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Originally published online as doi:10.1189/jlb.0403164 on September 12, 2003

Published online before print September 12, 2003
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(Journal of Leukocyte Biology. 2003;74:1015-1025.)
© 2003 by Society for Leukocyte Biology

A novel population of Gr-1+-activated macrophages induced during acute toxoplasmosis

Dana G. Mordue1 and L. David Sibley2

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri

2Correspondence: Department of Molecular Microbiology, Box 8230, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: sibley{at}borcim.wustl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages are potent mediators of parasite control following in vitro activation, yet the subsets of mononuclear cells that contribute to resistance in vivo remain poorly defined. To identify effector cells that contribute to the control of Toxoplasma gondii during the initial stages of disseminated infection, we developed a low-dose intraperitoneal challenge model. A population of unusual macrophage-like cells was recruited to the peritoneal cavity during the first 4 days postinfection. Surprisingly, these cells expressed the granulocyte marker Gr-1 and the macrophage marker CD68. They also expressed high levels of major histocompatibility complex class II and low levels of F4/80 and CD11b and were negative for the immature myeloid cell marker CD31, the dendritic cell marker CD11c, and the B cell marker B220. Gr-1+ macrophages produced interleukin-12 p40, generated reactive nitrogen intermediates during acute infection, and inhibited virulent type I and nonvirulent type II strains of the parasite in vitro. Gr-1+ macrophages were the primary cell type recruited in response to nonvirulent type II strain parasites, and large numbers of neutrophils (Gr-1+/CD68-) were also recruited to the peritoneum in response to virulent type I strain parasites. Our findings suggest that activated CD68+/Gr-1+ macrophages contribute to parasite control during infection by directly inhibiting parasite replication and through production of T helper cell type I cytokines.

Key Words: host defense • intracellular pathogen • neutrophils • cellular activation • dendritic cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Toxoplasma gondii is an obligate intracellular parasite that infects a variety of warm-blooded vertebrates including humans [1 ]. Acute toxoplasmosis induces a strong interferon-{gamma} (IFN-{gamma})-dependent cell-mediated immune response that typically leads to the control of the fast-growing tachyzoite form of the parasite. Chronic infection is a consequence of the conversion of the tachyzoite form of the parasite into the persistent bradyzoite stage, which is contained within tissue cysts [2 ]. In immunocompromised individuals, conversion of bradyzoites back to tachyzoites can result in fatal toxoplasmosis if left untreated [3 ]. During acute infection and reactivation of chronic infection, pathology occurs in part as a result of parasite-mediated destruction of cells and the ensuing inflammatory response.

Although strains of T. gondii are similar, the vast majority of isolates belongs to one of three distinct clonal lineages [4 , 5 ]. The outcome of acute toxoplasmosis in the mouse model is strongly dependent on the genotype of the parasite [5 6 7 ]. Type I strains of the parasite are unique in their extreme virulence in the murine host, where a single, viable parasite rapidly leads to death, irrespective of the genetic background of the host [5 6 7 ]. Virulence is in part a result of enhanced migration by type I strains that allows them to cross biological barriers more rapidly and consequently, to spread systemically [8 ]. The higher tissue burdens reached by type I strains ultimately lead to a cascade of proinflammatory mediators that induce pathology [9 ]. In contrast, types II and III strains of the parasite have 50% lethal doses of >=103 parasites and readily establish chronic infection, and the outcome of infection is dependent on the genetic background of the host [10 11 12 13 ]. Although the role of parasite genotype in disease severity is less clear in humans, several studies suggest that type I strains of parasite may have a greater propensity to cause ocular and congenital toxoplasmosis [14 15 16 ].

T. gondii has been used as a model intracellular pathogen to discover basic mechanisms of immune regulation and function in the murine host. These studies reveal that IFN-{gamma} is required to control infection [17 18 19 ] and that IFN-{gamma} receptors must be present on hematopoietic and nonhematopoietic cell populations [19 ]. IFN-{gamma} also serves to up-regulate expression of inducible GTPases that are required for parasite control [20 , 21 ]. Interleukin (IL)-12 is critically important for the proper induction of IFN-{gamma}, and during the initial infection, macrophages [22 ], neutrophils [23 ], and dendritic cells [24 ] are important sources of IL-12 production. Although CD4+ and CD8+ T cells are important for controlling acute infection, innate-immune responses, mediated in particular by natural killer (NK) cells, also contribute to early control of parasite replication [25 , 26 ]. Neutrophils also play an important role in control of acute infection, and they are rapidly recruited following high-dose challenge via intraperitoneal (i.p.) inoculation [27 , 28 ]. Notably, CXC chemokine receptor 2 knockout mice, which have defects in neutrophil chemotaxis, are more susceptible to infection by normally nonlethal challenge with type II strain T. gondii [29 ].

Previous infection studies using i.p. inoculation of parasites have relied on high-dose challenges [9 , 23 , 27 28 29 ]. This model is most appropriate for monitoring the cellular responses to high antigen loads that occur late during acute infection. Following oral infection, it is likely that disseminated toxoplasmosis results from a few organisms that penetrate across the intestinal barrier and spread systemically [30 31 32 ]. However, oral infection is inherently highly variable and thus difficult to use as a model for dissemination. To evaluate the early immune response to low-dose challenge, we developed a low-dose i.p. inoculation model that simulates the effects of early disseminated infection with T. gondii in the murine host.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
CD1 outbred mice were obtained from Charles River (Wilmington, MA). Animals were housed under specific pathogen-free conditions at Washington University School of Medicine (St. Louis, MO). Female mice between 8 and 12 weeks of age were used for experiments.

Parasites and infection
T. gondii strains were maintained by serial 2-day passage of tachyzoites in human foreskin fibroblast cells as described previously [9 ]. The RH strain [American Type Culture Collection (ATCC), Manassas, VA, #50838; ref. 33 ] was used as a representative of the type I genotype. A cloned line of the ME49 strain, PTG (ATCC #50841) [34 ], was used as a representative of the type II genotype. Infections were established using a low-dose challenge of 100 parasites administered by i.p. injection. RH strain parasites expressing green fluorescent protein (GFP; strain RH-GFP5S65T; ATCC #50940) and PTG strain parasites expressing GFP (strain PTG-GFP5S65T; ATCC #50941) were propagated as above. All strains were tested for mycoplasma contamination using the GenProbe kit (Fisher Scientific, Pittsburgh, PA) and remained negative throughout the experiments.

Antibodies (Ab)
The following cell markers were detected using labeled Ab obtained from BD PharMingen (San Diego, CA): Ly-6G (Gr-1) was detected with monoclonal Ab (mAb) RB6-8C5, NK cells were detected using mAb DX5, CD8+ cells were detected with mAb 53-6.7, CD4+ T cells were detected with mAb GK1.5, B cells were detected with mAb RA3-6B2 specific for CD45R/B220, {alpha}ß T cells were detected with mAb H57-597, {gamma}{delta} T cells were detected with mAb GL3, CD11b (Mac-1) was detected with mAb M1/70, major histocompatibility complex (MHC) class II was detected with mAb M5114, CD31 was detected with MEC 13.3 mAb. The following Ab were obtained from eBioscience (San Diego, CA): CD11c was detected using mAb N418, integrin {alpha}4 (CD49d) was detected with mAb R1-2, Ly-6G (Gr-1) was detected with mAb RB6-8C5 rat immunoglobulin G (IgG)2b served as a negative control. Intracellular IL-12 was detected with phycoerythrin (PE) or fluorescein isothiocyanate (FITC)-conjugated C15.6 mAb (BD PharMingen). The macrophage markers CD68 and F4/80 were detected with FITC-conjugated mAb FA/11 and PE-conjugated F4/80, respectively (Serotec, Oxford, UK). Dendritic cells were detected with mAb NLDC-145 against CD205 (DEC-205; Serotec). Unlabeled rabbit polyclonal antiserum against murine inducible nitric oxide synthase (iNOS) was obtained from BD Transduction Laboratories (Franklin Lakes, NJ). Cy-5-conjugated mAb RB6-8C5 against Ly-6G (Gr-1) was obtained from eBiosciences. Intracellular parasites were detected with rabbit polyclonal antiserum against T. gondii. For flow cytometry, rabbit anti-T. gondii IgG was directly conjugated to Alexa Fluor 647 (Molecular Probes, Eugene, OR). FcRs were blocked using mAb supernatant from the 24G2 hybridoma (ATCC) or purified anti-mouse CD16/CD32 from eBioscience. Lysosome-associated membrane protein-1 (LAMP-1) was detected with hybridoma supernatant of clone 1D4B (ATCC).

Cytokine measurement
Peritoneal cells were isolated from uninfected or infected mice and cultured in vitro at a concentration of 5 x 106 cells/ml in Dulbecco’s modified Eagle’s medium, supplemented with L-glutamine, HEPES, gentamicin, and 10% fetal calf serum (FCS; referred to as D10) at 37°C in 5% CO2. Supernatants were collected after 24 h of culture and analyzed for IFN-{gamma}, IL-12 p40, IL-12 p70, tumor necrosis factor-{alpha} (TNF-{alpha}), IL-4, and IL-10 using paired enzyme-linked immunosorbent assay Ab sets obtained from BD PharMingen.

Immunofluorescence microscopy
Peritoneal cells were deposited onto glass slides using a Cytopro cytocentrifuge (Wescor, Logan, Utah). For differential cell counts, cells were stained with Diff-Quick (American Scientific Products, McGraw Park, IL) and were examined by light microscopy. For immunofluorescence, cells were fixed with phosphate-buffered saline (PBS)-buffered formaldehyde for 10 min and permeabilized with 0.05% saponin (Sigma Chemical Co., St. Louis, MO) before staining. For experiments that required staining for Ly-6G (Gr-1), cells were stained with mAb RB6-8C5 before fixation, as the epitope was sensitive to formaldehyde. Stained slides were mounted using Vectashield with or without 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) or 50% PBS/glycerol. A minimum of 200 cells were counted per sample.

Flow cytometry and cell sorting
Peritoneal cells were isolated from infected or uninfected mice, and erythrocytes were lysed in 0.83% NH4Cl2, 0.01 M Tris HCl, pH 7.5. Peritoneal cell populations were stained with specific Ab and analyzed by flow cytometry to determine the relative percent of each cell type in the total population. For flow cytometric analysis, cell suspensions (1x106) were washed in PBS containing 3% FCS and 0.1% azide, blocked with mAb 24G2 to prevent nonspecific binding to FcR, and then stained with FITC, PE, or Cy-5-conjugated Ab in the presence of mAb 24G2. All steps were done at 4ºC. Detection of CD68 and intracellular parasites required fixation in PBS-buffered formaldehyde and permeabilization with saponin before staining with the appropriate Ab. After staining, cells were washed in PBS, and 5 x 104–1 x 105 cells were analyzed using a FACscan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). Data were analyzed using Cell Quest software (Becton Dickinson Immunocytometry Systems).

For intracellular cytokine staining, cells were initially stained with Cy-5-conjugated Gr-1 mAb (RB6-8C5), then fixed, and permeabilized as described above. Cells were then stained with FITC-conjugated anti-CD68 (mAb FA/11) and PE-conjugated anti-IL-12 (mAb C15.6). Cells were gated based on expression of IL-12 and analyzed for expression of CD68 or Gr-1 using the CellQuest software

For experiments that did not require viable cells, data were obtained by flow cytometry based on staining for Gr-1 and CD68. For functional studies, viable CD68+/Gr-1+ cells were sorted based on their high expression of Gr-1 and low-to-intermediate expression of F4/80. Differential cell counts of the sorted population confirmed that the population was 85–90% macrophages with 10–15% neutrophils.

Reactive nitrogen intermediates (RNI) and arginase detection
Peritoneal cells were isolated from uninfected or infected mice and plated at a concentration of 1 x 106 cells/ml in triplicate in 96-well plates and cultured in D10 at 37°C, 5% CO2. After 4 h, nonadherent cells were removed, and adherent cells were cultured for an additional 20 h at 37°C. Supernatants were collected at 20 h, nitrite in the supernatants was analyzed by the addition of equal volumes of Griess reagent (Sigma Chemical Co.) and culture supernatant, and the absorbance at 540 nm was determined. Nitrite concentration was determined from a standard curve generated with NaNO2. To determine the identity of iNOS+ cells, peritoneal cells were stained for expression of CD68, Gr-1, and iNOS and were examined by immunofluorescence. The percent of iNOS+ cells that were CD68+/Gr-1+ versus CD68+/Gr-1- was determined. The experiment was repeated twice with a minimum of three mice per group.

Arginase activity was measured from 106 peritoneal exudate cells from infected and uninfected mice as described previously [35 ]. Urea production was quantified by absorbance at 540 nm after addition of 40 µL {alpha}-isonitrosopropiophenone (dissolved in 100% ethanol) followed by heating at 100°C for 20 min. The experiment was repeated twice on day 4 postinfection using three to six infected mice per experiment.

Analysis of parasite inhibition
Peritoneal cells from control or infected mice were isolated 4 days after infection and cultured at 5 x 106 cells/ml for 4 h and were then briefly rinsed to remove nonadherent cells. Monolayers were challenged with 2 x 105 type I strain RH or type II strain PTG parasites that stably expressed GFP. Parasite survival and replication were analyzed after 20 h by determining the number of GFP-expressing parasites/macrophages. Results were normalized to control peritoneal cells to compare experiments. For neutralization of NO, 1 mM aminoguanidine was added to adherent cells before the addition of parasites. This concentration of aminoguanidine was sufficient to inhibit nitrite production below detectable levels (data not shown). To analyze antitoxoplasmic activity of Gr-1+ macrophages, cells from infected mice were sorted based on their high expression of Gr-1 and low-to-intermediate expression of F4/80. Sorted cells were cultured at a concentration of 2 x 106 cells/ml and challenged with 2 x 105 parasites. Parasite survival and replication were determined as above.

Statistical analysis
Data from separate experiments were combined and analyzed using one-way ANOVA to calculate the variance and statistical significance (Fisher’s F test, df=1, null hypothesis of equal means) of differences between sample means using the statistical package Mintab (release 12, <http://www.minitab.com/>). The fact that separate experiments were conducted on different days was considered a random variable.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Early cytokine responses following low-dose challenge
Cellular immune responses to T. gondii were analyzed following low-dose i.p. challenge with 100 parasites of the virulent type I strain RH or of the less-virulent type II strain PTG. As reported previously [9 ], this dose of RH strain parasites was lethal in 8-10 days, and infections with PTG strain were controlled. This difference was reflected in the proliferation of parasites within the peritoneal cavity following low-dose i.p. inoculum in outbred mice (Fig. 1A ). RH strain parasites increased at a greater rate and ultimately reached significantly (P<=0.05) higher levels than the PTG strain. To evaluate early differences in the immune response that might contribute to these divergent outcomes, peritoneal cells were harvested from infected mice and cultured overnight in vitro to assess production of cytokines during the first 4 days of infection. IL-12p40 was induced by infection with either strain, and the levels were slightly elevated in mice infected with PTG versus RH strain on day 2 postinfection (Fig. 1B) . Production of IL-12p70 was delayed and was not detected at significant amounts until day 4 (Fig. 1C) . Production of IFN-{gamma} occurred before this and was significantly (P<=0.05) elevated at day 2 following infection with PTG versus RH strain (Fig. 1D) . IL-10 and TNF-{alpha} were induced at similar levels in response to either strain over the first 4 days (data not shown). IL-4 levels were consistently below the level of detection (<10 pg/ml; data not shown).



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  		Figure 1. Parasite growth and local inflammatory cytokine levels during acute infection of mice with T. gondii. (A) Growth kinetics of parasite strains during infection. Type II strain PTG parasites decreased in the peritoneal cavity after day 6 postinfection, and type I strain RH continued to proliferate (*, P<=0.05). (B–D) Local production of inflammatory cytokines. Infection with type II strain PTG parasites induced slightly higher production of IL-12 p40 and significantly (*, P<=0.05) higher levels of IFN-{gamma} than RH on day 2 postinfection. (A, B, D) Data shown are the mean ± SEM from two or more experiments consisting of three mice per time point. (C) Data shown are from a single experiment with five mice per group.

 
Recruitment of CD68+/Gr-1+ and Gr-1+/CD68- cells
To determine the cell types involved in the early control of infection, the cellular composition in the peritoneum was followed daily for the first 5 days postinfection. To identify the cell types that were recruited, peritoneal cells were isolated and stained using a panel of Ab specific for various cell types and analyzed by flow cytometry. The proportion of cells that coexpressed the macrophage marker CD68 and the granulocyte marker Gr-1 increased dramatically on day 4 postinfection with either parasite strain (Fig. 2A ). The percentage of CD68+/Gr-1+ cells was typically greater in response to type II strain PTG parasites compared with type I strain RH parasites (P<=0.05 at day 4 postinfection). In addition to CD68+/Gr-1+ cells, the proportion of neutrophils (Gr-1+/CD68-) also increased dramatically between days 3 and 4 postinfection and was significantly (P<=0.001) higher in mice infected with the RH strain (Fig. 2B) . Infection with either parasite strain led to a substantial decrease in the overall percentage of CD68+/Gr-1- macrophages (60% in uninfected mice and <10% in infected mice; Fig. 2C ). Consistent alterations in other cell types including dendritic cells, {alpha}ß, {gamma}{delta}, NK-T cells, B cells, or NK cells were not observed during the first 5 days of infection with either parasite strain (data not shown). When the total number of cells of each type was analyzed on day 4, it was evident that the decrease in percentages of CD68+/Gr-1- cells was partially a result of loss of this cell population (Fig. 2D) . The predominant cells found in PTG-infected mice were CD68+/Gr-1+, and large numbers of neutrophils (Gr-1+/CD68-) were found in RH-infected mice (Fig. 2D) . The dramatic increase in CD68+/Gr-1+ cells was likely a result of recruitment of new cells, although it remains possible that existing CD68+ macrophages were induced to express Gr-1 (Fig. 2D) .



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  		Figure 2. Cellular compostion of inflammatory peritoneal infiltrate. (A) CD68+ cells that expressed high levels of Gr-1 were recruited to the peritoneum following infection. This population was slightly higher in response to PTG than to RH infection (*, P<=0.05). (B) Acute infection led to an influx of neutrophils (Gr-1+/CD68-). Neutrophil recruitment was higher in response to RH infection than to PTG (**, P<=0.001). (C) The percentage of macrophages that did not express Gr-1 (CD68+/Gr-1-) was decreased. (A–C) Results shown represent mean ± SEM from three to five experiments consisting of two to three mice per time point. (D) Total number of cell populations on day 4 postinfection. Infection with RH strain parasites induced an influx of Gr-1+/CD68- cells (neutrophils). Infection with either strain of parasite resulted in a decrease in CD68+/Gr-1- macrophages and a corresponding increase in CD68+/Gr-1+ cells. Data shown are the mean ± SD from one experiment consisting of three mice per group. (E) Identity of infected cell populations. PTG strain parasites predominantly infected CD68+ cells, and RH strain parasites also infected neutrophils. Data shown are the mean ± SEM from three or more experiments consisting of two or three mice per time point.

 
Type II strain PTG parasites predominantly infect CD68+ cells
To evaluate whether particular cell types were more likely to harbor parasites early during infection, peritoneal cells were stained with Ab to specific cell types along with an anti-T. gondii Ab to detect intracellular parasites. Type II strain PTG parasites were predominantly found in CD68+ cells by day 4 postinfection and were present in the Gr-1+ and Gr-1- populations (Fig. 2E) . In contrast, type I strain RH parasites were often present in neutrophils (Gr-1+/CD68-) in addition to CD68+ cells (Fig. 2E) . Neutrophils, as defined by nuclear morphology, typically contained vacuoles with only one to two parasites, suggesting they prevented parasite replication (data not shown). In contrast, host cells that contained greater than four parasites/vacuole had crescent-shaped nuclei characteristic of macrophages and did not stain for intracellular iNOS (data not shown), suggesting a subset of macrophages was permissive for parasite replication. Additionally, CD8+ T cells, CD4+ T cells, and B cells (B220-positive) each accounted for <5% of those infected by parasites of either strain type (data not shown). Collectively, this group of cells is shown in a single category labeled as "other" in Figure 2E .

To distinguish between uptake of parasites by phagocytosis versus active parasite invasion, peritoneal cells were stained for intracellular parasites and LAMP-1 before being analyzed by florescence microscopy [36 ]. Consistent with host cell entry by active invasion, parasite-containing vacuoles in macrophages and neutrophils were 90–95% LAMP-1-negative (data not shown), indicating that parasites were present in parasitophorous vacuoles and not in phagolysosomes.

CD68+/Gr-1+ cells induced by infection are activated macrophages
To determine whether the CD68+/Gr-1+ cells were macrophages or neutrophils, Gr-1+ cells on day 4 postinfection were sorted into CD68+ and CD68- populations, stained with Diff-Quick, and examined by light microscopy. The parameters used for sorting each cell population are shown in Figure 3A . CD68+/Gr-1+ cells were large cells with crescent-shaped nuclei and pale blue cytoplasm characteristic of macrophages (Fig. 3B) . As expected, the Gr-1+/CD68- cells were primarily composed of smaller cells with a ring-shaped nuclear morphology indicative of neutrophils (Fig. 3B) .



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  		Figure 3. Identity of CD68+/Gr-1+ cells. (A) Fluorescence-activated cell sorter (FACS) plot of CD68 versus Gr-1 expression showing gates used for sorting CD68+/Gr-1+ and Gr-1+/CD68- cells. (B) Nuclear morphology of Gr-1+/CD68- cells versus CD68+/Gr-1+ cells. CD68+/Gr-1+ cells had crescent-shaped nuclei characteristic of macrophages in contrast to Gr-1+/CD68- cells that had ring-shaped nuclei characteristic of neutrophils. Data shown are representative of three similar experiments. Original scale bars = 5 µm.

 
To evaluate the activation status of cells that were recruited to the peritoneal cavity following infection, the expression of cell-surface markers was analyzed by flow cytometry. Cells were isolated from mice at 4 days postinfection and stained with Ab to CD68 and Gr-1 and a battery of additional markers including B220, CD11b, CD11c, F4/80, CD31, {alpha}4 integrin, and MHC class II. Cell populations were gated based on expression of CD68 and Gr-1 into the following groups: CD68+/Gr-1+ cells (middle panel), CD68+/Gr-1- cells (right panel), and Gr-1+/CD68- cells (left panel; Fig. 4A ). CD68+/Gr-1+ and CD68+/Gr-1- macrophages from infected mice had low expression of the macrophage-specific antigen F4/80 in contrast to its high expression on naïve macrophages (Fig. 4A) . CD11b (Mac-1) expression was also decreased on CD68+/Gr-1+ and CD68+/Gr-1- macrophages from infected mice relative to naïve macrophages (Fig. 4B) . CD31, a marker associated with immature myeloid cells, was absent on CD68+/Gr-1+ and CD68+/Gr-1- cells induced during infection (Fig. 4C) . The majority of CD68+/Gr-1+ and CD68+/Gr-1- cells was also negative for the dendritic cell marker CD11c (Fig. 4D) and the B cell marker B220 (Fig. 4E) . CD68+/Gr-1+ cells also expressed high levels of {alpha}4 integrin (data not shown). Collectively, these results suggest that CD68+/Gr-1+ cells induced by infection with T. gondii represent a mature, activated population of macrophages.



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  		Figure 4. Expression of cell-surface markers on peritoneal macrophages examined by flow cytometry. CD68+ macrophages, regardless of Gr-1 expression, expressed decreased levels of F4/80 (A) and CD11b (B) compared with resident cells from uninfected mice and were negative for CD31 (C), CD11c (D), and B220 (E). Surface expression of markers was determined on gated cell populations by flow cytometric analysis. The dotted line represents CD68+ (right and center panels) cells or Gr-1+ cells (left panels) from uninfected mice. The intermediate line and bold line represent cells from mice infected with RH and PTG, respectively. Data shown are representative of at least two separate experiments consisting of at least three mice per group.

 
To determine the functional characteristics of CD68+/Gr-1+ cells, we examined the expression of MHC class II molecules on the cell surface by flow cytometry. MHC class II was up-regulated on CD68+/Gr-1+ cells (middle panel) relative to CD68+ cells from uninfected animals, suggesting that infection results in activation of CD68+/Gr-1+ macrophages (Fig. 5A ). The high level of expression of MHC class II on CD68+/Gr-1+ cells, in addition to their expression of the macrophage marker CD68, further distinguishes this population from neutrophils that were invariably MHC class II-negative (Fig. 5A , left panel). To determine whether macrophages infected with T. gondii also up-regulate MHC class II, peritoneal cells were isolated from type I strain RH-infected mice on day 4 postinfection and were stained with Ab against CD68, MHC class II, and T. gondii before analysis by flow cytometry. Cells that expressed CD68 and stained positive for intracellular parasites had elevated surface expression of MHC class II (Fig. 5B) . Similar increases in MHC class II were observed for PTG-infected cells (data not shown).



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  		Figure 5. Expression of MHC class II on CD68+ macrophages. (A) Expression of MHC class II on cells isolated at 4 days postinfection from control (dotted line), PTG-infected (bold line), and RH-infected (intermediate line) mice. (B) Analysis of CD68+ cells from RH-infected mice at 4 days postinfection. Cells that stained positive for intracellular parasites and expressed CD68 were selectively gated (left), and the expression of MHC class II was displayed as a histogram (right). Data shown are from a representative of two similar experiments consisting of three mice per group.

 
Uninfected Gr-1+ and Gr-1- macrophages produce IL-12 p40
To determine the identity of the cells that were responsible for IL-12 production, peritoneal cells were isolated on day 4 postinfection, costained for intracellular IL-12 p40, CD68, and Gr-1, and analyzed by flow cytometry. In noninfected animals, ~0.02% of cells expressed IL-12 p40; this level was increased by fivefold in infected mice (data not shown). The low level of Il-12 p40-positive cells is likely a reflection of the fact that no inhibitors of the secretory system were used to enhance intracellular staining. Cells that stained positive for intracellular IL-12 p40 were selectively gated, and the percent of cells that were Gr-1+/CD68-, CD68+/Gr-1+, or CD68+/Gr-1- was determined. CD68+ macrophages, regardless of their expression of Gr-1, were the primary producers of IL-12 p40 (Fig. 6A ). In contrast, few neutrophils (Gr-1+/CD68-) stained positive for intracellular IL-12 p40 on day 4 postinfection.



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  		Figure 6. Production of IL-12. (A) Cells that stain for intracellular IL-12 p40 were almost exclusively CD68+ macrophages on day 4 postinfection. Cells that stained positive for intracellular IL-12 p40 by FACS were gated, and their expression of CD68 and Gr-1 was evaluated. Data shown are the mean ± SEM from two experiments consisting of two or three mice per group. (B) Immunofluorescence analysis of IL-12-positive cells. Macrophages expressing IL-12 p40 did not contain visible parasites or parasite antigen. Original scale bars = 5 µm.

 
Peritoneal cells from infected mice were stained with IL-12 p40 and anti-T. gondii Ab and were then analyzed by immunofluorescence to evaluate whether IL-12 p40 was produced primarily by uninfected or infected macrophages. IL-12-p40-positive cells on day 4 postinfection were largely uninfected (greater than 95% had no staining for parasite antigen) and had crescent-shaped nuclei characteristic of macrophages (greater than 90%; Fig. 6B ).

CD68+/Gr-1+ macrophages produce RNI
Peritoneal cells were analyzed on day 4 postinfection to determine if CD68+/Gr-1+ macrophages produced arginase or RNI. Total peritoneal cells and adherent cells from mice infected with either strain of parasite spontaneously produced NO in vitro in the absence of additional stimulation (Fig. 7A ). In contrast, arginase activity was not elevated compared with controls in response to either strain of parasite (data not shown). To determine whether CD68+/Gr-1+ cells were responsible for the production of NO, peritoneal cells from infected mice were stained with Ab against Gr-1, CD68, and iNOS before analysis by immunofluorescence. The cells that expressed iNOS during infection were predominantly CD68+/Gr-1+ cells (80%; Fig. 7B ).



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  		Figure 7. Macrophage production of RNI. (A) Adherent peritoneal cells from infected mice spontaneously produced NO on day 4 postinfection. Data shown are the mean ± SEM from three experiments consisting of five mice per group. (B) Cells that expressed iNOS were primarily CD68+/Gr-1+ macrophages. Data shown are the mean ± SD from one experiment consisting of five mice per group.

 
CD68+/Gr-1+ macrophages inhibit parasite replication
To determine if CD68+ cells induced by infection were able to control parasite replication, peritoneal cells were isolated from infected or control mice and challenged in vitro with GFP-labeled parasites. Adherent peritoneal cells from mice infected with either strain were capable of inhibiting parasite replication in vitro. Inhibition of parasite growth was likely a result of decreased survival/entry, as overall, fewer cells were infected (Fig. 8 , left), and was also a result of direct inhibition of replication, as reflected by the decreased number of parasites per vacuole (Fig. 8 , right). Furthermore, this inhibitory activity was manifest when peritoneal cells were challenged in vitro with the same strain as used in vivo as well as the opposite strain (Fig. 8A) . The combination of adherent peritoneal cells from PTG-infected mice tested against GFP-expressing PTG parasites consistently showed the greatest inhibition of parasite replication/survival (>95% inhibition; Fig. 8A ). In contrast, adherent peritoneal cells from uninfected mice were permissive for growth of both strains of parasite (Fig. 8A) . Inhibition of NO with aminoguanidine had only a slight effect on the ability of adherent peritoneal cells to control parasite replication, suggesting that it was largely NO-independent (Fig. 8B) . When Gr-1+ macrophages were sorted based on their high expression of Gr-1 and low expression of F4/80, they were also capable of inhibiting GFP-expressing parasites of both strain types in vitro (Fig. 8C) .



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  		Figure 8. Parasite inhibition by peritoneal cells. (A) Adherent peritoneal cells isolated at day 4 postinfection were capable of inhibiting parasite survival/replication. Data shown are the mean ± SEM from three experiments consisting of two to four mice per group. In the panels on the right, data are expressed as mean number of parasites per vacuole, and on the left, they are expressed as mean number of parasites per 100 macrophages. (B) Inhibition of NO by treatment with aminoguanidine only slightly diminished the antitoxoplasmic activity of peritoneal cells from PTG-infected mice. Data shown are the mean ± SEM from two experiments consisting of two to four mice per group. (C) In vitro antitoxoplasmic activity of Gr-1+ macrophages, which were sorted and challenged in vitro with type I strain RH or type II strain PTG parasites expressing GFP. Data shown on the left are the mean ± SEM from two experiments with four mice pooled per group. Graphs on the right are from a single representative experiment and are plotted as mean ± SD.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our studies demonstrate that during low-dose i.p. infection with T. gondii, there is an early recruitment of CD68+ cells that also express high levels of the granulocyte antigen Gr-1. The appearance of this population of cells coincided with the induction of high levels of T helper cell type 1 (Th-1) cytokines and control of the proliferation of nonvirulent type II strain parasites. With the exception of Gr-1 expression, these macrophages shared phenotypic and functional characteristics with activated macrophages including up-regulation of surface MHC class II, production of IL-12, production of RNI, and control of parasite replication. CD68+/Gr-1+ cells likely contribute to control of toxoplasmosis in vivo through production of inflammatory cytokines and inhibition of parasite growth.

Following low-dose challenge, the population of CD68+/Gr-1+ cells that accumulated in the peritoneal cavity also expressed CD11b and F4/80 but at lower levels than peritoneal macrophages from uninfected mice. Our studies indicate that these cells are macrophages based on the following features: uniform expression of the marker CD68, a well-defined macrophage-specific lysosomal-associated protein that is expressed on all mature macrophage populations [37 ]; large size with crescent-shaped nuclei and pale blue cytoplasm characteristic of macrophages; expression of high levels of surface MHC class II, present on activated macrophages but generally absent on murine neutrophils [38 ]; a relative absence of dendritic cell markers CD205 (DEC-205) and CD11c.

A previous study showed that a high-dose i.p. challenge of T. gondii induces the recruitment of neutrophils that express intracellular IL-12 within 6 h, while F4/80-positive macrophages instead expressed intracellular IL-10 [27 ]. These authors concluded that neutrophils are the primary producers of IL-12 early during acute infection. However, the present study reveals that low-dose challenge results in IL-12 production by macrophages (i.e., CD68+/Gr-1+ and CD68+/Gr-1-), but not by neutrophils (Gr-1+/CD68-). The low-dose challenge model also revealed that neutrophil recruitment was strongly associated with type I strain RH infection and was only moderately increased by infection with type II strain PTG parasites. Thus, recruitment of high levels of neutrophils may only be associated with elevated numbers of parasites that are typical of high-dose challenge or infections with highly virulent type I strains. Instead, following with nonlethal challenge, CD68+/Gr-1+ macrophages were the predominant cells in the peritoneum. The low-dose challenge more closely resembles the natural dissemination of parasites out of the gut following oral infection [31 ], although further studies will be needed to determine if similar CD68+/Gr-1+ cells are also recruited during natural infections.

Previous studies using oral challenge with T. gondii have demonstrated that infection with a nonvirulent strain via this natural route induces protection against subsequent lethal challenge [39 ]. Recruitment of intraepithelial lymphocytes is important in the development of resistance to infection [40 ], and these cells stimulate production of inflammatory chemokines from mouse intestinal epithelial cells including macrophage-inflammatory protein-2, monocyte chemoattractant protein-1 (MCP-1), MCP-3, and IFN-inducible protein 10 [41 ]. To what extent these chemokines might recruit neutrophils, macrophages, or dendritic cells to the site of infection has not been investigated in this intestinal model of infection. Further studies will be necessary to define the chemokines and receptors that are important for the recruitment of Gr-1+ and CD68+ cells to the peritoneal cavity following i.p. infection.

Recruitment of CD68+/Gr-1+ macrophages and depletion of CD68+/Gr-1- macrophages were similar following infection with type I and type II strain T. gondii. Macrophages induced by both strains of parasite appeared to be classically activated as indicated by iNOS induction rather than arginase production and the absence of IL-4 and only low levels of IL-10 [42 , 43 ]. Consistent with their activation phenotype, a portion of CD68+/Gr-1+ cells expressed intracellular IL-12 in the absence of additional stimuli. Consequently, these cells may contribute to the induction of IFN-{gamma}, a critical mediator of activation and parasite control. Similar to the findings reported here, infection with bacillus Calmette-Guerin elicits a population of macrophages that express high levels of MHC class II, low CD11b, and low-to-moderate F4/80; these cells also produce RNI without further stimulation [38 , 44 ].

Activated CD68+/Gr-1+ macrophages are likely important for the control of toxoplasmosis, as they produce IL-12, generate RNI, and actively inhibit types I and II strains of T. gondii in vitro. Although these cells were capable of producing RNI, the inhibition of parasite survival was largely independent of NO. Previous studies have shown that RNI production is an important mediator of parasite inhibition following in vitro activation of macrophages [45 ] but that it is not crucial to survival during acute infection in vivo [46 , 47 ]. Consistent with their activation profile, CD68+/Gr-1+ cells expressed MHC class II at high levels, even in cells that contained intracellular parasites. This contrasts with previous reports that infection by T. gondii can suppress expression of MHC class II [48 , 49 ]. However, this discrepancy is likely a reflection of the timing of infection versus cellular activation. When macrophages are previously activated, they can efficiently kill T. gondii; however, infection of naive cells renders them less responsive to subsequent activation [50 ].

Our results show that type I strain parasites reached 20-fold higher parasite numbers by day 6 postinfection compared with type II strain parasites, and this may drive the overproduction of Th-1 cytokines seen late in infection [9 ]. Differences in parasite numbers in the peritoneum are in part likely a result of differences in doubling time, as described previously [51 ]. This enhanced rate of replication combined with a greater capacity for dissemination [8 ] may allow type I strains to escape control. Notably, our studies did not identify major differences in the cytokine response or cellular infiltration induced by infection with virulent type I versus nonvirulent type II strains of T. gondii during the early phase of acute infection. This result contrasts with our previous report showing that late in the acute infection, type I strains induce substantially higher levels of Th-1 cytokines, which are associated with tissue damage in mice that succumb to infection [9 ]. The present study indicates that the differences in cytokine levels that occur in late infection are not a result of an inherently greater cytokine response to type I parasites at the early stage. Instead, several features suggest that type II strains are more efficient at inducing protective responses early during infection. First, type II strain infections induced greater numbers of CD68+/Gr-1+ cells that may contribute to a more effective immune control of parasite replication. Second, in separate studies conducted in vitro, we have observed that type II but not type I strains are potent inducers of IL-12 p40 and p70 from bone marrow-derived macrophages (Robben, P. Pm., Mordue, D. G., Truscott, S. M., Takeda, K., Akira, S., Sibley, L. D., Induction of IL-12 by Toxoplasma gondii depends on the parasite genotype, submitted). Even a modest increase in IL-12 and related cytokines may be functionally important, as IFN-{gamma} was significantly higher at day 2 in mice infected with PTG versus RH strain parasites in the present study. Finally, our studies indicate that PTG strain parasites are also slightly more susceptible to inhibition by activated macrophages. Consequently, inherent differences in the efficiency of cell recruitment, the early induction of effective immune responses, and susceptibility to inhibition may combine to provide more effective control of type II strain infections in vivo.

IL-12 plays a critical role in the protection against T. gondii. Administration of recombinant (r)IL-12 prolongs survival to lethal challenge, and neutralization of IL-12 using polyclonal Ab increases mortality [52 , 53 ]. Additionally, mice that are deficient in p40-/- are unable to control the acute infection with normally nonlethal type II strains, and this defect is restored by administration of rIL-12 [54 ]. Recently, it has become apparent that p40 is a subunit of several cytokines including IL-12 and IL-23 [55 , 56 ]. Additionally, a related p40-like subunit is part of the cytokine IL-27, which is involved in early Th-1 responses [55 ]. The extent to which these different cytokines contribute to the control of toxoplasmosis is presently unknown, as the susceptibilities of mice with selective deletions in p35 and p19 have not yet been reported. In this regard, it may be significant that we observed induction of p40 before release of IL-12 p70. The p40 subunit may participate in IL-12 or IL-23 or could also play a role as homodimers, which have been shown to have agonistic and antagonistic effects in different systems [55 ].

Although Gr-1 is generally considered a hallmark for neutrophils, an increasing number of studies have revealed a wider distribution for this marker. Gr-1 is expressed by immature macrophages thought to function in immunosuppression [57 , 58 ]. These suppressive macrophages also express CD31 and have low levels of MHC class II and thus are distinct from the cells recruited in response to toxoplasmosis. Additionally, following an inflammatory stimulus, Gr-1+ monocytes are rapidly recruited to the peritoneal cavity in mice via a pathway that does not depend on prior migration of neutrophils [59 ]. Using an adoptive transfer model, it was recently shown that Gr-1+ monocytes specifically home to sites of inflammation and that given the proper stimulus, they can express dendritic cell markers [60 ]. Consistent with this, dendritic cells have been described that express Gr-1 in combination with markers such as CD11c [61 ]. The Gr-1+ macrophage cells that are recruited in response to T. gondii infection do not express the dendritic cell makers CD11c or CD205 and are negative for B220. However, given the plasticity of monocytes and dendritic cells, it is possible that these cells represent different stages in maturation of a common lineage. Overall, the CD68+/Gr-1+ cells recruited to the peritoneal cavity in response to T. gondii infection are phenotypically similar to recently described inflammatory monocytes [59 , 60 ], suggesting that this may be an excellent model to study the recruitment, differentiation, and function of inflammatory monocytic cells.

Because of its high expression on granulocytes [62 ], Ab to Gr-1 have been used in a variety of models to selectively deplete granulocytes and thereby evaluate neutrophil function. Ab neutralization studies have shown that Gr-1+ cells are required for protection against type II strains of T. gondii [28 ]. The current study indicates that infection by T. gondii induces a population of activated macrophages that also expresses Gr-1. Therefore, administration of anti-Gr-1 Ab during acute infection is likely to deplete inflammatory monocytes as well as neutrophils. Further studies are needed to decipher the relative roles of inflammatory macrophages versus neutrophils in controlling acute toxoplasmosis.


   ACKNOWLEDGEMENTS
 
This work was partially supported by a grant from the National Institutes of Health (AI 36629 to L. D. S.). L. D. S. is the recipient of a Scholar Award in Molecular Parasitology from the Burroughs Wellcome Fund. We thank Chunlei Su, Eric Denkers, Dan Goldberg, and Peter Dube for helpful discussions and/or comments on the manuscript.


   FOOTNOTES
 
1 Current address: Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, Madison, WI 53706. Back

Received April 18, 2003; revised July 28, 2003; accepted July 29, 2003.


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 DISCUSSION
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