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McGill University Centre for the Study of Host Resistance, and Montreal General Hospital Research Institute, Montreal, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA levels on days 3–5. However, IL-12R ß1 mRNA
levels in the spleen were similarly up-regulated in both mouse strains
by day 3. Taken together, these data suggest that high systemic IL-12
production, accompanied by an early and sustained up-regulation of both
IL-12R ß1 and ß2 mRNA levels in the spleen, as occurs in resistant
B6 mice, appears to preferentially induce protective Th1 responses
against blood-stage malaria. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 induces IFN- synthesis by both NK and T cells, augments NK
cell cytotoxicity and induces T cell proliferation, and is also a
potent stimulus for Th1 cell development both in vitro (9) and in vivo
(10). Th1 cells produce IL-2, IFN-, and TNF-
; provide activation
signals for macrophages; mediate delayed-type hypersensitivity
reactions; and provide B cell help for the production of IgG2a Abs.
Importantly, Th1 responses have been implicated in host protective
immunity against parasitic diseases, including Leishmania
major (10), Toxoplasma gondii (11), and blood-stage
malaria (12, 13, 14). Th2 cells, on the other hand, secrete IL-4, IL-5,
IL-6, IL-10, and IL-13, and provide B cell help for Ab synthesis of
other isotypes, especially IgE and IgG1 (15, 16, 17, 18).
Malaria poses a major global health threat today, a worrisome prospect
mainly attributable to the resurgence of drug-resistant forms of the
parasite and the lack of an effective vaccine (19). To address the host
immune mechanism(s) against blood-stage malaria, our laboratory has
used the model of Plasmodium chabaudi AS infection in
resistant C57BL/6 (B6) and susceptible A/J mice (20). Briefly,
resistant B6 mice mount an early Th1 response, show moderate levels of
acute primary parasitemia and anemia, and clear the infection by 4 wk.
In contrast, susceptible A/J mice mount an early Th2 response, show
very high levels of acute primary parasitemia and anemia, and infection
is fatal after 10–12 days. We have recently reported that systemic
administration of murine rIL-12 for 6 days beginning on the day of
infection could protect susceptible A/J mice against i.p. challenge
with 106 P. chabaudi AS parasitized
erythrocytes. Moreover, this IL-12-induced protection occurred by
IFN-, TNF-, and nitric oxide-dependent mechanisms.
Despite the pivotal role of IL-12 in mediating Th1 cell development, it
is presently unknown whether or not endogenous IL-12 synthesis, or lack
thereof, plays any crucial role in the induction of host immunity
against blood-stage malaria. To further understand the role of IL-12 in
host defense against malaria, we have analyzed in vivo IL-12 production
in resistant B6 and susceptible A/J mice during early blood-stage
P. chabaudi AS malaria. We have also analyzed splenic mRNA
expression of the two recently cloned IL-12R subunits (21, 22) in these
hosts after malaria infection. Our results show an important
correlation between resistance and an early presence of high levels of
systemic IL-12 p70, concomitant with significant up-regulation of mRNA
levels of both IL-12R ß1 and ß2, and IFN- in the spleen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice, 6–8 wk old, were age- and sex-matched in all experiments. A/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and B6 mice were from Charles River Breeding Laboratories (St. Constant, Quebec, Canada). P. chabaudi AS was maintained as described previously (23). Infection was initiated by i.p. injection of 106 P. chabaudi AS parasitized RBC and the course of infection was monitored by previously described procedures (23).
Reagents and sera
Murine rIL-12 was a generous gift of Dr. S. Wolf (Genetics Institute, Cambridge MA). Rat anti-murine IL-12 mAbs, C15.1 and C15.6 (IgG1 isotype), and C17.8 (IgG2a isotype) were generated as previously described (24). Hybridomas producing these mAbs were a kind gift of Drs. M. Wysocka and G. Trinchieri (Wistar Institute, Philadelphia, PA). All three mAbs detect the p40 subunit of IL-12. Red-T/G297–289, a mixture of mAbs against the p35/p70 subunit of IL-12, was purchased from PharMingen (Mississauga, Ontario, Canada). At indicated times, blood was obtained from A/J and B6 mice by cardiac puncture, allowed to clot, and sera were separated by centrifugation at 13,800 x g for 3 min. Sera were kept at 4°C and immediately analyzed for IL-12 levels by ELISA.
Splenectomy and spleen cell preparation
Splenectomy and sham-splenectomy procedures were performed using standard techniques published previously (25). Mice were rested for 3 wk after surgery before initiating P. chabaudi AS infection. At the indicated times, single cell suspensions of spleen cells in RPMI 1640 (Flow Laboratories, McLean, VA) supplemented with 5% heat-inactivated FCS (HyClone, Logan UT), 2% HEPES buffer (Flow Laboratories), and 0.12% gentamicin (Schering Canada, Montreal, Quebec) were prepared under aseptic conditions as previously described (26). To obtain nonadherent spleen cells, splenic adherent cells were depleted by 2 h adherence to plastic at 37°C in a humidified, CO2 incubator. Nonadherent spleen cells, pooled from three uninfected A/J or B6 mice, were adjusted to 4 x 106/ml and aliquots of 2–3 ml were placed in 6-well culture dishes (Nunc, Roskilde, Denmark). Cells were stimulated for 24 h in the presence of 5 µg/ml Con A (Calbiochem, La Jolla, CA), 1.8 ng/ml rIL-12, or medium as control.
ELISAs
Two-site sandwich ELISAs were used to measure serum levels of
IL-12; p40- and p70-specific ELISAs were used. The former detects all
species of IL-12 including p40 monomers, homodimers and p40-p35
heterodimers, whereas the latter detects only levels of the
heterodimer. For the p40-specific ELISA, the capturing Ab was C15.1 and
the detecting Ab was biotinylated C15.6. For the p70-specific ELISA,
the capturing Ab was Red-T/G297–289 and the detecting Ab was
biotinylated C17.8. Streptavidin-horseradish peroxidase
conjugate (Life Technologies, Grand Island, NY) was added for final
detection. Washing, blocking, incubation, and final detection of
products were performed using methods previously established in our
laboratory for the IFN- ELISA (27) with minor modifications. The
coating buffer for Red-T/G297–289 was 0.1 M NaHCO3 (pH
8.2). Incubation conditions for serum samples and standard rIL-12 were
overnight at 4°C, and after addition of biotinylated Ab, plates were
incubated for 4–5 h at room temperature. Plates were read in a
microplate reader (Fisher Scientific, Nepean, Ontario, Canada) at 405
m, with a reference wavelength of 492 m.
Cytokine mRNA determination by RT-PCR
Spleens were aseptically removed from uninfected or infected mice at indicated times, immediately frozen in liquid nitrogen and stored at -70°C. The procedure for RNA isolation was based on a modification of the single-step method described by Chomzynski and Sacchi (28). Briefly, frozen tissue samples were homogenized in TRIzol reagent (Life Technologies) using a polytron homogenizer (Brinkmann, Kimematica, Switzerland). Cells were directly lysed by adding TRIzol reagent. Total RNA was subsequently isolated following the manufacturer’s instructions.
RT-PCR was performed as previously described by Kichian et al. (29).
Cycling conditions and the number of PCR cycles were empirically
determined for each primer pair. Typically, 30 cycles were performed,
and the cycling conditions used were 94°C for 1 min, 54°C for
20 s, 72°C for 1 min, and a final extension at 72°C for 10
min. To increase the specificity of PCR amplification, the "hot
start" method, involving an initial denaturation of the reaction
mixture containing all reagents except the enzyme for 3 min at 94°C
was used. Taq polymerase (Life Technologies), in 1x PCR
buffer, was then added to the reaction mixture at 85°C before cycling
was initiated. Both positive and negative controls were included in
each assay to ensure efficacy of the reaction and to rule out possible
cDNA contamination of reagents. The housekeeping gene glyceraldehyde
6-phosphate dehydrogenase (G6PDH) was simultaneously amplified
in each assay to verify that equal amounts of cDNA were added in each
PCR. Nucleotide sequences for primers and probes for IL-12 p40 and
IL-12 p35 (30), IFN- (31), and G6PDH (29) were used as described
previously. Primers and probe sequences were designed in our laboratory
for IL-12R ß1 and ß2, based on the recently cloned cDNAs for these
genes (GenBank accession nos. U23922 and U64199, respectively). The
sequences were: IL-12R ß1 sense, 5'-TGA-AGA-CGG-CGC-GTG-GGA-GTC-A-3';
antisense, 5'-TCG-CGG-GTA-CAA-CAC-CTC-CGG-G-3'; probe,
5'-GCG-AGC-GGA-CAC-TGC-GAG-GC-3' (product size, 412 bp) and IL-12R ß2
sense, 5'-GGT-TGC-TGG-CTC-CTC-ACC-AGG-3'; antisense,
5'-ATG-CAG-CCC-CTT-TGC-TCC-GGG-3'; probe,
5'-TCC-CCC-ACA-CTG-GCT-GCG-GA-3' (product size, 424 bp).
Fifteen microliters of final PCR products were analyzed by
electrophoresis in a 1.2% agarose gel, denatured, neutralized, and
transferred onto a Hybond-N membrane (Amersham, Arlington Heights, IL)
by Southern blot analysis. After UV cross-linking (UV Stratalinker
1800, Stratagene, La Jolla, CA) and baking in a vacuum oven (2 h at
80°C), membranes were hybridized with cytokine-specific
[-32P]ATP (Amersham) end-labeled oligonucleotide
probes that hybridize to a portion of the amplified segment between the
primers. Probes were radioactively labeled with 10 U of T4
polynucleotide kinase (Life Technologies) in a 0.5x One-Phor-All
buffer supplied with the enzyme. After hybridization and washing,
cytokine or cytokine receptor mRNA was detected by autoradiography with
Biomax MR Kodak film (Rochester, NY). The intensity of bands
corresponding to specific cytokines was analyzed by high-resolution
optical densitometry (SciScan 500, United States Biochemical,
Cleveland, OH) and normalized to those of G6PDH. As shown in Fig. 1
, titration of input cDNA was performed
for each cytokine or cytokine receptor, followed by PCR and Southern
blot analysis to ensure linearity between input cDNA and final PCR
products.
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Statistical significance of differences in mean between groups of mice was determined by one-way ANOVA, followed by Bonferonni posttest. The Student-Newman-Keuls’ posttest was used in the analysis of serum IL-12 levels. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish possible differences in the in vivo level of systemic
IL-12 production between resistant B6 and susceptible A/J mice, sera
were collected from both strains during the first 5 days after P.
chabaudi AS infection. Sera were also obtained from uninfected
mice of either strain. All serum samples were analyzed for IL-12 levels
by the p70-specific ELISA, which only detects levels of the
biologically active IL-12 p40-p35 heterodimer, and by the p40-specific
ELISA, which detects all forms of the IL-12 p40 subunit including p40
monomers and dimers, as well as p40–p35 heterodimers. As shown in Fig. 2A, serum levels of p70 on day
2 were significantly higher in B6 compared with A/J mice
(p < 0.01), or compared with basal levels on
day 0 (p < 0.001). Serum p70 levels remained
substantially elevated in B6 mice on day 3 and decreased rapidly
thereafter, returning to basal levels by day 5. In contrast, A/J mice
showed no significant changes in serum p70 levels after P.
chabaudi AS infection on any of the days examined.
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B). In both strains,
p40 levels were significantly increased on day 3 compared with basal
levels on day 0 (p < 0.05). By day 5, serum
levels of total p40 had returned to basal levels in both strains.
Interestingly, there were no significant differences in total levels of
serum p40 between B6 and A/J on any of the days examined. It is of
interest to point out that levels of total p40 in the serum of either
strain were at least 10-fold in excess of p70 levels, in agreement with
previous observations (32) of excessive release of free p40 by IL-12
producing cells. Taken together, these data suggest the existence of
distinct and complex mechanisms for regulating p70 vs p40 synthesis and
secretion in vivo. The spleen is the major source of endogenous IL-12 p70
To determine the source of systemic IL-12 production, we examined
the spleen as well as the liver for up-regulation of mRNA levels of
IL-12 p40 and p35 subunits. Resistant B6 and susceptible A/J mice were
infected with P. chabaudi AS and during the first 5 days
postinfection, the spleen and liver were removed and analyzed for
tissue IL-12 mRNA levels by RT-PCR. Spleens and livers from uninfected
B6 and A/J mice were also analyzed. As shown in Fig. 3A, mRNA levels of p40 in the
spleen were significantly increased in A/J mice on day 3 compared with
basal levels on day 0 (p < 0.001). Moreover,
p40 mRNA levels in the spleen on day 3 were significantly higher in A/J
compared with B6 mice (p < 0.001). By days 4
and 5, p40 mRNA levels in the spleen of A/J mice had returned to basal
levels. In B6 mice, p40 mRNA levels in the spleen were slightly
increased on day 3, followed by a return to basal levels by days 4 and
5.
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B).
However, splenic p35 mRNA levels were significantly higher in A/J
compared with their B6 counterparts on day 2 (p
< 0.001), day 3 (p < 0.01), and day 4
(p < 0.05). Because A/J mice appeared to
express higher mRNA levels for both p40 and p35, p40 mRNA levels were
normalized against p35 mRNA levels for each mouse strain. The kinetics
of the ratios of p40/p35 mRNA levels in the spleen are shown in Fig. 3C. Interestingly, very similar patterns were seen in the
kinetics of p40/p35 mRNA ratios in A/J and B6 mice. In both strains,
there was a significant increase in p40/p35 mRNA ratios on day 3
compared with basal levels (p < 0.001 and
p < 0.01, in A/J and B6 mice, respectively). However,
no significant differences between strains in p40/p35 mRNA ratios in
the spleen were evident on any day postinfection examined. Compared with the spleen, p40 mRNA levels in the livers of B6 and A/J mice were very low (data not shown). Autoradiographic bands corresponding to p40 mRNA levels in the spleen were readily detectable after 2 h exposure. In contrast, under identical experimental conditions, bands corresponding to p40 mRNA levels in the liver required up to a week of exposure to be detectable. Moreover, mRNA levels of p35 in livers of both strains were virtually undetectable on all days examined (data not shown), in agreement with observations of Schoenhaut et al. (6). Taken together, these data suggest the spleen, rather than the liver, to be the likely source of systemic IL-12 production during blood-stage malaria.
To confirm the spleen as the major source of in vivo IL-12 production
during early blood-stage P. chabaudi AS infection,
experiments were conducted using splenectomized and sham-splenectomized
B6 mice. As shown in Table I,
sham-splenectomized controls had significantly elevated levels of serum
p70 on day 2, comparable with levels seen in intact B6 mice on this
day. In contrast, splenectomized animals failed to show the significant
increase in serum p70 levels seen in spleen-intact B6 mice on day 2
postinfection.
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mRNA levels in
the spleen
Recent studies indicate that full biological responses to IL-12
require the expression of both IL-12R ß1 and IL-12R ß2 at the
surface of cells responding to this cytokine (33, 34). Therefore, we
asked whether or not A/J and B6 mice differ in the levels of expression
of IL-12R mRNA in the spleen during early P. chabaudi AS
malaria. As shown in Fig. 4A,
the kinetics of IL-12R ß1 mRNA levels in the spleen were similar in
A/J and B6 mice, except on day 4 when IL-12R ß1 mRNA levels were
significantly higher in B6 compared with A/J mice
(p < 0.01). In A/J mice, IL-12R ß1 mRNA
levels in the spleen were significantly increased on day 3
(p < 0.001) and day 5
(p < 0.001), compared with basal levels on day
0. In B6 mice, significant increases over basal levels of IL-12R ß1
mRNA levels in the spleen occurred on day 3 (p
< 0.05), day 4 (p < 0.001), and day 5
postinfection (p < 0.001).
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B; p < 0.001).
However, by day 5, there was no significant difference between strains
in IL-12R ß2 mRNA levels in the spleen. In A/J mice, only on day 5
were IL-12R ß2 mRNA levels in the spleen significantly higher
(p < 0.001) than basal levels on day 0. In
contrast, IL-12R ß2 mRNA levels in the spleen were significantly
higher than basal levels in B6 mice on day 3 (p
< 0.001), day 4 (p < 0.001), and day 5
postinfection (p < 0.001). Taken together,
these data suggest that resistant B6 mice, compared with susceptible
A/J mice, show an earlier and sustained up-regulation of IL-12R ß2
mRNA levels in the spleen.
Earlier up-regulation of IL-12R ß2 mRNA in the spleen of B6 mice was
accompanied by higher up-regulation IFN- mRNA expression. As shown
in Fig. 4C, splenic IFN- mRNA levels were significantly
higher in B6 compared with A/J mice on days 4 and 5 postinfection
(p < 0.01). This finding is consistent with
previous observations from our laboratory (13) and others (12) of an
important correlation between early predominantly Th1 responses and
resistance to blood-stage P. chabaudi AS malaria.
A/J mice are not inherently deficient in up-regulation of IL-12R or
IFN- mRNA expression
We next addressed the question of whether or not A/J mice,
compared with their B6 counterparts, are as capable of up-regulating
IL-12R and IFN- mRNA expression in the absence of P.
chabaudi AS infection. Nonadherent spleen cells from uninfected
A/J and B6 mice were cultured in vitro for 24 h in medium alone,
or medium containing Con A or murine rIL-12. After the incubation
period, total RNA was isolated and mRNA levels of IL-12R and IFN-
were analyzed by RT-PCR. As shown in Fig. 5, treatment with Con A or rIL-12 in
vitro induced IL-12R ß1, and ß2 as well as IFN- mRNA expression
to comparable levels in nonadherent spleen cells from either A/J or B6
mice.
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mRNA expression in the spleen
could be modulated in vivo during infection by the administration of
exogenous rIL-12 to A/J mice. P. chabaudi AS infected A/J
mice were treated with either the standard protective dose of rIL-12
(0.1 µg/mouse/day for 6 days beginning on the day of infection) or
PBS as control. Fig. 6 shows that
treatment of P. chabaudi AS infected A/J mice with rIL-12
resulted in significant increases in mRNA levels of IL-12R ß2 and
IFN- expressed by unfractionated spleen cells on day 7 postinfection
compared with PBS-treated controls. These data suggest that the
apparent defect in up-regulation of IL-12R ß2 mRNA seen in the spleen
of A/J compared with B6 mice during early P. chabaudi AS
malaria may be due in part to reduced production of p70 in A/J mice.
Moreover, the deficiency in IL-12R ß2 mRNA expression during
blood-stage malaria in these hosts can be corrected by treatment with
exogenous rIL-12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this report, evidence is presented that demonstrates for the first
time significant differences between resistant B6 and susceptible A/J
mice in the level of systemic production of IL-12 during early
blood-stage P. chabaudi AS malaria. As early as day 2
postinfection, serum levels of p70, the form of IL-12 that accounts for
most of its known biological actions, were significantly higher in B6
compared with A/J mice. In B6 mice, the serum levels of IL-12 on day 2
represented a significant increase over basal levels seen in uninfected
mice. In contrast, no significant increases in serum p70 over basal
levels were observed in A/J mice during the first 5 days of infection.
Previous studies in our laboratory have shown that during early
P. chabaudi AS infection, spleen cells recovered from B6
compared with A/J mice, produced substantially higher levels of IFN-
in vitro after stimulation with Con A or parasitized RBC (13). In
addition, IFN- mRNA levels in the spleen were found to be
significantly higher in B6 compared with A/J mice, whereas IL-4 mRNA
levels in the spleen were significantly higher in A/J mice during the
first week of infection (39). The results reported here of significant
differences between A/J and B6 mice in systemic production of IL-12 are
consistent with these earlier observations and provide further
understanding of the underlying mechanism(s) for the observed dichotomy
of Th responses seen in the two inbred mouse strains during blood-stage
malaria.
Differences between resistant and susceptible mice in early in vivo IL-12 production were also recently reported in a model of Mycobacterium avium infection (40). BALB/c mice, which are genetically susceptible and mount predominantly Th2 responses to M. avium infection, exhibited reduced IL-12 p70 levels in aqueous spleen extracts on days 1 and 3 postinfection and developed large and numerous granulomas. In contrast, genetically resistant DBA/2 mice, which mount predominantly Th1 responses to this infection, had reduced mycobacterial burdens and increased IL-12 p70 levels in aqueous spleen extracts on these days postinfection. Furthermore, it was found that IL-12 p40 mRNA levels in the spleen were higher in resistant DBA/2 compared with susceptible BALB/c mice shortly (3–24 h) after the infection, whereas p35 mRNA levels were constitutively expressed. In our studies, detectable up-regulation in p40 mRNA levels was seen in the spleens of both A/J and B6 mice on day 3 postinfection, whereas p35 mRNA was constitutively expressed. The fact that mRNA synthesis would have to precede protein synthesis and release from the spleen suggests that this increase in splenic p40 mRNA levels on day 3 is probably not directly related to the increase in serum p70 levels in B6 mice on day 2. This elevated serum p70 levels in day 2 infected B6 mice could be related to transient increases in splenic p40 mRNA levels occurring h after initiating P. chabaudi AS infection that could have been missed at the days postinfection chosen for our study.
Despite this lack of direct correlation with serum p70 levels, the fact
that both p40 and p35 mRNA were readily detectable in the spleens of
infected A/J and B6 mice, but not in the liver, was important in
suggesting that the spleen might be an important source of IL-12
production during early P. chabaudi AS infection. It is
presently unclear why both p40 and p35 mRNA levels were higher in
spleens of A/J compared with B6 mice, although the ratios of p40/p35
mRNA levels were similar in both strains. Studies have shown that for
several IL-12 producing cells, mRNA levels for p40 are 
10-fold more
abundant than for p35, consistent with observations that p40 is often
produced 10–100-fold in excess of secreted p70 levels (4, 41, 42).
This wide range of excess in p40 over p70 levels might be involved in
the fact that, for similar serum p40 levels, serum p70 levels varied
significantly between P. chabaudi AS infected A/J and B6
mice.
The regulation of IL-12 mRNA expression and secretion of p40 vs p70
protein secretion appears to be complex. 1) The secretion of p70
requires that both p40 and p35 genes be expressed in the same cell. In
situ hybridization studies on sections of mouse spleen by Bette et al.
(43) revealed that large populations of spleen cells appear to express
either p40 or p35 mRNA but not both. 2) Even when both p40 and p35 mRNA
are expressed in the same cell, recent evidence suggests that different
signals can modulate secretion of p70 vs p40. After stimulation with
Salmonella dublin LPS, murine macrophages released
substantial amounts of p40 but very little p70 (44). However, LPS and
IFN- costimulation was accompanied by secretion of high levels of
both p40 and p70 (44). Similar results have been reported for IL-12 p70
vs p40 secretion by microglial cells in the central nervous system
(45).
The spleen has long been recognized as a key site for the induction of anti-plasmodia mechanism(s) (46). During early blood-stage P. chabaudi AS infection, resistant B6 mice develop massive splenomegaly (47), marked by significant changes in spleen cells expressing CD3, B220, and Mac-1 (48). Moreover, Yap and Stevenson (49) demonstrated that an architecturally intact spleen was necessary for resolving blood-stage P. chabaudi AS infection in resistant B6 mice. In this report, we show that the spleen is the major source of systemic IL-12 production in P. chabaudi AS infected B6 mice. The spleen was also the site of significant up-regulation of IL-12R mRNA levels in both A/J and B6 mice during early blood-stage malaria, suggesting an important mechanism by which the protective effects of IL-12 in the spleen are mediated.
Monocyte/macrophages are generally regarded as the major physiologic sources of IL-12 (32). However, recent studies indicate that other cell types, including neutrophils (42, 50) and dendritic cells (51), can produce IL-12. Our recent study demonstrated that splenic macrophages recovered from B6 compared with A/J mice acutely infected with P. chabaudi AS produced significantly greater quantities of IL-12 p70 in vitro (our unpublished data). Studies are currently in progress to identify the specific phenotype of cells in the spleen that express IL-12R mRNA. Evidence from FACs analysis studies suggests that the major cell types that express IL-12R are NK and T cells (52). In addition, recent analysis of lymph node cells from L.major-infected mice revealed significant up-regulation of IL-12R on CD4+, CD8+, and B220+ cells (53).
Significant differences between A/J and B6 mice were evident in the kinetics of up-regulation of splenic IL-12R mRNA levels after P. chabaudi AS infection. In B6 mice, splenic IL-12R ß2 mRNA levels were significantly elevated over basal levels by day 3 and levels remained significantly elevated on days 4 and 5 postinfection. In contrast, significant up-regulation of IL-12R ß2 mRNA over basal levels in the spleen of A/J mice was not evident until day 5 postinfection. On the other hand, IL-12R ß1 mRNA levels in the spleen were significantly up-regulated in both strains by day 3 postinfection.
The delayed kinetics of up-regulation of IL-12R ß2 mRNA levels in the
spleens of susceptible A/J mice could have important immunological
implications. Recent evidence suggests that the expression of both
IL-12R ß1 and ß2 is required in order for high-affinity interaction
between IL-12 p70 and the IL-12 receptor complex to occur (34, 54).
Importantly, the earlier and sustained expression of mRNA levels of
both IL-12R in the spleens of malaria-infected B6 mice correlated with
higher mRNA levels of IFN- in this organ on days 3–5 postinfection,
compared with A/J mice. These results are consistent with our earlier
observations that, for protective efficacy against blood-stage malaria,
rIL-12 treatment in A/J mice had to be initiated on or 1 day before
P. chabaudi AS infection (26). Treatment with rIL-12 was not
protective when initiated in A/J mice beginning on day 3 after
infection, emphasizing the importance of the presence of IL-12 early in
infection. In both A/J and B6 mice, up-regulation of IL-12R mRNA levels
in the spleen was accompanied by the disappearance of both p40 and p70
species of IL-12 from sera, possibly reflecting the increasing
predominance of membrane bound forms of IL-12 in the spleen.
Taken together, this study demonstrates significant differences between resistant B6 and susceptible A/J mice in the level of systemic IL-12 production that correlates with the developmental dichotomy of Th responses previously observed in these hosts during early P. chabaudi AS malaria. In addition, we show that the spleen is the major source of systemic IL-12 production in B6 mice, and that an early and sustained up-regulation of both IL-12R ß1 and IL-12R ß2 appears to be required for IL-12-induced protective immunity against blood-stage malaria.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mary M. Stevenson, Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, Quebec, Canada, H3G 1A4. E-mail address:
3 Abbreviations used in this paper: p35, p40, and p70, 35-, 40-, and 70- to 75-kDa subunits, respectively; B6, C57BL/6; G6PDH, glyceraldehyde 6-phosphate dehydrogenase.
Received for publication June 30, 1998. Accepted for publication October 8, 1998.
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References |
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 C. C. Keller, O. Yamo, C. Ouma, J. M. Ong'echa, D. Ounah, J. B. Hittner, J. M. Vulule, and D. J. Perkins Acquisition of Hemozoin by Monocytes Down-Regulates Interleukin-12 p40 (IL-12p40) Transcripts and Circulating IL-12p70 through an IL-10-Dependent Mechanism: In Vivo and In Vitro Findings in Severe Malarial Anemia Infect. Immun., September 1, 2006; 74(9): 5249 - 5260. [Abstract] [Full Text] [PDF]
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J. Poovassery and J. M. Moore Murine Malaria Infection Induces Fetal Loss Associated with Accumulation of Plasmodium chabaudi AS-Infected Erythrocytes in the Placenta. Infect. Immun., May 1, 2006; 74(5): 2839 - 2848. [Abstract] [Full Text] [PDF]
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Y. Maeno, S. Nakazawa, N. Yamamoto, M. Shinzato, S. Nagashima, K. Tanaka, J. Sasaki, S. R. Rittling, D. T. Denhardt, T. Uede, et al. Osteopontin Participates in Th1-Mediated Host Resistance against Nonlethal Malaria Parasite Plasmodium chabaudi chabaudi Infection in Mice Infect. Immun., April 1, 2006; 74(4): 2423 - 2427. [Abstract] [Full Text] [PDF]
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R. Ing, P. Gros, and M. M. Stevenson Interleukin-15 Enhances Innate and Adaptive Immune Responses to Blood-Stage Malaria Infection in Mice Infect. Immun., May 1, 2005; 73(5): 3172 - 3177. [Abstract] [Full Text] [PDF]
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Z. Su, M.-F. Tam, D. Jankovic, and M. M. Stevenson Vaccination with Novel Immunostimulatory Adjuvants against Blood-Stage Malaria in Mice Infect. Immun., September 1, 2003; 71(9): 5178 - 5187. [Abstract] [Full Text] [PDF]
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 S. Chaisavaneeyakorn, C. Othoro, Y. P. Shi, J. Otieno, S. C. Chaiyaroj, A. A. Lal, and V. Udhayakumar Relationship between Plasma Interleukin-12 (IL-12) and IL-18 Levels and Severe Malarial Anemia in an Area of Holoendemicity in Western Kenya Clin. Vaccine Immunol., May 1, 2003; 10(3): 362 - 366. [Abstract] [Full Text] [PDF]
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I. Aguilar-Delfin, P. J. Wettstein, and D. H. Persing Resistance to Acute Babesiosis Is Associated with Interleukin-12- and Gamma Interferon-Mediated Responses and Requires Macrophages and Natural Killer Cells Infect. Immun., April 1, 2003; 71(4): 2002 - 2008. [Abstract] [Full Text]
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I. Angulo and M. Fresno Cytokines in the Pathogenesis of and Protection against Malaria Clin. Vaccine Immunol., November 1, 2002; 9(6): 1145 - 1152. [Full Text] [PDF]
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Z. Su and M. M. Stevenson IL-12 Is Required for Antibody-Mediated Protective Immunity Against Blood-Stage Plasmodiumchabaudi AS Malaria Infection in Mice J. Immunol., February 1, 2002; 168(3): 1348 - 1355. [Abstract] [Full Text] [PDF]
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A. Elhofy, I. Marriott, and K. L. Bost Salmonella Infection Does Not Increase Expression and Activity of the High Affinity IL-12 Receptor J. Immunol., September 15, 2000; 165(6): 3324 - 3332. [Abstract] [Full Text] [PDF]
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Z. Su and M. M. Stevenson Central Role of Endogenous Gamma Interferon in Protective Immunity against Blood-Stage Plasmodium chabaudi AS Infection Infect. Immun., August 1, 2000; 68(8): 4399 - 4406. [Abstract] [Full Text] [PDF]
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A. J. F. Luty, D. J. Perkins, B. Lell, R. Schmidt-Ott, L. G. Lehman, D. Luckner, B. Greve, P. Matousek, K. Herbich, D. Schmid, et al. Low Interleukin-12 Activity in Severe Plasmodium falciparum Malaria Infect. Immun., July 1, 2000; 68(7): 3909 - 3915. [Abstract] [Full Text] [PDF]
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J. A. Martiney, B. Sherry, C. N. Metz, M. Espinoza, A. S. Ferrer, T. Calandra, H. E. Broxmeyer, and R. Bucala Macrophage Migration Inhibitory Factor Release by Macrophages after Ingestion of Plasmodium chabaudi-Infected Erythrocytes: Possible Role in the Pathogenesis of Malarial Anemia Infect. Immun., April 1, 2000; 68(4): 2259 - 2267. [Abstract] [Full Text] [PDF]
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H. Sam, Z. Su, and M. M. Stevenson Deficiency in Tumor Necrosis Factor Alpha Activity Does Not Impair Early Protective Th1 Responses against Blood-Stage Malaria Infect. Immun., May 1, 1999; 67(5): 2660 - 2664. [Abstract] [Full Text] [PDF]
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