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*
Department of Allergology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan;
Gunma Prefectural College of Health Sciences, Maebashi, Japan;
Department of Molecular Embryology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan; and
§
Department of Bacteriology, School of Medicine, Niigata University, Niigata, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and increased production of IL-4 and IL-10. Delayed-type
hypersensitivity response was also significantly reduced. Moreover,
these Tg mice showed increased susceptibility to the infection with an
intracellular pathogen, blood-stage Plasmodium berghei XAT,
which is an irradiation-induced attenuated substrain of P.
berghei NK65, presumably due to the decreased IFN- production.
These results suggest that p40 functions as an IL-12 antagonist in
vivo, and that Th1 responses in p40 Tg mice are significantly reduced.
Thus, these Tg mice could be a useful model to evaluate the inhibitory
effect of p40 on IL-12-mediated various immune responses in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production
by T cells and NK cells and promotes a generation of Th1 cells (1, 4, 5, 6, 7). The p40 expression is highly regulated by various stimuli in
monocytes/macrophages and dendritic cells, and the p40 is considered
the regulatory component for IL-12 production and responsible for
IL-12R binding (1, 4). For the generation of bioactive IL-12, the
expression of both subunits within one cell is required, although their
expression is differentially regulated (1, 4, 8). It was demonstrated that p40 is produced as monomer and homodimer in large excess over IL-12 p70 both in vitro (9, 10) and in vivo (11, 12). The p40 level in murine circulation was reported to remain high after LPS stimulation, whereas IL-12 production rapidly decreases (11, 12). These results indicate a critical role of p40 as a natural antagonist of IL-12. Indeed, mouse p40 has been demonstrated to bind to mouse IL-12R with an affinity similar to that of IL-12. However, it does not trigger biologic activation and inhibits IL-12-mediated responses by competitive binding to the IL-12R in vitro (13, 14). Human p40, which also exists in monomeric and dimeric forms, binds to the IL-12R and acts as a competitive antagonist of IL-12 in vitro (15). It has been demonstrated very recently that locally produced mouse IL-12 p40 from a transplanted myoblast cell line suppresses Th1-mediated immune responses and prevents the allogeneic rejection of the myoblast (16). However, the ability of p40 as an IL-12 antagonist in various immune responses remains to be elucidated further under physiologic conditions in vivo.
In the present study, we generated p40 transgenic (Tg)3 mice predominantly expressing the transgene in liver and found that IL-12-mediated Th1 responses were reduced significantly due to the antagonistic activity of p40 in these p40 Tg mice. Thus, these mice would be useful to evaluate the function of p40 as an IL-12 antagonist in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The open reading frame (1 kb) of murine IL-12 p40 cDNA (kindly
donated by Dr. M. Kobayashi, Genetics Institute, Cambridge, MA) was
amplified using PCR and inserted into the EcoRI site of
pLG1-SAP that contains the human serum amyloid P component (SAP)
promoter and the rabbit ß-globin gene (17), resulting in construction
of the plasmid pLG1-SAP-IL-12 p40 (Fig. 1
). Before injection into fertilized
mouse eggs, the plasmid was digested with HindIII and
XhoI, and the resultant 2.8-kb fragment of SAP-IL-12 p40
gene was isolated and used for microinjection.
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Fertilized eggs were obtained from C57BL/6 mice, and several
hundred molecules of the SAP-IL-12 p40 fragment (2.8 kb) were injected
into the pronucleus of the fertilized eggs, as described (18). When the
mice were at 4 wk of age, genomic DNA was extracted from a piece of the
tail of each mouse and used to detect the transgene by PCR using the
following two primers: one (ß2 sense primer,
5'-TGCTGTCTCATCATTTTGGC-3') corresponds to the 5' untranslated region
of rabbit ß-globin gene, and the other (p40 antisense primer,
5'-TTTGGTGCTTCACACTTCAGG-3') corresponds to the IL-12 p40 gene
(Fig. 1
).
Reverse-transcriptase (RT) PCR analysis
Total RNA was extracted from various tissues by using the
guanidine thiocyanate procedure (19). One microgram of total RNA was
reverse transcribed into cDNA using SuperScript RT (Life Technologies,
Gaithersburg, MD) by an incubation for 1 h at 42°C in a reaction
mixture of 50 mM Tris-HCl (pH 8.3) containing 75 mM KCl, 3 mM
MgCl2, 0.4 U/µl RNase inhibitor (WAKO Chemicals,
Osaka, Japan), 0.2 mM deoxynucleotide triphosphates, 1 mM DTT, and 0.8
U/µl RT from Moloney murine leukemia virus (Life Technologies) after
annealing with oligo(dT) primer (Promega Corp., Madison, MD). To avoid
detection of endogenous IL-12 p40 mRNA, the following two primers were
used: one (ß1 sense primer, 5'-GATCCTGAGAACTTCAGGCTC-3')
corresponds to the 5' untranslated region of rabbit ß-globin gene,
and the other (p40 primer) corresponds to the IL-12 p40 gene (Fig. 1).
The PCR was performed in 10 mM Tris-HCl (pH 9) containing 50 mM KCl,
1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 0.1%
Triton X-100, 0.5 µM each primer, and 0.025 U/µl Taq DNA
polymerase (Toyobo, Osaka, Japan). Reactions for the p40 transgene or
ß-actin were subjected to 40 cycles consisting of 94°C for 30
s, 55°C for 30 s, and 72°C for 90 s, or 35 cycles
consisting of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min,
respectively. All PCR mixtures were subjected to denaturation at 94°C
for 3 min before the first cycle, and to final extension at 72°C for
7 min after the last cycle. The amplified products were size
fractionated by electrophoresis on a 1.5% agarose gel, followed by
ethidium bromide staining for UV-assisted visualization and Southern
blot hybridization with 32P-end-labeled p40 internal
oligonucleotide probe (5'-TAAGACCTTTCTAAGATGCGAGGCC-3'), as
described (20).
ELISA for serum IL-12 p40 and p70
Blood was obtained from the eye artery, and serum was separated by centrifugation. The serum was assayed for murine IL-12 p40 by sandwich ELISA using rat anti-mouse IL-12 p40 mAbs (C15.6 and C17.8, kindly provided by Dr. G. Trinchieri, The Wister Institute, Philadelphia, PA), as described (16). Serial dilutions of murine rIL-12 (sp. act., 4.6 x 106 U/mg, kindly donated by Dr. M. Kobayashi, Genetics Institute) were used as the standard. To detect p40, microtiter plate (Becton Dickinson, Mountain View, CA) was coated with anti-IL-12 p40 mAb (C15.6), followed by an incubation with twofold serially diluted samples to be tested. After washing, the plate was incubated with biotinylated anti-IL-12 p40 mAb (C17.8) and subsequently with peroxidase-labeled streptavidin. The plate was then developed with o-phenylenediamine, and the level of p40 was determined photometrically by measuring OD490 in reference to the standard titration curve of murine rIL-12. Reagents for ELISA of murine IL-12 p70 were generously provided by Dr. D. H. Presky (Hoffmann-La Roche, Nutley, NJ). In this assay (21), rat anti-mouse IL-12 p70 mAb (9A5), peroxidase-labeled rat anti-mouse IL-12 p40 mAb (5C3), and rIL-12 were used as a coating Ab, developing Ab, and standard, respectively, and ELISA was performed as described above.
Western blot analysis
Serum IL-12 p40 was separated by SDS-PAGE (10%) under reducing or nonreducing conditions. The separated proteins were transferred to a polyvinilidene difluoride microporous membrane (Immobilon PVDF; Milipore Co., Bedford, MA). Transblots were incubated with biotinylated anti-mouse IL-12 p40 mAb (C17.8) and then with alkaline phosphatase-conjugated streptavidin. The Ab-reactive bands were visualized using 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitroblue tetrazolium (NBT) phosphatase substrate system.
FACS analysis
One million cells were analyzed on a FACScan using a Lysis II
software (Becton Dickinson) for data analysis. For two- or three-color
analysis of thymus, LN, and spleen cells, PE anti-CD8 (53-6.7, rat
IgG2a), PE anti-B220 (RA3-6B2, rat IgG2a), PE anti-NK1.1
(PK136, mouse IgG2a), FITC anti-Thy-1.2 (3O-H12, rat IgG2b), FITC
anti-CD3 (145-2C11, hamster IgG), biotinylated anti-TCR-
ß
(H57-597, hamster IgG) (all from PharMingen, San Diego, CA), and
biotinylated anti-CD4 (GK1.5, rat IgG2b), which was prepared using
purified mAb from ascites on a protein G column, and
streptavidin-Cy-chrome (PharMingen) were used. The mAbs specific for
heat-stable Ag (HSA, M1/69, rat IgG2b), Pgp-1 (KM201, rat IgG1),
IL-2R (PC61, rat IgG1), and c-kit (ACK-2, a gift from Dr.
S. Nishikawa, Kyoto University, Kyoto, Japan) (22) were used in the
form of culture supernatants of each hybridoma clone. FITC anti-rat
IgG F(ab')2 (Life Technologies) was used as a second Ab.
Dead cells positively stained with 7-aminoactinomycin D (Sigma Chemical
Co., St. Louis, MO) were gated out.
NK cell lytic activity
YAC-1 lymphoma target cells were incubated for 1 h with Na251CrO4 (Amersham, Arlington Heights, IL) and washed extensively. Spleen cells were used as effector cells and mixed with the target cells (1 x 104 cells) in 200 µl RPMI 1640 medium supplemented with 10% FCS, 5 x 10-5 M 2-ME, and 100 µg/ml kanamycin in 96-well round-bottom plates. Plates were incubated for 4 h at 37°C, and 51Cr release into the supernatants was determined in a gamma counter. The lytic activity of the cells was assayed at E:T ratios of 100:1, 50:1, 25:1, and 12.5:1. The specific 51Cr release (%) was determined as follows: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. The maximum release was obtained by target cell lysis with 1% Triton X-100.
Cytokine production
Mice were injected s.c. in the hind footpads with keyhole limpet
hemocyanin (KLH, 50 µg) emulsified with CFA. After 1 wk, popliteal
lymph nodes (LNs) were removed and the cells were cultured in the
medium containing 0.3 mg/ml of KLH at 6 x 106
cells/ml. Culture supernatants were harvested after 48 h and
assayed for the concentration of IFN-, IL-4, and IL-10 by ELISA kits
obtained from PharMingen, according to the manufacturer’s
instruction.
Delayed-type hypersensitivity (DTH) reaction
Mice were primed with 1 x 108 SRBC suspended in 0.2 ml PBS by s.c. injection in the back (23). After 7 days, mice were injected s.c. in the left footpad with 1 x 108 SRBC suspended in 20 µl PBS, and in the right footpad with 20 µl PBS as a control. Twenty four hours later, footpad swelling was measured with a caliper. The degree was calculated as the percentage of the swelling using the following formula: footpad swelling (%) = [(thickness of footpad injected with SRBC - thickness of footpad injected with PBS)/(thickness of footpad injected with PBS)] x 100.
Malarial infection
Plasmodium berghei XAT is an attenuated substrain of P. berghei NK65 obtained by an irradiation, as described (24, 25). Briefly, a pool of mouse blood infected with the original highly virulent P. berghei NK65 parasites was exposed to x-ray irradiation (400 Gy at 8 Gy/min), resultant parasites were inoculated into nude mice, and self-limiting parasites in immune competent mice were obtained (24). Mice were injected i.v. with an erythrocyte suspension containing 1 x 104 parasitized RBC (PRBC). Parasitemia was assessed by the microscopic examination of Giemsa-stained smears of tail blood. The percentage of parasitemia was calculated as follows: [(number of infected erythrocytes)/(total number of erythrocytes counted)] x 100. To see the protective effect of rIL-12, mice were injected i.p. with 100 ng of murine rIL-12 for 5 consecutive days beginning on the day -1 relative to the inoculation with P. berghei XAT.
Statistical analysis
Statistical analysis was performed by Student’s t test. p values < 0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three mice carrying the p40 transgene were generated: two males
(p40 Tg A and B lines) and one female (p40 Tg C line). These mice were
mated with C57BL/6 mice, and all of them transmitted the transgene to
their offsprings, which were used in the present experiments. To
examine tissue specificity of the p40 transgene expression, total RNA
was prepared from various tissues of these Tg mice, p40 Tg A and B
lines, when they were at 5 to 6 wk of age, and RT-PCR was performed,
followed by Southern blot analysis using the internal probe. The PCR
product (357 bp) was detected predominantly in liver of these Tg mice
(Fig. 2). The PCR product was also
observed slightly in kidney of p40 Tg A mouse, not of p40 Tg B mouse
(Fig. 2A), and in stomach of p40 Tg B mouse, not of
p40 Tg A mouse (Fig. 2B). No apparent band due to the
p40 transgene was detected when the RT-PCR was conducted using RNA
obtained from lymphoid organs such as spleen, LN, and thymus, and also
from lung and small intestine.
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To examine whether p40 molecule was circulating in p40 Tg mice, sera from each mice were assayed for p40 in sandwich ELISA when mice were at 6 to 7 wk of age. Surprisingly, the mean concentrations of p40 in p40 Tg A and B mice were extremely high: 46 ± 30.3 µg/ml (12–100.1 µg/ml, n = 12) and 14.9 ± 8.4 µg/ml (5–33.4 µg/ml, n = 22), respectively. In contrast, the mean concentration in control littermates was 0.78 ± 0.28 ng/ml (0.38–1.10 ng/ml, n = 7). Furthermore, we tried to detect serum IL-12 p70 in ELISA, and the mean concentration of p70 in control littermates was about 30 pg/ml or less, which was the lowest limit in the assay system. The concentration of p70 in p40 Tg mice was comparable with that in control littermates, indicating that the p70 production due to the p40 transgene appeared not to occur in the p40 Tg mice. In the following experiments, we mainly used p40 Tg A mice.
Next, p40 molecule in the sera of p40 Tg mice was examined by Western
blot analysis after separation by SDS-PAGE. The anti-p40 mAb
(C17.8) detected several bands ranging from 40 to 200 kDa in the sera
from these Tg mouse lines (p40 Tg A and B) under nonreducing conditions
(Fig. 3A). The pattern
in bands detected by the mAb was quite similar to each other in all p40
Tg mouse lines p40 Tg A and B, and also C (data not shown). In
contrast, no band was detected by the mAb in sera of control
littermates. Major serum p40 molecule was of monomer with apparent
Mr of approximately 45 k and triplet
probably due to the differential glycosylation (16, 26). Homodimer with
apparent Mr of approximately 120 k
was also detected together with other minor bands (approximately 80 and
100 k). In addition, higher m.w. complexes of p40
with apparent Mr of approximately 200
k were also seen. Under reducing conditions, all bands came
down to the position of monomer with triplet, confirming that those
bands were of complexed forms of p40 (Fig. 3B). Thus,
serum p40 consisted of mainly monomer and homodimer, and also of higher
m.w. complexes.
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Upon gross necropsy or histologic examination, no apparent
abnormality was detected in organs of p40 Tg A and B mice, including
liver. They were apparently of normal size and weight, and both sexes
were fully fertile. To see whether the high concentration of p40
affects the T cell subpopulations by affecting the development in the
thymus, flow-cytometric analysis of thymus, LN, and spleen cells at 5
to 6 wk of age was performed. No difference was observed in these cells
between p40 Tg mice and control littermates in the expression of CD3,
CD4, CD8, B220, Thy-1.2, and NK1.1 (data not shown). Thymocytes of Tg
mice were normal in expression of HSA, IL-2R, CD44, and
c-kit, typical markers for immature thymocytes (data not
shown). In addition, the size of thymus and the absolute number of
thymocytes were normal. Thus, no apparent abnormality of lymphocyte was
noted in ontogeny of these p40 Tg mice.
Diminished enhancement of NK cell lytic activity by rIL-12 administration to p40 Tg mice
Since administration of rIL-12 was reported to enhance NK cell
lytic activity in vivo (27), we first examined whether the lytic
activity of spleen cells would be increased by the administration of
rIL-12 in p40 Tg mice to evaluate the antagonistic activity of serum
p40 in vivo. p40 Tg A mice and control littermates were treated with
rIL-12 (30 ng) or PBS by i.p. injection for 2 consecutive days, spleens
were removed 24 h after the second injection, and these cells were
assayed for lytic activity on YAC-1 cells. rIL-12 administration
greatly enhanced the NK cell lytic activity in control littermates
(Fig. 4). However, no significant
enhancement of the lytic activity was observed in p40 Tg mice (Fig. 4).
Similar results were obtained when p40 Tg B mice were used (data not
shown). These results suggest that serum p40 in p40 Tg mice is
functional as an IL-12 antagonist in vivo.
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A number of evidences have been accumulated for a critical role of
IL-12 in promoting Th1 responses both in vitro and in vivo (1).
Therefore, we next investigated Ag-induced cytokine production of T
cells from p40 Tg A mice in comparison with that from control
littermates. Mice were immunized with KLH in CFA, draining LNs were
removed 7 days later, and cytokine production of LN cells stimulated
with KLH for 48 h was examined in ELISA. LN cells from KLH-primed
p40 Tg mice were reduced markedly in the production of IFN- on KLH
stimulation (Fig. 5). In contrast,
Ag-induced IL-4 and IL-10 production of these cells was significantly
increased (Fig. 5). Similar results were obtained when p40 Tg B mice
were used (data not shown). These results indicate that in p40 Tg mice
the response in Th1 cytokine production is decreased, and that in Th2
cytokine production is increased.
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). Similar results were obtained when
p40 Tg B mice were used (data not shown). These results further
indicate that Th1 responses in p40 Tg mice are reduced significantly,
and that p40 functions as an IL-12 antagonist in vivo.
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IL-12 has been demonstrated to play an important role in the
development of protective immunity involving Th1 responses, especially
IFN- production, against intracellular pathogens such as
Leishmania major, Listeria monocytogenes (1, 2),
and blood-stage Plasmodium chabaudai AS (28) and P.
berghei XAT (T. Yoshimoto et al., manuscript submitted).
Therefore, we finally examined the susceptibility of p40 Tg mice to the
infection with an intracellular pathogen, blood-stage P.
berghei XAT. p40 Tg A mice and control littermates were inoculated
i.v. with 1 x 104 PRBC, and blood smears were
examined for parasitemia. All control mice cleared PRBC in about 3 wk
after two peaks of parasitemia (Fig. 7A). In p40 Tg mice,
however, parasitized corpuscles were increased progressively in
percentage (Fig. 7A). Three of five p40 Tg mice could
not clear PRBC and eventually died of the infection, and the rest of
these mice scarcely recovered from the infection. This increased
susceptibility of p40 Tg mice would be attributable to the antagonistic
activity of p40 against the function of bioactive IL-12 produced by the
infection. To confirm this notion, p40 Tg A mice and control
littermates were administrated i.p. with 100 ng of rIL-12 for 5
consecutive days from day -1 to day 4 after the inoculation, and
examined for parasitemia. Appearance of parasitemia was greatly delayed
in the control littermates treated with rIL-12, and PRBC were cleared
in about 3 wk (Fig. 7B and T. Yoshimoto et al., manuscript
submitted). In contrast, the rIL-12 administration did not delay the
onset of the parasitemia in p40 Tg mice, and the parasitemia in these
mice progressed similarly to that in untreated p40 Tg mice (Fig. 7B). Eventually, two of five p40 Tg mice could not
clear PRBC and died, and the rest of these mice scarcely recovered. We
have found recently that IFN- production in spleen, which is
critically important for the protective immunity to blood-stage
P. berghei XAT infection, is highly dependent upon IL-12
produced by the infection (T. Yoshimoto et al., manuscript submitted).
Therefore, spontaneous in vitro IFN- secretion of spleen cells in
p40 Tg A mice infected with P. berghei XAT was examined at
various time intervals after the inoculation to see the in vivo effect
of p40 on the IFN- production during the infection. IFN-
production of spleen cells in p40 Tg mice was reduced significantly
compared with that of control littermates (Fig. 8), which presumably accounts for the
reduced ability of p40 Tg mice to establish the protective immunity to
blood-stage P. berghei XAT infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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level (mean concentration:
3.533 ng/ml) in IFN- Tg mice, which were generated similarly using
the SAP promoter (17). This may imply that p40 is stable in serum,
which is advantageous for the clinical application of p40 as a
therapeutic agent. The serum p40 consisted of mainly monomer
(Mr = approximately 45
k) and homodimer (Mr =
approximately 120 k), and also of higher m.w.
complexes (Mr = approximately 200
k). The electrophoretic mobility of serum p40
homodimer on nonreducing SDS-PAGE was much slower than that calculated
according to the m.w., as previously described for p40 homodimer (14, 26). Monomer and homodimer of p40 and the minor molecules were also
detected in the culture supernatant of murine myeloma cells (Ag8.653)
transfected with p40 cDNA ligated to the expression vector, BCMGSNeo
(30), whereas the higher m.w. complexes were not detected (data not
shown). These results may indicate the potential complex formation of
p40 with other serum molecules.
p40 Tg mice did not show any apparent phenotypic abnormality in
lymphoid cells, which is consistent with the results in IL-12 p40- or
p35-deficient mice that IL-12 seemed not to be required for normal T
lymphocyte differentiation and maturation (31, 32). To evaluate the
antagonistic activity of serum p40 in p40 Tg mice, we first examined
the NK cell lytic activity of spleen cells after administration of
rIL-12 and found that no significant enhancement of the lytic activity
was observed in p40 Tg mice. These results suggest that serum p40
functions as an IL-12 antagonist in vivo toward NK cells, which are
compatible with the in vitro results reported previously (14).
Furthermore, we clarified significantly reduced Th1 responses in p40 Tg
mice due to the antagonistic activity of serum p40 against IL-12 in
terms of Ag-induced cytokine production and DTH reaction, to an extent
comparable with that observed in IL-12 p40-deficient mice (31). In
addition, the p40 Tg mice showed increased susceptibility to the
infection with an intracellular pathogen, blood-stage P.
berghei XAT, presumably due to the decreased IL-12-dependent
IFN- production. We also examined the susceptibility of p40 Tg mice
to the infection with another intracellular pathogen, L.
monocytogenes. It was reported previously in murine L.
monocytogenes infection that neutralization of IL-12 resulted in
increased number of CFU recovered from spleens and livers on day 3 and
5 after the inoculation (33, 34). However, there were no significant
differences in spleen and liver burdens of L. monocytogenes
strain EGD between p40 Tg mice and control littermates on days 2, 4,
and 6 after the inoculation (data not shown). This is a contrast to the
results of P. berghei XAT infection. Although further
analysis is necessary, this different susceptibility of p40 Tg mice to
these pathogens might result from different IL-12 dependency to develop
the protective immunity to these pathogens. Thus, the p40 Tg mice would
be useful to evaluate the applicability of the treatment with p40 to
inhibit various immune responses mediated by IL-12 in vivo.
After this study was submitted, another report demonstrating the IL-12
antagonistic activity of rIL-12 p40 homodimer in vivo in the acute
endotoxemia was published (26). The study showed that endotoxemic mice
generate serum IL-12 p40 homodimer in quantities corresponding to
approximately one-third of the p40 monomer, whose ratio seems to be
roughly comparable with that in serum p40 molecules of p40 Tg mice
(Fig. 3). Treatment of mice with 40 µg of purified rp40 homodimer at
18 and 2 h before endotoxin challenge was shown to result in
increase (25 ng/ml) of serum p40, which was approximately 50-fold in
excess of the peak heterodimer concentration attained during
endotoxemia (0.5 ng/ml), and therefore in significant decrease of
IFN- circulation (26). The authors concluded that p40 homodimer is a
naturally occurring cytokine antagonist that is produced in vivo and
that specifically modulates systemic inflammatory responses dependent
on the paracrine effects of IL-12 heterodimer. Taken together with our
data, these results further suggest that p40 homodimer functions as an
IL-12 antagonist in vivo in IL-12-mediated various immune responses. We
cannot precisely determine the concentration of p40 homodimer alone in
p40 Tg mouse serum, whereas it should be much higher than that attained
by the administration of rp40 homodimer. Therefore, our p40 Tg mice
would be more useful to analyze the in vivo effect of p40 on
IL-12-mediated various immune responses. Considering that p40 is
potentially stable in serum and nonimmunogenic, p40 could be applied to
treat diseases associated with enhanced IL-12 production, such as
Th1-dependent autoimmune diseases and graft rejection in
transplantation. For the evaluation of applicability of p40 treatment
to these diseases, especially, in which administration of p40 over a
prolonged period of time may be required, p40 Tg mice, which
constitutively express high serum p40, would be an useful model. These
applicabilities are currently under investigation.
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Acknowledgments |
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Footnotes |
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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, Japan.
3 Abbreviations used in this paper: Tg, transgenic; DTH, delayed-type hypersensitivity; KLH, keyhole limpet hemocyanin; LN, lymph node; PE, phycoerythrin; PRBC, parasitized red blood cell; RT, reverse transcriptase; SAP, serum amyloid P component.
Received for publication May 12, 1997. Accepted for publication September 24, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by mouse T helper clones that are unresponsive to B7 costimulation. J. Exp. Med. 180:223. in the liver. Proc. Natl. Acad. Sci. USA 91:614. in vivo. Int. Immunol. 6:157. and TNF- and occurs via a nitric oxide-dependent mechanism. J. Immunol. 155:2545.[Abstract]
production and type 1 cytokine responses. Immunity 4:471.[Medline]
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 A. Hino, M.-N. Kweon, K. Fujihashi, J. R. McGhee, and H. Kiyono Pathological Role of Large Intestinal IL-12p40 for the Induction of Th2-Type Allergic Diarrhea Am. J. Pathol., April 1, 2004; 164(4): 1327 - 1335. [Abstract] [Full Text] [PDF]
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T. D. Russell, Q. Yan, G. Fan, A. P. Khalifah, D. K. Bishop, S. L. Brody, and M. J. Walter IL-12 p40 Homodimer-Dependent Macrophage Chemotaxis and Respiratory Viral Inflammation Are Mediated through IL-12 Receptor {beta}1 J. Immunol., December 15, 2003; 171(12): 6866 - 6874. [Abstract] [Full Text] [PDF]
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 H. Enomoto, S. Shiojiri, K. Hoshi, T. Furuichi, R. Fukuyama, C. A. Yoshida, N. Kanatani, R. Nakamura, A. Mizuno, A. Zanma, et al. Induction of Osteoclast Differentiation by Runx2 through Receptor Activator of Nuclear Factor-{kappa}B Ligand (RANKL) and Osteoprotegerin Regulation and Partial Rescue of Osteoclastogenesis in Runx2-/- Mice by RANKL Transgene J. Biol. Chem., June 20, 2003; 278(26): 23971 - 23977. [Abstract] [Full Text] [PDF]
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T. Kopp, P. Lenz, C. Bello-Fernandez, R. A. Kastelein, T. S. Kupper, and G. Stingl IL-23 Production by Cosecretion of Endogenous p19 and Transgenic p40 in Keratin 14/p40 Transgenic Mice: Evidence for Enhanced Cutaneous Immunity J. Immunol., June 1, 2003; 170(11): 5438 - 5444. [Abstract] [Full Text] [PDF]
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C. Berberich, J. R. Ramirez-Pineda, C. Hambrecht, G. Alber, Y. A. W. Skeiky, and H. Moll Dendritic Cell (DC)-Based Protection Against an Intracellular Pathogen Is Dependent Upon DC-Derived IL-12 and Can Be Induced by Molecularly Defined Antigens J. Immunol., March 15, 2003; 170(6): 3171 - 3179. [Abstract] [Full Text] [PDF]
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F. Huaux, M. Arras, D. Tomasi, V. Barbarin, M. Delos, J.-P. Coutelier, A. Vink, S. H. Phan, J.-C. Renauld, and D. Lison A Profibrotic Function of IL-12p40 in Experimental Pulmonary Fibrosis J. Immunol., September 1, 2002; 169(5): 2653 - 2661. [Abstract] [Full Text] [PDF]
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N. Ohta, T. Hiroi, M.-N. Kweon, N. Kinoshita, M. H. Jang, T. Mashimo, J.-I. Miyazaki, and H. Kiyono IL-15-Dependent Activation-Induced Cell Death-Resistant Th1 Type CD8{alpha}{beta}+NK1.1+ T Cells for the Development of Small Intestinal Inflammation J. Immunol., July 1, 2002; 169(1): 460 - 468. [Abstract] [Full Text] [PDF]
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A. M. Cooper, A. Kipnis, J. Turner, J. Magram, J. Ferrante, and I. M. Orme Mice Lacking Bioactive IL-12 Can Generate Protective, Antigen-Specific Cellular Responses to Mycobacterial Infection Only if the IL-12 p40 Subunit Is Present J. Immunol., February 1, 2002; 168(3): 1322 - 1327. [Abstract] [Full Text] [PDF]
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 M. J. Holtzman, J. D. Morton, L. P. Shornick, J. W. Tyner, M. P. O'Sullivan, A. Antao, M. Lo, M. Castro, and M. J. Walter Immunity, Inflammation, and Remodeling in the Airway Epithelial Barrier: Epithelial-Viral-Allergic Paradigm Physiol Rev, January 1, 2002; 82(1): 19 - 46. [Abstract] [Full Text] [PDF]
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 M. J. Walter, N. Kajiwara, P. Karanja, M. Castro, and M. J. Holtzman Interleukin 12 P40 Production by Barrier Epithelial Cells during Airway Inflammation J. Exp. Med., February 5, 2001; 193(3): 339 - 352. [Abstract] [Full Text] [PDF]
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H. Hochrein, M. O'Keeffe, T. Luft, S. Vandenabeele, R. J. Grumont, E. Maraskovsky, and K. Shortman Interleukin (Il)-4 Is a Major Regulatory Cytokine Governing Bioactive IL-12 Production by Mouse and Human Dendritic Cells J. Exp. Med., September 18, 2000; 192(6): 823 - 834. [Abstract] [Full Text] [PDF]
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D. A. Schmitt and S. E. Ullrich Exposure to Ultraviolet Radiation Causes Dendritic Cells/Macrophages to Secrete Immune-Suppressive IL-12p40 Homodimers J. Immunol., September 15, 2000; 165(6): 3162 - 3167. [Abstract] [Full Text] [PDF]
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A. Pagenstecher, S. Lassmann, M. J. Carson, C. L. Kincaid, A. K. Stalder, and I. L. Campbell Astrocyte-Targeted Expression of IL-12 Induces Active Cellular Immune Responses in the Central Nervous System and Modulates Experimental Allergic Encephalomyelitis J. Immunol., May 1, 2000; 164(9): 4481 - 4492. [Abstract] [Full Text] [PDF]
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S. J. Ha, C. H. Lee, S. B. Lee, C. M. Kim, K. L. Jang, H. S. Shin, and Y. C. Sung A Novel Function of IL-12p40 as a Chemotactic Molecule for Macrophages J. Immunol., September 1, 1999; 163(5): 2902 - 2908. [Abstract] [Full Text] [PDF]
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J. D. Marshall, J. Chehimi, G. Gri, J. R. Kostman, L. J. Montaner, and G. Trinchieri The Interleukin-12-Mediated Pathway of Immune Events Is Dysfunctional in Human Immunodeficiency Virus-Infected Individuals Blood, August 1, 1999; 94(3): 1003 - 1011. [Abstract] [Full Text] [PDF]
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H. Yamane, T. Kato, and H. Nariuchi Effective Stimulation for IL-12 p35 mRNA Accumulation and Bioactive IL-12 Production of Antigen-Presenting Cells Interacted with Th Cells J. Immunol., June 1, 1999; 162(11): 6433 - 6441. [Abstract] [Full Text] [PDF]
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Indicates the death day of
individual mice. Similar results were obtained in two independent
experiments.