, Virginia Commonwealth University) (31).
Virus stocks of CB4 strain E2 were prepared in monolayers of HeLa cells
using a multiplicity of infection (MOI) of 0.1 PFU/cell
in Dulbecco's
modified Eagle medium. Virus was harvested by freeze-thawing
and stored
at
80°C. Viral titers were determined on HeLa cell
monolayers by
using a standard plaque assay technique. Mice were
infected at 6 to 8 weeks of age intraperitoneally (i.p.) with
10
4 PFU of CB4.
The observed 50% lethal dose for this virus stock
in NOD mice was
calculated to be 100 PFU. Virus was plaque assayed
in tissue from
individual organs, aseptically weighed, and homogenized
in
diluent.
Immunohistochemical staining.
Mice were anesthetized before
their organs were removed, placed in 10% formaldehyde, and processed
for paraffin sectioning. Additionally, organs were snap frozen in
O.C.T. on dry ice. Immunohistochemistry was performed on 4-µm-thick
paraffin sections or 7-µm-thick frozen sections prepared and blocked
with avidin and biotin (Vector Laboratories, Burlingame, Calif.).
Staining was done with primary antibody to insulin (Dako, Carpinteria,
Calif.), inducible nitric oxide synthetase (iNOS) (Transduction
Laboratories, Lexington, Ky.), CB4 (American Type Culture Collection,
Rockville, Md.), or F4/80 (Serotec, Washington, D.C.). The second
antibody was a biotinylated anti-guinea pig immunoglobulin G (IgG),
anti-rabbit IgG, anti-rat IgG, or anti-horse IgG used in conjunction
with the Vectastain ABC (peroxidase) kits (Vector Laboratories).
Staining was detected by using diaminobenzidine as a chromagen.
Sections were counterstained in Mayer's hematoxylin (Sigma, St. Louis,
Mo.) and mounted in Permount (Fisher, Pittsburgh, Pa.).
AG treatment of mice.
Mice were divided into multiple
groups: CB4-infected NOD and NOD-IFN- mice receiving aminoguanidine
(AG) (Sigma) or phosphate-buffered saline (PBS) and sham-infected
animals receiving AG or PBS. AG (8 mg dissolved in 0.5 ml of PBS) or
0.5 ml of PBS was injected i.p. followed by i.p. CB4 inoculation as
described above. Thereafter, the mice were injected twice daily with 4 mg of AG or PBS for a total of 8 mg/day for 14 days. Mice were
sacrificed on days 4, 7, and 14, and tissues were divided in half for
plaque assay and histological analysis. In order to determine if AG was
able to block in vivo iNOS activity, mice were bled prior to treatment and 24 h postinfection and posttreatment. Plasma samples were obtained through Ultrafree-MC columns (Millipore Corp., Bedford, Mass.)
at 14,000 × g for 30 min at 4°C. Levels of
NO2
and NO3
(NO2/NO3), the
stable degradation products of nitric oxide (NO), were determined in
plasma by using the Cayman nitrite/nitrate assay kit (Alexis
Biochemicals, San Diego, Calif.). Treatment with AG in both NOD and
NOD-IFN- Tg mice did not lead to increased rates of mortality in
infected or uninfected mice.
Statistical analysis.
The unpaired Student t test
was performed for all viral plaque assay analyses, and significant
differences and P values are indicated in the figures and
figure legends.
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RESULTS |
Susceptibility to CB4 infection in mice lacking IFN-.
CB4
infects mice with a variety of clinical outcomes. While high doses of
virus (104 to 106 PFU per mouse) introduced
i.p. to SJL, C57BL/6, and B10 mice lead to viral clearance by 2 weeks
and survival, a similar inoculum in BALB/c and NOD mice led to rapid
death (within 1 week). The primary tissue target for destruction of CB4
is the pancreas, as mice are hypoglycemic following infection and
surviving mice suffer from severe pancreatitis with the loss of acinar
tissue. While the pancreatic islets appear infected, islet tissue loss is not observed. Additionally, other tissues are infected, and mice
suffer from mild myocarditis as well as inflammation in the lungs,
kidneys, and liver. As a result of the severe pancreatitis, all
infected mice exhibit an initial period of hypoglycemia following infection, from which susceptible mice (BALB/c and NOD) never recover.
To evaluate the role of IFN-
in susceptibility of mice to tissue
damage following CB4 infection, GKO (
7) and genetically
matched control mice [(129/SvJ × C57BL/6)F
2] were
infected with
CB4. Although initially hypoglycemic at 1 week after
infection,
the normally resistant control mice were healthy and
survived
for more than 3 months. On the other hand, the MHC-matched GKO
mice exhibited hypoglycemic blood glucose levels (<50 mg/dl;
n = 8), and none survived past day 5 postinfection
(Fig.
1A).
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FIG. 1.
(A) Survival of GKO mice following CB4 infection. Mice
were given 104 PFU of coxsackievirus B4 and were monitored
over time for survival. Both male (n = 4) and female
(n = 7) GKO mice ( ) were infected, while equal
numbers of male (n = 4) and female (n = 4)
C57BL/6 × 129 mice ( ) were infected (n = 8).
(B) Viral recovery from infected GKO mice. Mice were infected with CB4
and sacrificed at 3 days postinfection. Organs were harvested and
assayed for virus recovery by standard plaque assay. Four GKO ( ) and
eight C57BL/6 × 129 ( ) mice were assayed. Standard deviations
are indicated by error bars. No significant differences were
observed.
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We next measured the replication of CB4 to determine whether the poor
survival of mice lacking IFN-
correlated with an increased
virus
load. Quantitation of viral replication by plaque assay
demonstrated
that, despite the lack of IFN-
and differences in
clinical outcome,
no significant differences in viral titer were
observed at either 3 (Fig.
1B) or 4 (data not shown) days postinfection
for GKO and control
mice in the pancreata, spleens, or kidneys.
This indicated that IFN-
does not promote survival by limiting
virus
replication.
We further studied the histopathology of the pancreata from infected
GKO and control mice to assess the damage resulting from
CB4 infection.
Four days after infection, GKO and control mice
had similar tissue
pathology within the pancreas (Fig.
2).
Although
the dropout of acinar tissue was not typically observed by 5 days
postinfection, a dramatic loss of exocrine tissue structure and
atrophy of the exocrine pancreas was observed at this time.
Additionally,
significant numbers of infiltrating lymphocytes and
macrophages
were easily identified. Islets remained functionally intact
at
5 days postinfection in both GKO and control mice as demonstrated
by
immunostaining for insulin (Fig.
2), glucagon, and somatostatin
(data
not shown). By 2 weeks postinfection, the control mice had
significant
loss and dropout of their acinar tissue, although
islets remained
intact and produced functional insulin. Fat replacement
of some of the
acinar tissue could be observed by 3 months postinfection.
In general,
no obvious pathologic differences between CB4-infected
GKO mice and
other strains of mice were observed.
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FIG. 2.
Histological analysis of pancreata of GKO mice following
viral infection. Representative hematoxylin-and-eosin (H&E)-stained
sections of the pancreas from a GKO and C57BL/6 × 129 mouse
reveal the large inflammatory insult, necrosis, and damage to the
pancreas following infection (×172). The mice were sacrificed at 4 days postinfection. Islet integrity and function were confirmed by
immunohistochemistry using antibody to insulin (×344). Islets are
designated with an "i." Acinar tissue is designated with an
"a." Brown insulin staining is present over islets, demonstrating
functionality following infection.
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IFN-
induces a number of responses within the tissue to help clear
viral infections including increasing the expression of
MHC molecules
and iNOS. However, immunohistochemical analysis
of infected pancreatic
tissue from GKO mice revealed no obvious
difference in the cell surface
expression of MHC class I or II
or in the production of iNOS in the
coxsackievirus-infected, inflamed
tissue (data not
shown).
Local expression of IFN- protects from lethal CB4
infection.
The results described above demonstrate that mice
required a systemic source of IFN- to survive lethal viral
infection. Is local expression of the cytokine required for protection
of the pancreas, or is it protecting by modulating systemic immune
responses? To address this question, we infected transgenic mice
expressing IFN- in the cells of the pancreas (12)
with a lethal dose (104 PFU) of CB4. These NOD-IFN- Tg
mice were previously derived and offer a local concentration of IFN-
near the primary site of CB4 infection. Following infection with a
lethal dose of CB4, less than 50% of NOD mice survived to 6 days
postinfection, and the remaining mice did not survive to day 10 (20 of
20) (Fig. 3A). However, their
NOD-IFN- Tg counterparts were protected from lethal infection (18 of 18) and lived for more than 6 months. Additionally, at 3 days
postinfection non-Tg mice developed hypoglycemia (75 ± 10;
n = 6), while NOD-IFN- Tg mice remain normoglycemic (120 ± 12; n = 6). The ability to maintain normal
glycemic levels and survive infection was directly linked to -cell
expression of IFN-.
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FIG. 3.
(A) Survival of NOD () (n = 20),
NOD/SCID () (n = 20), and NOD-IFN- Tg ( )
(n = 18) mice following CB4 infection. Mice were given
104 PFU of coxsackievirus B4 and were monitored over time
for survival. Equal numbers of male (n = 9 or 10) and female
(n = 9 or 10) mice from each group were infected. (B)
Changes in pancreatic viral replication over time. NOD (), NOD/SCID
(), and NOD-IFN- Tg ( ) mice were infected with CB4 and
sacrificed 3, 4, or 7 days postinfection. Pancreata were harvested and
assayed for virus recovery by standard plaque assay. At least four mice
from each strain were assayed at each time point. Standard deviations
are indicated by error bars. At day 3 postinfection, NOD-IFN- Tg
mice were significantly lower in viral titer than both NOD and NOD/SCID
mice at a P value of <0.0001. Additionally, NOD/SCID mice
had a significantly higher titer at day 7 at a P value of
<0.003. Asterisks denote statistically significant differences. (C)
Viral recovery from infected NOD () (n = 8),
NOD/SCID () (n = 6), and NOD-IFN- Tg ()
(n = 5) mice. Mice were infected with CB4 and
sacrificed 3 days postinfection. Organs (pancreas, spleen, and kidney)
were harvested and assayed for virus recovery by standard plaque assay.
Standard deviations are indicated by error bars. Significant
differences in NOD-IFN- mice versus NOD mice were observed at
P values of <0.0001 for all three organs. Asterisks denote
statistically significant differences. (D) Viral recovery from infected
NOD () (n = 8), NOD/SCID () (n = 8), and NOD-IFN- Tg () (n = 4) mice at 7 days postinfection. Mice were infected with CB4 and sacrificed at 7 days following infection. Organs were harvested and assayed for virus
recovery by standard plaque assay. Standard deviations are indicated by
error bars. Significant differences in NOD/SCID mice versus the other
mice were observed at P values of <0.003 (pancreas) and
<0.05 (spleen and kidney). Asterisks denote statistically significant
differences.
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To determine whether this local expression of IFN-
protected mice
from lethal infection by reducing the viral load in the
pancreas, we
quantitated virus by plaque assays on tissues from
both Tg and non-Tg
mice 3, 4, and 7 days postinfection with CB4
(Fig.
3B). While high
levels of replicating virus were observed
in the pancreas of both Tg
and non-Tg mice following infection,
Tg mice had significantly lower
levels of virus at the peak of
infection (day 3). Despite this
difference at day 3 postinfection,
by day 4 equivalent levels of virus
were found in the pancreata
of both types of mice and ultimately virus
was cleared at the
same rate in both Tg and non-Tg mice. Analogously,
lower levels
of CB4 were found in the spleen and kidneys of Tg mice at
the
peak of infection (day 3), viral titers were relatively equivalent
by day 4 postinfection, and clearance of virus in Tg and non-Tg
mice
was similar. The differences in IFN-
expression and clinical
outcome
were associated with a difference in the level of viral
replication at
the peak of infection. Since the pancreas is the
primary site of
replication, reductions in the level of virus
in the pancreas most
likely reflect the ability of the virus to
circulate and replicate in
other
organs.
To better define the precise role of lymphocytes following CB4
infection, we compared NOD/SCID mice, which lack both T and
B cells, to
NOD mice with an intact immune response. Infection
with a lethal dose
of CB4 led to only 25% mortality in NOD/SCID
mice by day 21, although
greater than 95% succumbed by day 28
(Fig.
3A). In comparison, all the
NOD mice died within 10 days
of infection. Therefore, the lethal effect
of CB4 was to a great
extent lymphocyte mediated, although if left
unchecked, this cytopathic
virus eventually resulted in death. The
ability of CB4 to replicate
in NOD/SCID mice was also quantitated by
plaque assay. Although
similar levels of CB4 were observed in the
pancreata of NOD/SCID
and NOD mice at both 3 and 4 days postinfection,
considerably
less virus was recovered from both the spleens and kidneys
of
NOD/SCID mice (Fig.
3C and D). Additionally, virus levels decreased
over time in the surviving NOD mice, eventually leading to clearance
by
7 to 10 days postinfection. However, viral clearance was observed
in
the NOD/SCID mice with a quite substantial and significant
level of
virus remaining in pancreata at 7 days postinfection
(Fig.
3D).
Although virus levels were eventually reduced in the
pancreas,
clearance was never observed. Thus, the absence of a
competent immune
response with T and B cells resulted in at least
a temporary resistance
to lethal
infection.
To distinguish between the pathology of infection in NOD, NOD-IFN-
,
and NOD/SCID mice, mice were sacrificed 4 days after
infection for
histological examination of their pancreata. All
three groups of mice
presented with pronounced immune infiltration
of the pancreas, most
notably in the acinar tissue. Additionally,
loss of cells and damage
were observed in all three mouse strains,
but to the greatest extent in
the NOD mice (Fig.
4). Despite the
pronounced pathology, NOD-IFN-
mice did have areas with
significantly
less acinar tissue damage and many surviving acini.
Immunohistochemical
staining with antibody to insulin (Fig.
4) and to
somatostatin
and glucagon (data not shown) revealed that the infection
did
not compromise the production of islet hormones. Clearly, most
of
the damage was to the acinar tissue.
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FIG. 4.
Histological analysis of pancreata of NOD, NOD/SCID, and
NOD-IFN- Tg mice following CB4 infection. Representative
hematoxylin-and-eosin (H&E)-stained sections of the pancreas from NOD,
NOD/SCID, and NOD-IFN- Tg mice reveal the large inflammatory
insult, necrosis, and damage to the pancreas following infection
(×172). The mice were sacrificed at 4 days postinfection. Islet
integrity and function were confirmed by immunohistochemistry using
antibody to insulin (×344). Brown insulin staining is present over
islets, demonstrating functionality following infection.
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NOD-IFN-
mice have a unique pancreatic morphology as a result of
transgene expression alone; they exhibit a pronounced inflammatory
insult, which precludes the effects of viral infection (
12).
This IFN-
-mediated insult is directed at the islets, and despite
the
obvious inflammatory damage to the pancreas, blood glucose
balance is
maintained, for the most part due to islet cell regeneration
originating from proliferating ducts where the new islets are
sequestered. To determine whether these regenerated islets were
protected from CB4 infection as they had been from infiltrating
lymphocytes, we performed immunostaining of pancreatic sections
with
antibody to CB4. Infected islet cells were noted in the pancreata
of
both infected NOD and NOD-IFN-
Tg mice at 4 days postinfection
(Fig.
5). So while the duct wall
restricts lymphocytes from entering,
there is no noticeable block in
the penetration of CB4 into intraductal
islets.
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FIG. 5.
Immunohistological analysis of the islets from
CB4-infected NOD-IFN- Tg mice for viral antigen. A representative
immunostained section of the islets from the pancreas of a NOD-IFN-
Tg mouse reveals islet cells staining positively for viral antigen
(×344). The mice were sacrificed at 4 days postinfection. A serial
hematoxylin-and-eosin (H&E)-stained section is provided for comparison
(×344). Brown anti-CB4 staining is present over islets, demonstrating
viral infection.
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The mechanism of IFN-
-mediated protection was then investigated by
analyzing the constituents of the immune infiltration
as well as by
looking for changes in expression of the immune
response molecules, MHC
molecules, and iNOS. Prior to infection,
pancreatic expression of
IFN-
by NOD-IFN-
Tg mice induces the
expression of high levels
of endogenous MHC and iNOS as well as
an array of infiltrating immune
cells. Comparatively, NOD mice
exhibit a more modest level of
endogenous immune cell infiltration
and immune response molecule
expression, while NOD/SCID mice lack
both endogenous immune cell
infiltration and immune response molecule
expression. However,
following CB4 infection, a number of immune
response molecules are
upregulated and the number of infiltrating
inflammatory cells has
increased. While similar numbers of both
CD4
+ and
CD8
+ T cells were observed (data not shown) within the
infected pancreata
of NOD and NOD-IFN-
mice, the levels of
activated macrophages
were quite different (Fig.
6). Not only were significantly more
activated macrophages observed prior to infection in the Tg mice,
but
by day 3 postinfection, the level of activated macrophages
as measured
by F4/80 staining appeared to further increase in
both the pancreatic
acinar and islet tissue. After infection,
macrophages appeared to
mobilize out into the acinar tissue and
to encircle and possibly
protect individual acini. This activation
of macrophages was not
observed in infected NOD mice. The observation
of high numbers of
activated macrophages within the acinar tissue
at day 3 postinfection
is directly associated with the reduced
viral load in the pancreas at
day 3, survival, and transgene expression.
To determine whether
protection could also be associated with
increases in local and/or
macrophage expression of iNOS, pancreatic
tissue sections were,
further, immunostained for iNOS from Tg
and non-Tg mice following
infection. High levels of iNOS expression
were observed in both Tg and
non-Tg mice (Fig.
7). While no clear
difference was observed, it is quite possible that expression
of iNOS
in the activated macrophages is mediating protection from
the viral
infection.
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FIG. 6.
Immunohistological analysis of pancreata from
CB4-infected NOD and NOD-IFN- Tg mice for macrophages.
Representative immunostained sections of the pancreas from uninfected
and 3-day-postinfection NOD and NOD-IFN- Tg mice reveal differences
in the number and activation state of resident macrophages. Antibody to
the macrophage activation marker, F4/80, was used to identify activated
macrophages which, following infection, appear to have mobilized into
the acinar tissue and encircled groups of acini (×324). Islets are
designated with an "i." Brown antimacrophage staining is present
over macrophages. Activated macrophages can be observed encircling
acini, and some are marked with arrowheads.
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FIG. 7.
Immunohistological analysis of pancreata from
CB4-infected NOD and NOD-IFN- Tg mice for iNOS. Representative
immunostained sections of the islets from the pancreata of NOD and
NOD-IFN- Tg mice show cells of both the acinar and islet location
that stained positively for iNOS. Additionally, immune infiltrating
cells also stained positively for iNOS. The mice were sacrificed at 3 days postinfection (×348). Brown anti-iNOS staining is present over
many cell types in the three mouse strains, demonstrating upregulation
of iNOS following infection.
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NO not involved in protection from lethal infection.
To
address whether IFN- was able to protect the pancreas from the
effects of lethal coxsackievirus infection through upregulation of iNOS
and subsequent increases in the level of NO, mice were treated with AG
before and after infection. AG is a selective inhibitor of iNOS
activity, and treatment of rodents with AG has been shown to
effectively neutralize iNOS (6). Daily administration of AG
had no effect on the protection from lethal infection of NOD-IFN-
mice, since all seven of the mice tested survived past the 14th day of
CB4 infection (Fig. 8). Simultaneously,
all the non-Tg NOD littermate control mice treated with AG
(n = 6) and those mice mock-treated with PBS
(n = 6) died from lethal infection by day 6. Furthermore, AG treatment did not affect the clearance of virus from
either set of mice (data not shown). The AG treatment was sufficient to
block in vivo iNOS activity, as AG-treated animals were unable to
produce increased levels of
NO2/NO3 in plasma
following CB4 infection (data not shown). Additionally, histological
examination and immunohistochemical staining revealed no differences in
pathology between AG-treated and untreated mice (data not shown).
Therefore, neither iNOS nor NO plays a primary role in the protection
from lethal infection or clearance of CB4 in these mice.
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FIG. 8.
Survival of NOD () and NOD-IFN- Tg () mice
infected with CB4 during AG treatment. Six groups of mice were
examined. The figure presents the comparison of survival between two
groups: NOD (n = 6) and NOD-IFN- Tg (n = 7) mice given 104 PFU of coxsackievirus B4 and 8 mg of
AG per day. In addition, uninfected NOD (n = 4) and
NOD-IFN- Tg (n = 4) mice given 8 mg of AG and
infected NOD (n = 4) and NOD-IFN- Tg (n = 4) mice given PBS in place of AG were monitored for survival
over time. Of these, only the infected PBS-treated NOD mice succumbed
to lethal infection (4 of 4 died by day 7). The rest of the mice lived
longer than 21 days.
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DISCUSSION |
In this report, we demonstrate that IFN- is required to protect
against lethal coxsackievirus infection. The presence of systemic
IFN- was required for survival following infection as was
demonstrated by the lack of survival of GKO mice. This lack of IFN-
successfully switched an otherwise resistant mouse to one with
susceptibility to lethal infection, implying that IFN- and/or
IFN--induced host factors are responsible for the ability to resist
lethal infection. More significantly, we have demonstrated here that
local overexpression of IFN- in the pancreata of susceptible mice
offset the lethal effects of the virus. The mechanism of this
IFN--mediated protection is most likely the result of resident activated macrophages that are easily mobilized to defend the pancreas
from the invading virus. Additionally these studies demonstrate that
this defense is not dependent on NO, as the administration of the iNOS
inhibitor AG did not override protection. The importance of
inflammatory cytokines like IFN- in the control of cytopathic infections is further underscored by these results, which support the
possible therapeutic benefits of local administration to combat viral pathogenesis.
Coxsackievirus frequently infects humans, which results in debilitating
pancreatitis and myocarditis and has also been implicated in the
development of insulin-dependent diabetes mellitus (1, 30).
This report is the first study to describe organ-specific IFN--mediated protection from lethal viral infection, yet the mechanism by which coxsackievirus rapidly kills susceptible animals is
not known. The protective nature of the local transgene expression is
evidence for the cause of death as a result of pancreatic failure and/or destruction. Preceding death, systemic blood glucose levels dropped to less than 50 mg/dl, and this hypoglycemia alone may quite
possibly have led to loss of life. Even though the pancreatic islets
continued to function and produce high levels of insulin, glucagon, and
somatostatin, the much-affected acinar tissue was lost, decreasing the
production of digestive enzymes, including amylase, that directly
contribute to the blood glucose level provided by the gut.
Alternatively, the hypoglycemia may simply be indicative of the rapid
destruction of exocrine tissue that provides the necessary digestive
enzymes for the mice to gain nourishment and retain both their
metabolic homeo- and thermostasis. Further, this rapid cytolysis of
acinar tissue may have allowed large concentrations of digestive
enzymes to empty not into the gut but into the peritoneal cavity,
causing havoc to many of the organs that sustain life. This may well be
the case, as numerous organs from the peritoneal cavity (i.e., kidney,
spleen, and liver) were observed to exhibit degradation along their
outer edges (data not shown). Alternatively, protected mice maintained
intact portions of their acinar tissue and were subjected to a smaller
viral load.
On the basis of studies of several viruses, it has been proposed that
noncytopathic viruses are controlled by the host mainly by specific
lytic defense whereas cytopathic viruses like coxsackievirus are
controlled by cytokines and neutralizing antibodies (17). The results presented herein are consistent with the latter notion. In
considering mechanisms that account for the systemic protection, it
seems significant that NOD/SCID mice were temporarily protected from
lethal coxsackievirus but that genetically matched immunocompetent mice
succumbed quickly, implying a role for the innate response in mediating
protection. In general, SCID mice have a stronger innate response that
allows them to compensate for their lack of T and B cells with
increased levels of macrophages and natural killer (NK) cells. This in
turn may account for their increased survival and further suggests that
resistant strains of mice are likely to have stronger innate responses
than susceptible mice. Since IFN- was clearly required for survival,
we can postulate that the innate response including activated
macrophages and NK cells requires IFN- for its antiviral defense.
Thus IFN-, elicited by high levels of macrophages and NK cells, may
mediate early survival of CB4 infection, whereas lymphocytes are
eventually required for complete viral clearance. Current studies are
focused on examining the role of various immune cells through depletion analysis.
The mechanism of protection afforded by endogenous systemic IFN- and
Tg-directed pancreatic IFN- may be quite different. IFN- is
pluripotent in its function, inducing cellular processes that activate
the macrophage inflammatory response pathway (8, 16, 24, 25,
28) as well as direct the protection (9, 21) and even
growth of tissues (11, 12). The rapid nature of CB4-mediated
destruction and the ability of IFN- to protect from lethal infection
most likely reflect a strong dampening of the viral or immune-directed
cytolysis. Viral replication rates in the pancreas were affected by the
presence of IFN- by day 3, and although a considerably large and
equivalent viral load was still present at day 4, this may suggest that
protection is due to a simple reduction in early viral spread and
damage. However, this did not appear to be the case with the GKO mice
and their resistant MHC counterparts, as no difference in viral titer
was observed at these early time points. Nevertheless, reduction in early spread of virus may be one way to allow enough of the acinar tissue to escape infection and survive and/or conversely to reduce the
amount of degradative enzymes released from the damaged tissue. This
rapid protection is most likely explained by the predisposing environment of the Tg pancreas, which contains a large number of
activated macrophages and NK, T, and B cells ready and willing to
defend the tissue. In fact, the massive and rapid mobilization of
activated macrophages in the pancreas of the Tg mice implicates a
macrophage-dependent mechanism in protection. Further, the inability of
AG to alter such protection further implies that the mechanism of CB4
protection is NO independent. Nonetheless, macrophages have multiple
mechanisms by which to defend against infections. However, prior
studies with coxsackievirus B3 (CB3) have demonstrated a clear role for
NO in the antiviral mechanism, as inhibition of iNOS resulted in
increased viral replication and mortality (18). Although CB3
and CB4 are quite similar in structure, receptor usage, and the peptide
sequence of many gene products, they differ markedly in their primary
targets of pathology (1, 30), as well as the nature of their
induced immune responses (2, 15, 22). In fact, the patterns
of resistance and susceptibility of BALB/c and C57BL/6 mice are
reversed with respect to CB3 and CB4 (1, 30). Moreover,
differences of single amino acids distinguish CB4 variants resulting in
different degrees of virulence and pathology (22). Thus,
although the NO pathway was not responsible for the IFN--mediated
protection in this model, it certainly may participate in the defense
against other infections.
The IFN--mediated tissue protection in Tg mice may simply be
explained by this preactivated local innate response but may also
involve additional and distinct IFN--induced protective mechanisms.
For instance, the higher levels of MHC expression in Tg mice (data not
shown) may increase immune surveillance and recognition of infected
cells by immune cells within the pancreas tissue. Additionally, the
presence of increased numbers of T cells in the pancreata of
IFN--expressing Tg mice preceding infection is well established
(12). Along with the observed increases in inflammatory T
cells, increases in the numbers of regulatory T cells and gamma/delta T
cells are observed (data not shown). Such cells may reduce the activity
of the virus-specific T cells in the pancreas enough to decrease their
cytolytic activity while retaining their ability to clear virus.
Moreover, a number of IFN-induced cellular proteins, including PML,
2',5'-oligoadenylate synthetase, p68, and the Mx proteins, have been
shown to display antiviral properties (23, 26, 29).
Overexpression of some of these proteins can provide resistance to
certain viruses while having no effect whatsoever on others (3, 5,
19, 20). Similarly, IFN- has reduced the replication of
hepatitis B virus via distinct intracellular mechanisms
(13). IFN- may also induce host factors to stabilize the
acinar tissue during infection or downregulate viral virulence factors
and, in either case, limit the pathology of infection. Finally, IFN-
can alter expression of the apoptosis-related proteins Bcl-2, Bcl-x,
and Bax (10), which enable cells to survive
cytokine-mediated cell death. While our results point strongly to a
macrophage-mediated mechanism, they do not rule out the possibility
that multiple protective mechanisms are also in play.
| |
ACKNOWLEDGMENTS |
We thank Charles Gauntt (University of TexasSan Antonio) for
his generous gift of coxsackievirus B4 Edwards strain 2, Howard Fox for
the use of his virus facilities, and Gail Patstone for technical help.
We also thank Howard Fox, Michael Buchmeier, Cecile King, and Michelle
Krakowski for helpful discussions.
M.S.H. was a recipient of a Juvenile Diabetes Foundation International
Postdoctoral Fellowship Award and an American Diabetes Association
Career Development Award. N.S. was supported by a Diabetes
Interdisciplinary Research Program grant from the Juvenile Diabetes
Foundation International.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Immunology (IMM-23), The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (619) 784-9066. Fax: (619) 784-9083. E-mail: noras{at}scripps.edu.
This is manuscript number 11674-IMM from The Scripps Research Institute.
| |
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Journal of Virology, March 1999, p. 1756-1766, Vol. 73, No. 3
0022-538X/99/$04.00+0
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Top
Abstract
Introduction
