Neutrophils are well known to rapidly migrate to foci of infection, where they exert microbicidal functions. We sought to determine whether neutrophils responding to in vivo infection with the protozoan pathogen Toxoplasma gondii were capable of IL-12 production as suggested by recent in vitro studies. Intraperitoneal infection induced a neutrophil influx by 4 h, accompanied by ex vivo IL-12 p40 and p70 release. Approximately 85% of the neutrophils displayed intracellular stores of IL-12, as determined by flow cytometry and confocal fluorescence microscopy. Neutrophils from IFN-γ knockout mice also expressed IL-12, ruling out an IFN-γ-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.

Neutrophils are critical effector cells in the host’s response to microbial invasion (1). They play a role in recognizing and neutralizing bacteria, fungi, and parasites (2, 3, 4, 5, 6). To accomplish this, neutrophils must possess a coordinated and specialized set of functions that mediate detection and chemotaxis toward an invading micro-organism and effective killing mechanisms. Their ability to defend the body relies in part upon rapid mobilization of preformed compounds found in granules such as the degradative enzymes cathepsin G, elastase, and lysozyme (7, 8). Recent findings from several laboratories, including our own, have revealed that neutrophils also release several immunomodulatory cytokines in response to in vitro microbial stimulation (3, 6, 9, 10). Specifically, our work has focused on the role of neutrophils in the early immune response to the microbial pathogen, Toxoplasma gondii.

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-γ 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.

C57BL/6 female mice (6–12 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME). IFN-γ 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.

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 × 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).

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 × 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 × 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.

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.

Cells (2 × 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 κ 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).

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 × 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 κ 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 × 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.

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.

FIGURE 1.

Production of IL-12 by T. gondii-infected peritoneal cells. C57BL/6 mice were injected i.p. with 2 × 106 RH strain tachyzoites or PBS. After 4 h the peritoneal cells were obtained by lavage with PBS and incubated in medium alone at 4 × 106/well for 18 h. Cell-free supernatants were collected for cytokine measurement by ELISA as described in Materials and Methods. The infected population was composed of 46% neutrophils, 46.5% macrophages, 6.5% lymphocytes, and 1% eosinophils as determined by differential counts. The control population was composed of 6% neutrophils, 83% macrophages, 10.5% lymphocytes, and 0.5% eosinophils. Results are expressed as the mean ± SD. ND, none detected. Differences between infected and uninfected cultures were statistically significant.

FIGURE 1.

Production of IL-12 by T. gondii-infected peritoneal cells. C57BL/6 mice were injected i.p. with 2 × 106 RH strain tachyzoites or PBS. After 4 h the peritoneal cells were obtained by lavage with PBS and incubated in medium alone at 4 × 106/well for 18 h. Cell-free supernatants were collected for cytokine measurement by ELISA as described in Materials and Methods. The infected population was composed of 46% neutrophils, 46.5% macrophages, 6.5% lymphocytes, and 1% eosinophils as determined by differential counts. The control population was composed of 6% neutrophils, 83% macrophages, 10.5% lymphocytes, and 0.5% eosinophils. Results are expressed as the mean ± SD. ND, none detected. Differences between infected and uninfected cultures were statistically significant.

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FIGURE 9.

Systemic levels of IL-12 p40 are decreased in neutrophil-depleted, infected mice. Mice (five per group) were administered 200 μg of anti-Gr-1 mAb (depleted, infected) or a control rat Ig (control, infected) i.p. on days −2, 0, +2, and +4. Mice were infected with 100 ME49 cysts i.p. on day 0. Data from two uninfected mice were also included. Serum was obtained every 2 days beginning on day 0, and levels of IL-12 p40 were determined by ELISA. Results are expressed as the mean ± SD. Differences between control, infected, and depleted, infected mice were statistically significant on days 4, 6, and 8.

FIGURE 9.

Systemic levels of IL-12 p40 are decreased in neutrophil-depleted, infected mice. Mice (five per group) were administered 200 μg of anti-Gr-1 mAb (depleted, infected) or a control rat Ig (control, infected) i.p. on days −2, 0, +2, and +4. Mice were infected with 100 ME49 cysts i.p. on day 0. Data from two uninfected mice were also included. Serum was obtained every 2 days beginning on day 0, and levels of IL-12 p40 were determined by ELISA. Results are expressed as the mean ± SD. Differences between control, infected, and depleted, infected mice were statistically significant on days 4, 6, and 8.

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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 × 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.

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. 2,A 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. 2 D 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.

FIGURE 2.

Reciprocal expression of IL-12 and IL-10 by peritoneal neutrophils and macrophages, respectively. Cells from 4-h infected and PBS-injected mice were examined by confocal fluorescence microscopy after staining for IL-12 (A and D), IL-10 (B and E), or isotype control (C and F). Nuclei were counterstained with propidium iodide. A–C, Infected populations; D–F, control populations from uninfected animals. A, The arrows point to IL-12+ neutrophils. B, Numerous neutrophils (e.g., white arrow) that do not display IL-10 in contrast to the surrounding macrophages (e.g., green arrow). D, The white arrow indicates a neutrophil staining for IL-12 in an uninfected population. E, The white arrow shows a neutrophil from an uninfected population that does not stain for IL-10. The scale bar in A represents 20 μm. The infected population was composed of 36% neutrophils, 46% macrophages, 17% lymphocytes, and 1% eosinophils as determined by differential counts. The control population was composed of 3% neutrophils, 76% macrophages, 19% lymphocytes, and 1% eosinophils.

FIGURE 2.

Reciprocal expression of IL-12 and IL-10 by peritoneal neutrophils and macrophages, respectively. Cells from 4-h infected and PBS-injected mice were examined by confocal fluorescence microscopy after staining for IL-12 (A and D), IL-10 (B and E), or isotype control (C and F). Nuclei were counterstained with propidium iodide. A–C, Infected populations; D–F, control populations from uninfected animals. A, The arrows point to IL-12+ neutrophils. B, Numerous neutrophils (e.g., white arrow) that do not display IL-10 in contrast to the surrounding macrophages (e.g., green arrow). D, The white arrow indicates a neutrophil staining for IL-12 in an uninfected population. E, The white arrow shows a neutrophil from an uninfected population that does not stain for IL-10. The scale bar in A represents 20 μm. The infected population was composed of 36% neutrophils, 46% macrophages, 17% lymphocytes, and 1% eosinophils as determined by differential counts. The control population was composed of 3% neutrophils, 76% macrophages, 19% lymphocytes, and 1% eosinophils.

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Since neutrophils have been reported to be a source of IL-10 in C. albicans infection (3, 19), we also examined RH-infected and PBS-injected populations for expression of this cytokine. Fig. 2,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. 2,E, compare to Fig. 2,B). 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. 3,A). This was true even for tachyzoite-infected macrophages, as shown in Fig. 3,A. Additionally, macrophages appear to be preferentially infected over neutrophils. Fig. 3 B 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.

FIGURE 3.

Infected macrophages are apparent by 6 h p.i., but only neutrophils stain for IL-12. Six-hour infected peritoneal cells were obtained by lavage and stained for IL-12 by indirect immunofluorescence as described in Fig. 2. A, Arrows indicate tachyzoites within macrophages. These cells display little or no evidence of IL-12 production, in contrast to the surrounding neutrophils. B, Higher magnification of granulocytes showing punctate cytosolic staining for IL-12. The bar is equivalent to 5 μm. The population was composed of 26% neutrophils, 47% macrophages, 27% lymphocytes, and 0% eosinophils.

FIGURE 3.

Infected macrophages are apparent by 6 h p.i., but only neutrophils stain for IL-12. Six-hour infected peritoneal cells were obtained by lavage and stained for IL-12 by indirect immunofluorescence as described in Fig. 2. A, Arrows indicate tachyzoites within macrophages. These cells display little or no evidence of IL-12 production, in contrast to the surrounding neutrophils. B, Higher magnification of granulocytes showing punctate cytosolic staining for IL-12. The bar is equivalent to 5 μm. The population was composed of 26% neutrophils, 47% macrophages, 27% lymphocytes, and 0% eosinophils.

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To better quantitate IL-12-producing cells, flow cytometric analysis of peritoneal cells was performed. Infected and control peritoneal cells were obtained 4 h p.i., fixed and permeabilized, and stained with anti-Gr-1 and anti-IL-12 mAb. Fig. 4, 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. 4,C 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. 4,D). To determine whether injection of PBS alone provided sufficient stimulus to trigger the IL-12 expression shown in Fig. 4,C, we also examined peritoneal populations from noninjected animals. This cell population displayed virtually identical characteristics to those shown in Fig. 4 C (data not shown).

FIGURE 4.

Flow cytometric analysis of IL-12 production by peritoneal cells. C57BL/6 mice were injected with PBS or infected with 2 × 106 RH strain tachyzoites. Four hours later peritoneal cells were obtained, permeabilized, and stained for IL-12 and Gr-1 as described in Materials and Methods. A and B, Forward and side scatter plots for uninfected and infected populations, respectively. The Gr-1-expressing subsets have been gated, and expression of IL-12 and Gr-1 are shown in C and D. The numbers in each quadrant indicate the percentage of the total population shown. The infected population was composed of 56% neutrophils, 40% macrophages, 2% lymphocytes, and 2% eosinophils, while the control population was composed of 13% neutrophils, 78% macrophages, 9% lymphocytes, and 0% eosinophils.

FIGURE 4.

Flow cytometric analysis of IL-12 production by peritoneal cells. C57BL/6 mice were injected with PBS or infected with 2 × 106 RH strain tachyzoites. Four hours later peritoneal cells were obtained, permeabilized, and stained for IL-12 and Gr-1 as described in Materials and Methods. A and B, Forward and side scatter plots for uninfected and infected populations, respectively. The Gr-1-expressing subsets have been gated, and expression of IL-12 and Gr-1 are shown in C and D. The numbers in each quadrant indicate the percentage of the total population shown. The infected population was composed of 56% neutrophils, 40% macrophages, 2% lymphocytes, and 2% eosinophils, while the control population was composed of 13% neutrophils, 78% macrophages, 9% lymphocytes, and 0% eosinophils.

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To confirm that only the granulocytes produced IL-12, all the major subsets in an RH-infected population were gated, as demonstrated in Fig. 5,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. 5,C) and depleted one-half of the population of granulocytes with immunomagnetic beads coated with anti-Gr-1 mAb (Fig. 5,D). 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.

FIGURE 5.

Only the granulocyte subset stains for IL-12. Flow cytometric analysis of RH-infected peritoneal cells was performed. The major subsets of cells were gated as shown in A, and histograms demonstrating IL-12 staining are displayed in B. Only the subset delineated R2 and corresponding to the granulocytes (determined by back-gating) possessed IL-12. Solid lines indicate staining with anti-IL-12 mAb, and dashed lines indicate staining with an isotype control. The population was composed of 53% neutrophils, 42% macrophages, 2% lymphocytes, and 3% eosinophils as determined by differential counts. To demonstrate that the granulocytes fall within the R2 gate, a 4-h RH-infected population was obtained and is shown in C. One-half of the cells were depleted of granulocytes through the use of immunomagnetic beads bound to anti-Gr-1 mAb (D). Numbers in C and D refer to the percentage of cells within each gate. A decrease after granulocyte depletion was found only in R2. E and F, Stained cytospins of the populations in C and D, respectively, showing a loss of neutrophils. The composition of cells in C was 54% neutrophils, 41% macrophages, 5% lymphocytes, and 0% eosinophils. The composition of cells in D was 4% neutrophils, 91% macrophages, 5% lymphocytes, and 0% eosinophils.

FIGURE 5.

Only the granulocyte subset stains for IL-12. Flow cytometric analysis of RH-infected peritoneal cells was performed. The major subsets of cells were gated as shown in A, and histograms demonstrating IL-12 staining are displayed in B. Only the subset delineated R2 and corresponding to the granulocytes (determined by back-gating) possessed IL-12. Solid lines indicate staining with anti-IL-12 mAb, and dashed lines indicate staining with an isotype control. The population was composed of 53% neutrophils, 42% macrophages, 2% lymphocytes, and 3% eosinophils as determined by differential counts. To demonstrate that the granulocytes fall within the R2 gate, a 4-h RH-infected population was obtained and is shown in C. One-half of the cells were depleted of granulocytes through the use of immunomagnetic beads bound to anti-Gr-1 mAb (D). Numbers in C and D refer to the percentage of cells within each gate. A decrease after granulocyte depletion was found only in R2. E and F, Stained cytospins of the populations in C and D, respectively, showing a loss of neutrophils. The composition of cells in C was 54% neutrophils, 41% macrophages, 5% lymphocytes, and 0% eosinophils. The composition of cells in D was 4% neutrophils, 91% macrophages, 5% lymphocytes, and 0% eosinophils.

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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. 6,A. 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. 6 B, 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.

FIGURE 6.

Granulocytes express the maturation marker Gr-1 at different levels. Peritoneal cells were obtained from infected (A) and control (B) mice 4 h after i.p. infection with RH or PBS injection, respectively. Cells were stained with anti-Gr-1, and nuclei were counterstained with propidium iodide. This was followed by confocal fluorescence microscopy. Many neutrophils are visible in A, but levels of Gr-1 differ from cell to cell, as evidenced by the arrows. The white arrow in B indicates a neutrophil staining for Gr-1, but numerous Gr-1 neutrophils can be seen. The scale bar in B represents 20 μm. The infected population was composed of 84% neutrophils, 11% macrophages, 3% lymphocytes, and 2% eosinophils. The control population was composed of 11% neutrophils, 55% macrophages, 34% lymphocytes, and 0% eosinophils.

FIGURE 6.

Granulocytes express the maturation marker Gr-1 at different levels. Peritoneal cells were obtained from infected (A) and control (B) mice 4 h after i.p. infection with RH or PBS injection, respectively. Cells were stained with anti-Gr-1, and nuclei were counterstained with propidium iodide. This was followed by confocal fluorescence microscopy. Many neutrophils are visible in A, but levels of Gr-1 differ from cell to cell, as evidenced by the arrows. The white arrow in B indicates a neutrophil staining for Gr-1, but numerous Gr-1 neutrophils can be seen. The scale bar in B represents 20 μm. The infected population was composed of 84% neutrophils, 11% macrophages, 3% lymphocytes, and 2% eosinophils. The control population was composed of 11% neutrophils, 55% macrophages, 34% lymphocytes, and 0% eosinophils.

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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. 7,A. 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. 7 B shows similar results from GKO mice; 91% of the Gr-1+ cells stained for IL-12. This suggests that IFN-γ is not required for IL-12 production by peritoneal neutrophils during in vivo infection.

FIGURE 7.

Granulocyte expression of IL-12 is IFN-γ independent. C57BL/6 (A) and GKO (B) mice were infected i.p. with 2 × 106 RH strain tachyzoites. Four hours later, peritoneal cells were obtained, permeabilized, and stained for IL-12 and Gr-1 as described in Materials and Methods. The Gr-1-expressing subsets have been gated, and expression of IL-12 and Gr-1 is shown, with the percentages in each quadrant indicated. The C57BL/6 population was composed of 40% neutrophils, 49% macrophages, 11% lymphocytes, and 0% eosinophils, as determined by differential counts. The GKO population was composed of 45% neutrophils, 46% macrophages, 9% lymphocytes, and 0% eosinophils.

FIGURE 7.

Granulocyte expression of IL-12 is IFN-γ independent. C57BL/6 (A) and GKO (B) mice were infected i.p. with 2 × 106 RH strain tachyzoites. Four hours later, peritoneal cells were obtained, permeabilized, and stained for IL-12 and Gr-1 as described in Materials and Methods. The Gr-1-expressing subsets have been gated, and expression of IL-12 and Gr-1 is shown, with the percentages in each quadrant indicated. The C57BL/6 population was composed of 40% neutrophils, 49% macrophages, 11% lymphocytes, and 0% eosinophils, as determined by differential counts. The GKO population was composed of 45% neutrophils, 46% macrophages, 9% lymphocytes, and 0% eosinophils.

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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%.

FIGURE 8.

Peripheral blood granulocytes prestore IL-12. Peripheral blood was obtained from uninfected C57BL/6 mice as described in Materials and Methods. PBL were isolated and stained for IL-12 and Gr-1. Flow cytometric analysis was performed on the ungated population. The numbers in each quadrant indicate the percentage of the total population shown.

FIGURE 8.

Peripheral blood granulocytes prestore IL-12. Peripheral blood was obtained from uninfected C57BL/6 mice as described in Materials and Methods. PBL were isolated and stained for IL-12 and Gr-1. Flow cytometric analysis was performed on the ungated population. The numbers in each quadrant indicate the percentage of the total population shown.

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When mice were infected with RH, and PBL were analyzed by flow cytometry 4 h after infection, there was leukocytosis with neutrophilia compared with uninfected mice (Table I 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.

Table I.

Infection with RH leads to a neutrophilia with increases in both Gr-1low and Gr-1high subsets

Noninfecteda,bInfectedIncrease of Infected/Noninfectedc
Expt.Gr-1lowGr-1highGr-1lowGr-1highGr-1lowGr-1high
5.88 9.48 10.12 21.17 1.72 2.23 
5.14 8.01 11.97 14.24 2.33 1.78 
9.07 5.48 9.51 11.65 1.05 2.13 
Noninfecteda,bInfectedIncrease of Infected/Noninfectedc
Expt.Gr-1lowGr-1highGr-1lowGr-1highGr-1lowGr-1high
5.88 9.48 10.12 21.17 1.72 2.23 
5.14 8.01 11.97 14.24 2.33 1.78 
9.07 5.48 9.51 11.65 1.05 2.13 
a

Mice were i.p. infected with 2 × 106 RH strain tachyzoites. Four hours after infection, blood was collected from infected and noninfected mice and stained for flow cytometry as described in Materials and Methods.

b

Numbers indicate percentage of cells in the specified Gr-1 subset within the total PBL population.

c

Numbers indicate fold increase of infected cells over noninfected cells.

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.

In this paper, we have demonstrated that neutrophils produce and secrete bioactive IL-12 during early microbial infection. Within 4 h of inoculation, there was a 7-fold increase in the frequency of neutrophils in the peritoneal cavity, on the average, thus illustrating their capacity to rapidly respond to a biological insult (Figs. 1, 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-γ-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-α (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-γ 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.

We are grateful to Drs. T. G. Clark and E. J. Pearce for critical review of the manuscript.

1

This work was supported by National Institutes of Health Grant AI40540.

3

Abbreviations used in this paper: GKO, IFN-γ−/− mice; STAg, soluble tachyzoite Ag; p.i., postinfection; NMS, normal mouse serum.

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