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-Independent IL-121
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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when stimulated with
T. gondii Ag. Both cytokines are produced in the absence
of IFN-
. Production of IL-12 does not require TNFR p55, and release
of TNF- occurs independently of IL-12. We show that there is an
influx of neutrophils into the peritoneal cavity that peaks at 
8 h
in response to injection of live tachyzoites and that this is
correlated with increased transcription of IL-12 p40. Our results
establish that murine neutrophils possess the ability to produce
immunoregulatory cytokines during T. gondii infection
and suggest that this response may be important in early host defense
and in triggering cell-mediated immunity to the
parasite. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Successful immunity to T. gondii requires a strong
cell-mediated immune response, and IFN-, in particular, is required
to survive infection (3). Indeed, high levels of IFN-
present during both acute and chronic phases are a distinct feature of
infection. The major sources of this type 1 cytokine are believed to be
NK cells and T lymphocytes (4, 5). An important issue in
understanding the host-pathogen relationship with regard to T.
gondii and other microbial pathogens is to determine how a type 1
cytokine phenotype is established. Events occurring during first
encounter of the parasite with the innate immune system are likely to
be crucial in shaping the subsequent response.
Early IL-12 production is critical in triggering cell-mediated
immunity. Both dendritic cells and thioglycollate-elicited macrophages
produce this cytokine in response to T. gondii (6, 7). In combination with TNF- and IL-1ß, IL-12 induces NK
cell production of IFN- (8). The latter cytokine
promotes macrophage IL-12 production, as well as release of reactive
oxygen and nitrogen intermediates (9, 10, 11, 12). IFN- also
promotes MHC class I and II expression, presumably resulting in
enhanced Ag presentation (13, 14). Furthermore, in vitro
studies demonstrate that IFN- induces tryptophan degradation in
fibroblasts, resulting in cessation of parasite growth
(15).
Neutrophils are often the first cell type recruited to an area of
infection or inflammation. They are considered to be terminally
differentiated effector cells capable of phagocytosis and
intracellular killing via mechanisms dependent upon degradative
enzymes, reactive oxygen and nitrogen intermediates, and have been
shown specifically to kill T. gondii (16).
Moreover, recent evidence points to neutrophils as a source of
immunoregulatory cytokines, such as IL-12, IL-10, and TNF-, during
infection with the yeast pathogen, Candida albicans, and
during in vitro stimulation with LPS (17, 18, 19, 20, 21, 22, 23, 24, 25). Our recent
work demonstrates a crucial role for granulocytes in a model of
cytokine toxicity induced by T. gondii extracts
(26). Here, mice sensitized by
D-galactosamine injection undergo lethal hepatic
necrosis mediated by IL-12, TNF-, and IFN-, and this cytokine
response is prevented by mAb-mediated depletion of granulocytes. These
findings implicate granulocytes as important
cells in the cascade of reactions leading to
type 1 cytokine synthesis. Nevertheless, they do not establish whether
the parasite directly stimulates murine granulocyte cytokine release or
whether the cells are indirectly involved in the response.
In this paper, we directly demonstrate that murine granulocytes, and
particularly neutrophils, produce high levels of IL-12 and low, but
significant, levels of TNF- when stimulated with T.
gondii Ag. Both cytokines are produced in the absence of IFN-.
Production of IL-12 does not require TNFR p55, and release of TNF-
occurs independently of IL-12. We hypothesize that neutrophil
production of IL-12 and TNF- plays an important role in innate
immunity against T. gondii and may be important in
influencing the adaptive immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6, C57BL/6-IfngtmTs (GKO),3 C57BL/6-Il12atm1Jm (IL-12-/-), and C57BL/6-Tnfrsf1atm1Mak (TNFR p55-/-) female mice (6–12 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME). C3H-HeN female mice (6–12 wk of age) were obtained from Taconic Farms (Germantown, NY). The animals were housed under specific pathogen-free conditions at the College of Veterinary Medicine animal facility at Cornell University (Ithaca, NY).
Parasites and Ag
Tachyzoites from RH, ME49, and C strains were maintained on human foreskin fibroblast monolayers in DMEM (Life Technologies, Gaithersburg, MD), 1% FCS (HyClone, Logan, UT), 100 U/ml of penicillin, and 0.1 mg/ml of 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 Boehringer Mannheim, Indianapolis, IN, respectively), microbiological assay, and fluorescent DNA staining (performed by the Mycoplasma Testing Laboratory, Coriell Institute for Medical Research, Camden, NJ). Freeze-thaw tachyzoites (FTZ) were prepared by harvesting lysed fibroblast cultures and washing in PBS. The tachyzoites were counted and stored frozen in aliquots at -70°C until use. FTZ were thawed immediately before experiments.
To prepare soluble tachyzoite Ag (STAg), RH strain tachyzoites were sonicated in the presence of a protease inhibitor mixture consisting of 0.2 mM PMSF (Sigma, St. Louis, MO), 0.2 µM aprotinin (Boehringer Mannheim), 1 µM leupeptin (Boehringer Mannheim), and 1 mM EDTA (Sigma). The resulting sonicate was dialyzed into PBS, centrifuged at 10,000 x g for 1 h, and the supernatant was collected and filtered through a 0.2-µm pore-sized membrane (Corning Costar, Cambridge, MA). The protein concentration was determined by a Bradford assay (27), and the filtrate was stored at -70°C until use. Parasite extracts were found to be free of endotoxin contamination, as measured by the Limulus amebocyte assay. Soluble extracts of uninfected human foreskin fibroblasts were prepared in an identical manner to the preparation of STAg. Additionally, a Schistosoma mansoni soluble egg Ag (SEA) preparation, kindly provided by Dr. E. J. Pearce (Cornell University, Ithaca, NY), was also used in control experiments (28).
ME49 bradyzoites were maintained in vivo by i.p. inoculation of Swiss Webster mice (Taconic Farms) with 20 cysts obtained from the brains of mice that were infected 6–8 wk earlier.
Oral infection of neutrophil-depleted mice
Prior to oral infection of mice, brain suspensions were adjusted to administer 100 cysts per mouse. The cysts were given by gavage to ether-anesthetized mice. RB6C6.8C5 (29) or a control rat Ig (Accurate Chemical and Scientific, Westbury, NY) was injected i.p. on days -2, 0, +2, and +4 at 200 µg/mouse to deplete mice of granulocytes. The efficiency of depletion was determined by Diff-Quik stained blood smears.
RB6C6.8C5 (originally provided by Dr. R. L. Coffman, DNAX Research Institute, Palo Alto, CA) was purified from hybridoma supernatants using affinity chromatography (protein G-Sepharose; Pharmacia Biotech, Uppsala, Sweden). Eluates were dialyzed into PBS, filtered through a 0.2-µm pore-sized membrane, and protein concentration was determined by a Bradford assay.
Cell purification
For in vitro stimulations, either peripheral blood leukocytes or peritoneal exudate cells (PEC) were used. Peripheral blood was collected by cardiac puncture and placed in EDTA-containing tubes. The blood was then layered over Histopaque-1077 (Sigma) and centrifuged at 700 x g for 30 min at room temperature to separate leukocytes from the total cell population. To obtain PEC, mice were injected with 1 ml of 10% thioglycollate (Difco Laboratories, Detroit, MI) i.p., and a peritoneal lavage was performed with 10 ml of ice-cold PBS 18 h later. 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 was counted per slide. To obtain purified neutrophil populations, PEC were subjected to negative selection with immunomagnetic beads (Dynal, Oslo, Norway) coupled to mAb M5.114 or 10-3.6.2 (American Type Culture Collection, Manassas, VA), depending on the mouse strain, to remove macrophages. Cells were incubated with the conjugated beads for 15 min at 4°C with gentle mixing. Beads with attached macrophages were removed with the use of a magnet (MPC-2; Dynal). This cycle was repeated three times. The remaining, unattached cells were washed once in PBS, counted, and differential cell counts were performed. The populations obtained in this manner were routinely comprised of 93–96% neutrophils. Eosinophils often were not detectable and in no case did they exceed 2.8% of the population. Cell viability was determined to be 95% or greater by trypan blue exclusion (Sigma). Since we found that cell viability began to decrease slightly by 12 h, most of our experiments were conducted with only a 6-h incubation period. Macrophages were obtained by negative selection, as described above, using immunomagnetic beads coupled to mAb RB6C6.8C5. Macrophage purity was 96%.
Depletion of peripheral blood neutrophils was accomplished by two rounds of complement lysis, as previously described (30), using RB6C6.8C5-containing hybridoma supernatant or medium alone. Specificity of depletion was determined by FACS analysis.
Cell culture conditions
Cells were diluted in complete DMEM consisting of 10% FCS, 1 mM sodium pyruvate (Life Technologies), 0.1 mM nonessential amino acids (Life Technologies), 30 mM HEPES (Life Technologies), 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and 5 x 10-5 M 2-ME and cultured at a concentration of 4 x 106/ml in 96-well plates (Corning Costar) in triplicate. Cells were stimulated with medium alone, fibroblast lysate, LPS (S. minnesota Re 595; Sigma), SEA, FTZ, or STAg at various specified doses for the indicated times at 37°C with 5% CO2. Supernatants were harvested between 2 and 24 h after culture initiation and stored at -20°C until assayed.
Cytokine measurement
To measure IL-12 p40, cytokine-specific mAb C15.6 and C17.8
(kindly provided by Dr. G. Trinchieri, Wistar Institute, Philadelphia,
PA; Ref. 31) were employed. To perform the ELISA, 96-well
plates (Corning Costar) were coated overnight at 4°C with mAb C15.6
in PBS (10 µg/ml), followed by three washes in PBS containing 0.05%
Tween (PBST). Plates were blocked for 2 h at 37°C in PBS with
1% BSA (Sigma). After washing in PBST, sample supernatants and a
rIL-12 standard (Genzyme, Cambridge, MA) were added, and plates were
incubated overnight at 4°C. After washing in PBST, biotinylated mAb
C17.8 was added, and plates were incubated at 37°C for 90 min.
HRP-conjugated streptavidin (Genzyme) was then added, and plates were
incubated for an additional 60 min at 37°C. Finally, 100 µl of
2,2-azinodi-(3-ethylbenzthiazoline-6-sulfonate) substrate (ABTS;
Kirkegaard & Perry Laboratories, Gaithersburg, MD) were added to each
well, and sample absorbances were measured on a Microplate Bio Kinetics
Reader (Bio-Tek Instruments, Winooski, VT) at 405 nm. TNF- levels
were determined using a murine-specific ELISA kit, according to the
manufacturer’s instructions (Genzyme). IL-10 levels were determined as
described previously (32). The detection sensitivities in
the ELISAs were 10 pg/ml (IL-12), 15 pg/ml (TNF-), and 30 pg/ml
(IL-10).
RNA isolation and RT-PCR
Following supernatant collection, cells were resuspended in RNA STAT 60 at a ratio of 2.5 x 106 cells/500 µl RNA STAT 60 (Tel-Test, Friendswood, TX) and placed in sterile 1.5-ml Eppendorf tubes. To obtain sufficient RNA for analysis, triplicate samples from 96-well plates were pooled. Alternatively, freshly isolated PEC were resuspended in RNA STAT 60 at the same ratio. Then, 100 µl of chloroform/500 µl RNA STAT 60 (Fisher Scientific, Pittsburgh, PA) were added, the solution vortexed and then centrifuged at 13,000 x g for 15 min at 4°C. The aqueous phase was transferred to a fresh tube containing an equal volume of isopropanol (Sigma) and incubated at -20°C overnight. To precipitate RNA, a one-tenth volume of 3 M sodium acetate was added to the RNA/isopropanol mixture. The contents were vortexed and centrifuged at 4°C for 15 min at 13,000 x g. The RNA pellet was washed once in 75% ethanol, resuspended in water, and the concentration was determined using a spectrophotometer equipped with a UV lamp (Beckman DU-50; Beckman Instruments, Palo Alto, CA). A total of 6 µg of RNA was reverse transcribed using oligo-dT primers (Promega, Madison, WI). After heating samples to 72°C on an automated thermocycler (MJ Research, Watertown, MA) and chilling on ice to permit hybridization, a master mix was added that contained 5x RT buffer (Life Technologies), 0.1 M DTT (Life Technologies), 2.5 mM dNTP (Sigma), 40 U/ml RNasin (Promega), and 200 U/ml Superscript II (Life Technologies). The samples were incubated at 45°C for 60 min, followed by a 10-min incubation at 94°C. The resulting cDNA was diluted 1:4 with water and stored at -20°C until use.
PCR was performed using a master mix containing 2.5 mM dNTP, 10x PCR buffer with 1.5 mM MgCl2 (Promega), 0.2 µM primers, and 5 U/µl Taq polymerase (Life Technologies). The nucleotide sequences for sense and anti-sense primers, respectively, were: HPRT, GTT-GGA-TAC-AGG-CCA-GAC-TTT-GTT-G and GAT-TCA-ACT-TGC-GCT-CAT-CTT-AGG-C; IL-12 p40, CGT-GCT-CAT-GGC-TGG-TGC-AAA-G and GAA-CAC-ATG-CCC-ACT-TGC-TG (7). A total of 10 µl of cDNA was added to 40 µl of master mix, and samples were incubated for 3 min at 95°C, then subjected to PCR amplification with an experimentally determined optimal thermocycler program for each cytokine cDNA (94°C for 1 min, 54°C for 1 min, 72°C for 2 min, with a final extension of 7 min at 72°C). A total of 33 cycles was used for HPRT, while 35 cycles were used for IL-12 p40. PCR products were resolved on 2% agarose gels, and bands were visualized by staining with ethidium bromide (Sigma). The sizes of the bands were compared with a m.w. marker to ensure that each was consistent with its predicted size.
Statistical analyses
Significant differences were determined by the Wilcoxon rank sum
test or ANOVA for Figs. 1
and 3,
respectively. For all other figures noted, the Student’s t
test was used. Probability values < 0.05 were considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated a role for granulocytes in a model of
lethal cytokine toxicity triggered by T. gondii
(26). However, as shown in Fig. 1
and as demonstrated
elsewhere (9, 33), granulocytes are required to survive
acute infection with the parasite. Thus, animals administered depleting
anti-granulocyte mAb are unable to survive beyond 11 days following
oral infection with cysts of the low virulence parasite strain, ME49.
While it is possible that eosinophils, which are recognized and
depleted by anti-GR-1 mAb, contribute to resistance, we believe
that neutrophils are more likely to be the pivotal cell type in this
response. Thus, in our own studies, infected
IL-5-/- mice, which display an impaired
eosinophil response, survive acute stage disease (48). This contrasts
with the case of the anti-GR-1 mAb-treated mice, which fail to
survive beyond 11 days of infection (Fig. 1). Furthermore,
Scharton-Kersten et al. (9) reported that administration
of anti-IL-5 mAb failed to affect mortality in ME49-infected
mice.
Elicited PEC produce inflammatory cytokines in response to T. gondii Ag
Given the observation that neutropenia is correlated with
increased susceptibility to T. gondii (Fig. 1 and Refs.
9, 33) and our previous findings that granulocytes are
capable of mediating a lethal proinflammatory cytokine response
(26), we sought to determine whether neutrophils,
themselves, serve as a cytokine source during parasite stimulation.
Table I demonstrates that peripheral
leukocytes respond to T. gondii Ag by producing both IL-12
and TNF-. When granulocytes were depleted by mAb and complement
administration, there was an 60% reduction in IL-12 and 80%
decrease in TNF- production, suggesting that peripheral blood
granulocytes do indeed elaborate cytokines in response to T.
gondii. Further support for this comes from our recent studies
indicating that human neutrophils produce IL-12 p70 and TNF- in
response to T. gondii (47).
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shows that stimulation with increasing concentrations of STAg elicited
a strong IL-12 response with saturation occurring at 20 µg/ml. In
contrast, LPS failed to induce a response of similar magnitude.
Interestingly, this pattern of cytokine production was reversed when we
measured TNF- in the same supernatants. Thus, STAg was a very weak
TNF- stimulus, whereas LPS stimulation induced high levels of this
cytokine. This dichotomy of response suggests that STAg and LPS provide
distinct signals to the responder population, a hypothesis supported by
the observation that neutrophil-enriched populations from LPS
nonresponder mouse strains produce IL-12 and TNF- after T.
gondii stimulation at levels equivalent to those observed in LPS
responder strains (data not shown). The finding that cultures
stimulated with LPS contain high TNF- but low IL-12 levels may, in
part, be explained by an inhibitory effect of TNF- on IL-12
production (34).
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, C
and D, fibroblast lysate did not elicit detectable cytokine
production at 2 or 20 µg/ml, whereas in parallel cultures, the same
concentration of STAg elicited both IL-12 and TNF-. We also examined
the response to Schistosoma mansoni SEA, an unrelated
parasite Ag preparation. SEA failed to elicit cytokine production (Fig. 2, C and D). IL-12 production in response to high and low virulence T. gondii strains
It has long been known that different strains of T.
gondii display different levels of virulence in the murine host
(35, 36, 37, 38). Since IL-12 is critical in the development of a
type 1 phenotype, we asked if high and low virulence parasite strains
differ in their IL-12-inducing capability. FTZ from RH (high
virulence), ME49 (low virulence), and C (low virulence) strains were
used to stimulate PEC in vitro. As shown in Fig. 3, the three strains elicited equivalent
levels of IL-12 in culture supernatants collected at 6 h.
Moreover, we were unable to detect a difference in samples cultured up
to 24 h (data not shown). Therefore, differing abilities to induce
neutrophil IL-12 are unlikely to account for the virulence pattern of
the parasite.
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Since macrophages were present in our thioglycollate-elicited cell
populations, it was possible that these cells, and not neutrophils,
were responsible for the cytokines produced in response to STAg.
Therefore, we sought to develop a method to select and eliminate
macrophages from our preparations. Macrophages were removed by
successive rounds of binding to immunomagnetic beads coupled to
anti-MHC class II mAb. Fig. 4
demonstrates a typical stained cytospin preparation from an elicited
PEC population before and after macrophage removal. As can be seen in
Fig. 4B, the overwhelming majority of macrophages has been
eliminated after the immunomagnetic bead depletion, leaving a
population of highly purified neutrophils. Notably, eosinophils
consistently comprised <2.8% of the cell population.
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shows the results of dose response
and kinetics experiments using purified neutrophils. As demonstrated in
Fig. 5A, considerable IL-12 production was detected, which
again saturated at 20 µg/ml of STAg. In contrast to IL-12, TNF-
levels were low over the dose range employed (Fig. 5B).
Additionally, we often detect a very small amount of TNF- in the
supernatants of peritoneal neutrophils cultured in medium alone (Fig. 5B). This may be an artifact of culture or a response to the
thioglycollate. We next sought to define the kinetics of cytokine
production in vitro. As shown in Fig. 5C, purified
neutrophil populations produced IL-12, which was first detectable after
6 h of STAg stimulation, and levels peaked at 12 h. We were
unable to detect IL-12 in supernatants from neutrophils cultured with
medium alone. TNF- protein was detected by 2 h, and levels
increased slightly throughout the time course. Low levels of TNF-
were found in the medium-stimulated neutrophil cultures, but the levels
were always significantly less than that obtained in the presence of
STAg (Fig. 5D). The finding that TNF- release occurred
within 2 h of STAg stimulation suggests that at least some of this
cytokine may be stored in a preformed state in neutrophils, as it is in
other cell types, such as the mast cell (39, 40). We are
currently exploring this hypothesis.
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, STAg induced increased expression of
neutrophil IL-12 p40 transcripts as early as 2 h post stimulation.
This response diminished over time and was near background by 24
h. This profile is consistent with the protein data in Fig. 5C; stimulation with STAg up-regulated transcription of p40
by 2 h with protein being detected by 6 h. We also examined
transcriptional expression of TNF-, but were unable to demonstrate a
consistent pattern of up-regulation (data not shown). While this may be
taken as evidence that TNF- is stored preformed in neutrophils, such
an interpretation must be treated with caution since regulation of this
cytokine occurs largely at the posttranscriptional level (41, 42).
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clearly demonstrates that the majority
of IL-12 and TNF- was elaborated by neutrophils. Thus, <33% of the
total protein measured in either case was contributed by
macrophages.
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). Interestingly, the corresponding
macrophage population produced much higher levels of IL-10, and indeed,
we cannot rule out the possibility that the low amount of IL-10 in the
neutrophil population derived solely from contaminating
macrophages. Inflammatory cytokine production by STAg-stimulated neutrophils from TNFR p55-/-, IL-12-/-, and GKO mice
To explore the role of endogenous cytokines in the IL-12 and
TNF- neutrophil responses, cytokine and cytokine receptor knockout
mice were i.p. injected with 10% thioglycollate, and neutrophils were
purified out of the resultant PEC population. Neutrophils were
stimulated with medium or 200 µg/ml of STAg for 6 h, and IL-12
and TNF- were measured in the supernatants. As shown in Fig. 8, A and B, IFN-
and TNFR p55 are not required for IL-12 production. However, the role
of endogenous IFN- in neutrophil TNF- production appears more
complex. In GKO mice, neutrophils produced TNF-, regardless of
stimulation (Fig. 8C). The level of TNF- from
medium-stimulated cultures from the knockout animals was consistently
higher than wild type over several experiments. While we do not yet
understand the basis for this result, similar findings have been
reported during Histoplasma capsulatum infection in GKO mice
(43). Endogenous IL-12 was found not to influence
neutrophil TNF- production (Fig. 8D). While we often
detected more TNF- in the knockout medium-stimulated cultures as
compared with wild type, the differences were never statistically
significant.
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We next asked if neutrophils would respond to infection with
T. gondii in vivo. Mice were i.p. injected with PBS or live
RH strain tachyzoites suspended in PBS. In response to the parasite,
there was an influx of neutrophils into the peritoneal cavity that
peaked by 8 h (Fig. 9A).
As Fig. 9B shows, the neutrophil influx was accompanied by
increased levels of IL-12 p40 gene transcripts. Parasite-induced IL-12
was apparent at 4 h postinfection, a time preceding a significant
neutrophil influx. This early response is likely to originate from
resident cells present in the peritoneal cavity. In this regard, it
should be noted that neutrophils comprised 38% of the total resident
cell population. Because the neutrophil influx correlated with
increased levels of IL-12 p40 transcripts, these results suggest that
T. gondii triggers neutrophil IL-12 synthesis in an in vivo
situation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Importantly, these
responses occur using neutrophils isolated from GKO mice, which are
defective in IFN- production. Although most of the studies presented
here employed thioglycollate-elicited neutrophils, we do not believe
that preactivation with this inflammatory stimulus is a prerequisite
for parasite-triggered cytokine production. First, neutrophils from the
peripheral blood of humans (47) and mice (Table I) also
produce IL-12 and TNF- when stimulated with parasite Ag. Second,
i.p. injection of tachyzoites results in a rapid neutrophil influx, and
this correlates with the appearance of IL-12 p40 transcripts in the
peritoneal cavity. Because neutrophil depletion renders mice
susceptible to normally nonlethal infection, a hypothesis we are
currently exploring is that these cells are an important early cytokine
source, triggering cell-mediated immunity and driving macrophage
activation. The concept of neutrophils fulfilling a crucial role during microbial infection is underscored by studies in mice infected with Candida albicans. In this system, neutrophil production of IL-12 or IL-10 determines self-healing or non-healing disease progression, respectively. Whether IL-12 or IL-10 is produced is determined by the particular Candida strain used. Mice that are neutrophil-depleted at the time of infection with a low pathogenicity strain of C. albicans die rapidly with an ongoing type 2 response, unless rescued by rIL-12. Thus, neutrophils are pivotal in shaping the immune response to this pathogen. Our results suggest that T. gondii stimulates a robust neutrophil IL-12 response with minimal amounts of IL-10. This finding is consistent with the well-known ability of the parasite to stimulate a strong type 1 cytokine response.
Nevertheless, we do not yet know if neutrophils serve a pivotal immunoregulatory role in toxoplasmosis as they appear to do during murine candidiasis. It is well known that an effective cell-mediated immune response requires early IL-12 production. While dendritic cells and macrophages produce IL-12, our data establish that neutrophils also contribute to this response. Our in vivo results demonstrate that murine neutrophils respond to live tachyzoites by rapidly migrating to the area of infection, and the influx of these cells generally correlates with appearance of IL-12 p40 transcripts.
The first appearance of IL-12 and TNF- in culture supernatants of
parasite Ag-stimulated neutrophils occurred at 6 and 2 h,
respectively, and levels saturated at 20 µg/ml of STAg for both
cytokines. The protein kinetics profile and PCR data suggest that IL-12
is made de novo. The rapid appearance of TNF- protein could be due
to secretion of preformed, stored cytokine, and, indeed, mast cells
store ready-made TNF- in their granules that is rapidly released
upon appropriate stimulation (39, 40). It is also possible
that neutrophils store inactive TNF- transcripts that become
modified and are translated rapidly in response to STAg stimulation
(44). We are currently examining this issue further.
Our data demonstrate that the neutrophil response to STAg is distinct
from that induced by LPS. Parasite Ag was a much stronger stimulus for
IL-12 production but only elicited low levels of TNF-. In contrast,
LPS induced much less IL-12 while eliciting very high levels of
TNF-. Therefore, our results suggest that each stimulus induces a
distinct set of biochemical pathways in the neutrophil. In support of
this hypothesis, neutrophils from C3H-HeJ mice, an LPS nonresponder
strain, maintain the ability to produce high levels of IL-12 and low
levels of TNF- when stimulated with STAg (data not shown). An
alternative, and not mutually exclusive, model is that LPS rapidly
induces much higher TNF- levels, and this cytokine then inhibits
simultaneous IL-12 production. Such an immunoregulatory mechanism has
recently been demonstrated using elicited peritoneal macrophages
(34). Indeed, we have consistently found that neutrophils
from TNFR p55-/- mice produce higher levels of
IL-12 in response to STAg stimulation when compared with wild type.
While the data are not conclusive, they support the concept that
TNF- may exert anti-inflammatory control under certain
conditions of neutrophil stimulation.
From our data, it is clear that neither IL-12 nor IFN- is required
for TNF- production. Levels of the latter cytokine in response to
STAg, while low, were not significantly different between knockout and
wild-type neutrophils. Curiously, we found that medium-stimulated
neutrophils from GKO mice produced higher levels of TNF- than did
wild-type cells. Zhou et al. (43) reported that TNF- in
GKO mice secondarily infected with H. capsulatum were highly
increased over wild-type mice. These investigators found that TNF-,
in the absence of IFN-, served a compensatory and protective role
because neutralizing anti-TNF- mAb treatment rendered the mice
susceptible to reinfection (43). Our studies provide some
evidence for increased TNF- levels in GKO mice. Nevertheless, in the
absence of IFN-, unlike the case of murine H. capsulatum
infection, TNF- cannot compensate since GKO animals uniformly
succumb to acute toxoplasmosis (45).
We do not yet know whether neutrophils provide the initial source of
IL-12 that triggers cell-mediated immunity during infection. In
addition to neutrophils, both activated macrophages and dendritic cells
produce IL-12 in response to T. gondii. Our data show that
neutrophils can serve as an IFN--independent source of IL-12.
Moreover, the finding that IL-12, itself, acts directly on dendritic
cells to prime for IL-12 production raises the possibility that
neutrophils are important in early dendritic cell activation by
T. gondii (46). The high number of circulating
neutrophils, as well as their ability to rapidly migrate in large
numbers to lesions within the host, lends support to a model wherein
neutrophil IL-12 production plays an important role in the pathogenesis
of toxoplasmosis and other microbial infections.
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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, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. E-mail address:
3 Abbreviations used in this paper: GKO, IFN--/- mice; FTZ, freeze-thaw tachyzoites; PEC, peritoneal exudate cells; SEA, soluble egg Ag; STAg, soluble tachyzoite Ag.
Received for publication February 24, 1999. Accepted for publication June 7, 1999.
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