A Pathogenic Role of IL-12 in Blood-Stage
Murine Malaria Lethal Strain Plasmodium
berghei NK65 Infection
This information is current as
of June 18, 2017.
Takayuki Yoshimoto, Yasuhiro Takahama, Chrong-Reen Wang,
Toshihiko Yoneto, Seiji Waki and Hideo Nariuchi
J Immunol 1998; 160:5500-5505; ;
http://www.jimmunol.org/content/160/11/5500
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References
A Pathogenic Role of IL-12 in Blood-Stage Murine Malaria
Lethal Strain Plasmodium berghei NK65 Infection1
Takayuki Yoshimoto,2* Yasuhiro Takahama,* Chrong-Reen Wang,* Toshihiko Yoneto,*
Seiji Waki,† and Hideo Nariuchi*
I
L-12 is a heterodimeric cytokine produced by monocytes/
macrophages and dendritic cells in response to bacteria and
bacterial products and also by the stimulation of APC
through CD40-CD40 ligand (L)3 interaction, and induces IFN-g
production by NK cells and T cells (1– 4). IL-12 has been demonstrated to be crucial for the generation of a protective Th1 response against a variety of intracellular pathogens (5–9) and also
for the pathogenesis of some Th1-associated autoimmune
disorders (10).
An abundance of evidence has been accumulated for the importance of Th cells in the resolution of blood-stage malarial infection
(11–14). Protective immunity against the malarial infection with
Plasmodium chabaudi was shown to be mediated by the activation
of Th1 cells, and IFN-g was demonstrated to play a critical role in
the control of malarial infection (15–17). Plasmodium berghei
NK65 is a lethal murine malarial strain, and P. berghei XAT is its
irradiation-induced attenuated variant (18). Parasitemia in mice infected with blood-stage P. berghei NK65 increases progressively,
and all mice die in 2 to 3 wk, while the P. berghei XAT parasites
are spontaneously cleared in immune competent mice in 3 wk after
*Department of Allergology, Institute of Medical Science, The University of Tokyo,
Tokyo, Japan; and †Gunma Prefectural College of Health Sciences, Maebashi, Japan
Received for publication August 18, 1997. Accepted for publication January 30, 1998.
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 study was supported by a Grant-in-Aid for Scientific Research on Priority
Areas, by a Grant-in-Aid for International Scientific Research (Joint Research), and
by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture, Japan, and from the Japanese Ministry of Public
Health and Welfare.
2
Address correspondence and reprint requests to Dr. Takayuki Yoshimoto, Department of Allergology, Institute of Medical Science, The University of Tokyo, 4-6-1
Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
3
Abbreviations used in this paper: L, ligand; PRBC, parasitized RBC; iNOS, cytokine-inducible nitric oxide synthase; GOT, glutamic-oxaloacetic transaminase; GPT,
glutamic-pyruvic transaminase; CM, cerebral malaria.
Copyright © 1998 by The American Association of Immunologists
two peaks of parasitemia. Moreover, mice recovered from the P.
berghei XAT infection exhibit a strong resistance to the following
challenge with the lethal P. berghei NK65, indicating that P.
berghei XAT is a good model for live vaccine. Therefore, comparison of immune responses induced by these lethal and attenuated parasites would lead us to elucidate the mechanisms of protective immunity and pathogenesis. We previously demonstrated
that IFN-g produced by CD41 T cells plays a pivotal role in the
protective immunity against P. berghei XAT infection (19, 20).
We have recently demonstrated that the attenuated P. berghei XAT
infection induces IL-12 production in spleen and that the IL-12
plays an important role in the protective immunity via IFN-g production (21). However, since anti-IFN-g treatment was demonstrated to prolong the survival of mice infected with the lethal P.
berghei NK65, IFN-g was considered to be potentially involved in
the pathogenesis of P. berghei NK65 infection (19). In the present
study, therefore, we asked whether the lethal P. berghei NK65 also
induces IL-12 production and analyzed the role of IL-12 in the
protection or pathogenesis. Here, we show that the infection induces IL-12 production similar to that of the P. berghei XAT infection, but on the contrary the IL-12 production is involved in the
pathogenesis of liver injury via IFN-g production rather than the
protection.
Materials and Methods
Mice and parasite infection
Female C57BL/6 mice (6 – 8 wk old) were purchased from Japan SLC
(Hamamatsu, Japan). Mice were injected i.v. for malarial infection with
a RBC suspension containing 1 3 104 RBC parasitized (PRBC) with a
lethal strain, P. berghei NK65 (18). Parasitemia was assessed by the
microscopic examination of Giemsa-stained smears of tail blood. The
percentage of parasitemia was calculated as follows: parasitemia (%) 5
[(number of infected erythrocytes)/(total number of erythrocytes
counted)] 3 100.
0022-1767/98/$02.00
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We studied whether the infection with a blood-stage murine malaria lethal Plasmodium berghei NK65 induces IL-12 production,
and if so, how the IL-12 production is involved in the protection or pathogenesis. The infection of C57BL/6 mice enhanced mRNA
expression of IL-12 p40 and also IFN-g, IL-4, and IL-10 in both spleen and liver during the early course of the infection. It also
enhanced the mRNA expression of TNF-a, Fas ligand, and cytokine-inducible nitric oxide synthase. Increased IL-12 p40 production was also observed in the culture supernatant of spleen cells and in sera of infected mice. In addition, the infection caused
massive liver injury with elevated serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities
and body weight loss. Treatment of these infected mice with neutralizing mAb against IL-12 prolonged the survival and diminished
the liver injury with reduced elevation of serum serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and decreased body weight loss. However, the anti-IL-12 treatment did not affect parasitemia, and all these mice
eventually died. Similar results were obtained when infected mice were treated with neutralizing mAb against IFN-g. Moreover,
anti-IL-12 treatment greatly reduced the secretion and mRNA expression of IFN-g in both spleen and liver. These results suggest
that the lethal P. berghei NK65 infection induces IL-12 production and that the IL-12 is involved in the pathogenesis of liver injury
via IFN-g production rather than the protection. The Journal of Immunology, 1998, 160: 5500 –5505.
The Journal of Immunology
5501
RT-PCR analysis
Total RNA was extracted from spleen and liver by using a guanidine thiocyanate procedure (22). One microgram of total RNA was reverse transcribed into cDNA using SuperScript RT (Life Technologies, Gaithersburg, MD) in a reaction mixture of 50 mM Tris-HCl (pH 8.3) containing
75 mM KCl, 3 mM MgCl2, 0.4 U/ml RNase inhibitor (Wako Chemicals,
Osaka, Japan), 0.2 mM deoxynucleotide triphosphates, 1 mM DTT, and 0.8
U/ml reverse transcriptase (RT) from Moloney murine leukemia virus (Life
Technologies) after annealing with oligo(dT) primer (Promega, Madison,
WI). The PCR was performed in 10 mM Tris-HCl (pH 9.0) containing 50
mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 0.1%
Triton X-100, 0.5 mM each primer, and 0.025 U/ml Taq DNA polymerase
(Toyobo, Osaka, Japan). Reaction conditions and nucleotide sequences for
sense and antisense primers and internal probes for IFN-g, IL-4, IL-10,
IL-12 p40 and p35, TNF-a, cytokine-inducible nitric oxide synthase
(iNOS), FasL, and hypoxanthine phosphoribosyltransferase were the same
as those described (23–27). The amplified products were size fractionated
by electrophoresis on a 2% agarose gel, followed by ethidium bromide
staining for UV-assisted visualization and Southern blot hybridization with
32
P-end labeled-internal oligonucleotide probes as described (28).
Detection of IL-12 p40 and IFN-g
Histologic examination
Livers were removed from mice, fixed in 4% paraformaldehyde, embedded
in paraffin, and cut into 3-mm sections. Individual sections were examined
after staining with hematoxylin and eosin. For the histologic scoring of
liver injury, 10 fields that cover the almost whole section were selected, the
necrotic-like area in each field was measured at 3100 magnification, and
the percentage of necrotic-like area was calculated compared with all areas
observed. We also measured the necrotic-like cell number and calculated
the percentage of necrotic-like cell compared with all cells observed.
Glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic
transaminase (GPT) activities
The serum GOT and GPT activities were measured in the Automatic Analyzer 7250 (Hitachi, Tokyo, Japan).
intervals, and mRNA expression of various cytokines was examined by RT-PCR (Fig. 1). IL-12 p40 mRNA expression was increased from as early as 2 days after the parasite inoculation and
peaked on days 4 to 6 in both spleen and liver. mRNA expression
of IFN-g and IL-4 was also increased in similar time kinetics in
both spleen and liver, and IL-10 mRNA expression was also increased, but slightly behind IL-4 mRNA expression in time kinetics. IL-12 p35 mRNA expression was detected even in spleen of
noninfected mice and was not affected by the infection, while the
expression was hardly detected in liver of noninfected mice and
slightly increased by the infection. mRNA expression of TNF-a
and FasL was also increased in both spleen and liver. iNOS mRNA
expression was detected on days 6 to 8 in liver, but slightly in
spleen. Thus, the P. berghei NK65 infection enhanced mRNA expression of IL-12 p40, IFN-g, IL-4, IL-10, and TNF-a, and also of
FasL in spleen and liver. In contrast to these mRNA expression,
iNOS mRNA was detected slightly in spleen but more in liver in
a short time from 6 to 8 days after the inoculation and became
undetectable.
The production of IL-12 p40 and IFN-g by spleen cells was
analyzed at a protein level. After i.v. inoculation with PRBC,
spleens were obtained at various time intervals, spontaneous IL-12
Neutralization of cytokines in vivo with mAbs
To neutralize endogenous IL-12 or IFN-g, mice were injected i.p. with of
rat anti-mouse IL-12 p40 (C17.8, IgG2a) or rat anti-mouse IFN-g
(XMG1.2, IgG1) (31), 0.3 mg/injection/mouse, for 4 consecutive days
from the day of the parasite inoculation and then twice a week until day 14.
These mAbs were purified from ascites on a protein G column. As a control
Ab, normal rat IgG (Sigma, Chemical, St. Louis, MO) was used.
Statistical analysis
Statistical analysis was performed by Student’s t test, except for survival.
Survival was evaluated by generation of Kaplan-Meier plots and log rank
analysis. p , 0.05 was considered statistically significant.
Results
Induction of IL-12 expression in spleen and liver by blood-stage
P. berghei NK65 infection
To examine whether the infection of C57BL/6 mice with a bloodstage lethal P. berghei NK65 induces IL-12 production, we first
examined mRNA expression of IL-12 and other cytokines in
spleen and liver of infected mice. Mice were inoculated i.v. with
1 3 104 PRBC, spleen and liver were obtained at various time
FIGURE 2. Enhanced IL-12 p40 and IFN-g production by blood-stage
P. berghei NK65 infection. After i.v. inoculation with 1 3 104 PRBC,
spleens were obtained at various time intervals, and these cells were cultured in vitro without addition of parasite Ag for 48 h. The culture supernatants were assayed for measurement of IL-12 p40 (A) and IFN-g (B) in
ELISA. Data are shown as mean 6 SD of 3–10 mice. * and ** indicate p ,
0.01 and p , 0.001, respectively, compared with day 0.
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Spleen cells were cultured at 6 3 106 cells/ml without addition of parasite
Ag in RPMI 1640 medium supplemented with 10% FCS, 5 3 1025 M
2-ME and 100 mg/ml kanamycin. Liver mononuclear cells were prepared
as described previously (19) and cultured at 2 3 106 cells/ml without
addition of parasite Ag in the medium. After incubation for 48 h, culture
supernatants were harvested and assayed for IL-12 p40 and IFN-g in sandwich ELISA using rat anti-mouse IL-12 p40 (C17.8 and C15.6, provided
by Dr. G. Trinchieri, Wistar Institute, Philadelphia, PA) as described (29)
and rat anti-mouse IFN-g (R4 – 6A2 and XMG1.2, PharMingen, San Diego, CA) according to the manufacturer’s instruction (30), respectively.
Murine rIL-12 and rIFN-g were gifts from Dr. M. Kobayashi (Genetics
Institute, Cambridge, MA) and Dr. M. Moriyama (Toray Industries, Kamakura, Japan), respectively.
FIGURE 1. Cytokine mRNA expression in spleen and liver during the
early course of blood-stage P. berghei NK65 infection. After i.v. inoculation with 1 3 104 PRBC, spleens and livers were obtained at various time
intervals, and total RNA was prepared and subjected to RT-PCR analysis.
The amplified products were size fractionated by electrophoresis on 2%
agarose gels followed by Southern blot hybridization with 32P-end-labeled
internal oligonucleotide probes. Similar results were obtained in two repeated experiments. HPRT, hypoxanthine phosphoribosyltransferase.
5502
PATHOGENIC ROLE OF IL-12 IN MALARIA INFECTION
FIGURE 3. Reduced elevation of serum
GOT and GPT activities and decreased body
weight loss in infected mice by treatment with
neutralizing mAb against IL-12. After i.v. inoculation with 1 3 104 PRBC, endogenously
produced IL-12 was neutralized by treatment
with anti-IL-12 or PBS for 4 consecutive days
from the day of inoculation and then twice a
week. Normal rat IgG was used as a control
Ab. Sera were obtained from these mice 7
days after the inoculation and assayed for
GOT (A) and GPT (B) activities. Body weight
(B) was measured 14 days after the inoculation. Data are shown as mean 6 SD of 4 to 6
mice. * and ** indicate p , 0.01 and p ,
0.001, respectively, compared with PBS alone
or control Ab. Similar results were obtained in
two repeated experiments.
Prolonged survival and diminished liver injury by treatment of
infected mice with neutralizing mAb against IL-12
We previously demonstrated that blood-stage P. berghei NK65
infection causes mononuclear cell infiltration into liver and that the
treatment of infected mice with anti-IFN-g prolongs the survival
(19). To examine whether IL-12 produced by the parasite infection
is involved in protection or pathogenesis, infected mice were
treated with neutralizing mAb against IL-12 for 4 consecutive days
from the day of parasite inoculation and then twice a week until
day 14. After i.v. inoculation of mice with PRBC, serum GOT and
GPT activities were elevated, and their body weight was gradually
decreased (Fig. 3), with severe anemia. In addition, massive liver
injury with necrotic cells forming focal necrosis-like lesions and
mononuclear cell infiltration were observed by histologic examination of livers of infected mice compared with that of noninfected
mice (Fig. 4, A and B). Treatment of infected mice with neutralizing anti-IL-12 significantly prolonged the survival compared
with that with PBS or control Ab (Fig. 5A). However, the parasitemia in these mice was not affected by these treatments in level
and time kinetics (Fig. 5B). Similarly, neutralization of IFN-g by
injecting its mAb resulted in prolonged survival (Fig. 5A) as reported previously (19). Concomitant with the prolonged survival,
elevation of serum GOT and GPT activities and the body weight
loss were significantly reduced by the anti-IL-12 treatment, but not
with control Ab (Fig. 3). Similar reduction of serum GOT and GPT
activities was observed when infected mice were treated with antiIFN-g (data not shown). Moreover, the liver injury was significantly diminished by treatment with anti-IL-12, but not with control Ab (Fig. 4). These results suggest that IL-12 and IFN-g are
involved in the pathogenesis of liver injury rather than the protection in the P. berghei NK65 infection.
Reduced IFN-g production by treatment of infected mice with
neutralizing mAb against IL-12
To further examine whether the pathogenic effect of IL-12 is mediated by IFN-g, we next analyzed the effect of anti-IL-12 treatment on IFN-g production in spleen and liver. Spleen cells of
anti-IL-12-treated mice were obtained at various time intervals and
cultured in vitro, and IFN-g secreted spontaneously in culture was
assayed. The IFN-g production was greatly reduced by the treatment with anti-IL-12, but not with control Ab (Fig. 6A). IFN-g
production by liver mononuclear cells, which were prepared from
livers obtained 6 days after the inoculation, was also significantly
reduced (Fig. 6B). We then examined the expression of IFN-g and
other cytokines at mRNA level in spleen and liver. Consistent with
the above results, RT-PCR analysis revealed that IFN-g mRNA
expression was greatly reduced by the treatment with anti-IL-12,
but not with control Ab, in both spleen and liver (data not shown).
In contrast, the expression of IL-4 and IL-10 mRNA appeared not
to be greatly affected by the treatment (data not shown). These
results suggest that IFN-g production in spleen and liver of the
infected mice is dependent on IL-12 produced by the infection.
Discussion
Recently we demonstrated that the infection with blood-stage attenuated P. berghei XAT induces IL-12 production in spleen,
which is important for the development of protective immunity via
IFN-g production (21). However, we previously found that IFN-g
production might be involved in the pathogenesis of blood-stage
lethal P. berghei NK65 infection (19). In the present study, we
therefore further extended these studies and elucidated that the
lethal P. berghei NK65 infection also induces IL-12 production,
but the IL-12 is involved in the pathogenesis of liver injury via
IFN-g production rather than the protection. This is in strong contrast with our previous results showing the protective effect of
IL-12 in P. berghei XAT infection (21). Thus, IL-12 seems to have
both protective and pathogenic effects depending on the immune
responses elicited by the attenuated and lethal parasites via IFN-g
production. The difference between these immune responses,
which determines the outcome of the infection, might be due to the
quantity of IL-12 produced, the tissue in which it is produced, the
time period during which its production is sustained, and the presence of other molecules regulating its production. Further study is
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p40 and IFN-g production by these cells without addition of parasite Ag in culture were assayed in ELISA (Fig. 2). IL-12 p40
production was enhanced as early as 2 days after the parasite inoculation, peaked on day 4, and sharply decreased, while IFN-g
production was sharply enhanced on day 4 and gradually decreased. These results are consistent with those in mRNA expression mentioned above. Moreover, similar enhancement of IL-12
p40 production was observed in sera of infected mice (data not
shown). These results again suggest that the P. berghei NK65 infection enhances the production of IL-12 p40 and IFN-g.
The Journal of Immunology
5503
currently under investigation to elucidate more precise mechanisms by which the attenuated parasite evokes protective immunity
and on the contrary the lethal parasite evokes pathogenesis.
Pathogenic effects of IL-12 have recently been reported (9, 32–
34). rIL-12 administration induced mononuclear cell infiltration in
C57BL/6 mouse liver, occasionally associated with hepatocyte necrosis (32). It was also demonstrated that low doses of rIL-12
administration into C57BL/6 mice exhibit anti-viral effect against
lymphocytic choriomeningitis virus infection, whereas high doses
of rIL-12 administration are detrimental to the resistance largely
mediated by TNF-a and induce necrotic lesions in the spleen (9,
33). Moreover, IL-12 has recently been demonstrated to be a key
cytokine in Th1-dependent liver injury involving IFN-g, which
induced by the injection of Propionibacterium acnes and LPS in
sensitive C57BL/6 mice (34). Thus, these phenomena are highly
similar to those seen in mice infected with the P. berghei NK65,
further suggesting the crucial role of IL-12 in the pathogenesis of
liver injury in the infection.
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FIGURE 4. Diminished liver injury in infected mice by treatment with neutralizing mAb
against IL-12. After i.v. inoculation with 1 3 104
PRBC, endogenously produced IL-12 was neutralized by treatment with anti-IL-12 (C) or PBS
(B) for 4 consecutive days from the day of inoculation, then twice a week. Normal rat IgG was
used as a control Ab (D). Livers were obtained
from these mice 14 days after inoculation and
also from a noninfected mouse (A), histologic
analysis was conducted after staining with hematoxylin and eosin (3100), and typical results are
shown. Histologic scoring of liver injury was also
performed, and the percentage of necrotic-like
area was determined (E). Data are shown as
mean 6 SD of three noninfected mice, seven
PBS-treated infected mice, seven antiIL-12-treated infected mice, and six control Abtreated infected mice. * indicates p , 0.001, compared with PBS alone or control Ab. Similar
results were obtained when the percentage of necrotic-like cell number was evaluated (data not
shown).
IFN-g and TNF-a were reported to be critically involved in
causing liver injury, because active hepatitis was seen in IFN-gtransgenic mice (35), Con A-induced hepatitis was suppressed in
mice lacking IFN-g (36), and liver injury during endotoxemia was
blocked by anti-TNF-a (37). A small amount of rTNF-a injection
was also shown to cause a variety of pathologic changes including
liver injury in mice infected with Plasmodium vinckei (38). These
results greatly support the present conclusion that the pathogenic
effect of IL-12 to cause liver injury is mediated by IFN-g. Moreover, the infection enhanced TNF-a mRNA expression and neutralization of TNF-a led to the prolonged survival and reduced
liver injury (data not shown). However, anti-IL-12 treatment affected much less or little mRNA expression of TNF-a (data not
shown), although the treatment significantly reduced the mRNA
expression and production of IFN-g (Fig. 6). Further studies are
necessary to clarify the involvement of TNF-a in the IL-12-mediated liver injury. In addition, the P. berghei NK65 infection enhanced the mRNA expression of iNOS and FasL in both spleen
5504
PATHOGENIC ROLE OF IL-12 IN MALARIA INFECTION
FIGURE 5. Prolonged survival by treatment of infected mice with neutralizing mAbs against IL-12 or IFN-g. After i.v. inoculation with 1 3 104
PRBC, endogenously produced IL-12 or IFN-g were neutralized by the
treatment with respective mAbs for 4 consecutive days from the day of
inoculation and then twice a week until day 14. Mice (n 5 5) were monitored for survival (A) and for parasitemia (B). Normal rat IgG was used
as a control Ab. Data in parasitemia are shown as mean 6 SD of 5 mice.
Survival was prolonged significantly by the treatment with mAbs against
IL-12 or IFN-g, compared with PBS or control Ab ( p , 0.03). Similar
results were obtained in two repeated experiments.
and liver, and the iNOS mRNA expression was higher in liver than
in the spleen of infected mice (Fig. 1). The latter result may indicate that NO would be involved in the pathogenesis of liver injury
as discussed previously (39). Indeed, iNOS and FasL have been
implicated in the pathogenesis of diseases such as hypotension,
immunosuppression, and cerebral malaria (CM) (40 – 42) and of
autoimmune diseases and hepatitis (43– 45), respectively. However, since liver injury was also observed in the P. berghei NK65infected CBA/Kl-lprcg/lprcg mice (46), which do not have any
functional Fas by point mutation, the apoptosis induced by FasFasL interaction in liver seems not to be necessary for liver injury
in infected mice (T. Yoshimoto et al., unpublished data). Further
studies are required to elucidate precisely the mechanism by which
to cause the pathogenesis of liver injury by the infection with P.
berghei NK65.
The reason mice die by the infection with P. berghei NK65
remains unknown but could be complicated. In the infection with
P. berghei ANKA (47) and human malaria (48), CM is considered
to be one major cause of mortality, although P. berghei NK65 has
not been reported to cause CM. In the early course of the infection,
IFN-g production was induced and peaked at 4 to 6 days after
inoculation, which resulted in liver injury and thus in acceleration
of the mortality. This is because that infected mice that were
treated with anti-IL-12 or anti-IFN-g showed diminished liver injury and thus prolonged survival, but these mice eventually died
presumably due to another reason such as severe anemia. As a
matter of fact, preliminary results showed that even infected mice
treated with anti-IL-12 or anti-IFN-g appeared to still show anemia, and thus the anemia seems to correlate with the extent of
parasitemia more than that of liver injury, although further quantitative measurement of hematocrit is necessary to examine the
effect of these mAb treatment on the extent of anemia.
The experimental CM model induced by blood-stage P. berghei
ANKA infection in mice was demonstrated to be associated with
elevated blood TNF-a levels resulting in high production of NO
(49). Moreover, IFN-g, IL-3, and granulocyte-macrophage-CSF
were reported to be required for CM development (50 –52). Recently, development of CM has been demonstrated to be blocked
in mice lacking IFN-g or TNF-a (53, 54). Considering that IFN-g
is central in the regulation of TNF-a and NO production (53–55)
and that IL-12 has an ability to induce IFN-g and TNF-a production (9, 32), IL-12 might play a critical role in the development of
CM in the cytokine cascade as in the P. berghei NK65 infection.
Taken together, the present results suggest that the infection
with blood-stage lethal P. berghei NK65 induces IL-12 production, which is critically involved in the pathogenesis of liver injury
via IFN-g production rather than in the protection. To our knowledge, this is the first report on the pathogenic role of endogenous
IL-12 in the parasite infection. Thus, the murine malaria models of
lethal P. berghei NK65 and its attenuated P. berghei XAT would
provide useful and valuable tools for investigation of the mechanisms by which to develop the protective immunity and cause the
pathogenesis in blood-stage malarial infection.
Acknowledgments
We thank Drs. G. Trinchieri (The Wister Institute), M. Kobayashi (Genetics Institute), and M. Moriyama (Toray Industries) for kindly providing rat
anti-mouse IL-12 p40 mAbs (C17.8 and 15.6), murine rIL-12, and murine
IFN-g, respectively. We also thank Drs. A. Tsubura (Kansai Medical University, Osaka, Japan) and H. Maeda (Sankyo, Tokyo, Japan) for suggestions on histologic analyses for and measurement of serum GOT and GPT
activities, respectively.
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FIGURE 6. Reduced IFN-g production in spleen and liver by treatment
with neutralizing mAb against IL-12. After i.v. inoculation with 1 3 104
PRBC, endogenously produced IL-12 was neutralized by treatment with
anti-IL-12 for 4 consecutive days from the day of inoculation and then
twice a week. Normal rat IgG was used as a control Ab. Spleens were
obtained at various time intervals, livers were obtained 6 days after the
inoculation, and their mononuclear cells were prepared. These cells were
cultured in vitro without addition of parasite Ag for 48 h, and the culture
supernatants were assayed for IFN-g in ELISA. Data are shown as mean 6
SD of three to four mice. * indicates p , 0.01, compared with PBS alone
or control Ab.
The Journal of Immunology
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