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Plays a Critical Role in Maintaining Secondary Immunity in the Absence of IFN-
*
Lymphokine Regulation Unit, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, and
Veterinary Resources Program, National Center for Research Resources, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussion References |
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, and TNF-
were important factors
in mediating primary protection. To study whether these and other
factors were involved in maintaining a protective immune response
following secondary infection, normal C57BL/6 mice were first infected
with a sublethal dose of H. capsulatum (1 x
105) and then reinfected 3 wk later with a lethal dose of
H. capsulatum (6 x 105). Under these
conditions, all mice developed an effective immune response with
sterilizing immunity. Moreover, normal C57BL/6 mice treated with
neutralizing Abs against either IL-12, TNF-, or IFN-, depleted of
neutrophils or treated with aminoguanidine at the time of reinfection,
maintained an effective immune response. The ability of animals to
survive a secondary infection in the absence of IFN- was verified by
showing that IFN--/- mice previously immunized with
H. capsulatum and treated with amphotericin B at the time
of primary infection had prolonged survival following reinfection with
a normally lethal dose. It was further shown that enhancement of
TNF- production in IFN--/- mice was the major
mechanism by which these mice were effective in controlling secondary
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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There is a substantial amount of evidence in many intracellular
infections including H. capsulatum that IL-12, through its
ability to regulate IFN- production, is critical in mediating a
protective immune response following primary infection (5, 6). In
addition, there has been interest in the role that IL-12 and IFN-
have in maintaining immunity during a secondary infection (7). In
experimental models of toxoplasmosis (8) and listeriosis (9), it was
shown that mice treated with anti-IL-12 at the time of secondary
infection maintained an effective immune response, while those treated
with anti-IFN- at the time of reinfection had a fatal outcome.
These results established that while memory immunity could be
maintained independent of IL-12, IFN- as the critical effector
molecule was still required. There are now several reports showing that
effective immunity against certain intracellular pathogens can be
achieved in the absence of IFN-. In this regard, it was initially
reported that IFN--/- mice infected with an attenuated
strain of Listeria monocytogenes had prolonged survival
following reinfection to a virulent strain of L.
monocytogenes (10). Moreover, in a separate study, an effective
primary immune response was seen from IFN-R-/- mice
following a primary infection with L. monocytogenes if IL-4
was neutralized or exogenous TNF- was administered at the time of
infection (11). Finally, there is a very recent report showing that
IFN--/- mice infected with Leishmania
donovani had a reduction in parasitic burden 12 wk after
infection mediated through endogenous production of TNF- (12). Taken
together, these results suggest that alternative mechanisms exist in
vivo by which effective immunity to an intracellular infection can be
achieved in the absence of IFN-. Thus, since disseminated
histoplasmosis has emerged as a difficult clinical problem in
individuals such as those infected with HIV, it was of interest for us
to study the mechanisms involved in the maintenance of a protective
immune response and, in particular, to focus on the roles of IL-12,
IFN-, and TNF- in mediating these memory responses.
In the studies reported here, as a prelude to understanding the factors
involved in maintaining memory immunity, we extended our previous
observations in defining the factors involved in mediating an effective
primary immune response to H. capsulatum. Similar to our
previous work (5), animals treated with either anti-IL-12,
anti-IFN-, or anti-TNF- at the time of primary infection
had accelerated mortality compared with the infected control. In
addition, animals depleted of neutrophils or treated with an inhibitor
of nitric oxide synthase (aminoguanidine) also had accelerated
mortality. In striking contrast, normal C57BL/6 mice that were
initially infected with a sublethal dose of H. capsulatum
(1 x 105) and then reinfected 3 wk later with a
lethal dose of H. capsulatum (6 x 105) had
prolonged survival and developed sterilizing immunity. Furthermore,
these animals maintained an effective immune response even if treated
at the time of secondary infection with neutralizing Abs against either
IL-12, IFN-, or TNF- or depleted of neutrophils or treated with
aminoguanidine. The ability of mice to develop an effective memory
response in the absence of IFN- was confirmed using
IFN--/- mice. Moreover, it was shown that, in the
absence of IFN-, TNF- was required to mediate an effective immune
response against H. capsulatum. It was noted, however, that
in some IFN--/- mice with little evidence of H.
capsulatum following secondary infection, there was an increase in
the inflammatory response in several organs, consistent with their
fatal outcome. Thus, while TNF- may be important in mediating
effective memory immunity to H. capsulatum in the absence of
IFN-, it is possible that its overproduction is deleterious to
the host.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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Virus-free female C57BL/6 mice were purchased from Harlan
Sprague-Dawley (Dublin, VA). IFN--/- mice (13)
were obtained from Taconic (Germantown, NY) and kept in the National
Institute of Allergy and Infectious Diseases (NIAID) Animal Care
Facility under pathogen-free conditions. Mice used were between 6 and
10 wk of age. Mice were inoculated i.v. with 0.5 ml sterile PBS with
varying doses of H. capsulatum yeast cells.
Media
HBSS (Biofluids, Inc., Rockville, MD) was used as a wash medium. Complete medium consisting of RPMI 1640 (Biofluids, Inc.) supplemented with 10% FCS (Biofluids), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), sodium pyruvate (1 mM), and 2-ME (0.05 mM) was used for culturing spleen cells.
Preparation and quantitation of H. capsulatum
Yeast phase of H. capsulatum (strain GS-57) was used in all the experiments as previously described (5). Quantitation of H. capsulatum was performed as previously described (5). Briefly, spleens from mice infected with H. capsulatum and treated with various cytokine antagonists were killed at various times postinfection. On some experiments, one-third of each spleen from two or three separate animals in each group was combined and homogenized in a sterile mortar using PBS to prepare a 1:10 w/v suspension. Tenfold dilutions in PBS were plated in duplicate at 0.05 ml/plate on BHI-SAGC medium and incubated for 7 days at 30°C. Colonies were enumerated, and the counts are recorded as CFUs. In additional experiments, three individual spleens, rather than pooled spleens, were quantitated for CFU. In these experiments, the SEM was <10%. In over 20 experiments, we found that combining three pooled spleens gave similar quantitative results to those seen using three individual spleens.
In vivo treatment of mice
Most Abs were purified from ascites by ammonium sulfate
precipitation. Rat anti-mouse IFN- mAb (XMG1.2) (14),
anti-CD4 (GK1.5) (15), and anti-CD8 (2.43) (16) were used to
neutralize IFN- and to deplete CD4 and CD8 T cells, respectively.
Purified mAbs against murine IL-12 (C17.8) (17) and TNF- (HT-11–22)
(18) were obtained from Drs. Giorgio Trinchieri and Mary Stevenson,
respectively, and have been shown to be effective in neutralizing IL-12
and TNF- in vivo. Anti-neutrophil Ab (RB6-8C5) (19) was a generous
gift of Dr. Robert Coffman and was used to selectively bind and deplete
neutrophils. Mice were injected i.p. with anti-IFN- (1 mg) at
the time of primary infection or secondary infection. In some
experiments, mice were injected with 2 mg i.p. 2 days before, at the
time of, and 3 days after reinfection. In both primary and secondary
infection, anti-IL-12 (1 mg), anti-TNF- (1 mg), and
anti-neutrophil (500 µg) were administered i.p. at the time of
infection. Anti-CD4 and anti-CD8 Abs (1 mg) were injected 5 days
before, at the time of, and 5 days after reinfection. This treatment
resulted in a >90% depletion of CD4 or CD8 T cells from spleen at 1
wk after reinfection as assessed by FACS. In experiments designed to
inhibit nitric oxide production in vivo, mice were treated with
aminoguanidine (10 mg/day) given i.p. five times per wk for 2 wk after
reinfection. Amphotericin B
(AmB)2, purchased from
Pharma-Tek Inc. (Huntington, NY), was dissolved in distilled water
shortly before use. Mice were treated with 100 µg of AmB at day 3
postinfection and subsequently three times a wk for a total of nine
doses. Mice were then reinfected 10 days after the last treatment
with AmB.
Cytokine mRNA measurement
Cytokine mRNA levels were determined by semiquantitative RT-PCR
techniques as described previously (5). In brief, total RNA was
isolated from spleen cells by resuspending in RNAzol B (Tel-Test,
Friendswood, TX) and recovering the aqueous phage after addition of
chloroform. RNA was precipitated with alcohol and resuspended in
RNase-free H2O. One µg of total RNA was reverse
transcribed by Moloney murine leukemia virus reverse transcriptase
(Life Technologies, Gaithersburg, MD). The reaction mixture was then
diluted 1:8, and 10 µl was used for specific semiquantitative
amplification of cytokine mRNA with Taq DNA polymerase
(Promega, Madison, WI) and specific cytokine sense and antisense
primers. The number of amplification cycles were as follows: 24,
hypoxanthine phosphoribosyltransferase; 28, IFN-; 30, IL-12 p40; and
35, IL-4, IL-10, NO, GM-CSF, and TNF- (17). Southern transfers of
PCR products were subsequently probed with internal cytokine-specific
oligonucleotides and visualized using the ECL (enhanced
chemiluminescent) detection system (Amersham, Arlington Heights,
IL).
Histology
Mice were necropsied, and sections of lung, liver, kidney, spleen, and heart were immersed in 10% neutral buffered formalin, trimmed, and embedded in paraffin blocks. Sections (6 µm thick) were cut on a microtome and stained with hematoxylin and eosin.
Statistics
Statistical evaluation of differences between means of experimental groups was done by analyses of variance and multiple Student’s t tests. The log-rank was used for statistical analysis of mortality. A value of p < 0.05 was considered to be significant.
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Results |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
|---|
The intent of these studies was first to establish the factors
involved for a protective immune response in a primary challenge to
infection with H. capsulatum and then to determine whether
these same factors were important in maintaining a memory response. In
a previous study (5), it was demonstrated that mice infected with
H. capsulatum (6 x 105) and treated at the
time of infection with anti-IL-12 or anti-IFN- had
accelerated mortality, while animals infected with a lower dose of
H. capsulatum (1 x 105) developed an
effective immune response that protected them from a fatal outcome. We
extended these findings by examining the role of several additional
factors shown to be important in mediating protective immunity against
various intracellular pathogens.
As shown in Figure 1
A, in data
combined from three independent experiments, animals infected with
H. capsulatum (6 x 105) had a mean
survival time of 19.45 ± 2.91 days. Mice treated with
anti-IFN- or anti-IL-12 at the time of infection had
accelerated mortality with mean survival times of 9.7 ± 0.82 and
11.33 ± 1.58 days, respectively (p <
0.0001). Furthermore, in these same experiments, depletion of
neutrophils at the time of infection also resulted in accelerated
mortality (7.89 ± 1.54 days) compared with the infected controls.
This latter effect is consistent with other reports showing that
neutrophils have an important role in controlling primary infection to
other microbial pathogens such as L. monocytogenes (20, 21)
or Candida albicans (22, 23). In an additional group of
experiments, the role of endogenous TNF- and nitric oxide was also
evaluated. As shown in Figure 1B, in data combined from two
independent experiments, animals treated with anti-TNF- at the
time of infection or with aminoguanidine daily also had accelerated
mortality with mean survival times of 10.25 ± 3.20 and 9.125
± 3.02 days, respectively, compared with the mean survival of
control-infected mice of 19.28 ± 4.39 days
(p < 0.0001).
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A, there was a four- to sixfold increase in
H. capsulatum detected from mice treated with
anti-IFN- (p < 0.0001), anti-IL-12
(p < 0.0001), or neutrophil depletion
(p < 0.0001), while in Figure 1B,
there was a two- to threefold and 1-log increase in CFU from mice
treated with aminoguanidine (p < 0.001) and
anti-TNF- (p < 0.0001), respectively.
These results demonstrate a strong correlation between infectious load
and accelerated mortality and underscore the multifactorial regulation
required for protective immunity in a primary response. In addition,
they provide a basis for understanding which factors might be required
for effective immunity in a secondary response to H.
capsulatum.
Protective immunity is maintained in a secondary response to
H. capsulatum in an IL-12- and IFN--independent mechanism
As noted above, while cytokines such as IL-12, IFN-, and
TNF- are critical in controlling primary infection to H.
capsulatum, the factors involved in maintaining an effective
immune memory response may be less stringent (6, 8, 9). Thus, in the
following experiments, we were interested in determining which factors
were required to maintain an effective immune response to reinfection
with H. capsulatum and, in particular, in focusing on the
role of IFN-, since it seems to be an important effector molecule in
mediating inhibition of intracellular growth of H.
capsulatum (24, 25, 26, 27).
In these experiments, mice were initially infected with a low dose of
H. capsulatum (1 x 105) and then
reinfected 3 wk later with a dose normally lethal to naive mice (6
x 105). As shown in Figure 2A, neutralization of IL-12 or
IFN- or depletion of neutrophils at the time of reinfection did not
lead to a fatal outcome in any of the animals in a secondary infection.
As a control (Fig. 2B), additional groups of naive
mice infected simultaneously with the same dose (6 x
105) of yeast cells and treated in a similar manner to
those undergoing reinfection were found to have accelerated mortality,
consistent with the findings in Figure 1.
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is such a critical effector molecule in mediating
protection in a primary infection, it was somewhat surprising that
effective immunity could be maintained in its absence. One explanation
for these results could be the fact that there is an increase in
IFN- at the initiation of a memory response compared with a primary
response (see below). In this regard, it is possible that we did not
achieve effective neutralization using 1 mg of anti-IFN- at the
time of reinfection. Thus, as shown Figure 3, in data combined from two independent
experiments, treatment of mice with 2 mg of anti-IFN- given at
day -2, day 0, and day 3 after reinfection did not lead to a fatal
outcome in any of the mice. Moreover, mice treated with a combination
of Abs against IFN-, IFN-R, and IL-12 at the time of reinfection
still maintained a protective response. Finally, to verify that
anti-IFN- was having a biologic effect in vivo, peritoneal
macrophages from animals treated with anti-IFN- were shown to
have a decrease (>50–70%) in staining for MHC class II compared with
the reinfected controls. These results suggest that effective memory
immunity against H. capsulatum is maintained not only in the
absence of neutrophils, TNF-, or nitric oxide but is also
independent of IL-12 and IFN-.
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To determine whether effective immunity to a secondary challenge
was due to control of H. capsulatum in vivo, the infectious
burden of H. capsulatum was assessed from spleen cells at
various time points following reinfection. As shown in Table I, there was approximately 3 logs
less H. capsulatum from all groups of mice at 6 or 14 days
after reinfection compared with the amount detected following primary
infection. Moreover, there was essentially no H. capsulatum
detected from spleen cells at 60 or 90 days after reinfection in all
groups in either experiment 1 or 2. These results provide further
evidence that IFN- may be dispensable to maintain an effective
immune response following secondary infection.
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To identify any qualitative or quantitative changes in cytokine
production that may be correlative with protection following
reinfection, mRNA expression for several cytokines was assessed by
semiquantitative PCR at various time points postinfection. As shown in
Figure 4, mice reinfected with H.
capsulatum alone had a modest increase in mRNA expression at days
2 and 6 postreinfection compared with mice not reinfected. Moreover, as
might be expected, increased expression of mRNA for IFN- occurred
relatively early (day 2) postreinfection and was diminished 14 days
postinfection, while expression for IFN- in primary infection
occurred later (day 6) and persisted at 14 days after infection.
Interestingly, mice treated with anti-IFN- had an increase in
mRNA for IFN- at 6 days postreinfection and maintained this
expression up to 14 days after reinfection compared with the infected
control. These results are consistent with previous studies showing
that in the course of infection, IFN-R-deficient mice have increased
production of IFN- in response to infection (28, 29), suggesting
that IFN- production in vivo may actually down-regulate its own
induction. Of additional interest was the fact that mice treated with
anti-IFN- also showed an increase in TNF- expression at 6
days postinfection. The fact that TNF- is increased in the absence
of IFN- may be biologically important in this infection and is
further elaborated on below. Finally, mice treated with anti-IL-12
alone had a modest decrease in IFN- at day 6 after reinfection
compared with control-infected mice, demonstrating that IFN-
production in a secondary infection is not dependent on IL-12.
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-/- mice have prolonged survival
following reinfection with H. capsulatum
To provide definitive proof that IFN- is not required to
maintain an effective immune response following reinfection with
H. capsulatum, IFN--/- mice were used for
assessing whether effective immunity could be achieved in a secondary
infection with H. capsulatum. In these experiments, since
IFN- is required to protect animals in a primary response, mice were
first infected with 1 x 105 yeast cells and treated
with AmB starting 3 days after primary infection and then three times
per week for 3 wk. Mice were then reinfected with a lethal dose of
H. capsulatum (6 x 105 yeast cells) 10
days later. As shown in Figure 5A, in data combined from two
independent experiments, IFN--/- mice that were
reinfected had a mean survival time of 32.00 ± 5.65 days, while
similar mice undergoing primary infection had a mean survival time of
9.00 ± 3.00 days (p < 0.0001).
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. As observed
from the data presented in Figure 4, it appeared that wild-type mice
secondarily infected with H. capsulatum and treated with
anti-IFN- had an increase in mRNA expression for TNF-.
Furthermore, since TNF- has been shown to have a role in host
defense against H. capsulatum (5, 6, 30, 31), we reasoned
that TNF- may have a compensatory role in mediating protection
following reinfection in the IFN--/- mice. As shown in
Figure 5B, in data combined from two additional independent
experiments, IFN--/- mice treated with
anti-TNF- at the time of reinfection had accelerated mortality
with a mean survival time of 10.77 ± 3.28 days compared with
reinfected controls with a mean survival time of 29.27 ± 5.41
days (p < 0.0001). This observation, when combined
with the data discussed below, provides strong evidence that TNF-
has a major role in mediating memory immunity to H.
capsulatum in the absence of IFN-. Quantitative burden of H. capsulatum following secondary infection
To verify that prolonged survival in IFN--/-
mice following reinfection was due to control of H.
capsulatum infectious burden, quantitative cultures were performed
on spleen cells (similar results were seen using liver cells) at
various time points after reinfection. As shown in Table II (Expt. 1), relatively low CFU of
H. capsulatum were detected from spleens of
IFN--/- mice at both 7 and 14 days following
reinfection compared with primary infection of IFN--/-
mice. Furthermore, in experiment 2, there was little to no CFU of
H. capsulatum detected from mice at 12 days or even 50 days
after reinfection. Finally, in experiment 3, IFN--/-
mice treated with anti-TNF- at the time of reinfection had an
approximately 3-log increase in the amount of H. capsulatum
(p < 0.005), consistent with their rapid
demise (Fig. 5B).
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-/- mice undergoing primary and
secondary infection simultaneously with various amounts of H.
capsulatum. As shown in Table III,
IFN--/- mice had a 2- to 3-log reduction in CFU from
mice undergoing secondary infection compared with primary infection at
all doses tested (p < 0.001). Furthermore, it
was interesting that depletion of either CD4 or CD8 T cells at the time
of reinfection led to a 2-log increase in the infectious burden, which
correlated with a fatal outcome in all of these mice by 10 days after
infection (data not shown). A potential mechanism for this is discussed
below.
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is significantly enhanced in
IFN--/- mice compared with wild-type controls at
the time of secondary infection
Since the data shown above support a critical role for TNF- in
maintaining an effective immune response in the absence of IFN-, we
assessed mRNA expression for TNF- and other cytokines from spleen
cells of IFN--/- and IFN-+/+ mice
following both primary and secondary infection at different time
points. As shown in Figure 6, relatively
low TNF- expression from IFN--/- mice was noted
both 2 and 7 days after primary infection compared with that induced
following secondary infection. Furthermore, while TNF- mRNA
expression from cells of IFN--/- mice was further
increased at 7 days after primary infection, there was a significant
increase at this time point following secondary infection. Perhaps the
most striking finding was the relative increase in TNF- mRNA
expression seen 7 days postinfection from the IFN--/-
mice compared with the IFN-+/+ mice. These data provide
additional evidence for TNF- having a compensatory role in the
absence of IFN-. It is also important to note that
IFN--/- mice treated with anti-CD4 or anti-CD8
had a marked reduction in mRNA expression of TNF- at 7 days after
secondary infection, consistent both with the increase in infectious
burden (Table III) and with the accelerated mortality in these mice
(data not shown). These data further demonstrate the importance of
TNF- production in maintaining an effective immune response in
IFN--/- mice and demonstrate that both CD4 and CD8 T
cells are potent sources of TNF- production. Since nitric oxide
appears to be an important mediatory in primary infection to H.
capsulatum (Fig. 1) and has been suggested to be involved in
mediating antiparasitic activity in IFN--/- mice
infected with L. donovani (12), its expression was assessed
in this experiment. As shown in Figure 6, there was little detectable
mRNA for nitric oxide 2 days after primary or secondary infection in
the IFN--/- mice compared with the
IFN-+/+ mice, underscoring the importance of IFN- in
eliciting nitric oxide production. Finally, since there is also
evidence that GM-CSF has a role in host defense against H.
capsulatum (32), we assessed whether there was an preferential
increase in GM-CSF expression in IFN--/- mice
following secondary infection. As shown in Figure 6, there does appears
to be an increase in mRNA for GM-CSF in IFN--/- mice
compared with IFN-+/+ mice 2 days after primary and
secondary infection; however, similar expression of GM-CSF was found 7
days after both primary and secondary infection in all mice. Thus,
while we cannot exclude a role for GM-CSF in mediating a protective
response in IFN--/- mice, it appears that the most
obvious difference in mRNA expression from IFN--/- and
IFN-+/+ mice is TNF-.
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-/- mice following secondary infection withH. capsulatum
As noted above, while there was an increase in survival of a
number of IFN--/- mice following reinfection in
several experiments, many of the mice ultimately succumbed. In
evaluating the cause of death, we found that in some experiments,
mortality was associated with disseminated H. capsulatum
infection; however, we were surprised to note that some of the mice
that had died had no evidence of H. capsulatum in the
spleens, livers, or lungs as assessed by quantitative cultures. In
these mice, autopsies were performed to assess the histopathologic
changes in the organs in an attempt to determine the cause of death. As
shown in Figure 7, in wild-type mice
vaccinated with a sublethal dose of H. capsulatum (1 x
105) and reinfected 3 wk later with a lethal dose (6
x 105), inflammation was limited to the liver at 7 days
post-second inoculation. Histopathologically, there was a mild
lymphohistiocytic hepatitis, characterized by multifocal infiltrates
with small numbers of lymphocytes and macrophages in the liver (Fig. 7a). The spleen (Fig. 7b), heart,
and other sampled organs were normal, and there was no evidence of
H. capsulatum yeast. In contrast, IFN--/-
mice treated in a similar manner as above had pale, swollen livers,
splenomegaly, and multiple pale foci scattered in the myocardium when
necropsied 7 days post-second inoculation. Histopathologically, there
was moderate acute and histiocytic hepatitis characterized by moderate
infiltrate of macrophages, neutrophils, and lymphocytes in the liver
(Fig. 7c). In the spleen, red and white pulp were
effaced by an infiltrate of macrophages (Fig. 7d). In
addition, there was mild multifocal acute and lymphohistiocytic
myocarditis (data not shown). Again, H. capsulatum yeast was
not present. Finally, inflammation was most severe in the
IFN--/- mice treated with anti-TNF-. When
necropsied 7 days post-second inoculation, mice had swollen pale livers
and kidneys. The spleen was enlarged (5 times normal) and had discrete
white nodules scattered in the parenchyma. Histopathologically, there
was severe pyogranulomatous disseminated hepatitis, with 40 to 50% of
the hepatic parenchyma effaced by dense infiltrates of macrophages and
neutrophils (Fig. 7e). Splenic parenchyma was largely
effaced by similar inflammatory infiltrates (Fig. 7f). There was multifocal, moderate to severe
pyogranulomatous inflammation affecting the heart, kidneys, adrenal
glands, lymph nodes, lungs, and thymuses (data not shown). Numerous
H. capsulatum yeasts were present in all areas of
inflammation. Taken together, these data suggest that the overall
inflammatory response in the IFN--/- mice is increased
compared with that of wild-type mice and that TNF- plays a central
role in mediating protection against disseminated infection.
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|
Discussion |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
|---|
The intent of these studies was to define the factors involved in
maintaining systemic immunity to H. capsulatum following a
secondary challenge. The initial experiments were aimed at defining the
factors involved in primary immunity and then examining whether these
same factors were required to maintain an effective immune response
following reinfection. Our results showing the importance of endogenous
IFN-, IL-12, and TNF- in mediating protection against primary
i.v. challenge with H. capsulatum are consistent with both
our earlier studies and that of Allendoerfer et al., using a pulmonary
inhalation model (5, 6). In addition, we now demonstrate that mice
treated with an anti-neutrophil Ab at the time of infection had
accelerated mortality and an increase in the infectious burden of
H. capsulatum. These data are in accordance with studies by
other investigators showing that depletion of neutrophils at the time
of primary infection to either L. monocytogenes (20, 21) or
C. albicans (22, 23) led to an increase in the infectious
burden and mortality in these mice. Furthermore, these data are
consistent with other studies showing that neutrophils have some
fungistatic activity against H. capsulatum (33, 34, 35).
Finally, since production of nitric oxide by murine macrophages is
important for the killing of various intracellular pathogens (36), we
evaluated its role in primary immunity to H. capsulatum. We
found that treatment of mice at the time of infection with
aminoguanidine, a potent inhibitor of nitric oxide synthase, resulted
in accelerated mortality. Based on these data, we would postulate that
a protective response to primary infection to H. capsulatum
is multifactorial. Similar to what has been reported for
Listeria infection (21), the initial response may require
neutrophils that would control infection either through a direct
phagocytic mechanism and/or through induction of IL-12 (23). Once IL-12
is produced (IL-12 may also be induced through direct activation of
macrophages), it mediates the generation of an effector Th1 response
involving enhanced production of IFN-, leading to increased TNF-
and nitric oxide production. These latter mediators, working directly
or in concert, would then mediate the intracellular killing of the
pathogen.
Factors involved in secondary immunity to H. capsulatum:
IFN- is not essential for an effective memory immune response
As highlighted above, while effective immunity to primary
infection requires a coordinated response requiring many factors, in a
secondary infection there appear to be compensatory mechanisms that can
mediate protection in the absence of these same factors required for
primary immunity. In this regard, our studies show that neutrophils are
not essential for the maintenance of immunity. Moreover, treatment of
mice at the time of reinfection with inhibitors against either nitric
oxide or TNF- did not alter survival or the ability of mice to
control the infection. In addition, neutralization of IL-12 at the time
of reinfection did not abrogate protection, consistent with previous
work showing that IL-12 was not required for the maintenance of
immunity following secondary challenge to either T. gondii
(8), L. monocytogenes (9), or H. capsulatum (6).
It was striking, however, that inhibition of IFN- at the time of
reinfection to H. capsulatum did not abrogate the
development of an effective immune response. The ability of mice to
maintain a protective immune response in the absence of IFN- was
verified in several ways. We first demonstrated that treating normal
C57BL/6 mice with a neutralizing Ab against IFN- before, during, and
after reinfection did not alter survival. Furthermore, treatment of
mice with a mixture of Abs including anti-IFN-, IFN-R, and
anti-IL-12 at the time of reinfection did not alter survival.
Finally, the observation that IFN--/- mice showed
enhanced survival with control of infection provided conclusive
evidence that IFN- was not absolutely essential for an effective
memory immune response.
It is important to note that IFN--/- or
IFN-R-/- mice have been used to assess the role of
IFN- in mediating a protective immunity to both primary and
secondary challenges with a variety of infectious pathogens. In several
of these studies, IFN-R-/- or IFN--/-
mice can develop an effective immune response leading to prolonged
survival if treated at the time of infection with various immune
manipulations (11, 37) or challenged with a nonlethal pathogen (38).
The ability of IFN--/- mice to develop a protective
immune response in a secondary infection without any additional immune
interventions was first reported by Harty and Bevan (10), who showed
that vaccination of mice with an attenuated strain of L.
monocytogenes induced a protective immune response up to 14 days
following reinfection with a lethal strain of L.
monocytogenes. Moreover, they demonstrated that CD8 T cell
depletion diminished the resistance of these mice, consistent with
their previous work showing that adoptive transfer of Ag-specific CD8 T
cells can provide antilisterial resistance. Thus, our studies are
complementary to the report of Harty and Bevan in providing evidence
that IFN--/- mice have an increase in survival
following reinfection; however, we show that vaccination with a
normally lethal strain of H. capsulatum provides protection
for at least 3 wk and up to 50 days following reinfection. In addition,
while cytolytic CD8 T cells were shown to have an important role in
mediating protective immunity to L. monocytogenes, we noted
that perforin-/- mice maintained an effective immune
response following infection even if treated with anti-IFN- at
the time of reinfection (data not shown). Taken together, these results
suggest that perforin-mediated killing and IFN- are not required for
effective intracellular killing of H. capsulatum following
reinfection.
TNF- plays a critical role in mediating a protective immune
response to secondary infection to H. capsulatum in the
absence of IFN-
While IFN- is an important regulator of intracellular killing
in many experimental models of intracellular infection, information is
now emerging that TNF- may be a critical regulator in control of
intracellular infections in the absence of IFN- (11, 12). The
evidence that TNF- has a prominent role in mediating a protective
immune response is supported by our studies examining the role of
cytokine and/or cell depletion at the time of secondary infection using
both IFN--/- and wild-type mice. First, using
IFN--/- mice, it was shown (Table II) that mice
treated with anti-TNF- at the time of reinfection had a striking
increase in the infectious burden of H. capsulatum that
correlated with accelerated mortality. In addition,
IFN--/- mice depleted of either CD4 or CD8 T cells at
the time of reinfection had accelerated mortality (data not shown) that
was also associated with an increase in the infectious load of H.
capsulatum and a decrease in mRNA expression for TNF-.
Furthermore, in experiments not shown, wild-type mice depleted of both
CD4 and CD8 T cells at the time of secondary infection had accelerated
mortality due to an increase in the infectious burden (our manuscript
in preparation). It is of interest that mRNA expression for both
IFN- and TNF- was markedly diminished from mice depleted of both
CD4 and CD8 T cells compared with that of infected control mice (data
not shown), suggesting that T cell- rather than NK-derived IFN- or
TNF- is mediating host protection. Finally, treatment of wild-type
mice with both anti-IFN- and anti-TNF- at the time of
reinfection resulted in accelerated mortality (data not shown). The
fact that depletion of both IFN- and TNF- led to a fatal outcome
following secondary infection in wild-type mice is consistent with the
finding above that, in the absence of IFN-, TNF- is required to
maintain an effective immune response.
Several studies have shown that the optimal conditions for effective
control of intracellular pathogens in a primary infection involves the
concerted effects of both IFN- and TNF- to augment production of
nitric oxide (39, 40, 41, 42, 43); however, it is possible that there exist
distinct independent pathways for these mediators to exert their
biologic effect in the absence of one another in secondary infection.
Thus, with regard to the data presented in this paper, the mechanism by
which TNF- mediates its effector function in
IFN--/- mice could be twofold. One possibility is that
TNF- acts indirectly by enhancing production of nitric oxide, which
would lead to effective intracellular killing. This mechanism is
supported by a recent study in which IFN--/- mice
infected with Leishmania major and treated with IL-12 had a
reduction in parasitic load that was associated with an increase in
TNF- production (12). Furthermore, in that study, the decrease in
the infectious burden mediated by IL-12 treatment was abrogated by
treating mice with aminoguanidine, suggesting that TNF- was
mediating its biologic effects indirectly through nitric oxide.
Alternatively, TNF- may exert a direct fungicidal and/or fungistatic
effect on intracellular H. capsulatum that is independent of
nitric oxide. A nitric oxide-independent effect was suggested by
studies showing that treatment of IRF1-/- mice (which are
markedly deficient in nitric oxide production) with IL-12 was able to
prolong survival of these mice following infection with
Toxoplasma gondii (44). While not directly assessed, it is
possible that effective immunity in these mice was mediated through an
IL-12 increase in TNF- production, as was demonstrated in the study
by Taylor and Murray (12). To conclude, in the studies reported here,
treatment with aminoguanidine at the time of reinfection did not alter
survival in wild-type mice, suggesting that the presence of IFN-
and/or TNF- alone is sufficient to provide effective intracellular
killing in the absence of nitric oxide. Furthermore, since
anti-TNF- treatment resulted in accelerated mortality in
IFN--/- mice due to overwhelming infection, it would
seem that TNF- is required to mediate an effective biologic response
in the absence of IFN-. Studies are under way to determine whether,
in the absence of IFN-, TNF- exerts its effects directly and/or
through enhancement of nitric oxide.
Increased TNF- production can have both protective and
deleterious effects on host survival
While IFN--/- mice were able to control the
growth of H. capsulatum in vivo following reinfection, it
was of interest that mice in several different experiments had a fatal
outcome 2 or 3 wk following reinfection without evidence of appreciable
H. capsulatum as assessed by quantitative cultures. In
performing autopsies on these mice, while there was no gross evidence
of yeast, there was an increase in the inflammatory response in several
organs including liver, spleen, and heart. Moreover, the fact that
there was myocarditis and myocardial necrosis in some of these mice
provided a mechanism for the fatal outcome. Thus, since TNF- appears
to have an important role in mediating an effective immune response in
these mice, it leads us to speculate that, in IFN--/-
mice, the increased production of TNF- leads to dysregulation of the
normal immune counter-regulatory mechanisms. In this regard, in
wild-type mice at the initiation of a primary infection, the induction
of IFN- leads to enhancement of TNF-, which in combination with
IFN-, leads to optimal nitric oxide production; however, once an
effective primary response is generated, both TNF- and/or nitric
oxide induce a potent negative feedback mechanism capable of inhibiting
further expansion of the response (45). We speculate that the normal
counter-regulatory mechanisms in our reinfection model are perturbed in
the IFN--/- mice, leading to an increase in the
inflammatory response. Furthermore, while the increase in TNF- may
contribute to eradication of H. capsulatum, its ability to
induce cellular necrosis and impair vascular integrity (46) may lead to
a deleterious outcome independent of infectious burden.
One final important point that should be noted is that an increase in
the inflammatory response was also noted following reinfection of
wild-type mice that had been treated with anti-IFN- (data not
shown); however, none of the wild-type mice that were treated with
anti-IFN- had a fatal outcome due to infectious burden or
inflammation. These data point out that there may be differences in
IFN--/- mice compared with wild-type mice treated with
anti-IFN- at the time of reinfection and suggest that
compensatory mechanisms exist in the IFN--/- mice
(i.e., increased TNF-) that are not fully replicated in wild-type
mice treated with anti-IFN-. Nevertheless, while the increased
inflammation in the IFN--/- mice is of interest, the
important point is that prolongation of survival is noted following
reinfection in both groups of mice.
Clinical implications for treating immunocompromised hosts with disseminated H. capsulatum
These studies provide a further understanding of the factors
regulating both primary and secondary infection to H.
capsulatum. One potential caveat to the findings observed from our
model is that mice are infected systemically through the i.v. route.
Since the natural route of infection occurs through pulmonary exposure,
our model may be considered to be less physiologic than the pulmonary
model; however, in a recent report by Allendoerfer et al., using a
pulmonary inhalation model to study primary infection against H.
capsulatum, they found that similar factors (i.e., IFN-, IL-12,
and TNF-) were involved in mediating a protective immune response
(6). Furthermore, since disseminated histoplasmosis that occurs in
immunocompromised hosts is often acquired through reactivation of a
previous primary pulmonary infection, we believe that our findings for
the regulation of memory immunity are indeed physiologically relevant.
Finally, to apply these findings for clinical use, we have shown in a
previous study that SCID mice infected with H. capsulatum
and treated with a combination of AmB three times per week and IL-12
(500 ng) once a week for 3 wk only led to prolonged survival and
sterilizing immunity at 60 days postinfection (47). Furthermore, these
treatments led to an increase in both IFN- and TNF- production
from spleen cells following in vitro stimulation, consistent with the
importance of these molecules in mediating intracellular killing of the
organism. Thus, if the potential toxicities of IFN- and TNF- can
be managed, we propose that IL-12 in combination with AmB would be an
efficacious regimen for treatment of disseminated H.
capsulatum in immunocompromised hosts.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: AmB, amphotericin B; GM-CSF, granulocytemacrophage CSF.
Received for publication August 8, 1997. Accepted for publication October 15, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
|---|
. J. Immunol. 155:785.[Abstract]
synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol. 153:2533.[Abstract]
but is partially independent of IL-12. J. Immunol. 155:3427.[Abstract]
. Immunity 3:109.[Medline]
treatment ameliorate disease by an intracellular pathogen in IFN- receptor-deficient mice. J. Immunol. 157:4746.[Abstract]
: effect of IL-12 in experimental visceral leishmaniasis in IFN- gene-disrupted mice. J. Exp. Med. 185:1231. genes. Science 159:1725.
by an intracellular parasite and induces resistance in T cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115. and TNF- and occurs via a nitric oxide-dependent mechanism. J. Immunol. 155:2545.[Abstract]
is not due to a limitation of the supply of methionine or cysteine to the fungus. Infect. Immun. 60:698.. Infect. Immun. 55:1014. cooperates with lipopolysaccharide to activated mouse splenic macrophages to an antihistoplasmal state. Infect. Immun. 61:1468. receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response. J. Exp. Med. 181:961. receptor-deficient mice generate antiviral Th1-characteristic cytokine profiles but altered antibody responses. J. Immunol. 153:2029.[Abstract]
plays a role in host defense against Histoplasma capsulatum. J. Infect. Dis. 162:1349.[Medline]
knockout mice. J. Exp. Med. 180:43. receptor. J. Immunol. 154:5338.[Abstract]
: central regulatory cytokine in the induction of macrophage antimicrobial activities. Pathobiology 59:182.[Medline]
-lipopolysaccharide-activated macrophages from Lshs and Lshr congenic mouse strains. Infect. Immun. 59:3935.-induced L-arginine-dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous TNF-. J. Immunol. 148:568.[Abstract]
and TNF- blocks in vivo synthesis of nitrogen oxides from L-arginine and protection against Francisella tularensis infection in Mycobacterium bovis BCG-treated mice. Infect. Immun. 61:689. and TNF- in mice with Rickettsia conorii-infected endothelium: impairment of rickettsicidal nitric oxide production resulting in fatal, overwhelming rickettsial disease. Infect. Immun. 62:1952.This article has been cited by other articles:
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|
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 F. S. Dhabhar and K. Viswanathan Short-term stress experienced at time of immunization induces a long-lasting increase in immunologic memory Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2005; 289(3): R738 - R744. [Abstract] [Full Text] [PDF]
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P. Zhou, B. L. Freidag, C. C. Caldwell, and R. A. Seder Perforin Is Required for Primary Immunity to Histoplasma capsulatum J. Immunol., February 1, 2001; 166(3): 1968 - 1974. [Abstract] [Full Text] [PDF]
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G. H. Chen, R. C. Reddy, M. W. Newstead, K. Tateda, B. L. Kyasapura, and T. J. Standiford Intrapulmonary TNF Gene Therapy Reverses Sepsis-Induced Suppression of Lung Antibacterial Host Defense J. Immunol., December 1, 2000; 165(11): 6496 - 6503. [Abstract] [Full Text] [PDF]
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R. Allendoerfer and G. S. Deepe Jr. Regulation of Infection with Histoplasma capsulatum by TNFR1 and -2 J. Immunol., September 1, 2000; 165(5): 2657 - 2664. [Abstract] [Full Text] [PDF]
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G. Yap, M. Pesin, and A. Sher Cutting Edge: IL-12 Is Required for the Maintenance of IFN-{gamma} Production in T Cells Mediating Chronic Resistance to the Intracellular Pathogen, Toxoplasma gondii J. Immunol., July 15, 2000; 165(2): 628 - 631. [Abstract] [Full Text] [PDF]
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H. W. Murray and S. Delph-Etienne Roles of Endogenous Gamma Interferon and Macrophage Microbicidal Mechanisms in Host Response to Chemotherapy in Experimental Visceral Leishmaniasis Infect. Immun., January 1, 2000; 68(1): 288 - 293. [Abstract] [Full Text] [PDF]
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G. S. Deepe Jr., R. Gibbons, and E. Woodward Neutralization of Endogenous Granulocyte-Macrophage Colony-Stimulating Factor Subverts the Protective Immune Response to Histoplasma capsulatum J. Immunol., November 1, 1999; 163(9): 4985 - 4993. [Abstract] [Full Text] [PDF]
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S. K. Bliss, Y. Zhang, and E. Y. Denkers Murine Neutrophil Stimulation by Toxoplasma gondii Antigen Drives High Level Production of IFN-{gamma}-Independent IL-12 J. Immunol., August 15, 1999; 163(4): 2081 - 2088. [Abstract] [Full Text] [PDF]
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R. Allendorfer, G. D. Brunner, and G. S. Deepe Jr. Complex Requirements for Nascent and Memory Immunity in Pulmonary Histoplasmosis J. Immunol., June 15, 1999; 162(12): 7389 - 7396. [Abstract] [Full Text] [PDF]
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B. A. Wu-Hsieh, W. Chen, and H.-J. Lee Nitric Oxide Synthase Expression in Macrophages of Histoplasma capsulatum-Infected Mice Is Associated with Splenocyte Apoptosis and Unresponsiveness Infect. Immun., November 1, 1998; 66(11): 5520 - 5526. [Abstract] [Full Text] [PDF]
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R. Allendoerfer and G. S. Deepe Jr. Blockade of Endogenous TNF-{alpha} Exacerbates Primary and Secondary Pulmonary Histoplasmosis by Differential Mechanisms J. Immunol., June 15, 1998; 160(12): 6072 - 6082. [Abstract] [Full Text] [PDF]
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 P. Zhou and R. A. Seder CD40 Ligand Is Not Essential for Induction of Type 1 Cytokine Responses or Protective Immunity after Primary or Secondary Infection With Histoplasma capsulatum J. Exp. Med., April 20, 1998; 187(8): 1315 - 1324. [Abstract] [Full Text] [PDF]
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L. Stobie, S. Gurunathan, C. Prussin, D. L. Sacks, N. Glaichenhaus, C.-Y. Wu, and R. A. Seder The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge PNAS, July 18, 2000; 97(15): 8427 - 8432. [Abstract] [Full Text] [PDF]
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