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Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, Ithaca, NY 14853
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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knockout
mice also expressed IL-12, ruling out an IFN--priming requirement.
Neither infected nor uninfected peritoneal macrophages displayed
intracellular IL-12, but these cells were strongly IL-10+.
Infection per se was unnecessary for IL-12 production because
peritoneal and peripheral blood neutrophils from uninfected animals
contained IL-12+ populations. Expression of the granulocyte
maturation marker Gr-1 (Ly-6G) was correlated with IL-12 production.
Mice depleted of their granulocytes by mAb administration at the time
of infection had decreased serum levels of IL-12 p40. These results
suggest a model in which neutrophils with prestored IL-12 are rapidly
mobilized to an infection site where they are triggered by the parasite
to release cytokine. Our findings place neutrophils prominently in the
cascade of early events leading to IL-12-dependent immunity to
T. gondii. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T. gondii is an obligate, intracellular protozoan parasite
that causes morbidity and mortality in a broad range of host species.
Indeed, a recent survey indicated that T. gondii is the
leading cause of human death among food-borne diseases in the United
States (11). As an opportunistic organism, its importance
as a pathogen has resurfaced with the AIDS epidemic, and it has been
estimated that 
30% of AIDS patients suffer from reactivation of
infection (12). Moreover, the parasite poses a serious
threat to the unborn fetus when transmitted placentally during maternal
infection (13).
Like many intracellular pathogens, T. gondii induces a
strong protective cell-mediated immune response that is driven by early
IL-12 production (14, 15). The source of this cytokine
during T. gondii infection has been attributed to both
macrophages and, more recently, dendritic cells (16, 17).
Previously, we reported that human and murine neutrophils elaborate
high levels of IL-12 upon in vitro stimulation with a T.
gondii Ag extract (6, 18), results that are in broad
agreement with the findings of others investigating Candida
albicans infection (19). Notably, experiments in
cytokine gene and cytokine receptor knockout animals demonstrated that
parasite-triggered IL-12 release occurred independently of
endogenous IFN- and did not require signaling through the TNF p55
receptor. We and others also found that C57BL/6 mice succumb acutely to
infection when depleted of their granulocytes, confirming the
importance of these cells during in vivo infection (6, 20, 21).
Given that neutrophils prestore and rapidly release microbicidal molecules when appropriately stimulated and that these cells can be triggered to release immunomodulatory cytokines during in vitro stimulation, we sought to determine whether neutrophils serve as an IL-12 source during in vivo infection. Furthermore, we examined whether neutrophils prestore this cytokine in the absence of an ongoing infection. Our results demonstrate that i.p. infection with tachyzoites induces a rapid influx of neutrophils into the peritoneal cavity expressing the maturation marker Gr-1 (Ly-6G), and these cells display high levels of intracellular IL-12 protein. Remarkably, we found that peripheral blood as well as peritoneal neutrophils from noninfected mice contain a population of Gr-1-expressing neutrophils that also display high levels of intracellular IL-12. Mice depleted of their granulocytes at the time of infection with bradyzoite cysts have decreased levels of serum IL-12. These results emphasize neutrophils as an important in vivo source of IL-12 during T. gondii infection. Moreover, the finding that intracellular IL-12 acquisition is associated with the normal neutrophil maturation program places these cells prominently in the cascade of early events that initiate cellular immunity to infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 female mice (6–12 wk of age) were obtained from The
Jackson Laboratory (Bar Harbor, ME). IFN- knockout female mice
(GKO)3 on a C57BL/6
background (6–12 wk of age) were obtained through a National Institute
of Allergy and Infectious Diseases contract with Taconic Farms
(Germantown, NY). The animals were housed under specific pathogen-free
conditions at the College of Veterinary Medicine animal facility at
Cornell University; the college maintains an animal facility that is
accredited by the American Association for Accreditation of Laboratory
Animal Care.
Parasites and infections
Tachyzoites of the RH strain were maintained on human foreskin fibroblast monolayers in DMEM (Life Technologies, Gaithersburg, MD), 1% FCS (HyClone, Logan, UT), and 100 U/ml penicillin and 0.1 mg/ml streptomycin (Life Technologies). Parasite cultures were free of contamination by Mycoplasma spp. as determined by RT-PCR, ELISA (kits from Stratagene (La Jolla, CA) and Roche (Indianapolis, IN), respectively), microbiological assay, and fluorescent DNA staining (performed by the Mycoplasma Testing Laboratory, Coriell Institute for Medical Research, Camden, NJ). Before infection, tachyzoites were washed in endotoxin-free PBS. Mice were i.p. infected with 2 x 106 tachyzoites or were injected with PBS.
ME49 bradyzoite cysts were maintained in Swiss-Webster mice as described previously (6). Mice were rendered neutropenic with an anti-Gr-1 mAb (RB6C6.8C5 hybridoma originally provided by R. L. Coffman (DNAX Research Institute, Palo Alto, CA) and were infected with 100 ME49 cysts i.p. as described previously (6).
Cell culture conditions
Peritoneal cells were obtained from mice 4 or 6 h postinfection (p.i.) by lavage with PBS. Cells were cultured in medium alone at 4 x 106/well for 18 h at 37°C with 5% CO2. Medium consisted of DMEM with 10% FCS, 1 mM sodium pyruvate (Life Technologies), 0.1 mM nonessential amino acids (Life Technologies), 30 mM HEPES (Life Technologies), 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 5 x 10-5 M 2-ME. Supernatants were harvested and stored at -20°C until assayed for IL-12 levels. To deplete peritoneal cell populations of granulocytes, immunomagnetic beads (Dynal, Oslo, Norway) coupled to anti-Gr-1 mAb were used as described previously (6). To determine the composition of isolated cells, differential cell counts were performed on Diff-Quik (American Scientific Products, McGraw Park, IL)-stained cytocentrifuge slides. A minimum of 300 cells were counted per slide.
Cytokine measurement
IL-12 p40 was measured in cell-free supernatants or plasma by ELISA as described in detail previously (6). To measure IL-12 p70, the protocol for determining p40 levels was followed, except that anti-IL-12 p70 (clone 9A5, PharMingen, San Diego, CA) was used as the coating Ab at 5 µg/ml. Detection sensitivities for IL-12 p40 were 10 and 50 pg/ml for IL-12 p70.
Confocal fluorescence microscopy
Cells (2 x 105/sample) were
centrifuged onto 12-mm microscope coverglasses (VWR Scientific,
Rochester, NY) resting on glass microscope slides (VWR Scientific)
using cytofunnels (Shandon, Pittsburgh, PA) and a cytospin centrifuge
(Shandon). Coverslips were then placed in wells of a 24-well tissue
culture plate (Corning Costar, Cambridge, MA) for subsequent staining.
Cells were fixed for 20 min at room temperature in PBS containing 3%
formaldehyde (Sigma, St. Louis, MO) with 0.1 mM
CaCl2 (Sigma) and 0.1 mM
MgCl2 (Sigma), then washed in permeabilization
buffer (0.075% saponin dissolved in PBS). Blocking was subsequently
performed (20 min at room temperature) using 5% normal mouse serum
(NMS; Accurate, Westbury, NY) diluted in permeabilization buffer. Next,
rat anti-mouse cytokine mAb were added (5 µg/ml), and cells were
incubated for 30 min at room temperature. Anti-IL-12 p40 (clone C17.8,
provided by G. Trinchieri, Wistar Institute, Philadelphia, PA)
(22), anti-IL-10 (clone JES5-16E3; PharMingen), and a
control rat Ig (Accurate Chemical, Westbury, NY) were used to stain
cells. After washing three times in permeabilization buffer,
FITC-conjugated mouse anti-rat 
light chain (clone OX-12;
Serotec, Raleigh, NC) diluted 1/500 in permeabilization buffer was
added, and cells were incubated for an additional 30 min at room
temperature. In some experiments, an FITC-conjugated anti-Gr-1 mAb
was employed (clone RB6C6.8C5; PharMingen). During the last 10 min of
incubation, propidium iodide (Sigma) was added (15 µg/ml final
concentration). The cells were washed three times in permeabilization
buffer, followed by four washes in PBS. Coverslips were mounted on
glass microscope slides in ProLong Anti-Fade (Molecular Probes, Eugene,
OR). The slides were examined with a Bio-Rad MRC600 confocal laser
scanning microscope, and images were collected using Comos software
(Bio-Rad, Hercules, CA).
Flow cytometric analysis
Peripheral blood was obtained by cardiac puncture, and
erythrocytes were lysed by a method described previously
(23). Briefly, 300 µl of whole blood was placed in 4 ml
of lysis buffer (156 mM ammonium chloride, 20 mM sodium bicarbonate,
and 1 ml of 0.5 M EDTA at pH 8 in water; reagents from Sigma). The
cells were incubated at room temperature for 5 min and then spun at
1500 rpm for 5 min. The resulting leukocyte pellet was washed once in
wash buffer composed of 0.1% albumin (Sigma) and 1 ml of 0.5 M EDTA at
pH 8 in Ca+2-, Mg+2-, and
phenol red-free HBSS (Life Technologies). Cells (1 x
106/sample) were fixed in 200 µl of 3%
formaldehyde with 0.1 mM CaCl2 and 0.1 mM
MgCl2 at room temperature for 20 min. Cells were
then washed twice in permeabilization buffer and blocked in 5% NMS
diluted in permeabilization buffer for 15 min at room temperature.
C17.8 mAb or control rat Ig were then added at 15 µg/ml. All Ab were
used at saturating concentrations. The cells were incubated at room
temperature for 20 min and then washed in permeabilization buffer three
times. Cells were resuspended in 5% NMS in permeabilization buffer,
and 1 µg/ml of PE-conjugated anti-rat light chain (clone
MRK-1, PharMingen) was added. The cells were incubated for 20 min at
room temperature, followed by two washes in permeabilization buffer.
Cells were then washed once in PBS and resuspended in 5% NMS diluted
in PBS with either FITC-conjugated anti-Gr-1 or isotype control
(clone A95-1; PharMingen) and incubated at room temperature for 20 min.
Finally, cells were washed three times in PBS and used for flow
cytometric analysis. Cells (1 x 105/sample)
were collected, and data were analyzed using CellQuest software and a
FACScalibur flow cytometer (Becton Dickinson Immunocytometry Systems,
San Jose, CA). The lower than expected side scatter of the granulocyte
population was a consequence of the fixation and permeabilization
protocol. It should be noted that no inhibitors of secretion, such as
monensin or brefeldin A, or cell activation factors, such as PMA or
ionomycin, were used in these studies.
Statistical analysis
Significant differences were determined using Student’s
t test in Figs. 1
and 9.
Values of p 
0.05 were considered significant. All
experiments were performed on a minimum of three independent
occasions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously established that 18-h thioglycolate-elicited murine
peritoneal neutrophils produce IL-12 p40 in response to in vitro
stimulation with a soluble tachyzoite Ag (STAg) preparation
(6). We also demonstrated that i.p. tachyzoite injection
induced an influx of neutrophils that correlated with increased levels
of IL-12 p40 gene transcripts. However, these data did not indicate
whether IL-12 protein was released, nor did they directly demonstrate
neutrophil IL-12 production. To address these issues, mice were
injected i.p. with 2 x 106 RH strain
tachyzoites in PBS or PBS alone. After 4 h, peritoneal cells were
obtained. Differential cell counts revealed that infection induced a
neutrophilic influx, such that 46% of the peritoneal cells were of
this cell type (data not shown). In contrast, injection of PBS failed
to elicit a similar influx. The unseparated cell populations were
placed in culture and incubated for 18 h without further
stimulation. Cells obtained from infected animals released both IL-12
p40 and the bioactive p70 form of the cytokine as determined by ELISA
(Fig. 1
). Approximately one-third of the p40 contributed to the
formation of p70. No IL-12 was detected from uninfected cultures. Since
this result represents a mixed populational response, we next examined
cytokine production at the single-cell level to definitively define the
IL-12 cell source during this model of early infection.
Mutually exclusive expression of IL-12 and IL-10 by neutrophils and macrophages, respectively
Cells obtained 4 h after RH infection or PBS injection were
immediately fixed and stained intracellularly for IL-12 or IL-10 by
indirect immunofluorescence, and nuclei were counterstained with
propidium iodide. The resulting samples were examined by confocal
fluorescence microscopy. Fig. 2A shows that the majority of
granulocytes, here distinguished by their characteristic nuclear
morphology, possess IL-12. Although eosinophils display similar nuclear
characteristics (24), Diff-Quik staining of parallel
samples revealed that eosinophils only constituted 1% of the
peritoneal population, ruling out the latter cells as the major IL-12
source. Notably, there is no evidence for expression of IL-12 in the
remainder of the population, most of which is composed of macrophages.
Fig. 2D shows an IL-12+ cell within
the population of peritoneal cells from uninfected animals.
Interestingly, while neutrophils are much less common in this
population, some stain for IL-12, suggesting that infection per se is
not a prerequisite for neutrophil IL-12 expression.
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B demonstrates that there is no evidence for expression of
IL-10 in the neutrophil population derived from infected animals.
However, macrophages present in this population displayed strong
staining for IL-10. FACS analysis indicated that
IL-10+ cells were also
F4/80+, confirming that these cells belong to the
monocyte/macrophage lineage (data not shown). In no experiment did we
find IL-12 expression by F4/80+ cells. This is
important because of a recent paper by Biermann et al.
(24), who found that morphological distinction between a
subset of granulocytes and monocytes/macrophages can be difficult due
to similar ring-shaped nuclear morphology. Of note, peritoneal
macrophages from uninfected mice appear to contain IL-10, although
levels are clearly lower than those found in cells from infected mice
(Fig. 2E, compare to Fig. 2B). The isotype
control mAb shown in Fig. 2, C and F, confirm the
specificity of the cytokine mAb staining.
Fig. 3 demonstrates IL-12 staining in
peritoneal cells 6 h after infection. Neutrophils remained
strongly IL-12+, while macrophages continued to
display little or no evidence for the presence of intracellular IL-12
(Fig. 3A). This was true even for tachyzoite-infected
macrophages, as shown in Fig. 3A. Additionally, macrophages
appear to be preferentially infected over neutrophils. Fig. 3B is a higher magnification image of
IL-12+ neutrophils. The punctate appearance of
intracellular IL-12 suggests the possibility that the cytokine is
located within cytoplasmic granules.
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, A and B, show
the forward and side scatter plots for control and infected
populations, respectively. The granulocyte populations, as determined
by Gr-1 back-gating, are demarcated. An influx of granulocytes was
apparent; 13% of the total population in A represented the
granulocyte population, which increased to 56% upon infection. Fig. 4, C and D, demonstrates IL-12 staining by these
populations. In Fig. 4C the majority of cells were
Gr-1- (80%). Of the Gr-1+
subset, 75% of the cells stained for IL-12, while only 4.9% of the
Gr-1- cells possessed IL-12. Infected
populations contained more Gr-1+ cells, with 85%
staining for IL-12 (Fig. 4D). To determine whether injection
of PBS alone provided sufficient stimulus to trigger the IL-12
expression shown in Fig. 4C, we also examined peritoneal
populations from noninjected animals. This cell population displayed
virtually identical characteristics to those shown in Fig. 4C (data not shown).
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B. Only the granulocyte
population, labeled R2, stained for IL-12. To demonstrate that the
granulocytes fell only within the R2 gate, we obtained a 4-h-infected
peritoneal cell population (Fig. 5C) and depleted one-half
of the population of granulocytes with immunomagnetic beads coated with
anti-Gr-1 mAb (Fig. 5D). Only cells contained within the
R2 gate decreased in percentage. Stained cytospin preparations of these
populations, shown in Fig. 5, E and F, revealed a
decrease in neutrophils (from 54 to 4% after depletion). Based upon
these data, we conclude that RH infection induces rapid recruitment of
neutrophils, the vast majority of which contain intracellular IL-12 and
express Gr-1.
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Since gated granulocyte populations in Fig. 4 included
Gr-1- cells, we examined Gr-1 expression in infected and
control populations (Fig. 6). Different
levels of Gr-1 expression are evident in Fig. 6A. The white
arrow indicates a granulocyte that stained strongly for Gr-1, while the
red arrow points to a cell displaying comparatively lower levels. In
Fig. 6B, the white arrow indicates a strongly staining
granulocyte within the uninfected population, but many
Gr-1- granulocytes may be visualized in this
field. Overall, we found more Gr-1- granulocytes
in control populations.
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is not required for IL-12 production by peritoneal
neutrophils
Our previous work has shown that thioglycolate-elicited
neutrophils derived from GKO mice secrete IL-12 p40 in response to STAg
at levels comparable to those in wild-type mice (6). To
confirm that in vivo infected peritoneal neutrophils from GKO mice
produce levels similar to those in wild-type mice, we obtained 4-h
infected cells and performed a flow cytometric analysis for IL-12 and
Gr-1 expression. Granulocytes from C57BL/6 mice were gated, and
staining for IL-12 and Gr-1 is shown in Fig. 7A. Once again, there were
Gr-1- cells within this population that stained
poorly for IL-12. Ninety-two percent of the Gr-1+
cells stained for IL-12. Fig. 7B shows similar results from
GKO mice; 91% of the Gr-1+ cells stained for
IL-12. This suggests that IFN- is not required for IL-12 production
by peritoneal neutrophils during in vivo infection.
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Since our data indicate that a population of peritoneal
neutrophils from noninfected animals contains intracellular IL-12, we
wondered whether they synthesized and stored the cytokine before
extravasation from the circulation into the peritoneal cavity. To
answer this question, PBL from normal C57BL/6 mice were subjected to
intracellular cytokine staining and flow cytometric analysis. As shown
in Fig. 8, two populations of
Gr-1+ cells are apparent.
Gr-1low cells, presumably the more immature
granulocytes (25), did not stain for IL-12. However, the
Gr-1high subset clearly expressed a high level of
intracellular IL-12, which indicates that
Gr-1high granulocytes synthesize and prestore
IL-12 as part of their normal developmental program independently of
pathogenic stimulation. It is unlikely that the
Gr-1low cells are eosinophils, as
Diff-Quik-stained parallel samples revealed that this cell type
represented only 5%.
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and data not shown). Frequencies
increased for both Gr-1low and
Gr-1high subsets. In two of three experiments,
the Gr-1high subset increased more dramatically
than the Gr-1low subset.
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Our previous work and that of others established that neutrophils
are necessary to survive acute infection with the low virulence strain
ME49 (6, 20, 21). To ascertain whether their impact may be
explained at least in part by an immunoregulatory function, we measured
serum IL-12 levels in neutrophil-depleted mice from the time of
infection until they became clinically ill, which occurred 8 days p.i.
Infected mice administered control rat Ig never appeared clinically
ill. Fig. 9 demonstrates a systemic
impairment in the ability to produce IL-12 by neutrophil-deleted mice,
a result that strongly implicates these cells as an important IL-12
source during infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 2, 4, and 6). The majority of influxing neutrophils contained IL-12, as
shown by confocal fluorescence microscopy and flow cytometry (Figs. 2 and 4). Moreover, we found that expression of IL-12 by neutrophils was
an IFN--independent process (Fig. 7). Importantly, in this model of
early infection we were unable to demonstrate IL-12 production by
macrophages (Figs. 2, 3, 5, and 6). This was true despite active
infection of this cell type (Fig. 3).
Our previous work demonstrated that the influx of neutrophils into the
peritoneal cavity after infection with RH is correlated with increased
levels of IL-12 p40 gene transcripts (6). Figs. 2, 4, and 8 indicate that neutrophils are capable of prestoring IL-12 protein,
since both peritoneal and peripheral blood neutrophils from uninfected
animals displayed strong intracellular IL-12 staining. The precedence
for this finding is that other cell types, such as mast cells, prestore
cytokines and are also capable of de novo synthesis under appropriate
conditions (26, 27). Evidence that neutrophils display an
ability to up-regulate cytokine mRNA levels in response to T.
gondii comes from recent studies in our laboratory
(6). Thus, thioglycolate-elicited murine neutrophils show
increased IL-12 p40 transcript levels by 2 h in response to in
vitro parasite Ag stimulation. Currently, a concept we favor is that
neutrophils prestore cytokines such as IL-12, which may be secreted
upon appropriate stimulation, and they also replenish their repository
through increased gene transcription.
The data in this manuscript contribute to an emerging view of
neutrophils as central regulators of immunity through their ability to
serve as an early IL-12 source during infection (28).
Thus, C. albicans strains that induce healing infection are
capable of stimulating neutrophil IL-12 production, and this is
required to control infection (3, 19, 29). Recent data
also suggest that control of murine Mycobacterium
tuberculosis infection is dependent upon an immunoregulatory
function of neutrophils (30). Here, we show that depriving
mice of their neutrophils at the time of infection results in decreased
systemic levels of IL-12 p40 (Fig. 9). Our work clearly establishes
that neutrophils produce IL-12 during in vivo infection, and more
importantly, the data show that intracellular cytokine is accumulated
as part of the normal developmental program of these cells.
Gr-1, also termed Ly-6G, is a molecule found on the surface of
granulocytes. Its expression is correlated with maturation; immature
granulocytes are Gr-1- or
Gr-1low and retain the capability of
proliferating in response to growth factors such as IL-3 and GM-CSF
(25, 31). As the cell matures, Gr-1 levels increase, and
the cell no longer responds to growth factors. Our current hypothesis
is that Gr-1 expression and IL-12 production increase with the
differentiation state of neutrophils and that infection or activation
facilitates differentiation. Experimental support for this comes from
finding Gr-1low subsets in peripheral blood that
did not possess IL-12, while Gr-1high cells in
the same population were IL-12+ (Fig. 8).
Furthermore, the finding that in two of three experiments, the
Gr-1high subset in peripheral blood increased
over the Gr-1low subset suggests that infection
may accelerate neutrophil maturation (Table I). The neutrophilia
present in the PBL population from infected animals probably represents
a dynamic situation in which parasite-triggered neutrophil
extravasation into the peritoneal cavity is counterbalanced by
increased hemopoietic release from the bone marrow.
Although a strong type 1 response is critical for surviving infection
with T. gondii, it is clear that overproduction of
proinflammatory cytokines can be pathological.
IL-10-/- mice succumb to normally nonlethal
infection with ME49, and death is associated with high levels of IL-12
and TNF-
(32, 33). Romani et al. (19)
reported that a subset of neutrophils produces IL-10 in response to a
virulent strain of C. albicans. We were not able to identify
a subset of neutrophils, either in blood or in the peritoneal cavity
regardless of infection, that produced IL-10 (Fig. 2 and data not
shown). Thus, whether neutrophils produce IL-10 is likely to depend
upon the specific pathogenic stimulus. Nevertheless, our data indicate
that macrophages synthesize IL-10 (Fig. 2). Classically, macrophages
have been thought to be an important source of early IL-12 during
infection with T. gondii. Our results here and those
reported by others suggest differently (17). Additionally,
our data demonstrate that IL-10 is prestored. We have previously shown
that when macrophage-enriched populations are stimulated with STAg or
medium, IL-10 can be detected only in supernatants from cells
stimulated with parasite Ag (6). Currently, control of
cytokine secretion is an active area of research in our laboratory. We
favor the hypothesis that macrophages, in the early stages of
infection, help control the inflammatory process when cells such as
neutrophils and dendritic cells are triggered by the parasite to
release high levels of IL-12.
Establishment of protective immunity and early control of infection
through innate immune responses are essential for survival during
toxoplasmosis (15). It has become clear that events
occurring early, during the innate response, shape acquired immunity
(34). It is well known that early IL-12 production is
necessary for Th1 cell differentiation. Sustained and protective
immunity against T. gondii requires a type 1 response with
subsequent IFN- production. Thus, it is important to understand how
the immune response is initiated. Our hypothesis is that neutrophils
play an immunomodulatory role in infection, and furthermore, that they
are involved in the earliest recognition of the parasite and generation
of immunity. The capacity to prestore IL-12 may allow for rapid
mobilization and secretion after stimulation. Also, the ability to
increase transcript levels suggests a sustained response to the
parasite. The mechanisms by which neutrophil-derived IL-12 is released
and its influence on T cell differentiation are currently being
explored in our laboratory.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric Denkers, Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, Ithaca, NY 14853-6401.
3 Abbreviations used in this paper: GKO, IFN--/- mice; STAg, soluble tachyzoite Ag; p.i., postinfection; NMS, normal mouse serum.
Received for publication April 18, 2000. Accepted for publication July 26, 2000.
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References |
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by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115., and the chemokines macrophage inflammatory protein-1 and -1ß in response to Toxoplasma gondii antigens. J, Immunol. 162:7369. production and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672.[Medline]
response of human mast cells following exposure to Gram-negative and Gram-positive bacteria. Infect. Immun. 66:6030., and TNF-. J. Immunol. 157:798.[Abstract]
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R. S. Goldszmid, A. Bafica, D. Jankovic, C. G. Feng, P. Caspar, R. Winkler-Pickett, G. Trinchieri, and A. Sher TAP-1 indirectly regulates CD4+ T cell priming in Toxoplasma gondii infection by controlling NK cell IFN-{gamma} production J. Exp. Med., October 29, 2007; 204(11): 2591 - 2602. [Abstract] [Full Text] [PDF]
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W. Sukhumavasi, C. E. Egan, and E. Y. Denkers Mouse Neutrophils Require JNK2 MAPK for Toxoplasma gondii-Induced IL-12p40 and CCL2/MCP-1 Release J. Immunol., September 15, 2007; 179(6): 3570 - 3577. [Abstract] [Full Text] [PDF]
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R. T. Sasmono, A. Ehrnsperger, S. L. Cronau, T. Ravasi, R. Kandane, M. J. Hickey, A. D. Cook, S. R. Himes, J. A. Hamilton, and D. A. Hume Mouse neutrophilic granulocytes express mRNA encoding the macrophage colony-stimulating factor receptor (CSF-1R) as well as many other macrophage-specific transcripts and can transdifferentiate into macrophages in vitro in response to CSF-1 J. Leukoc. Biol., July 1, 2007; 82(1): 111 - 123. [Abstract] [Full Text] [PDF]
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A. Rydstrom and M. J. Wick Monocyte Recruitment, Activation, and Function in the Gut-Associated Lymphoid Tissue during Oral Salmonella Infection J. Immunol., May 1, 2007; 178(9): 5789 - 5801. [Abstract] [Full Text] [PDF]
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 B. A. Maletto, A. S. Ropolo, D. O. Alignani, M. V. Liscovsky, R. P. Ranocchia, V. G. Moron, and M. C. Pistoresi-Palencia Presence of neutrophil-bearing antigen in lymphoid organs of immune mice Blood, November 1, 2006; 108(9): 3094 - 3102. [Abstract] [Full Text] [PDF]
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L. Kim, B. A. Butcher, C. W. Lee, S. Uematsu, S. Akira, and E. Y. Denkers Toxoplasma gondii Genotype Determines MyD88-Dependent Signaling in Infected Macrophages J. Immunol., August 15, 2006; 177(4): 2584 - 2591. [Abstract] [Full Text] [PDF]
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 S. Bennouna, W. Sukhumavasi, and E. Y. Denkers Toxoplasma gondii Inhibits Toll-Like Receptor 4 Ligand-Induced Mobilization of Intracellular Tumor Necrosis Factor Alpha to the Surface of Mouse Peritoneal Neutrophils Infect. Immun., July 1, 2006; 74(7): 4274 - 4281. [Abstract] [Full Text] [PDF]
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C.-H. Liu, Y.-t. Fan, A. Dias, L. Esper, R. A. Corn, A. Bafica, F. S. Machado, and J. Aliberti Cutting Edge: Dendritic Cells Are Essential for In Vivo IL-12 Production and Development of Resistance against Toxoplasma gondii Infection in Mice J. Immunol., July 1, 2006; 177(1): 31 - 35. [Abstract] [Full Text] [PDF]
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S. J. Park, M. T. Wiekowski, S. A. Lira, and B. Mehrad Neutrophils Regulate Airway Responses in a Model of Fungal Allergic Airways Disease J. Immunol., February 15, 2006; 176(4): 2538 - 2545. [Abstract] [Full Text] [PDF]
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 K. Ali, M. Middleton, E. Pure, and D. J. Rader Apolipoprotein E Suppresses the Type I Inflammatory Response In Vivo Circ. Res., October 28, 2005; 97(9): 922 - 927. [Abstract] [Full Text] [PDF]
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S. T. Smiley, P. A. Lanthier, K. N. Couper, F. M. Szaba, J. E. Boyson, W. Chen, and L. L. Johnson Exacerbated Susceptibility to Infection-Stimulated Immunopathology in CD1d-Deficient Mice J. Immunol., June 15, 2005; 174(12): 7904 - 7911. [Abstract] [Full Text] [PDF]
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P. M. Robben, M. LaRegina, W. A. Kuziel, and L. D. Sibley Recruitment of Gr-1+ monocytes is essential for control of acute toxoplasmosis J. Exp. Med., June 6, 2005; 201(11): 1761 - 1769. [Abstract] [Full Text] [PDF]
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S. Bennouna and E. Y. Denkers Microbial Antigen Triggers Rapid Mobilization of TNF-{alpha} to the Surface of Mouse Neutrophils Transforming Them into Inducers of High-Level Dendritic Cell TNF-{alpha} Production J. Immunol., April 15, 2005; 174(8): 4845 - 4851. [Abstract] [Full Text] [PDF]
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L. Kim, L. D. Rio, B. A. Butcher, T. H. Mogensen, S. R. Paludan, R. A. Flavell, and E. Y. Denkers p38 MAPK Autophosphorylation Drives Macrophage IL-12 Production during Intracellular Infection J. Immunol., April 1, 2005; 174(7): 4178 - 4184. [Abstract] [Full Text] [PDF]
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M. N. Kelly, J. K. Kolls, K. Happel, J. D. Schwartzman, P. Schwarzenberger, C. Combe, M. Moretto, and I. A. Khan Interleukin-17/Interleukin-17 Receptor-Mediated Signaling Is Important for Generation of an Optimal Polymorphonuclear Response against Toxoplasma gondii Infection Infect. Immun., January 1, 2005; 73(1): 617 - 621. [Abstract] [Full Text] [PDF]
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C. Borg, A. Jalil, D. Laderach, K. Maruyama, H. Wakasugi, S. Charrier, B. Ryffel, A. Cambi, C. Figdor, W. Vainchenker, et al. NK cell activation by dendritic cells (DCs) requires the formation of a synapse leading to IL-12 polarization in DCs Blood, November 15, 2004; 104(10): 3267 - 3275. [Abstract] [Full Text] [PDF]
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 S. P. Matzer, F. Rodel, R. M. Strieter, M. Rollinghoff, and H. U. Beuscher Constitutive expression of CXCL2/MIP-2 is restricted to a Gr-1high, CD11b+, CD62Lhigh subset of bone marrow derived granulocytes Int. Immunol., November 1, 2004; 16(11): 1675 - 1683. [Abstract] [Full Text] [PDF]
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C. R. Brown, V. A. Blaho, and C. M. Loiacono Treatment of Mice with the Neutrophil-Depleting Antibody RB6-8C5 Results in Early Development of Experimental Lyme Arthritis via the Recruitment of Gr-1- Polymorphonuclear Leukocyte-Like Cells Infect. Immun., September 1, 2004; 72(9): 4956 - 4965. [Abstract] [Full Text] [PDF]
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 P. Athanassopoulos, L. M.B. Vaessen, A. P.W.M. Maat, A. H.M.M. Balk, W. Weimar, and A. J.J.C. Bogers Peripheral blood dendritic cells in human end-stage heart failure and the early post-transplant period: evidence for systemic Th1 immune responses Eur. J. Cardiothorac. Surg., April 1, 2004; 25(4): 619 - 626. [Abstract] [Full Text] [PDF]
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D. G. Mordue and L. D. Sibley A novel population of Gr-1+-activated macrophages induced during acute toxoplasmosis J. Leukoc. Biol., December 1, 2003; 74(6): 1015 - 1025. [Abstract] [Full Text]
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S. Bennouna, S. K. Bliss, T. J. Curiel, and E. Y. Denkers Cross-Talk in the Innate Immune System: Neutrophils Instruct Recruitment and Activation of Dendritic Cells during Microbial Infection J. Immunol., December 1, 2003; 171(11): 6052 - 6058. [Abstract] [Full Text] [PDF]
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G. Rotta, E. W. Edwards, S. Sangaletti, C. Bennett, S. Ronzoni, M. P. Colombo, R. M. Steinman, G. J. Randolph, and M. Rescigno Lipopolysaccharide or Whole Bacteria Block the Conversion of Inflammatory Monocytes into Dendritic Cells In Vivo J. Exp. Med., October 20, 2003; 198(8): 1253 - 1263. [Abstract] [Full Text] [PDF]
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K. G. Hogg, S. Kumkate, S. Anderson, and A. P. Mountford Interleukin-12 p40 Secretion by Cutaneous CD11c+ and F4/80+ Cells Is a Major Feature of the Innate Immune Response in Mice That Develop Th1-Mediated Protective Immunity to Schistosomamansoni Infect. Immun., June 1, 2003; 71(6): 3563 - 3571. [Abstract] [Full Text] [PDF]
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S. Buonocore, F. Paulart, A. Le Moine, M. Braun, I. Salmon, S. Van Meirvenne, K. Thielemans, M. Goldman, and V. Flamand Dendritic cells overexpressing CD95 (Fas) ligand elicit vigorous allospecific T-cell responses in vivo Blood, February 15, 2003; 101(4): 1469 - 1476. [Abstract] [Full Text] [PDF]
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 B. Mehrad, M. Wiekowski, B. E. Morrison, S.-C. Chen, E. C. Coronel, D. J. Manfra, and S. A. Lira Transient Lung-Specific Expression of the Chemokine KC Improves Outcome in Invasive Aspergillosis Am. J. Respir. Crit. Care Med., November 1, 2002; 166(9): 1263 - 1268. [Abstract] [Full Text] [PDF]
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A. C. Kirby, U. Yrlid, and M. J. Wick The Innate Immune Response Differs in Primary and Secondary Salmonella Infection J. Immunol., October 15, 2002; 169(8): 4450 - 4459. [Abstract] [Full Text] [PDF]
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B. A. Butcher and E. Y. Denkers Mechanism of Entry Determines the Ability of Toxoplasma gondii To Inhibit Macrophage Proinflammatory Cytokine Production Infect. Immun., September 1, 2002; 70(9): 5216 - 5224. [Abstract] [Full Text] [PDF]
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D. W. Pascual, T. Trunkle, and J. Sura Fimbriated Salmonella enterica Serovar Typhimurium Abates Initial Inflammatory Responses by Macrophages Infect. Immun., August 1, 2002; 70(8): 4273 - 4281. [Abstract] [Full Text] [PDF]
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M. Rodriguez-Sosa, A. R. Satoskar, R. Calderon, L. Gomez-Garcia, R. Saavedra, R. Bojalil, and L. I. Terrazas Chronic Helminth Infection Induces Alternatively Activated Macrophages Expressing High Levels of CCR5 with Low Interleukin-12 Production and Th2-Biasing Ability Infect. Immun., July 1, 2002; 70(7): 3656 - 3664. [Abstract] [Full Text] [PDF]
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C. A. Scanga, J. Aliberti, D. Jankovic, F. Tilloy, S. Bennouna, E. Y. Denkers, R. Medzhitov, and A. Sher Cutting Edge: MyD88 Is Required for Resistance to Toxoplasma gondii Infection and Regulates Parasite-Induced IL-12 Production by Dendritic Cells J. Immunol., June 15, 2002; 168(12): 5997 - 6001. [Abstract] [Full Text] [PDF]
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N. Mason, J. Aliberti, J. C. Caamano, H.-C. Liou, and C. A. Hunter Cutting Edge: Identification of c-Rel-Dependent and -Independent Pathways of IL-12 Production During Infectious and Inflammatory Stimuli J. Immunol., March 15, 2002; 168(6): 2590 - 2594. [Abstract] [Full Text] [PDF]
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M. J. Skeen, E. P. Rix, M. M. Freeman, and H. K. Ziegler Exaggerated Proinflammatory and Th1 Responses in the Absence of gamma /delta T Cells after Infection with Listeria monocytogenes Infect. Immun., December 1, 2001; 69(12): 7213 - 7223. [Abstract] [Full Text] [PDF]
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L. Del Rio, S. Bennouna, J. Salinas, and E. Y. Denkers CXCR2 Deficiency Confers Impaired Neutrophil Recruitment and Increased Susceptibility During Toxoplasma gondii Infection J. Immunol., December 1, 2001; 167(11): 6503 - 6509. [Abstract] [Full Text] [PDF]
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S. P. Matzer, T. Baumann, N. W. Lukacs, M. Rollinghoff, and H. U. Beuscher Constitutive Expression of Macrophage-Inflammatory Protein 2 (MIP-2) mRNA in Bone Marrow Gives Rise to Peripheral Neutrophils with Preformed MIP-2 Protein J. Immunol., October 15, 2001; 167(8): 4635 - 4643. [Abstract] [Full Text] [PDF]
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B. A. Butcher, L. Kim, P. F. Johnson, and E. Y. Denkers Toxoplasma gondii Tachyzoites Inhibit Proinflammatory Cytokine Induction in Infected Macrophages by Preventing Nuclear Translocation of the Transcription Factor NF-{kappa}B J. Immunol., August 15, 2001; 167(4): 2193 - 2201. [Abstract] [Full Text] [PDF]
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S. K. Bliss, L. C. Gavrilescu, A. Alcaraz, and E. Y. Denkers Neutrophil Depletion during Toxoplasma gondii Infection Leads to Impaired Immunity and Lethal Systemic Pathology Infect. Immun., August 1, 2001; 69(8): 4898 - 4905. [Abstract] [Full Text] [PDF]
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L. R. Hall, E. Diaconu, and E. Pearlman A Dominant Role for Fc{{gamma}} Receptors in Antibody-Dependent Corneal Inflammation J. Immunol., July 15, 2001; 167(2): 919 - 925. [Abstract] [Full Text] [PDF]
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M. Wysocka, S. Robertson, H. Riemann, J. Caamano, C. Hunter, A. Mackiewicz, L. J. Montaner, G. Trinchieri, and C. L. Karp IL-12 Suppression During Experimental Endotoxin Tolerance: Dendritic Cell Loss and Macrophage Hyporesponsiveness J. Immunol., June 15, 2001; 166(12): 7504 - 7513. [Abstract] [Full Text] [PDF]
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L. R. Hall, E. Diaconu, R. Patel, and E. Pearlman CXC Chemokine Receptor 2 But Not C-C Chemokine Receptor 1 Expression Is Essential for Neutrophil Recruitment to the Cornea in Helminth-Mediated Keratitis (River Blindness) J. Immunol., March 15, 2001; 166(6): 4035 - 4041. [Abstract] [Full Text] [PDF]
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