CXCR2 Deficiency Confers Impaired Neutrophil Recruitment and Increased Susceptibility During Toxoplasma gondii Infection1 Laura Del Rio,† Soumaya Bennouna,* Jesus Salinas,† and Eric Y. Denkers*2 Neutrophil migration to the site of infection is a critical early step in host immunity to microbial pathogens, in which chemokines and their receptors play an important role. In this work, mice deficient in expression of the chemokine receptor CXCR2 were infected with Toxoplasma gondii and the outcome was monitored. Gene-deleted animals displayed completely defective neutrophil recruitment, which was apparent at 4 h and sustained for at least 36 h. KitW/KitW-v animals also displayed defective polymorphonuclear leukocyte migration, suggesting mast cells as one source of chemokines driving the response. Tachyzoite infection and replication were accelerated in CXCR2ⴚ/ⴚ animals, resulting in establishment of higher cyst numbers in the brain relative to wild-type controls. Furthermore, serum and spleen cell IFN-␥ levels in infected, gene-deleted mice were reduced 60 –75% relative to infected normal animals, and spleen cell TNF-␣ was likewise reduced by ⬃50%. These results highlight an important role for CXCR2 in neutrophil migration, which may be important for early control of infection and induction of immunity during Toxoplasma infection. The Journal of Immunology, 2001, 167: 6503– 6509.
T
he CXC chemokine IL-8 is an important chemoattractant and activator of neutrophils (1–3). In humans, there are two high-affinity receptors for IL-8, designated CXCR1 and CXCR2 (4, 5). Although mice do not possess a homologous IL-8 gene, they express a CXCR with similarity to human CXCR2 which is expressed predominantly on neutrophils. The murine receptor binds several IL-8-like CXC chemokines, most notably macrophage inflammatory protein (MIP)3-2 and KC (6). Recently, mice with a targeted deletion of CXCR2, also known as murine IL-8R homolog (IL-8RL), were constructed (7). The animals display defective neutrophil migration in response to thioglycolate, although the killing function of intracellular and extracellular bacteria remains intact. In a model of urinary tract infection, transepithelial polymorphonuclear leukocyte (PMN) migration is defective, resulting in bacteremia and systemic disease (8 –10). The murine IL-8R homolog has also been implicated in increased susceptibility during infections with pathogens such as Candida albicans and Legionella pneumophila (11, 12), but its role during protozoan infection has hitherto remained unexplored. In our laboratory, we use the intracellular protozoan Toxoplasma gondii as a model to study initiation of immunity and early host resistance during microbial infection. Toxoplasmosis is a widespread parasitic infection among human and animal popula*Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; and †Departamento de Patologia Animal (Microbiologia e Immunologia), Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain Received for publication June 8, 2001. Accepted for publication October 1, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant AI47888. L.D.R. was supported by Ministerio de Educacio´n y Cultura of Spain. 2 Address correspondence and reprint requests to Dr. Eric Y. Denkers, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. E-mail address:
[email protected] 3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; PMN, polymorphonuclear leukocyte; KO, knockout; STAg, soluble tachyzoite lysate Ag; WT, wild type; PEC, peritoneal exudate cell; IL-8Rh, IL-8R homolog.
Copyright © 2001 by The American Association of Immunologists
tions worldwide. In situations of immunodeficiency and during congenital infection, Toxoplasma emerges as a major opportunistic pathogen that can be lethal if not appropriately treated (13, 14). T. gondii is well known as a potent type 1 cytokine inducer, and while these cytokines are required to survive infection, their overproduction can lead to pathology and death (15–23). We and others recently reported a requirement for neutrophils in early resistance to T. gondii (24 –27). Although these cells display microbicidal activity through phagocytosis and release of superoxides and peroxides, it is also clear that PMN can serve as a source of several proinflammatory cytokines during infection. For the case of Toxoplasma, PMN release IL-12 and TNF-␣, as well as chemokines such as MIP-1␣ and MIP-1, in response to parasite stimulation (25, 28, 29). In an in vitro model of infection, tachyzoites induced recruitment of large neutrophil numbers into the peritoneal cavity within 4 h of injection (30). The PMN were found to be the major source of IL-12 in this model system, and Ab-mediated neutrophil depletion resulted in early death of the animals, associated with defective type 1 cytokine responses (24). These results, and similar findings by others (31–36), led us to hypothesize that PMN, by virtue of their ability to rapidly migrate to a site of infection and release proinflammatory cytokines, may be important immunoregulatory cells during the immune response to Toxoplasma. In the present report, we examined the ability of CXCR2⫺/⫺ mice to respond to T. gondii infection. Our results reveal a profound defect in the ability of neutrophils to migrate into the peritoneal cavity following tachyzoite inoculation. Production of proinflammatory cytokines, in particular TNF-␣ and IFN-␥, was lower in CXCR2-deficient mice. The gene-deleted mice harbored more parasites in the peritoneal cavity during early infection and greater brain cyst numbers during chronic infection. Mast celldeficient (KitW/KitW-v) mice also displayed a defective ability to recruit PMN during early infection, suggesting that these cells serve as a major chemokine source involved in neutrophil recruitment. Our results suggest that CXCR2 is required for early neutrophil recruitment, and that this chemokine receptor and its 0022-1767/01/$02.00
6504 ligands play an important protective role in resistance to Toxoplasma.
Materials and Methods Mice Female BALB/c and 129/J mice (6 – 8 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME). Heterozygous CXCR2 knockout (KO) mice (C.129S2(B6)-Cmkartm1/Mwm) were purchased from The Jackson Laboratory and bred at the College of Veterinary Medicine animal facility (Cornell University, Ithaca, NY). The KO animals were originally engineered by homologous recombination of a defective CXCR2 gene into 129 embryonic stem cells, followed by transplantation into C57BL/6 blastocysts. Resulting chimeric animals were backcrossed onto a BALB/c background for 10 generations (7). After genotyping for the CXCR2 gene, a colony of homozygous KO mice was established and used in the studies described. Female KO animals were age-matched to wild-type (WT) controls. Mast cell-deficient KitW/KitW-v and congenic normal WBB6F1⫹/⫹ littermates were obtained from The Jackson Laboratory. The mice were housed under specific pathogen-free conditions in the College of Veterinary Medicine animal facility, which is accredited by the American Association for Accreditation of Laboratory Care.
Mouse genotyping To isolate DNA, tail snips were digested in lysis buffer (80 l of 10% sodium dodecyl sulfate, 400 l of 1 M Tris (pH 7.4), 400 l of 1 M NaCl, 80 l of 500 mM EDTA, 4 mg of proteinase K in 3040 ml of H2O per tail snip). Following an 18-h incubation at 56°C, 800 l of H2O was added and digest-centrifuged, and resulting supernatant was added to premixed phenol/CHCl3/isoamylalcohol (25:24:1; Sigma-Aldrich, St. Louis, MO). After vortexing and centrifugation, the aqueous phase was collected and re-extracted with CHCl3. DNA precipitation was achieved by addition of 50 l of 3 M sodium acetate and 1 ml of 100% ethanol followed by incubation at ⫺70°C overnight. After centrifugation (10 min at 4°C), pellets were washed in 70% ethanol and resuspended in Tris-EDTA buffer, pH 8. DNA was quantitated on a UV spectrophotometer (Bio-Rad, Hercules, CA). PCR amplification was accomplished as described elsewhere (25) using primers specific for the CXCR2 gene and the neomycin cassette, which replaces the single exon encoding the IL-8Rh in KO mice (7). Primer sequences used were GGTCGTACTGCGTATCCTGCCTCA (CXCR2, forward), TAGCCATGATCTTGAGAAGTCCAT (CXCR2, reverse), CT TGGGTGGAGAGGCTATTC (neomycin, forward), and AGGTGAGAT GACAGGAGATC (neomycin, reverse). The amplification cycle consisted of 2 min at 94°C followed by 30 cycles at 94°C for 30 s, 57°C for 30 s, and 72°C for 5 min. Chain elongation at 72°C was continued for 5 min after the last cycle. Amplification of CXCR2 and neomycin gene fragments results in 350- and 280-bp products, respectively. An ethidium bromide gel showing PCR amplification products from a representative typing experiment is shown in Fig. 1. Genotyping of Nramp1 was performed essentially as described above. The primers used were TGGACGCATCCCGCTGTGGGG (forward primer specific for 129-derived Nramp1), TGGACGCATCCCGCTGT GGGA (forward primer specific for BALB/c-derived Nramp1), and GCATGATGATGGCACCGACGAT (common reverse primer). The amplification cycle consisted of 2 min at 94°C followed by 26 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 5 min. Chain elongation at 72°C was continued for 5 min after the last cycle. RT-PCR amplification of this portion of the Nramp1 gene results in a 790-bp amplicon, as predicted from sequence data (37) and as confirmed by agarose gel electrophoresis.
CXCR2 DEFICIENCY DURING T. gondii INFECTION PECs Peritoneal exudate cells (PEC; 2 ⫻ 105 per sample) collected by peritoneal lavage with 10 ml of PBS were cytospun (700 rpm for 5 min) onto glass microscope slides (VWR Scientific, Rochester, NY) using cytofunnels (Thermo Shandon, Pittsburgh, PA). To determine the composition of PEC and the number of parasites, differential counts were performed on DiffQuick-stained (American Scientific Products, McGraw Park, IL) cytocentrifuge slides. A minimum of 300 cells were counted per slide.
Spleen cell culture Spleens were collected from mice 7 days after i.p. infection with 100 ME49 cysts. After gentle mashing, cells were suspended in complete DMEM consisting of 10% FCS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10 mM HEPES buffer, 100 U/ml penicillin, 0.1 mg/ml streptomycin (all from Life Technologies), and 50 mM 2-ME (Sigma-Aldrich). The resulting single cell suspension was centrifuged for 7 min at 1,000 ⫻ g, supernatant was decanted, and erythrocytes were lysed using erythrocyte lysis buffer (Sigma-Aldrich). Cells were cultured (37°C; 5% CO2) in duplicate wells of 96-well plates (Costar, Cambridge, MA) at a concentration of 5 ⫻ 106 cells/ml with medium or STAg (2 g/ml). After 24 or 48 h, supernatants were collected and stored at ⫺20°C until assayed.
Cytokine measurement To measure IFN-␥, the ELISA was performed. Ninety-six-well plates (Costar) were coated overnight at 4°C with mAb HB170 in ELISA coating buffer (0.1 M Na2CO3, 0.1 M NaHCO3, 1 mM NaN3, pH 9.6). After removing supernatant, plates were blocked for 1 h at 37°C with 3% nonfat dry milk in PBS. After five washes with PBS containing 0.05% Tween (PBST), samples and rIFN-␥ standard (R&D Systems, Minneapolis, MN) were added in 3% nonfat dry milk, and the plates were incubated for 1 h at 37°C. Plates were washed five times in PBST, biotinylated anti-IFN-␥ mAb XMG1.2 (BD PharMingen, San Diego, CA) was added, and the plates were incubated at 37°C for 1 h. After washing five times, HRPlabeled streptavidin (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added and plates were incubated for 30 min at 37°C. Finally, plates were washed 10 times and 100 l of ABTS substrate (Kirkegaard & Perry Laboratories) was added to each well. Sample absorbances were measured at 405 nm on a Microplate BioKinetics reader (Bio-Tek Instruments, Winoosky, VT). IL-12(p40) was measured in a similar manner, using plate-bound antiIL-12 mAb C15.6 and biotinylated anti-IL-12 mAb C17.8 (kindly provided by G. Trinchieri, Wistar Institute, Philadelphia, PA). Plates were coated with C15.6 diluted in PBS, then blocked with 1% BSA (Sigma-Aldrich) in PBS. After washing, HRP-labeled streptavidin addition (Kirkegaard & Perry Laboratories), washing, and ABTS addition (Kirkegaard & Perry Laboratories), sample absorbances were measured at 405 nm. TNF-␣ levels were measured by a mouse-specific TNF-␣ ELISA kit according to the manufacturer’s instructions (R&D Systems).
Flow cytometric analysis PEC and RBC-lysed splenocytes were washed with FACS buffer (1% FCS, 0.01% NaN3 in PBS) and blocked with anti-mouse CD16/CD32 (BD PharMingen) in PBS with 5% normal mouse serum for 30 min on ice. After the cells were washed with FACS buffer, FITC-conjugated anti-B220 (Caltag Laboratories, Burlingame, CA), anti-CD4, anti-Gr-1 (both from BD PharMingen), and PE-conjugated anti-F4.80 (Caltag Laboratories), antiCD8 (BD PharMingen), and anti-CD11b (clone M1/70; BD PharMingen) were added, and the cells were incubated on ice for 30 min. The cells were analyzed on a FACSCalibur flow cytometer, and CellQuest software (BD Immunocytometry Systems, San Jose, CA) was used to analyze the data.
Statistical analysis Parasites, Ag, and infection Tachyzoites of the virulent RH strain were maintained in vitro by infection of human foreskin fibroblasts and biweekly passage in complete medium consisting of DMEM (Life Technologies, Gaithersburg, MD) supplemented with 1% FBS (HyClone Laboratories, Logan, UT), penicillin (100 U/ml), and streptomycin (100 g/ml) (both from Life Technologies). Tachyzoites from freshly lysed fibroblast cultures were washed once with endotoxin-free PBS (Sigma-Aldrich) and resuspended in endotoxin-free PBS for i.p. injection. ME49 bradyzoite cysts were maintained in SwissWebster mice and infections were conducted as described previously (25). Soluble tachyzoite lysate Ag (STAg) was prepared by sonication of RH strain tachyzoites in the presence of protease inhibitors as described elsewhere (25). The STAg solution was stored at ⫺70°C until use.
Significant differences were determined using Student’s t test. Values of p ⬍ 0.05 were considered significant. All experiments were performed on at least two independent occasions, and responses of individual animals were analyzed throughout.
Results
CXCR2⫺/⫺ mice display increased susceptibility to infection We initially confirmed the genotype of the F1 generation resulting from a cross between heterozygous CXCR2 WT and KO mice on a BALB/c genetic background (Fig. 1). The homozygous KO animals were selected to establish a colony of CXCR2⫺/⫺ animals. Resulting homozygous KO and WT control mice were infected by
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FIGURE 1. Genotypic analysis of mice from a heterozygous cross between WT and CXCR2 KO mice. DNA was isolated from tail snips of F1 progeny from ⫹/⫺ ⫻ ⫹/⫺ crosses and subjected to RT-PCR-mediated amplification of the endogenous CXCR2 gene and neomycin (Neo), which replaces CXCR2 in the KO chromosome. Amplified DNA was subjected to agarose gel electrophoresis and products were visualized by viewing ethidium bromide-stained gels under UV illumination.
i.p. injection of 100 ME49 cysts, and 30 days later brains were removed for cyst enumeration. As shown in Fig. 2, brains from CXCR2 KO animals harbored approximately five-fold greater cyst numbers than WT mice. Neutrophils fail to migrate into the peritoneal cavity during early infection in CXCR2 KO mice Because IL-8 is a major neutrophil chemotactic cytokine, we assessed the ability of PMN to migrate into the peritoneal cavity following parasite inoculation. WT and KO animals were i.p. injected with 2 ⫻ 106 RH strain tachyzoites. We previously showed that the latter results in a rapid PMN influx, detectable in C57BL/6 mice within 4 h of infection (30). A similar result was found in BALB/c animals, with an increase in neutrophils from 11% in noninfected mice to 45% in animals infected 4 h previously (Fig. 3). In a situation of CXCR2 deficiency, the percentage of PMN was lower in noninfected animals (4%), and there was a complete failure to recruit these cells following infection (Fig. 3). Parasite numbers are increased and PMN influx remains defective 36 h postinfection Chemokines in general are regarded as highly redundant mediators; therefore, it was of interest that recruitment of neutrophils in CXCR2⫺/⫺ mice was totally defective during early infection. Nevertheless, it was possible that PMN could be recruited by CXCR2independent cytokines later in infection. Accordingly, we examined peritoneal cell populations 36 h after tachyzoite infection. At
FIGURE 2. Cyst numbers are elevated in brains of CXCR2 KO relative to WT mice. Animals were infected by i.p. inoculation of 100 ME49 cysts. After 30 days, brains were removed and homogenized, and cyst numbers enumerated. Each circle represents an individual mouse. This experiment was repeated three times with equivalent results.
FIGURE 3. Defective PMN influx in CXCR2 KO mice early after T. gondii infection. Animals were infected by i.p. injection of 2 ⫻ 106 RH strain tachyzoites. After 4 h, PECs were harvested, centrifuged onto glass microscope slides, and stained with Diff-Quik. Percentages of eosinophils (EO), neutrophils (NEU), lymphocytes (LY), monocytes (MO), and mast cells (MC) were determined by differential counting. The results are expressed as mean ⫾ SD of individual mice. Results are representative of two different experiments.
this time point, large numbers of PMN were present in cell populations from WT mice (Fig. 4, A and C). In contrast, few or no neutrophils were evident in populations from CXCR2⫺/⫺ animals (B and D). In Fig. 5, cells from the peritoneal cavity were isolated and stained with the granulocyte-associated marker Gr-1 (Ly6G) (38) and the macrophage marker F4/80 (39) 36 h after PBS injection or RH infection. In WT mice, five distinct populations could be distinguished: Gr-1lowF4/80low, Gr-1lowF4/80high, Gr-1intF4/80int, Gr-1highF4/80low, and Gr-1highF4/80int (Fig. 5A). In response to parasite infection, there was a large increase in GR-1highF4/80low and Gr-1highF4/80int populations in cells from WT animals. Highlevel Gr-1 expression is known to be granulocyte restricted, and fluorescence microscopy confirmed that the GR-1highF4/80low and Gr-1highF4/80int populations were composed of neutrophils (data not shown). In CXCR2⫺/⫺ mice, there was an almost complete absence of Gr-1high cells, consistent with a failure in neutrophil
FIGURE 4. Defective PMN response at 36 h is associated with high tachyzoite numbers in CXCR2 KO mice. Tachyzoites (RH strain, 2 ⫻ 106) were i.p. inoculated into WT and KO animals, then 36 h later PECs were collected, centrifuged onto glass slides, and stained with Diff-Quik. A and C, Cells from WT animals, showing large PMN numbers. B and D, Cells from KO mice, demonstrating near complete absence of neutrophils but large tachyzoite numbers. Original magnification of A and B was ⫻40; C and D, ⫻100. The arrows in D point to individual tachyzoites. This experiment was repeated three times with equivalent results.
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FIGURE 5. Flow cytometric analysis of peritoneal cells from WT and mutant mice. A, Cells were collected 36 h after i.p. inoculation of 2 ⫻ 106 RH strain tachyzoites or an equivalent volume of PBS, then stained with mAb to Gr-1 (Ly6G) and F4/80. Percentages lying in each circled population are indicated. Each fluorescence plot shows the results from an individual mouse (n ⫽ five per group). B, CXCR2 KO (KO), BALB/c controls (BA), KitW/KitW-v (W/W-v), and WBB6F1⫹/⫹ controls (⫹/⫹) were infected as in A, then the number of Gr-1⫹ cells at 36 h postinfection was quantitated by flow cytometry. These experiments were repeated twice with identical results.
recruitment during infection (Fig. 5, A and B). Interestingly, tachyzoite infection resulted in a complete loss of Gr-1lowF4/80high cells, with a concomitant increase in Gr-1intF4/80int cells from WT and KO populations (Fig. 5A). The latter displayed a macrophage morphology, as determined by fluorescence microscopy (data not shown). We also performed double staining for F4/80 and CD11b in populations derived from infected and PBS-injected WT mice. As shown in Fig. 6, the F4/80high population present in PBS-injected animals also expressed high levels of CD11b. Likewise, after RH infection, the F4/80int cells coexpressed intermediate levels of CD11b. The F4/80lowCD11bint population appearing after infection most likely represents PMN, as these cells also expressed high levels of Gr-1 (data not shown). Increased tachyzoite numbers appeared to be present in preparations from the KO mice (Fig. 4). We confirmed that this was the case by enumerating parasite numbers and percentage of infected cells, as shown in Fig. 7. Thus, in terms of intracellular and ex-
CXCR2 DEFICIENCY DURING T. gondii INFECTION
FIGURE 7. Quantitation of tachyzoites and infection in peritoneal cavities of WT and CXCR2 KO mice. Peritoneal cells and extracellular tachyzoites were obtained by lavage 36 h after infection with 2 ⫻ 106 RH tachyzoites. Parasite and cell counts were determined by microscopic examination of Diff-Quik-stained cells. A, Extracellular tachyzoites were counted in four fields (17,000 m2 per field). B, The number of intracellular tachyzoites per infected cell was determined in four fields. C, The percentage of infected cells was estimated by counting four fields from each sample. In these experiments, 91% of infected cells are monocytes (two to eight tachyzoites per cell). Approximately 5% of the remaining infected cells are PMN with one to two intracellular tachyzoites. The data show mean ⫾ SD from individual mice (n ⫽ 5 per group) and are representative of two experiments. ⴱ, p ⬍ 0.05.
tracellular parasite numbers, as well as percentage of infected cells, there was a dramatic increase in parasite levels in CXCR2⫺/⫺ mice. These data confirm that the defect in neutrophil infiltration is sustained for at least 36 h, and that the absence of CXCR2 increases susceptibility to infection. CXCR2-dependent neutrophil influx is defective in mast celldeficient KitW/KitW-v mice Mast cells are capable of serving as a source of MIP-2 and TNF-␣, mediators which have been implicated in PMN recruitment in models of bacterial host defense and T cell-mediated delayed-type hypersensitivity (40, 41). The MIP-2 chemokine binds with high affinity to CXCR2 (6). Therefore, to determine whether mast cells were involved in the PMN influx during Toxoplasma infection, we examined the response of mast cell-deficient KitW/KitW-v mice. As shown in Fig. 5B, the influx of Gr-1⫹ cells was reduced approximately three-fold in mast cell-deficient mice relative to control littermates. Nevertheless, this decreased PMN influx was not as drastic as that seen in CXCR2 KO mice, in which there was an ⬃90% reduction in neutrophil numbers (Fig. 5B). We conclude that mast cells are an important chemokine source driving PMN recruitment during T. gondii infection, but that other cells are also likely to play a role in providing these mediators in this infection model. Parasite-induced type 1 cytokine responses are defective in CXCR2⫺/⫺ animals
FIGURE 6. Expression of F4/80 and CD11b on peritoneal cells from infected animals. Peritoneal cells were collected 36 h after i.p. injection of PBS or 2 ⫻ 106 RH strain tachyzoites. Cells were analyzed as described in Fig. 5, using Abs to F4/80 and CD11b (clone M1/70). Percentages lying in each circled population are indicated. This experiment is representative of two performed.
We next evaluated spleen cell responses in animals undergoing acute Toxoplasma infection. The spleen cell phenotype was evaluated by flow cytometry (Table I). Noninfected KO mice displayed increased levels of Gr-1⫹ cells in the spleen, as previously described (7), and this was also the case for 7-day infected animals. The remaining populations of T lymphocytes, B lymphocytes, macrophages, and NK cells appeared similar for both mouse strains, with increases in all populations following infection, although there was a small but statistically significant increase in F4/80⫹ cells in CXCR2 KO animals. Because we and others have found that PMN can serve as an IL-12 source that may play a role in type 1 cytokine response induction (24, 30, 34), we examined spleen cell cytokine release
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Table I. Phenotypic composition of splenocytes from T. gondii-infected and non-infected WT and CXCR2 KO micea Mouse
Gr-1⫹
WT noninfected KO noninfected WT infected KO infected
0.1 ⫾ 0.0 5.0 ⫾ 0.7c 3.3 ⫾ 0.8 13.6 ⫾ 3.9
b
B220⫹
CD4⫹
CD8⫹
F4/80⫹
Total
11.7 ⫾ 2.3 16.7 ⫾ 1.4 53.5 ⫾ 7.0 45.9 ⫾ 6.2
5.4 ⫾ 1.6 8.0 ⫾ 0.3 15.5 ⫾ 1.8 26.8 ⫾ 5.1
1.9 ⫾ 0.7 3.0 ⫾ 0.0 6.5 ⫾ 0.9 9.5 ⫾ 1.3
0.6 ⫾ 0.1 2.2 ⫾ 0.2c 7.4 ⫾ 0.9 12.5 ⫾ 2.5
22.4 ⫾ 5.4 46.4 ⫾ 6.8 103.3 ⫾ 12 142.5 ⫾ 20
a
Seven days after i.p. infection with 100 T. gondii cysts, splenocytes were isolated and stained for flow cytometric analysis. Data are expressed as means ⫾ SD (⫻ 10⫺6) of the total cell number expressing the indicated phenotypic marker. Each group was composed of five mice, and results are representative of two independent experiments. c p ⬍ 0.05 relative to cells from infected KO mice. b
after in vitro stimulation with STAg. Both IFN-␥ and TNF-␣ responses were lower using cells from 7-day infected KO mice relative to WT controls (Fig. 8). Nevertheless, IL-12 levels produced by spleen cells from KO and WT animals were not significantly different. This is likely to be the result of the presence of splenic PMN and dendritic cells, which produce IL-12 in response to STAg (25, 42). When we examined serum cytokine levels we found that CXCR2⫺/⫺ mice displayed five-fold less serum IFN-␥ than WT controls, although there was not a significant difference in TNF-␣ or IL-12 serum levels (Fig. 7). Similar results were found in mice undergoing infection with RH strain parasites. Nramp1 genotype of CXCR2⫺/⫺ animals Genetic studies have mapped the Nramp1 gene to within 50 kb of the murine IL-8Rh gene (43, 44). The Nramp1 gene is capable of influencing resistance to intracellular bacteria (44). Because the 129 mouse strain carries a WT Nramp1 allele and the BALB/c strain carries a mutant Nramp1 allele, it was possible that CXCR2 KO animals carried a 129-derived Nramp1 gene. We used primers specific for Nramp 783, the single nucleotide position differing between the two parental mouse strains (44), to amplify an Nramp1 amplicon in an allele-specific manner. As shown in Fig. 9A, the KO strain retains a 129-derived Nramp1 gene, despite 10 generations of backcrossing to the BALB/c strain. To confirm that presence of this allele was not affecting our results, we evaluated
FIGURE 8. Cytokine production by infected WT and KO animals. WT and CXCR2 KO mice were infected (100 ME49 cysts), then 7 days later spleens were isolated and serum was collected. Spleen cells were stimulated with STAg (2 g/ml), supernatants were harvested 48 h later, and cytokines were quantitated by ELISA. The data are expressed as means ⫾ SD of individual mice (n ⫽ 5 per strain). ⴱ, Significant differences between WT and KO (p ⬍ 0.05). The results are representative of three individual experiments.
the neutrophil influx in parental 129 and BALB/c mice. As shown in Fig. 9B, a strong PMN response was associated with both strains, despite carrying different Nramp1 alleles. In addition, the percentage of infected cells in the peritoneal cavity did not differ between the strains (Fig. 9B). These results strongly argue that the disrupted CXCR2 gene itself, rather than the 129-derived Nramp1 allele, accounts for the impaired neutrophil response in the KO mice.
Discussion The results of our study argue that mouse CXCR2 is essential for early neutrophil recruitment during T. gondii infection. Animals deficient in mast cells also displayed an impaired PMN influx, suggesting these cells as a source of mediators that bind to CXCR2. The defective neutrophil influx was associated with higher parasite numbers in the peritoneal cavity during early stages of infection, and this was reflected by increased cyst numbers later during chronic infection. Earlier studies suggested that tachyzoites release factors chemotactic for neutrophils (45). Our studies argue that host CXCR2 expression is necessary for PMN recruitment. While it is possible that the parasite releases CXCR2-binding ligands, we think it more likely that the chemotactic mediators are host derived. Indeed, in our laboratory we have been unable to detect tachyzoite-derived neutrophil chemotactic factors (S. K. Bliss and E. Y. Denkers, unpublished observations). Although mice do not express an IL-8 homolog, several CXC chemokines bind to the IL-8Rh, including MIP-2 and KC (6, 46). Of these, KC is ⬃10-fold less potent than MIP-2, arguing that the latter may be more important as a CXCR2 ligand. Nevertheless, in pulmonary infections with Legionella pneumophila and Pseudomonas aeruginosa, neutralization of either MIP-2 or KC resulted in only partial blockade of the PMN influx, whereas Ab blocking of CXCR2 completely inhibited neutrophil recruitment (12, 47). Thus, both MIP-2 and KC chemokines, by binding to CXCR2 on neutrophils, may be involved in neutrophil trafficking during T. gondii infection. Previous studies on IL-12⫺/⫺ and IFN-␥⫺/⫺ mice have shown that T. gondii infection induces abnormally high levels of PMN recruitment into the peritoneal cavity (18, 27). Given our results, it would be of interest to determine whether these cytokines play a down-regulatory role in expression of either CXCR2 or ligands of this chemokine receptor. We are currently examining MIP-2 and KC production, as well as neutrophil CXCR2 expression in IL-12 and IFN-␥ KO mice to determine levels of these mediators in the presence and absence of IL-12 and IFN-␥. Our results showing that KitW/KitW-v mice display a defective neutrophil influx during T. gondii infection implicate mast cells as an important chemokine source driving CXCR2-dependent PMN migration into the peritoneal cavity. Because the KitW/KitW-v mice and their WT counterparts are on a different genetic background than that of CXCR2 KO animals and the KitW/KitW-v mice also display other mast cell-independent abnormalities, this result must
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CXCR2 DEFICIENCY DURING T. gondii INFECTION
FIGURE 9. Nramp1 genotype of KO mice. A, An Nramp1 gene fragment was subjected to RT-PCR-assisted amplification using a primer specific for BALB/ c-derived (A primer) and 129-derived (G primer) Nramp1. B, Mice were infected with 2 ⫻ 106 RH strain tachyzoites, then 36 h later peritoneal cells were harvested, centrifuged onto glass slides, and stained with Diff-Quik. The percentage of PMN and infected cells was calculated by counting 300 cells in randomly selected fields.
be interpreted with some caution. Nevertheless, our findings are in accord with those of others suggesting that mast cells drive neutrophil recruitment through release of TNF-␣ and MIP-2 in models of bacterial clearance and T cell-mediated delayed-type hypersensitivity (40, 41). In our experiments, the defective neutrophil migration in mast cell-deficient mice was less profound than that occurring in CXCR2 KO animals. The latter finding may indicate that mast cells are not the sole source of CXCR2-binding chemokines in the peritoneal cavity during infection. In this regard, macrophages are capable of producing both MIP-2 and KC, and human neutrophils themselves are a potent IL-8 source (48 –50). Although mice do not express IL-8, it is nevertheless possible that murine PMN produce CXCR2-binding chemokines in response to T. gondii, as has been shown for the CC chemokines MIP-1␣ and MIP-1 (29). In a related study to that reported in this work, it was shown that CCR1 KO mice display increased susceptibility to T. gondii (51). Unlike CXCR2, the ligands for CCR1 are CC chemokines such as MIP-1␣, MIP-1, and RANTES (52). Indeed, the phenotype for T. gondii-infected CCR1 and CXCR2 KO mice appears distinct. Thus, CCR1⫺/⫺ animals display PMN trafficking to sites of infection but an impaired ability to mobilize neutrophils and their precursors from the bone marrow (51, 53). In contrast, our studies and those of others (8, 10) show a defective ability of CXCR2⫺/⫺ neutrophils to migrate to sites of infection. In addition, while we and others (12) found evidence for defective proinflammatory cytokine responses in the absence of a functional CXCR2, this was not the case in CCR1⫺/⫺ mice. Differences in the chemotactic effects of CCR1 and CXCR2 ligands on PMN may underlie these disparate effects. In our experiments, we found a dramatic infection-induced loss of F4/80 strongly positive cells in the peritoneal cavity of both WT and KO animals. This was accompanied by an increase in cells expressing intermediate amounts of F4/80 and Gr-1. Fluorescence microscopy suggested that the latter population was composed mainly of macrophages/monocytes (data not shown), and, indeed, macrophages/monocytes have been previously found to express low levels of Gr-1 during models of inflammation (54). The apparent loss of F4/80 bright-staining cells may reflect infectioninduced maturation of monocytes, as down-regulation of this Ag is associated with the latter process (39). The results of our study show that defective neutrophil recruitment is associated with increased host susceptibility and is detectable within 36 h of infection as increased tachyzoite numbers in the peritoneal cavity, and at 30 days postinfection as increased numbers of cysts establishing within the brain. The reason for this increased susceptibility is not known; however, we and others have presented evidence for an immunoregulatory role of PMN in initiating type 1 cytokine responses (12, 24, 31, 33, 55). The present data are consistent with this concept. Thus, CXCR2 KO mice displayed dysregulated IFN-␥ and TNF-␣ responses in the spleen, and serum IFN-␥ levels were dramatically lower in the IL-8Rh KO
mice. The fact that IFN-␥ responses were not totally absent likely accounts for the ability of CXCR2 KO mice to survive infection, albeit with higher cyst numbers. Nevertheless, while these data are consistent with a role for PMN in triggering type 1 cytokine responses, we cannot exclude the possibility that susceptibility of the CXCR2⫺/⫺ mouse strain reflects decreased microbicidal activity at the site of infection. The results of this study indicate that, in addition to an inactive CXCR2 gene, the KO animals differ from BALB/c WT mice by retaining an Nramp1 allele associated with resistance to Mycobacterium bovis. It is also of note that the Nramp1 gene may influence susceptibility to T. gondii (56). However, we think it unlikely that differences in the Nramp1 allele account for the defective PMN responses between WT and KO strains for the following reasons. First, our results show that the parental strains, which differ in Nramp1 alleles, display an identical neutrophil influx during infection and an equivalent parasite level in the peritoneal cavity. Second, while the Nramp1 gene may influence macrophage production of the CXCR2 ligand KC, the particular allele carried by the KO strain is associated with increased, rather than decreased, KC gene expression (57). Therefore, our data suggest that absence of CXCR2 itself, rather than an allele-specific Nramp1 influence, accounts for the effects reported in this work. In sum, our results point to an important function for CXCR2 in trafficking PMN to the site of protozoan infection. In this study, such newly recruited cytokine-secreting neutrophils may play a role in macrophage and dendritic cell activation, as well as displaying direct microbicidal activity. In this manner, PMN recruitment is likely to be an essential early step in controlling microbial infection and may underlie induction of acquired immunity to Toxoplasma and many other infections.
Acknowledgments We thank Dr. B. A. Butcher for critical review of the manuscript and J. Olaya for technical assistance.
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