By
From the * Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892; and Department of
Inflammation and Autoimmune Diseases, Hoffmann-La Roche, Inc., Nutley, New Jersey, 07110
The induction by IFN- of reactive nitrogen intermediates has been postulated as a major
mechanism of host resistance to intracellular pathogens. To formally test this hypothesis in vivo,
the course of Toxoplasma gondii infection was assessed in nitric oxide synthase (iNOS)
/
mice.
As expected, macrophages from these animals displayed defective microbicidal activity against
the parasite in vitro. Nevertheless, in contrast to IFN-
/
or IL-12 p40
/
animals, iNOSdeficient mice survived acute infection and controlled parasite growth at the site of inoculation.
This early resistance was ablated by neutralization of IFN-
or IL-12 in vivo and markedly diminished by depletion of neutrophils, demonstrating the existence of previously unappreciated
NO independent mechanisms operating against the parasite during early infection. By 3-4 wk
post infection, however, iNOS knockout mice did succumb to T. gondii. At that stage parasite expansion and pathology were evident in the central nervous system but not the periphery suggesting that the protective role of nitric oxide against this intracellular infection is tissue specific rather than systemic.
Reactive nitrogen intermediates (RNI)1, including nitric
oxide (NO), have been identified as important effector molecules which restrict pathogen growth in infected
hosts (1). Expression of distinct NO synthase enzymes results in synthesis of RNI by constitutive and/or inducible
pathways in a number of cell types including endothelial
cells, epithelial cells, fibroblasts, hepatocytes, muscle cells,
neutrophils, and phagocytes (1, 2). Murine macrophages
are one of the best characterized of these RNI sources. In
the latter cells, NO synthesis is not constitutive but requires stimulation by lipopolysaccharide and/or cytokines which
enhance transcription of the inducible nitric oxide synthase
(iNOS) enzyme, leading to the conversion of L-arginine to
L-citrulline and NO. Induction of the high output NO
pathway in macrophages is preferentially driven by Type I
immune responses in which production of IFN- A well studied inducer and target of the NO pathway is
the intracellular protozoan Toxoplasma gondii (17). This opportunistic pathogen is able to infect and propagate in virtually all nucleated host cells (18). Nonetheless, in immunocompetent individuals infection is largely asymptomatic
and is characterized by a brief acute stage in which rapidly
replicating tachyzoites disseminate to peripheral host tissues
(19). Fulminant infection is prevented by a potent innate
immune response that is largely T cell independent and leads
to the transformation of the parasite into a dormant bradyzoite
form, which is confined primarily to the central nervous system (CNS). Parasite latency in the chronic stage of infection is maintained by an adaptive T cell response. IFN- Several hypotheses have been proposed to explain the
role of IFN- Recently mice with a targeted disruption of the NOS2
(iNOS) gene have been generated by homologous recombination technology (32). Macrophages from these mutant
animals fail to express detectable iNOS mRNA, protein or
enzyme activity and consequently are unable to produce
significant levels of nitrite (NO2 Experimental Animals.
Mice with a targeted disruption of the
NOS2 gene (iNOS ko) were generously provided by Drs. J.D.
MacMicking, C. Nathan (Cornell University Medical College, New
York), and J.S. Mudgett (Merck Research Laboratories, Rahway,
NJ). These mice were generated as previously described (32) with
a gene replacement vector pINOS-RV1 that was designed to delete the 5
Parasites and Experimental Infection.
Tachyzoites of the virulent RH were maintained in vitro by infection of human foreskin fibroblasts and biweekly passage in DMEM (GIBCO BRL,
Gaithersburg, MD) supplemented with 1% FCS (Hyclone Laboratories, Logan UT), penicillin (100 U/ml), and streptomycin (100 ug/ml). Cysts of the avirulent ME49 strain (initially provided by Dr. J. Remington, Palo Alto Research Foundation) were
harvested from the brains of C57BL/6 mice which had been inoculated with ~20 cysts intraperitoneally 1 mo prior. For experimental infections, mice received 20 ME49 cysts or PBS (Biowhitaker, Walkersville, MD) by either the intraperitoneal (i.p.) or
peroral (p.o) route. Control inoculations with normal brains suspensions failed to elicit detectable inflammatory responses, NK
cell cytotoxicity or significant increases in cytokine levels (data not
shown). Soluble tachyzoite antigen (STAg) was prepared as described previously (24).
In Vivo Assessment of Acute and Chronic Infection.
Acute tachyzoite
growth was assessed using cytocentrifuge smears of cells from infected animals (22). Samples were prepared from 1.5 × 105 peritoneal exudate cells in a Cytospin (Shandon Lipshaw, Pittsburgh, PA) set for 5 min at 1,000 RPM. Slide preparations were fixed in
absolute methanol for 5 min and then stained with Diff-Quik (Baxter Healthcare Corporation, McGaw Park, IL), a modified
Wright-Giemsa stain, as specified by the manufacturer. Differential analyses, including assessment of the number of infected cells,
were performed on 400-500 cells using an oil immersion (100×
objective). In the experiments indicated, the presence of parasites
in heart, lung, liver, spleen, and peritoneum was assessed by microscopic examination of impression smears made from tissues at
5-6 d after infection.
is prominent. Interruption of the RNI pathway with enzyme antagonists inhibits the ability of macrophages to kill pathogens both in vitro and in vivo (3). Based on the above
evidence, a paradigm of host resistance has emerged in
which RNI, produced by cytokine activated macrophages,
are important effectors of the Type 1 immune response
against bacterial, fungal, helminth, and protozoan infectious agents (1).
has been shown to be crucial both for the early control of
tachyzoite expansion and for preventing reactivation of dormant parasite stages. Thus, anti-IFN-
mAb-treated, as well
as IFN-
/
mice rapidly succumb to primary infection
with normally avirulent parasite strains and this enhanced
susceptibility is associated with uncontrolled tachyzoite replication in the periphery (20). Similarly, acute disease can
be triggered in chronically infected animals by treatment with the same IFN-
-neutralizing mAb (23, 24). Finally,
exogenous administration of rIFN-
increases resistance to
acute infection while decreasing the incidence of encephalitis in the chronic stage (25, 26).
in host resistance to T. gondii. One mechanism that is readily demonstrable in vitro is the ability of
the cytokine to activate macrophages to kill intracellular
parasites (27, 28). The involvement of the RNI in this
IFN-
-mediated protection is based on the observation
that L-NMMA, a competitive analog of L-arginine, simultaneously inhibits NO synthesis and intracellular tachyzoite
killing by cytokine activated peritoneal and bone marrow- derived macrophages as well as microglial cells (9, 29, 30). A key function for NO in control of T. gondii infection is
also supported by in vivo observations. Mice in which NO
synthesis is impaired as a result of genetic disruptions of the
IFN-
or interferon regulatory factor-1 (IRF-1) genes succumb to acute infection within 14 d of parasite exposure
(22, 31). Similarly, animals treated with the RNI inhibitor
aminoguanidine also display enhanced susceptibility (11).
However, in the latter case, the mice survive the acute
stage but develop accelerated disease progression later in infection as assessed by the presence of increased parasite numbers and inflammatory infiltration in CNS tissue. The
basis of this discrepancy is not clear but may relate to the
impairment of effector functions unrelated to NO synthesis
in the IFN-
and IRF-1 knockout (ko) mice or to incomplete inhibition of NO in the drug-treated animals.
) or nitrate (NO3
). iNOS
ko animals have been shown to display increased susceptibility to infection with the gram-positive bacterium Listeria
monocytogenes (32) as well as the intracellular protozoan,
Leishmania major (33). In the present study, we have used
iNOS ko mice to formally assess the requirement for RNI
in host resistance to T. gondii. As predicted, macrophages
from these mutant animals were defective in parasite killing
in vitro. Surprisingly, however, control of acute infection
in vivo was unaffected by the iNOS deficiency. Our data
thus challenge the view that synthesis of RNI, an established correlate of in vitro killing, is a primary mechanism
of innate resistance to T. gondii in vivo and argue instead
that the major role of this effector function is to maintain
control of established infections.
end of the NOS2 gene (proximal 585 bases of the promoter and the exons 1-4). The iNOS ko animals used for our experiments were obtained from homozygous inbreeding in the F2
generation (129SvEv × C57BL/6). As shown in Fig. 1 (inset),
initial experiments comparing C57BL/6 (Division of Cancer Treatment, National Cancer Institute, Frederick, MD) and C57BL/6 × 129/J F1 (The Jackson Laboratory, Bar Harbor, ME) mice revealed no difference in the outcome of Toxoplasma infection over
the time period analyzed (3 mo). The latter finding is consistent with previously published data on the susceptibility of the parental strains, 129 and C57BL/6, to avirulent T. gondii (34, 35). Furthermore, in unrelated experiments other 129 × C57BL/6 hybrid strains (129 Sv Ev or 129/SvJ or 129/Ola) survived at least 3 mo after i.p. infection with 20 cysts of ME49 (data not shown). Due to
their greater availability, C57BL/6 mice were used as controls in
all subsequent experiments. As previously described (36), IL-12
p40
/
mice were generated by genetically disrupting exon 3 through a homologous recombination event between the wildtype gene sequence and a mutant allele carrying the PGK-1 neo
gene. Mice carrying the IL-12 p40 mutant allele were backcrossed five times to the C57BL/6 genetic background followed
by intercross of the heterozygotes in order to generate mice homozygous for the targeted mutation (IL-12 p40
/
). Breeding
pairs of mice with a targeted disruption of the IFN-
gene were
originally provided by Dyana Dalton and T. Stewart (Genentech,
San Bruno, CA) (37). The IFN-
knockout mice used were at
the seventh generation of back-crossing to the C57BL/6 strain.
Animals were housed in specific pathogen-free conditions and
both male and female mice were used for experiments at 5-12 wk
of age.
Fig. 1.
In contrast to IFN- ko and IL-12 p40
/
mice, iNOS ko
animals survive acute infection with T. gondii (ME49). Mice were infected either by the i.p. (A) or p.o. (B) route of infection with 20 ME49
cysts. The data shown in A are pooled from three independent experiments and involve a minimum of 10 ko mice per group. The experiment
presented in B involved five mice per group and is representative of three
performed. As described in Materials and Methods, infected C57BL/6 × 129/J F1 and C57BL/6 animals displayed similar survival patterns (inset).
[View Larger Version of this Image (20K GIF file)]
In Vitro Assessment of Tachyzoite Killing.
Resident macrophages
and inflammatory macrophages were harvested from animals
which were untreated or inoculated i.p. 4-5 d previously with either 1.5 ml of 3% thioglycollate (Sigma) or 20 cysts of the ME49
strain. Cells were harvested by injecting cold RPMI into the
peritoneal cavity and plated at 2 × 105 per well in 96-well plates
for 2 h in the presence or absence of rMuIFN- 100 U/ml (generously provided by Genentech, Inc., San Francisco, CA). Cultures were incubated overnight in the presence of medium alone
or RH tachyzoites (0.2 or 1.0 per cell). At this time, an aliquot of
supernatant was harvested from the cultures for measurement of
nitrite (NO2
) levels (see below) and the remaining cells were
pulsed with Uracil-[5,6-3H] (ICN Pharmaceuticals, Inc., Irvine,
CA) at 0.5 µCi/well for an additional 12 h to measure T. gondii
proliferation (38, 39). An incubation period of 24 h followed by a
12-18-h pulse with uracil-[5,6-3H] was found to be optimal in
our assay. The incorporation of radioactive uracil was determined
by liquid scintillation counting. In indicated experiments NGmonomethyl-L-arginine (L-NMMA; Calbiochem-Novabiochem
Corporation, LaJolla, CA) was added (1 mM) during the initial
IFN-
activation period. The percentage of killing was determined by the following calculation:
Cell Cultures and Serum Preparation.
Single cell suspensions
were prepared from spleen and peritoneal cells harvested at various time points post infection. Peritoneal cells were cultured at
4 × 105 cells and spleen cells at 8 × 105 per well in a total volume
of 200 µl in a medium consisting of RPMI-1640 (Bio Whittaker)
supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), Hepes (10 mM), and
2-mercaptoethanol (5 × 105 M) in the presence or absence of
STAg (5 µg/ml). Supernatants were harvested 72 h later for IFN-
,
IL-12, and nitrite determinations.
NO, IFN-, and IL-12 Measurements.
Nitrite (NO2
) levels
were used as an indicator of reactive nitrogen intermediates in
samples and were measured by the Griess assay (42). In brief, 100-µl
aliquots of supernatant were added to 96-well plates followed by
a 100 µl of a 1:1 mixture of 1% sulfanilamide dihydrochloride
(Sigma) in 2.5% H3PO4 and 0.1% naphthylethylenediamide dihydrochloride (Sigma) in 2.5% H3PO4. After a 10-min incubation at room temperature, the absorbance of the samples (A550) was read spectrophotometrically and units of nitrite (range of sensitivity: 4-250 µM) determined by comparison with a standard curve
generated with sodium nitrite (NaNO2) (Sigma). Levels of IFN-
and IL-12 were assayed by 2-site ELISA as previously described
(22). Cytokine levels were quantitated by reference to standard
curves generated with rIFN-
(Genentech) or rIL-12 (provided
by Genetics Institute, Cambridge, MA).
In Vivo Anti-Granulocyte, Anti-IFN-, Anti-IL-5, and Anti-
IL-12 treatments.
For cytokine depletion, mice were treated 1 d
before infection with 1 mg anti-IFN-
mAb (rat IgG1, XMG6
[43]), 2 mg anti-IL-5 mAb (rat IgG1, TRFK-5 [44]), or 1 mg
anti-IL-12 mAb (C17.8, rat IgG2a [45]). The anti-granulocyte
IgG2b mAb, RB6-8C5, originally derived by Robert Coffman,
was administered initially on d 0 at 0.5 mg and subsequently on
day 2 and 4 at 0.25 mg per mouse. The ascites employed was produced by Harlan Bioproducts for Science, Inc. (Indianapolis, IN)
from nude mice inoculated with the hybridomas and partially purified by ammonium sulfate precipitation. Normal rat Ig (Sigma)
was used as a control.
Statistical Analyses. Statistical determinations of the difference between means of experimental groups was determined using an unpaired, two tailed Student's t test.
We and others have previously demonstrated that mice
with impaired IFN- function succumb to acute infection
with T. gondii (21, 22). Loss of NO synthesis is one striking
immune defect that is apparent in these mice and could
contribute to the observed lack of parasite control. To assess the role for RNI in host resistance against this pathogen, iNOS ko mice were infected with 20 cysts of the
ME49 strain and their survival compared with that of IFN-
ko and C57BL/6 control mice. As previously reported,
IFN-
ko mice succumbed to i.p. infection within 9 d of
parasite inoculation, whereas 100% of the control animals
remained alive for the 40 d of the experiment (Fig. 1 A).
Similarly, mice with a targeted disruption of the IL-12 p40
subunit (IL-12 p40
/
) that are also defective in both
IFN-
(36) and NO synthesis (Fig. 2 A) failed to survive
the acute stage of infection (Fig. 1 A). In striking contrast,
iNOS ko animals infected under the same conditions survived for 19-24 d after exposure and thus displayed an intermediate pattern of susceptibility. It was formally possible
that the difference in survival between iNOS
/
and
C57BL/6 animals might be due to the different genetic
backgrounds of the two strains. However, this appears extremely unlikely since both parental strains, C57BL/6 and
129, are known to control avirulent T. gondii infection for at least 4 wk longer than the iNOS-deficient animals (34,
35). Moreover, the analysis performed here of survival rates
in C57BL/6 and 129 × C57BL/6 F1 mice receiving the
same ME49 challenge as the iNOS animals failed to reveal
a difference in mortality over the first 90 d of infection
(Fig. 1 A, inset).
One concern raised by our data was that i.p. inoculation
of the parasites does not induce the same mechanisms of
immunity as the natural, p.o. route of infection. However,
as shown in Fig. 1 B, p.o. infection with ME49 cysts led to
a mortality pattern similar to that observed after i.p. inoculation (Fig 1 A). Thus, control animals exhibited no mortality during the observation period whereas IFN- ko
mice succumbed within 1 wk and iNOS mice at ~3 wk
after parasite challenge.
Previous studies have demonstrated that T. gondii stimulates NO production in infected animals and have suggested that iNOS (NOS2) rather than NOS1 or NOS3 is
the primary enzyme involved in its induction. However, it
was formally possible that the latter isoforms might compensate for the deficiency in the iNOS ko animals. Measurement of the NO derivative, nitrite (N02) in supernatants of peritoneal cells harvested from 5-d infected animals
revealed synthesis of NO in C57BL/6 but not iNOS ko
cultures and thus failed to demonstrate a compensatory mechanism (Fig. 2 A). A similar deficit in NO synthesis was also observed in cultured spleen cells from these animals (C57BL/6
animals: naive,
3 µM, 5-day infected = 29 ± 6 µM; 5-d
infected iNOS ko mice,
3 µM). Interestingly, peritoneal
and spleen cells from IL-12 p40
/
and IFN-
ko animals
were also unable to synthesize significant levels of nitrite even
after stimulation with STAg. The latter findings are likely
to reflect the impaired IFN-
synthesis in these animals.
Based on the observed time of death, we postulated that
iNOS ko mice effectively restrict growth of the acute
tachyzoite form. To test this hypothesis, peritoneal cells
were recovered from mice at 5 d or brain tissue at 15 and
20 d after parasite challenge and the number of intracellular
tachyzoites or cysts determined. As shown in Fig. 2 B, the
percentage of infected exudate cells recovered from iNOS
and C57BL/6 was comparable and considerably less than
that detected in samples from IFN- ko or IL-12 p40-deficient mice (1-2% versus 30-40%, respectively) indicating
that tachyzoite growth is restricted during the acute stage in
the former mouse strains. Consistent with this result, differential counts of the peritoneal exudate cells from the infected mice revealed a comparable inflammatory response
in C57BL/6 and iNOS ko animals (Fig. 2 C). In contrast,
corresponding exudates from both IL-12 p40
/
and IFN-
ko mice contained over twice the number of cells
(data not shown) and significantly more granulocytes than
either the iNOS ko or C57BL/6 samples that were equivalent (Fig. 2 C). Since we clearly observed an induction of
nitrite in the C57BL/6-derived PEC and spleen cell cultures, but not in comparable cultures of iNOS ko cells (Fig.
2 A and text), these data indicate that the control of parasites in the acute stage of infection is not dependent upon
the microbicidal activity of nitric oxide. In support of this
conclusion, impression smears of heart, liver, lung, and spleen tissues revealed detectable tachyzoite replication in 5 d
infected IFN-
ko but not iNOS ko mice (data not shown)
arguing against the possibility that loss of parasite control
occurs in the iNOS-deficient animals but at a location distinct from the peritoneal inoculation site.
The mortality of iNOS
ko animals at 3-4 wk after infection (Fig. 1) suggested that
the control of parasite replication in the CNS, particularly
the brain, might be impaired in these mice. To investigate
this hypothesis, cysts were quantitated in brain homogenates or in tissue sections from infected iNOS ko mice as well as the parental C57BL/6 and C57BL/6 × 129 F1
strains. As shown in Fig. 3, more than twice as many cysts
were apparent in the brains of iNOS ko as compared to
C57BL/6 animals at days 12 and 21 after infection. In contrast, the parental strains, C57BL/6 and C57BL/6 × 129, displayed comparable cyst counts (1,600 ± 368 versus 1360 ± 305 cysts per brain, respectively, [n = 5]) as late as 30 d after infection. Moreover, inflammation was clearly more extensive in brain sections of infected iNOS ko as compared to C57BL/6 animals (Fig. 3, C-F). In addition, the
sections from the iNOS ko, as opposed to control mice,
displayed numerous necrotizing lesions (Fig. 3 E) that in
many cases were associated with active tachyzoite replication. Nonetheless, immediately before developing an obvious moribund state, peripheral tissues (lung, liver, and spleen) from infected iNOS ko and C57BL/6 controls displayed indistinguishable histologic changes consisting of
moderate peribronchial and periarterial inflammation in the
lung, slight periportal inflammation, granulomatous hepatitis with poorly formed granulomas and occasional necrotic hepatocytes in the liver, and extramedullary hematopoiesis
in the spleen. Closer to the time of death, the iNOS ko animals were severely depressed, demonstrated a weakness in
all four limbs with the rear limbs more strongly affected,
and had closed eyes. These symptoms are consistent with a
severe necrotizing encephalitis leading to mortality.
Macrophages from iNOS ko Mice Fail to Control In Vitro Replication of T. gondii Tachyzoites.
The low frequency of
tachyzoites in peritoneal exudates at 5 d after parasite challenge indicated that iNOS ko animals control T. gondii replication in this site during the acute stage of infection. From
the latter observation and the predominance of large mononuclear cells in the peritoneal cavity, it might be predicted
that iNOS ko macrophages are capable of intracellular parasite killing. To test this hypothesis, we evaluated the toxoplasmacidal activity of elicited and resident peritoneal cell
populations from C57BL/6 and iNOS ko mice. In our initial experiments, inflammatory cells were collected from
iNOS ko and C57BL/6 animals that were i.p. inoculated
with thioglycollate (Table 1). As previously described (46),
differential counts revealed that macrophage/monocytes were
the primary cell type in the exudates from both strains (80- 90% macrophage, 10% lymphocyte, 5% eosinophil, 2% mast
cell). Tachyzoites were added to the cultures and the
amount of parasite growth monitored by measurement of
3H-uracil incorporation. As expected, unactivated cultures
from either iNOS ko or C57BL/6 mice exhibited a 70-
100-fold increase in nucleotide incorporation after the addition of tachyzoites, suggesting that parasite replication
had occurred. Microscopic evaluation confirmed the latter
assumption: at 48 h after the addition of tachyzoites, the
number of parasites per cell increased from 1.7 to 6.3 in
C57BL/6 and from 2.1 to 4.5 in iNOS ko-derived cultures. Tachyzoite proliferation in the cells from C57BL/6
mice was dramatically reduced when the cells were pretreated with IFN- as measured by the 3H-Uracil assay (Table 1, >90% reduction ) or microscopic examination (t = 0, 1.7; t = 48 h, 1.5 tachyzoites per cell) and, in agreement with previous studies, this inhibition was reduced to less
than 3% by the inclusion in the assay of L-NMMA, an established nitric oxide antagonist (9, 10, 29). Consistent
with the postulated toxoplasmacidal role for RNI, IFN-
-
treated macrophages from iNOS ko animals were markedly
impaired in the ability to kill exogenously added parasites
(Table 1). The mean killing in activated cells from iNOS
ko animals was 13.9 ± 5.8% versus 91.7 ± 2.6% in cultures
from C57BL/6 mice (P
0.001, n = 10).
We have previously reported that i.p. challenge with T. gondii induces an influx of macrophages, neutrophils and
lymphocytes into the peritoneal cavity and that the cellular
composition of exudates from naive and 5-d infected animals is significantly different. One interpretation of our
data is that the cells required to kill T. gondii in vivo are recruited to the site of infection and are thus not normally
present in either resident or thioglycollate elicited populations. To test this hypothesis we assessed the microbicidal
activity of resident and T. gondii elicited cell populations
against exogenously added tachyzoites (Table 2). Cells from
naive C57BL/6 mice, although initially unable to kill T. gondii, efficiently limited parasite replication when IFN- was added to the cultures. As expected, cells from infected
C57BL/6 mice limited tachyzoite growth even in the absence of exogenously added IFN-
, a finding consistent
with the substantial endogenous production of this cytokine by PEC from infected animals (47) (Table 3). Neither
resident nor T. gondii elicited cells from iNOS ko animals
mediated appreciable parasite killing in the presence or absence of exogenously added cytokine. In these experiments, the mean killing in iNOS ko cultures was 13.2 ± 5.2%
versus 88.1 ± 3.7% for C57BL/6 cells (P
0.01, n = 10).
Together, our data indicate that both in vitro and ex vivo
macrophages from iNOS ko mice are defective in their
ability to kill the parasite although the same animals clearly
control early infection in vivo.
|
|
We and others have previously demonstrated that control of acute T. gondii infection in normal hosts is highly dependent upon the induction of IL-12 and IFN- (22, 47). To determine whether the same effector mechanism operates in the
absence of iNOS, we first assessed the production of these
cytokines during early infection with the ME49 strain (Table 3). IL-12 p40 levels measured in sera or cell cultures
(peritoneal or spleen) were not significantly different in 5-d
infected iNOS ko and C57BL/6 animals. Similarly, levels
of IFN-
were comparable in sera and splenic cell supernatants of cultures from knockout and control mouse strains.
Nevertheless, peritoneal cell cultures from iNOS ko animals produced higher levels of IFN-
than cultures from
control mice. The latter results suggest that the iNOS deficiency may have a localized rather than systemic effect on
the regulation of IFN-
synthesis. In vitro restimulation of
spleen and peritoneal cell cultures substantially augmented
production of both cytokines but failed to reveal further differences between the knockout and control animals (Table 3).
As might be predicted by the intact production of both
IL-12 p40 and IFN-, these cytokines were found to play a
critical role in the control of acute T. gondii infection by
the iNOS ko animals. Thus, treatment with neutralizing
mAb against IL-12 or IFN-
led to enhanced mortality of
the infected knockout mice with a kinetics comparable to
that previously observed in antibody treated wild-type animals (Fig. 4). Taken together, the above experiments argue
that iNOS-deficient mice survive acute infection as a result
of an IL-12/IFN-
-dependent mechanism indistinguishable from that arising in conventional mice and not because
of the induction of a normally inactive pathway of host resistance.
Neutrophils Contribute to Acute Resistance against T. gondii in both iNOS ko and Control Mice.
The ability of iNOS ko
mice to control T. gondii infection in vivo despite their impaired macrophage-toxoplasmacidal function suggested that
effector cells other than activated macrophages/monocytes may be crucial in mediating host resistance at the acute
stage. One alternative effector cell we considered is the
PMN since purified human PMN have been shown to restrict T. gondii replication in vitro (50) and these cells are
rapidly recruited to the site of infection in mice (22, 51).
To test the proposed contribution of PMN to acute resistance, both iNOS ko and C57BL/6 mice were treated with a
mAb (RB6-8C5) against the GR-1 antigen, a protein which
is expressed at high levels on murine neutrophils and eosinophils and at much lower levels on cells of the myeloid
lineage (52). In vivo administration of the RB6-8C5 mAb
significantly reduced neutrophil infiltration in the peritoneal cavity of both iNOS ko and C57BL/6 animals at 5 and 7 d after parasite challenge (Fig. 5 A). More importantly, a majority of the animals injected with the RB68C5 mAb succumbed during the acute phase of T. gondii
infection. Thus, by 14 d after infection 75% of mAb treated
iNOS ko and 40% of treated C57BL/6 mice had succumbed to infection with the ME49 strain (Fig. 5, B and
C). In contrast, in vivo administration of the IL-5 neutralizing mAb, TRFK-5, ablated the low level eosinophil response in T. gondii infected C57BL/6 and iNOS ko strains
but failed to affect mortality (data not shown). Taken together these experiments suggest that granulocytes, and in
particular neutrophils, contribute to acute resistance against
the parasite and may account for the ability of iNOS ko animals to control infection in the apparent absence of macrophage killing function.
IFN- is known to be a critical mediator of innate resistance to T. gondii infection in vivo (20). Based on its
readily demonstrated function in activating macrophages to
kill tachyzoites in vitro (27), IFN-
has been assumed to
restrict parasite growth in the host primarily through the action of this effector cell. Nevertheless, it is clear that T. gondii
can productively infect a wide range of different nucleated
host cells (18) and therefore, in contrast to pathogens such
as Leishmania, need not be controlled through contact with
macrophages. In the present study, we have addressed this
issue by examining the development of innate resistance to
Toxoplasma in iNOS-deficient animals that exhibit markedly impaired macrophage toxoplasmacidal activity in vitro
as a result of their inability to generate inducible NO.
Surprisingly, these mice displayed normal control of acute
T. gondii infection arguing that NO-dependent killing by
macrophages is not an essential mechanism of innate resistance against the parasite.
A trivial explanation for the observed results is that differences in background genes, rather than the absence of
iNOS, are responsible for the failure of the knockout mice
to die during the acute phase or, alternatively for their early
death relative to C57BL/6 controls. However, several lines
of evidence argue against this possibility. First, with regard to
the acute stage of infection, we know of no examples, from
our own experience or the literature, in which background
genes rescue or protect mice from T. gondii induced, acute
mortality. For example, IFN- ko mice succumb with the
same rapid kinetics whether they are back-crossed on to a
C57BL/6 or BALB/c background (22) although the same
backgrounds have a major influence on survival during the
chronic stage of infection. Moreover, in direct contrast to the iNOS ko animals, ICSBP ko mice (lacking the interferon
consensus sequence-binding protein) at a similar 129 × C57BL/6 back-cross generation rapidly succumb to acute
infection with a phenotype virtually indistinguishable from
infected IFN-
ko mice (Scharton-Kersten, T., A. Sher,
and K. Ozato, manuscript in preparation). Similarly, it is
unlikely that contaminating 129 genes are responsible for the earlier CNS associated death of iNOS ko relative to the
C57BL/6 mice since C57BL/6 × 129 F1 animals show
enhanced, rather than decreased survival when compared
to C57BL/6 mice (Fig. 1, inset). Thus, although it would
have been preferable to use more fully back-crossed iNOS
ko animals for these experiments, it is difficult to escape the
conclusion that the observed phenotype in the knockout mice is a direct effect of the absence of iNOS rather than a
difference in genetic background.
A central role for NO as the primary toxoplasmacidal
mediator of activated macrophages has been established from
in vitro studies employing NO synthase inhibitors (9, 29,
30). Our results strongly support this concept by demonstrating that peritoneal macrophages harvested from naive,
thioglycollate injected, or ME49 infected iNOS ko mice
are grossly impaired in their ability to control parasite replication in vitro even after IFN- activation (Tables 1 and 2).
Nevertheless, iNOS ko mice were clearly able to control
early infection such that few, if any, tachyzoites were apparent in peritoneal macrophages obtained from the animals during the acute stage (Fig. 2 B). One interpretation
of this major discrepancy between the in vitro and in vivo
findings is that activated macrophages are not crucial for innate resistance against T. gondii. Instead, other IFN-
-dependent effector mechanisms may prevent parasite growth before productive infection of macrophages can occur.
Alternatively, it is possible that macrophages are indeed the
major effector cells of tachyzoite killing but use a microbicidal mechanism which is operative in vivo but not in vitro. For example, macrophages in vitro may not receive
the appropriate costimuli or other accessory factors needed
to induce NO-independent parasite control. Given that peritoneal cells, freshly isolated ex vivo, from infected iNOS
ko animals show the same defect in tachyzoite killing as
resident or elicited macrophages from naive donors, the latter explanation seems unlikely. A final possibility not ruled
out by our experiments is that macrophages are indeed the
major effector cells of innate resistance but that the relevant subset is NO independent and thus, distinct from the NOdependent peritoneal population assayed in our experiments.
Regardless of the particular effector cell involved, it is
clear from the antibody neutralization experiments (Fig. 4)
that the mechanism of innate resistance in both wild-type
and iNOS-deficient animals is IFN- as well as IL-12 dependent. In addition to RNI-mediated killing by macrophages, three additional tachyzoite killing functions have
been identified that are IFN-
dependent and thus could
explain the acute resistance of the iNOS ko mice. First, reactive oxygen intermediates (ROI; e.g., H2O2 and O2
)
have been implicated in the toxoplasmacidal activity of certain murine and human macrophage populations in vitro
(53, 54). However, the physiologic significance of the ROI
pathway remains controversial since other laboratories have
reported that T. gondii tachyzoites fail to trigger the oxidative burst in the same cells (55) or that the parasites are resistant to the metabolites produced (56). We have recently
addressed this issue by studying T. gondii infection in p47
phox-deficient animals, which lack an inducible oxidative
burst (57). These knockout mice were found to efficiently
control both acute and chronic infection in vivo and macrophages from the mutant animals were fully capable of limiting tachyzoite replication in vitro arguing against a crucial
role for the ROI in host resistance (Scharton-Kersten, T.,
S. Jackson, and S. Holland, unpublished observations). A
second alternative control mechanism is tryptophan starvation of the parasite, an IFN-
-induced pathway used by
human fibroblasts and macrophage populations to inhibit
T. gondii replication in vitro (58, 59). Although clearly
functional in the human immune response, this microbicidal mechanism cannot be demonstrated in mice (60). Finally, an IFN-
-dependent toxoplasmacidal activity, which
is independent of RNI, ROI, or tryptophan starvation, has recently been described in human endothelial cells (61).
Again however, a murine equivalent of this uncharacterized human effector mechanism has not yet been identified.
In addition to IFN--activated macrophages, fibroblasts
and/or endothelial cells, we have also considered the possible contribution of granulocytes to parasite control since
human peripheral blood PMN have been shown to kill intracellular tachyzoites in vitro (50). Moreover, neutrophils
have recently been shown to play an important role in innate resistance to both L. monocytogenes (62, 63) and Candida albicans (64) in murine models. Depletion of granulocytes by treatment with RB6-8C5 resulted in enhanced mortality of ME49 infected iNOS ko as well as wild-type
animals. Since depletion of eosinophils by neutralization of
IL-5 failed to affect host resistance, the neutrophil is likely
to be the relevant effector cell in the granulocyte population. Nevertheless, because peritoneal cell populations from
infected iNOS ko animals do not display significant levels
of microbicidal activity, it has so far been difficult to demonstrate a direct effect of murine neutrophils on parasite
survival in vitro. It is possible however, that the role of
neutrophils in host resistance does not involve direct lysis
of tachyzoites but rather, indirect anti-microbial functions
such as scavenging infected cells (65), secretion of toxic
products leading to metabolic poisoning of the parasite, or
the production of chemokines required for recruitment of
other effector cell populations (66).
A critical issue raised by our findings is whether the control of acute T. gondii infection in iNOS ko animals is due
to a normally occurring, but previously unrecognized, effector mechanism or instead reflects the induction of an aberrant compensatory host response. Numerous instances of
the expression of such compensatory mechanisms have been
documented in knockout mice. For example, mice lacking
the 2-microglobulin chain of the MHC class I complex develop an abnormal expansion of NK 1.1+, IFN-
-producing effector cells following T. gondii infection (67). We
believe that such an interpretation is an unlikely explanation for the behavior of iNOS ko animals described here. Thus, in almost every parameter examined, infected iNOS
and wild-type mice were indistinguishable during the acute
stage of infection. For instance, control and ko mouse strains
exhibited essentially identical local inflammatory responses
(Fig. 2 C) as well as systemic IL-12 and IFN-
synthesis
(Table 3). Moreover, the effect of granulocyte depletion,
although more dramatic in iNOS ko mice, was apparent in
both mutant and wild-type strains (Fig. 5). A minor difference noted was the elevated, local production of IFN-
in
infected knockout mice, an alteration which was not evident in either spleens or sera of the same animals and which
may reflect the absence of NO mediated suppression of
lymphocyte function (68). A final argument is that iNOS
ko mice clearly show defective resistance to other intracellular pathogens such as L. major and L. monocytogenes indicating the absence of compensatory immune responses at
least in these models (32, 33). We therefore postulate that
the mechanism of innate immunity operating in iNOS ko
animals is the same as that induced in conventional mice
and thus represents an as yet unappreciated pathway of host
resistance against the parasite.
Although iNOS ko animals were clearly able to control
acute infection, they did eventually succumb to T. gondii at
3-4 wk after inoculation (Fig. 1) and, when compared to
control mice, harbored significantly higher numbers of
brain cysts at 12 and 21 d (Fig. 3, A and B). Histopathological examination of the brains of infected iNOS ko mice on
d 20 and beyond revealed the development of severe necrotizing lesions in the CNS (Fig. 3, C-F) and the presence
of unchecked tachyzoite replication in the affected areas. These features were reminiscent of the reactivation-associated toxoplasmic encephalitis observed in chronically infected mice treated with neutralizing mAb against TNF
(34) or IFN-
(23) and, to some extent, in animals treated
with the nitric oxide synthase inhibitor, aminoguanidine
(11). In the mouse, IFN-
-activated microglial cells, but
not astrocytes, have been shown to inhibit tachyzoite replication by means of NO-dependent mechanisms (29). In
addition to microglial cells, macrophages also infiltrate the CNS and may exhibit NO-mediated toxoplasmacidal activity. Our results suggest that these NO-dependent effector cells are essential for controlling tachyzoite replication
and dissemination in the CNS. This is in direct contrast to
the situation in the periphery, where as discussed above,
other NO-independent effector cells or mechanisms play
the dominant role in mediating resistance. One explanation
for why inducible NO is more critical for the control of
toxoplasma in the CNS is that the NO-independent mechanism operative in the periphery is excluded from or cannot function within nervous tissue. Further studies in the
iNOS ko infection model described here should be useful
in defining the basis of this stage specificity in effector function as well as the uncharacterized mechanism which limits
parasite growth during acute infection.
Address correspondence to Dr. Tanya Scharton-Kersten, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, Bldg. 4, Rm. 126, NIH, Bethesda, MD 20892.
Received for publication 20 December 1996 and in revised form 3 February 1997.
1 Abbreviations used in this paper: CNS, central nervous system; iNOS, inducible nitric oxide synthase; IRF, interferon response factor; ko, knockout; LMC, large mononuclear cell; L-NMMA, NG-monomethyl-L-arginine; NO, nitric oxide; p.o., peroral; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates; SMC, small mononuclear cell; STAg, soluble tachyzoite antigen.We are grateful to Dr. Allen Cheever for his expert evaluation of histopathologic sections. We also thank Pat Caspar, Ricardo Dreyfus, and Sara Hieny for technical assistance, Drs. John MacMicking, Carl Nathan (Cornell University Medical College, New York, NY) and John Mudgett (Merck Research Laboratories, Rahway, NJ) for providing breeding pairs of the iNOS ko animals, and Drs. Stephanie James, Warren Leonard, and Tom Wynn for critical reading of the manuscript.
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