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Departments of
*
Medicine,
Microbiology, and
Pathology, Dartmouth Medical School, Lebanon, NH 03756; and
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secreted by both of these cell types plays a pivotal role in acute
infection and recrudescence in the chronically infected host (6, 7). Recent studies have suggested that, in addition to T cells and NK cells, polymorphonuclear leukocytes (PMNs)3 may play a role in the generation of early resistance to T. gondii infection (8, 9) and an exacerbation of Toxoplasma infection has been reported in PMN-depleted mice (10, 11). Scharton-Kersten et al. (12) have demonstrated that mice deficient in inducible NO synthase succumbed to Toxoplasma infection after Ab depletion of PMNs. Antimicrobial effects of neutrophils are, in part, mediated by their ability to scavenge infected cells and secrete toxic products such as NO and reactive oxygen metabolites (13, 14, 15). These molecules have been shown to be involved in the intracellular killing of T. gondii (16, 17). Neutrophils also secrete chemokines, which are responsible for recruitment of other effector cell populations (9, 18, 19).
Chemokines are a large family of secreted proteins involved in
regulating multiple steps in host defense mechanisms and during
inflammatory responses (20). Chemokine classification is
based on the arrangement of cysteine residues in their amino-terminal
domains (21). CXC or 
-chemokines mainly target
neutrophils and T cells, whereas CC or 
-chemokines target monocytes,
eosinophils, and basophils with variable selectivity but usually do not
affect neutrophils (22). Chemokines activate leukocytes by
binding to G1-type G proteins (23). The 18 identified
receptor subtypes can be divided into 2 major groups, CXCR and CCR,
based on the 2 major classes of chemokines. One of the CCR receptors,
CCR1, is expressed on neutrophils, monocytes, lymphocytes, and
eosinophils and binds the leukocyte chemoattractant and hemopoiesis
regulator macrophage-inflammatory protein (MIP-1), as well as
several other related chemokines (24, 25, 26). Previous
studies have shown that mice deficient in the CCR1 gene had
defects in the trafficking of neutrophils to the blood (27, 28). In the present study, we tested the ability of
CCR1-/- mice to resolve acute T.
gondii infection. CCR1-/- mice showed a
higher susceptibility to Toxoplasma infection and had higher
tissue parasite loads than did immunocompetent controls.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of CCR1-/- mice has been described previously (27). The CCR1-/- mice used in this study were backcrossed for six generations onto the C57BL/6 background. Age- and sex-matched control C57BL/6 were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were challenged perorally (p.o.) with the 76K strain of T. gondii (kindly provided by Daniel Bout, Tours, France). This strain is maintained by continuous oral passage of cysts. Unless otherwise stated, each mouse was infected p.o. with 10–15 cysts.
Antigenic preparation
Toxoplasma lysate Ag (TLA) was prepared from tachyzoites of the RH strain of T. gondii. Parasites were cultured in human fibroblasts and isolated from monolayers by forced extrusion through a 27-gauge needle. Host cell debris was removed using a Percoll gradient (1.04 g/ml). Purified parasites, essentially free of any fibroblast contamination, were washed twice and resuspended in PBS. The resulting preparation was pulse sonicated at 4°C (eight times for 10-s intervals at 18 kHz) before centrifugation at 10,000 x g for 15 min to remove insoluble material. The protein concentration of the Ag preparation was determined using a commercial assay kit (Bio-Rad Laboratories, Cambridge, MA) and stored aliquoted at -20°C.
T cell proliferation
After general anesthesia, spleens were removed and homogenized in a petri dish, and contaminating erythrocytes were lysed in RBC lysis buffer (Sigma, St. Louis, MO). After two to three washes in HBSS (Life Technologies, Gaithersburg, MD) supplemented with 3% FBS (HyClone Laboratories, Logan, UT). Cells were suspended in RPMI 1640 (Life Technologies) containing 10% FBS and cultured in 96-well flat-bottom plates in a 200-µl volume at a starting concentration of 2 x 105 cells/well. Cells were stimulated with either 5 µg/ml Con A or 15 µg/ml TLA. After culture for 72 h, [3H]thymidine (0.5 µCi/well; Amersham, Arlington Heights, IL) was added to the wells. Pulsed splenocytes were harvested onto glass filters using an automated multiple sample harvester and dried. Incorporation of radioactive thymidine was determined by liquid scintillation counting according to a standard protocol.
Phenotypic analysis
Spleen cells from infected mice were isolated as described above, washed, and then suspended in 3% BSA-PBS. Phenotypic analysis of splenocytes was conducted using direct immunofluorescent staining and FACS analysis (Becton Dickinson, San Jose, CA). Cells (1 x 106/ml) were incubated with FITC- and/or PE-labeled Ab (all from PharMingen, San Diego, CA) on ice for 45 min. After incubation, cells were washed several times in buffer, fixed in 1% methanol-free formaldehyde, and stored at 4°C in dark before FACS analysis.
Quantitation of parasite burden
Tissues (intestine, spleen, liver, lung, and brain) from T. gondii-infected animals were collected at day 7 postinfection (p.i.). DNA was extracted from tissues using the Qiamp tissue kit (Qiagen, Chatsworth, CA), and 400 ng of each sample were analyzed by quantitative PCR. Amplification of parasite DNA was performed using primers specific for a 35-fold repetitive sequence of the Toxoplasma B1 gene (5'-GGAACTGCATCCGTTCATGAG-3' and 5'-TCTTTAAAGCTTCGTGGT C-3'), which is found in all known parasite strains (29). A 134-bp competitive internal standard containing the same primer template sequences as the 194-bp B1 PCR fragment was also synthesized (30). Amplification of this 194-bp segment of the B1 gene and the 134-bp segment of the internal standard was performed using in a 50-µl reaction mixture containing 1.25 U Amplitaq DNA polymerase; 1x buffer (Perkin-Elmer, Norwalk, CT); 0.2 mM concentrations each of dGTP, dATP, dTTP, and dCTP; and 0.4 µM concentrations of each B1 primer. For each reaction, a known amount of DNA from the tissues was amplified with varying amounts of the internal standard. Levels of parasite load were estimated by comparison to the internal controls. To determine the parasite load in infected tissues, PCR was performed under the same conditions using a known number of parasites. The level of internal control was calculated per parasite (30).
Histopathological analysis
Tissues from infected CCR1-/- animals and parental control animals were fixed in 10% buffered formalin, paraffin processed, and used to prepare 5-µm histological sections. Sections were stained with hematoxylin and eosin and photographed on an Olympus Van Ox microscope with Kodak Elite 100 film. The resulting images were digitized with a Polaroid Sprint scanner and processed using Adobe Photoshop software.
Cytokine PCR
Tissues from T. gondii-infected animals were
collected at day 7 p.i. RNA from the tissue samples was collected
using Trizol (Life Technologies) according to the manufacturer’s
instructions. Reverse transcription was performed using Moloney murine
leukemia virus reverse transcriptase (Life Technologies) and random
hexamer primers. (Promega, Madison, WI). Levels of mRNA for IFN- and
IL-10 were measured by quantitative PCR using the PQRS quantitative
method (31). The tissues from uninfected mice were used to
establish a baseline value of 1.0 against which the level of message
for cytokine in the test mice was quantitated.
IFN- assay
The splenocytes from infected wild-type and
CCR1-/- mice were cultured in a 24-well plate
in presence of 15 µg/ml TLA. After incubation for 72 h, culture
supernatants were collected and stored at -70°C. These supernatants
were analyzed for IFN- production by ELISA (Endogen, Woburn,
MA).
Neutrophil mobilization to peripheral blood
Relative PMNS numbers in uninfected and infected, wild-type and homozygous CCR1 knockout mice (three or four animals per group) were determined by dual-color immunofluorescent staining. Whole mouse blood (100 µl) was incubated for 1 h at 4°C with FITC-labeled anti-CD45.2 (clone 104; PharMingen), a pan leukocyte marker, and PE-labeled anti-mouse GR-1 (RB6-8C5; PharMingen), a PMN-specific marker. Samples were then washed three times by centrifugation, and the erythrocytes were lysed using FACS lysing solution (Becton Dickinson) before fixation with 1% paraformaldehyde. Duplicate cytospin slides were then prepared for each sample and analyzed using a Bio-Rad MR1000 Confocal Scanning Laser Microscope system as described previously (32). At least 300 cells on each slide were counted. Relative PMN numbers were then calculated as a percentage of the total blood leukocytes.
Determination of neutrophil index
To estimate the relative index of PMNs in the tissues of the infected animals, the livers and intestines from wild-type and CCR1-/- mice were collected at days 1, 2, and 3 p.i. Histological sections were stained with hematoxylin and eosin and examined by Olympus Van Ox microscope.
Statistical analysis
Statistical analysis of lymphoproliferation studies and relative PMN numbers were conducted by unpaired Student t tests (33).
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Results |
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To determine whether the deletion of the CCR1 gene altered
susceptibility to T. gondii infection,
CCR-1-/- and parental C57BL/6 mice were
challenged p.o. with 15 cysts of T. gondii. As shown in Fig. 1
, all of the knockout mice succumbed to
infection by day 14 postchallenge. In contrast, almost 80% of the
animals from the control group survived Toxoplasma
infection. None of the survivors exhibited any signs of clinical
sickness, and they continued to live until the termination of the
experiment.
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Tissues from both CCR1-/- and
parental mice were analyzed by quantitative PCR to determine whether
levels of parasite multiplication are increased in knockout animals.
The relative abundance of the B1 gene, a genetic marker for
T. gondii, was determined at 12 days p.i. The majority of
organs from knockout mice (spleen, liver, lungs, and brain) exhibited a
10- to 20-fold increase in parasite burden as compared with parental
C57BL/6 mice (Fig. 2). A nearly 6-fold
increase in the parasite load was noted in the intestines of
CCR1-/- mice (Fig. 2).
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Sections of the liver of day 8 p.i.
CCR1-/- mice (Fig. 3A) showed preservation of
overall tissue architecture, with multiple scattered foci of individual
hepatocyte necrosis accompanied by a mixed lymphocyte infiltrate and
the occasional granulocyte. Many small foci of intracellular T.
gondii replication were seen throughout the liver (Fig. 3A, inset, arrows). Parental control
mice (Fig. 3B) showed the expected small lymphocytic foci
scattered throughout the parenchyma and the focal fatty metamorphosis
typical of T. gondii-infected C57BL/6 mice
(34). The small intestine of both the
CCR1-/- mice and the parental controls (Fig. 3, C and D) showed scattered focal necrosis. This
necrosis was more pronounced and more likely to be full-thickness in
the parental controls as compared with the
CCR1-/- mice. Thus, it appears that the
inflammatory intestinal pathology reported in wild-type C57BL/6 mice
(34, 35) is comparatively less severe in the
CCR1-/-animals. The spleens of the
CCR1-/- mice (Fig. 3E) showed
lymphocyte depletion of the primary follicles, with evidence of
intracellular replication of T. gondii in macrophages and
stromal cells (Fig. 3E, inset, arrows). The parental
controls (Fig. 3F) showed preservation of the basic splenic
architecture with scattered perifollicular large clear cells typical of
the pattern seen in infected C57BL/6 mice.
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Cytokine analysis of the splenocytes from infected animals was
performed at day 7 p.i. using quantitative PCR. Message for
IFN- was increased in both the wild-type and the knockout mice in
response to T. gondii infection. Almost equal levels of
IFN- message were observed in the spleens of infected
CCR1-/- and control wild-type animals at day
7 p.i. (Fig. 4A). IL-10
levels were elevated in both knockout and wild-type infected mice as
compared with uninfected mice. No noticeable differences in the
production of IL-10 message were observed in the spleens of infected
knockout vs the spleens of wild-type mice (Fig. 4B). Both of
these cytokines play an important immunoregulatory role in infectious
diseases (36, 37, 38, 39).
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message, levels of IFN- protein produced by
the splenocytes of infected animals in response to antigenic
stimulation were determined by ELISA. As shown in Table I, minimal differences in the IFN-
release were noted between wild-type and
CCR1-/- mice.
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To determine whether the mortality observed in the
CCR1-/- mice was due to their inability to
elicit a normal T cell response, phenotypic analysis of splenocytes
from infected animals was performed. As shown in Table II, similar increases in both the
percentage and the absolute numbers of T cells were observed in the
infected CCR1-/- and wild-type mice. No
significant differences were seen within either the
CD4+ or the CD8+ T cell
populations of infected animals. As NK cells play an important role in
acute T. gondii infection, the splenocytes were analyzed for
the presence of NK cells. A nominal difference in the NK cell
population was observed between the two strains of mice.
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NO-mediated immunosuppression is induced in the infected CCR1-/- mice
We and others have demonstrated that acutely infected mice exhibit
an immune down-regulation at 7 days p.i., which manifests as a
decreased proliferative response to Con A stimulation (41, 42). It has been reported that this altered response is mediated
by NO released by IFN--activated macrophages (41, 42, 43).
To further evaluate the immune response generated against T.
gondii in CCR1-/- mice, lymphocyte
proliferation assays were performed. Splenocytes were harvested from
CCR1-/- mice at day 7 p.i., and
proliferative responses to Con A and TLA were compared with those of
age-matched wild-type controls. As shown in Fig. 5A, splenocytes from both
CCR1-/- and wild-type infected mice failed to
respond to Con A stimulation. In contrast, spleen cells from uninfected
control groups showed significant mitogenic response
(p < 0.001). However, when the NO synthase
antagonist, N-monomethyl-L-arginine
(L-NMMA), was added to the splenocyte cultures,
mitogenic responses of splenocytes from both
CCR1-/- and wild-type mice were partially
restored. Similarly, spleen cells from infected
CCR1-/- mice failed to proliferate in response
to TLA stimulation in the absence of L-NMMA (Fig. 5B). Treatment with the NO antagonist significantly
neutralized the suppression in the CCR1-/-
splenocyte culture (p = 0.03). Similar
observations were made with the spleen cell cultures from infected
wild-type controls (p = 0.02).
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Relative numbers of peripheral blood PMNs were estimated in both
uninfected and orally infected wild-type and homozygous CCR1 knockout
mice. When expressed as a percentage of total leukocytes, PMNs were not
significantly different in the peripheral blood of uninfected
CCR1-/- mice vs uninfected wild-type mice (Fig. 6). At day 1 p.i., PMNs as a
percentage of peripheral blood leukocytes dropped significantly in the
infected CCR1-/- mice vs uninfected
CCR1-/- mice (p <
0.05, unpaired t test), compared with the uninfected
wild-type mice (p = 0.1, NS) and the day 1
p.i. wild-type mice (p < 0.05, unpaired
t test). A similar drop in PMN numbers at this time point
was not observed in the control wild-type mice. However, numbers in the
wild-type mice were reduced at day 2 p.i. as compared with the day
1 p.i. levels (p < 0.05, unpaired
t test). In contrast, the percentage of PMNs in the knockout
mice had increased from the day 1 p.i. levels (not significant).
At 3 day p.i., numbers in the wild-type mice remained at day 2 levels
and were significantly lower than levels at day 1 p.i.
(p < 0.05, unpaired t test). PMN
numbers in the CCR1-/- mice were now
significantly higher than the day 1 p.i. levels
(p < 0.05, unpaired t test), having
recovered to levels equivalent to those seen in uninfected
mice.
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) and returned to the levels of
wild-type mice by day 3 p.i. The reduced tissue load is probably
due to a significant reduction in the levels of circulating PMNs
observed in the blood at day 1 p.i. in the
CCR1-/- mice. However, by day 3 p.i. both
relative peripheral blood PMN numbers and tissue PMN load had
recovered, indicating that redundant mechanisms of PMN maturation and
mobilization eventually lead to an increase in PMN production and
mobilization in the CCR1-/- mice.
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Discussion |
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-producing CD4+ T
cells (35). Because CCR1-/- mice
showed a normal CD4+ T cell immune response, it
is possible that other cell types such as neutrophils may play a
secondary role in the hyperimmune response in the intestine during
T. gondii infection. In the present study, a reduced PMN
index in the intestines of CCR1-/- mice in
comparison with parental wild-type mice was observed at day 2 p.i.
The role of neutrophils in the gut inflammation, although well
described during other infectious diseases (46, 47), needs
to be evaluated in the T. gondii model.
Although CCR1 is expressed on wide variety of lymphoid and myeloid
cells, the overt phenotypic difference in the knockout mice is one of
impaired myeloid progenitor cell function. Normal T cell responses in
infected CCR1 -/- mice suggest that CCR1 has a
redundant function in these cells. However, recent studies with a
cardiac allograft model have reported that mice lacking CCR1 receptor
showed significant prolongation of allograft survival
(48). The lack of allograft rejection by
CCR1-/- mice was attributed to a defect in the
influx of IFN--producing CD4+ T cells in these
animals. Contrary to these findings, lung lymph nodes from
CCR1-/- mice injected with
Schistosoma eggs showed increased IFN- production
(27). These reports raise the possibility that the role of
chemokines may vary according to the disease model. Earlier studies
have shown that neutrophils from CCR1-/- mice
are nonresponsive to MIP-1 suggesting that CCR1 may be the dominant
MIP-1 receptor subtype in these cells (27). CCR1 may
have evolved to play a more important role in mouse neutrophils to
compensate for the lack of other chemokine receptors, such as CXCR1
which is present on human neutrophils. Based on our current
observations with T. gondii and earlier findings with
MIP1--treated mice it can be postulated that CCR1 is important for
the mobilization of bone marrow PMN pool.
Recent reports have demonstrated that neutrophils have the
ability to produce both IL-12 and IFN- after stimulation with
microbial Ags (11). IL-12 is an important cytokine that
leads to type 1 cytokine (IFN-) synthesis in the infected host
(49, 50). IFN- is known to be critical for survival
against Toxoplasma infection (5, 6). Our
results showed no differences in the levels of IFN- message in the
spleens of the T. gondii-infected parental and
CCR1-/- mice. These findings are in agreement
with those of Sayles et al. (10), who demonstrated that
depletion of neutrophils had no effect on IFN- production of
T. gondii-infected animals. However, Sayles et al. reported
reduced numbers of CD4+ and
CD8+ T cells in response to T. gondii
infection in neutrophil-depleted mice. The differences between our
findings and those of Sayles et al. can be attributed to the fact that
unlike neutrophil depletion, lack of CCR1 does not result in the
complete blockade of neutrophil migration in these animals. This is
also further proved by the fact that at later stages of infection (day
3 p.i.), no apparent differences in the neutrophil count between
the tissues of knockout and wild-type mice were observed. Moreover, it
is likely that there are other, redundant, mechanisms by which T cells
can be activated in these mice.
Studies over the last few years have emphasized an important role for neutrophils as mediators of effective immune responses against microbial infections (11, 51). Neutrophils are often the first cell type recruited to an area of infection or inflammation. Neutrophil-depleted mice succumb to a mildly virulent strain of Candida albicans (51). Neutrophils have also been reported to contribute to early resistance against T. gondii infection (52). Depletion of PMNs with antigranulocyte Ab impairs the ability of mice to survive acute infection with a low virulence strain of T. gondii (12). In the present study, CCR1-/- mice, known to have disordered trafficking and mobilization of PMNs (28), exhibited increased susceptibility to T. gondii infection. However, based on our findings, it appears that CCR1-/- mice do not have any major problem in the trafficking of PMNs from the blood to the tissues. The relative PMN indices in the tissues of knockout and parental wild-type mice are more or less similar at day 1 p.i. Nevertheless, there is a very obvious decrease in the percentage of PMNs in the blood at this time point. This can be explained by an inability of the knockout mice to replenish the peripheral blood PMNs from the bone marrow pool, as circulating PMNs are recruited into the infected tissues. The decreased PMN levels in the blood of CCR1-/- mice results in a reduced PMN index in the tissues at day 2 p.i. However, the levels of PMNs in the peripheral blood and tissues are restored, respectively, at days 2 and 3 p.i. Thus, it seems that in the absence of CCR1, mobilization of PMNs to the peripheral blood during T. gondii infection can occur by alternative mechanisms. Recent studies have demonstrated that G-CSF plays an important role in granulopoiesis during infections (53). The role of G-CSF in the mobilization of PMNs during T. gondii infection is currently being investigated in our laboratory. A decrease in the percentage of PMNs was also observed in the peripheral blood of the wild-type C57BL/6 mice. However, the fall in PMN levels took place at day 2 p.i. as compared with day 1 p.i. in the knockout mice. Moreover, it appears that circulating PMNs levels in the wild-type mice are rapidly restored because the PMN index in the tissues remains unchanged. An interesting feature of these findings is that a very short delay in the mobilization of PMNs during T. gondii infection can have a pronounced effect on the outcome of infection. Consistent with our data, earlier studies have demonstrated that CCR1-/- mice had accelerated mortality when challenged with Aspergillus fumigatus; a fungal infection that is principally controlled by neutrophils (27).
Based on reports from other laboratories and our current observations, the hypothesis we propose is as follows. Natural immunity against T. gondii is dependent on the induction of strong parasite-specific immunity in the host (1). However, before adaptive immunity is established, the rapid multiplication of T. gondii tachyzoites in the infected host needs to be contained. Because neutrophils migrate very early during infection, they may be necessary to restrict the replication of the parasites. Earlier findings by Gao et al. (27) suggest that CCR1 may be essential for influx of neutrophils to the infected tissues. The absence of CCR1 probably results in defective neutrophil migration from bone marrow to the blood. This could lead to uncontrolled parasitic replication in T. gondii-infected mice, which ultimately overwhelms the capacity of the adaptive immune system to contain the infection.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Imtiaz A. Khan, Department of Microbiology, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70122.
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; TLA, Toxoplasma lysate Ag; MIP-1, macrophage-inflammatory protein; p.i., postinfection; L-NMMA, N-monomethyl-L-arginine; p.o., perorally.
Received for publication January 28, 2000. Accepted for publication November 8, 2000.
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L. Del Rio, S. Bennouna, J. Salinas, and E. Y. Denkers CXCR2 Deficiency Confers Impaired Neutrophil Recruitment and Increased Susceptibility During Toxoplasma gondii Infection J. Immunol., December 1, 2001; 167(11): 6503 - 6509. [Abstract] [Full Text] [PDF]
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) and homozygous CCR1
knockout mice (
) in the presence and absence of
Toxoplasma infection. Wild-type and homozygous knockout
mice, three animals/group, were either mock-infected or infected p.o.
with 15 cysts of T. gondii. Animals were sacrificed at
days 1, 2, and 3 p.i., and peripheral blood PMNs were estimated as
a percent of total leukocytes. Bars in each group represent the mean
percentage (±SEM) of the total leukocytes that were PMNs. Significant
differences (p < 0.05, unpaired t
test) between treatment pairs are indicated by brackets. The experiment
was performed three times with similar results.