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2 (IL-12R2)-Deficient Mice Are Defective in IL-12-Mediated Signaling Despite the Presence of High Affinity IL-12 Binding Sites
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Department of Inflammation/Autoimmune Diseases, Hoffmann-LaRoche, Nutley, NJ 07110; and
Lymphocyte Cell Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis, Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and
IL-12R2, have been identified and cloned. Previous studies
demonstrated that the IL-12R1 subunit was required for mouse T and
NK cells to respond to IL-12 in vivo. To investigate the role of
IL-12R2 in IL-12 signaling, we have generated IL-12R2-deficient
(IL-12R2-/-) mice by targeted mutation in embryonic
stem (ES) cells. Although Con A-activated splenocytes from
IL-12R2-/- mice still bind IL-12 with both high and
low affinity, no IL-12-induced biological functions can be detected.
Con A-activated splenocytes of IL-12R2-/- mice failed
to produce IFN-
or proliferate in response to IL-12 stimulation. NK
lytic activity of IL-12R2-/- splenocytes was not
induced when incubated with IL-12. IL-12R2-/-
splenocytes were deficient in IFN- secretion when stimulated with
either Con A or anti-CD3 mAb in vitro. Furthermore,
IL-12R2-/- mice were deficient in vivo in their
ability to produce IFN- following endotoxin administration and to
generate a type 1 cytokine response. IL-12-mediated signal transduction
was also defective as measured by phosphorylation of STAT4. These
results demonstrate that although mouse IL-12R1 is the subunit
primarily responsible for binding IL-12, IL-12R2 plays an essential
role in mediating the biological functions of IL-12 in
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by both T and NK cells, stimulation of
the proliferation of activated T and NK cells, and enhancement of T and
NK cell-mediated cytolytic lymphocyte responses (reviewed in Refs.
1, 2).
The biological functions of IL-12 are mediated through specific
receptors on T and NK cells. To date, two IL-12 receptor subunits have
been identified in mouse and humans (3, 4, 5). These receptor
subunits are members of the cytokine receptor superfamily. Their
cytoplasmic regions contain the box1 and box2 motifs that exist in
other cytokine receptors, and they are related closely over their
entire length to the " type" cytokine receptor glycoprotein 130
and the receptors for leukemia-inhibitory factor and G-CSF. Thus, the
two IL-12 receptor subunits were designated IL-12R1 and IL-12R2.
Analyses of IL-12 binding to these receptors demonstrated that, when
expressed in COS-7 cells individually, human IL-12R1 and IL-12R2
bind IL-12 with low affinity; however, when the two subunits are
coexpressed, they confer both high and low affinity binding of IL-12
and IL-12 responsiveness (5). In contrast to the human
IL-12 receptors, IL-12R1 is the primary binding component in the
mouse conferring both high and low affinity binding sites, whereas
IL-12R2 only binds weakly to IL-12 (4, 6).
IL-12R1-deficient mice have been generated previously
(7). IL-12R1 has been found to be required for high
affinity binding of IL-12 and for mouse T and NK cells to respond to
IL-12 in vitro and in vivo. To investigate the role of IL-12R2 in
mediating IL-12 biological functions, mice deficient in IL-12R2 were
generated using homologous recombination in embryonic stem
(ES)5 cells.
IL-12R2-deficient (IL-12R2-/-) mice were
visually indistinguishable from their wild-type littermates.
Development of T and B cells in IL-12R2-/-
mice was not affected as compared with wild-type littermates. Con
A-activated splenocytes from IL-12R2-/- mice
still bind IL-12 with both high and low affinity. Nevertheless, no
IL-12-mediated biological functions can be detected in the
IL-12R2-/- mice. The data presented in this
paper demonstrate that although mouse IL-12R1 binds IL-12 with both
high and low affinity, IL-12R2 plays an essential role in mediating
the biological functions of IL-12 in mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the animal facilities at Hoffmann-LaRoche (Nutley, NJ). YAC-1 lymphoma cells were maintained in culture as previously described (8).
Tissue culture medium (TCM) was a 1:1 mixture of RPMI 1640 and DMEM
(Life Technologies, Grand Island, NY) supplemented as previously
described (9) with 5% heat-inactivated FBS (Life
Technologies). Complete culture medium used in the experiments
consisted of RPMI 1640 (Life Technologies) supplemented as previously
described (10). Ab to mouse CD3 was obtained from
PharMingen (San Diego, CA). Purified mouse rIL-12 was prepared as
previously reported (9). Purified human rIL-2 was supplied
by Dr. F. Khan (Hoffmann-LaRoche). mAbs R4–6A2 and XMG1.2 to mouse
IFN- were obtained from PharMingen. Purified mouse rIFN- was
provided by Dr. G. Alber (Hoffmann-LaRoche, Basel, Switzerland). Con A
and Salmonella enteritidis LPS were obtained from Sigma (St.
Louis, MO).
Construction of the targeting vector
Five mouse IL-12R2 genomic clones were isolated from a 129/Sv
library. The inserts were digested with NotI and
EcoRI, and several digested fragments were subcloned into
pBluescript KS(+) (Stratagene, La Jolla, CA). Restriction mapping,
sequencing, and PCR were used to determine the genomic structure.
Analysis of the subclones to determine the genomic structure and
generation of the targeting vector were performed using standard
protocols (11). The targeting vector contains four DNA
fragments: 5'-flanking sequence, 3'-flanking sequence, the
PGK-1 neo gene (12), and the pMC1-tk
gene (13). A 3.2-kb EcoRI-EcoRI
fragment located upstream of the 4.2-kb
EcoRI-EcoRI fragment containing exons 2 and 3
from the IL-12R2 gene was isolated from the genomic subclone in
pBluescipt KS(+) and inserted into the NotI site of
pPGKneotk (14). The resulting plasmid, pGKneotk/3.2, was
then digested by HindIII, filled in with T4 DNA polymerase
(New England Biolabs, Beverly, MA) in the presence of all four
deoxyribonucleotides, and then treated with calf intestinal alkaline
phosphatase (Boehringer Mannheim, Indianapolis, IN). Finally, a 6.6-kb
EcoRI-EcoRI fragment containing exons 4–6 was
inserted into the HindIII-digested pPGKneotk/3.2 to
generate the targeting vector. The orientation of the 4.2- and 6.6-kb
fragments in the resulting plasmid was checked by further restriction
digestion. The targeting vector was digested with SalI to
linearize the plasmid before electroporation into ES cells.
Identification of targeted ES cell clones
The W9.5 ES cell line was maintained on irradiated primary murine embryonic fibroblasts as previously described (15) in medium containing 15% FBS and 100 U/ml leukemia-inhibitory factor (Life Technologies). ES cells from a confluent dish were harvested and electroporated with 25 µg of linearized targeting vector using a Gene Pulser (Bio-Rad, Richmond, CA). The cells were then cultured in medium containing G418 (350 µg/ml; Life Technologies) for 1 day after electroporation. Ganciclovir (2 mM; a gift from Syntex, Palo Alto, CA) was added 2 days after electroporation, and selection was conducted for 7 days. Colonies were then picked and expanded.
Southern blot analysis of ES cell DNA was used to determine which of
the colonies contained a correctly targeted event. ES cell DNA was
extracted as previously described (16). DNA was digested
with HindIII and StuI, fractionated by agarose
gel electrophoresis, transferred to Hybond-N membrane (Amersham,
Arlington Heights, IL) by a electroblot apparatus (Hoefer Scientific
Instrument, Canberra Packard, Vancouver, Canada), and UV cross-linked
using a Stratalinker (Stratagene). A 1.2-kb
EcoRI-BglII probe 3' to the targeted region was
labeled using Prime-It II Random Primer Labeling Kit (Stratagene) as
per the manufacturer’s protocol. The membrane was hybridized with the
probe for 1 h in Rapid-Hyb buffer (Amersham) at 65°C. The
membrane was then washed in 2x SSC/0.1% SDS at room temperature
followed by 0.1x SSC/0.1% SDS at 65°C. The membrane was analyzed by
a Molecular Dynamics phosphorImager (Sunnyvale, CA). Additional
Southern blot analyses using probes from the neomycin gene and the 5'
end of the IL-12R1 gene were performed to confirm the structure of
the targeted allele.
Generation of chimeric animals
Four correctly targeted ES cell clones, G4, J1, J3, and I1, were used to generate chimeric mice. ES cells were injected into host C57BL/6J blastocysts, and embryos were transplanted into the uterine horns of pseudopregnant C57BL/6J x CBA/J F1 females. Highly chimeric agouti males, as judged by agouti coat color, were bred to C57BL/6J and BALB/cByJ females. Progeny carrying one copy of the targeted allele were identified by PCR. Heterozygotes were intercrossed to obtain mice homozygous for the mutation.
PCR analysis of tail DNA was used to identify mice carrying the mutant allele. Tail DNA was isolated as described (16). Two sets of primers were used for the analysis: 5'-GAAGCGGGAAGGGACTGGCTGCTA-3' (PGK-1 neo); 5'-CGGGAGCGGCGATACCGTAAAGC-3' (PGK-1 neo); 5'-GTGTGCAAGCTTGGCACTGTGACCGTCCAG-3' (exon 3); and 5'-GTTTAGCTTGCAGACAAACAAGGTCATACC-3' (exon 3). An Invitrogen (Carlsbad, CA) Optimized Buffer J Kit was used as per the manufacturer’s protocol. The amplification parameters used in these experiments were: incubation at 94°C for 7 min (1 cycle); denaturation at 94°C for 1 min, annealing at 64°C for 1 min, and extension at 72°C for 1 min (35 cycles); and incubation at 72°C for 6 min (1 cycle). Amplicons were fractionated in gels containing 0.8% NuSieve (FMC BioProducts, Rockland, ME) and 0.8% agarose (Life Technologies) and visualized by ethidium bromide staining.
Binding assays
Recombinant mouse IL-12 was expressed, purified, and labeled with 125I as previously described (17). Activation of splenocytes with Con A (18) and mouse 125I-IL-12 binding assays were performed as previously described (17, 19). All binding assays were performed in triplicate. Receptor binding data were analyzed by using the nonlinear regression program RADLIG 4.0 (20) and plotted by the method of Scatchard (21).
Proliferation assay
Con A-activated splenocytes were incubated in 96-well plates (Costar, Cambridge, MA) at a final density of 5 x 105 cells/ml in TCM in the presence or absence of the indicated concentrations of cytokines. After incubation at 37°C for 48 h, cells were pulsed with 0.5 µCi/well [3H]TdR (New England Nuclear, Boston, MA) overnight at 37°C. The incorporation of [3H]TdR into cellular DNA was measured by harvesting the contents of each well onto glass fiber filters using a Tomtec cell harvester (Wallac, Gaithersburg, MD). All samples were assayed in triplicate.
Cytolytic assays
Cytolytic assays using 51Cr-labeled YAC-1 target cells were performed as previously described (9). Briefly, splenocytes were cultured at 4 x 106 cells/ml in the presence of indicated concentrations of IL-12 or IL-2 for 4 days at 37°C. Control cultures contained splenocytes without added cytokines. The cells were then harvested and tested for lytic activity. Lytic assays were performed in quadruplicate, and spontaneous 51Cr release ranged from 2.1 to 13.1%.
Induction of IFN- production in vivo and in vitro
LPS-induced IFN- production in vivo was performed as
previously described (10). For IL-12-induced IFN-
production in vivo, mice were injected with 1 µg/mouse IL-12 i.p.
once daily for 5 days and bled 6 h after the final dose was given
(9). For the induction of IFN- production in vitro, Con
A-activated splenocytes were incubated at a density of 3 x
106 cells/ml in TCM in increasing doses of IL-12.
Cultures were conducted in 48-well plates in duplicate wells. After
incubation at 37°C for 48 h, cell-free culture fluids were
harvested. IFN- in sera or culture fluids was assayed using an ELISA
as previously described (10).
Induction of Th1 responses in vivo
For induction of Th1 responses in vivo, mice were immunized with
keyhole limpet hemocyanin (KLH; Calbiochem, La Jolla, CA), followed by
culture of the immune lymph node cells (LNCs) with KLH to elicit
cytokine production in vitro. Mice were immunized s.c. at the base of
the tail with 100 µg alum-precipitated KLH together with 100 µg
heat-killed Propionibacterium acnes (Elkins-Sinn, Cherry
Hill, NJ). On day 5, the subinguinal, axillary, and para-aortic lymph
nodes were removed aseptically, passed through a wire mesh, washed, and
cultured in TCM supplemented with 10% FBS (Life Technologies) and 0 or
100 µg/ml of KLH. For measurement of IFN- production, LNCs were
incubated in 1-ml cultures in 24-well plates (Costar) at 6 x
106 cells/well. Cell-free culture fluids were
harvested by centrifugation after 48 h for IFN- ELISA and
stored at -20°C until assayed. IFN- production by LNCs cultured
without KLH was below the level of detection (data not shown).
Immunoprecipitation and Western blotting
After resting overnight in RPMI 1640 with 1% BSA, 2.5 x 107 Con A-activated splenocytes were plated in 1 ml TCM per well and stimulated with the indicated cytokines for 15 min. Following stimulation, lysis, immunoprecipitation, PAGE, and immunoblotting were performed as previously described (22).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-/- mice
To introduce a null mutation in the
IL12R2 gene, a replacement targeting vector
was constructed as shown in Fig. 1
A. Upon homologous
recombination, exons 2 and 3 were replaced with a neo
cassette. This deletion removes the ATG translation initiation codon
and deletes both the signal peptide and the N-terminal immunoglobulin
domain of the receptor and thus would be expected to result in a null
allele. The targeting vector was electroporated into W9.5 ES cells
(23), and colonies that were both G418- and
ganciclovir-resistant were isolated. Southern blot analysis using a
probe outside the targeting vector was used to identify correctly
targeted homologous recombination events by detecting a restriction
fragment length polymorphism between wild-type and mutated alleles
(data not shown). Approximately 25% of the isolated ES cell clones
contained a correctly targeted mutation. Four of these clones were used
to generate chimeric animals by injection into C57BL/6J blastocysts.
All clones gave rise to highly chimeric animals that were bred to
wild-type mice to generate mice heterozygous for the mutation. These
heterozygous mice were then intercrossed, and the resulting progeny
were screened by PCR using the primers shown in Fig. 1A. A
500-bp fragment amplified from the neo gene is diagnostic of
the mutant allele. A 265-bp fragment amplified from exon 3 of the
IL-12R2 gene is diagnostic for the wild-type
allele. Thus, the presence of only the 500-bp PCR fragment indicates
that the mouse is homozygous for the mutant allele
(IL-12R2-/-); both PCR fragments are
detected in DNA from IL-12R2+/- heterozygous
mice, and wild-type mice (IL-12R2+/+) are
characterized by the presence of only the 265-bp PCR fragment.
IL-12R2-/- mice were generated from each of
the four ES cell clones in the expected Mendelian frequency (Fig. 1B and data not shown). Experiments using mice generated
from each of the four different ES cell clones gave similar results and
therefore will not be described separately.
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2 mRNA expression by
IL-12R2-/- and wild-type mouse splenocytes.
A shortened 3.5-kb transcript was detected at significantly reduced
levels in Con A-activated splenocytes from
IL-12R2-/- mice compared with wild-type mice
(data not shown). The targeted allele results in a 0.5-kb deletion
consistent with the decrease in mRNA length observed in the mutant
mouse. Because exon 2 contains the ATG translation initiation codon and
exons 2 and 3 encode both the signal peptide and the N-terminal
immunoglobulin domain of the receptor, a truncated transcript would be
expected to result in a null allele. Lack of cell surface expression of
IL-12R2 was substantiated by flow cytometric analysis of activated
splenocytes from wild-type and mutant mice stained with a mAb that
recognizes the mouse IL-12R2 subunit (data not shown).
IL-12R2-/- mice were visually
indistinguishable from their IL-12R2+/+
littermates. Quantitative analysis of splenic
CD3+ T cells, CD4+ T cells,
CD8+ T cells, B220+ B
cells, and 2B4+ NK cells demonstrated that there
are no obvious differences between
IL-12R2-/- mice and wild-type controls (data
not shown). These data are in agreement with previously reported
results from characterization of IL-12p40- (10) and
IL-12R1-deficient mice (7), suggesting that IL-12
signaling is not required for development of T and B cells in
vivo.
Both high and low affinity IL-12-binding sites are still
expressed on Con A-activated splenocytes from
IL-12R2-/- mice
Splenocytes from IL-12R2-/- mice were
activated with Con A in the presence of IL-2. After 3 days of
stimulation, activated splenocytes were tested for
125I-IL-12 binding. Results are shown in Fig. 2. Mouse IL-12 binds to Con A blasts from
IL-12R2-/- mice with a high affinity
(Kd) of 12.8 pM, 27 sites/cell, and a
low affinity (Kd) of 8.45 nM, 1553
sites/cell, which is very similar to the results obtained from Con
A-activated splenocytes from wild-type mice (7). These
results demonstrate that deletion of IL-12R2 does not diminish the
number of high or low affinity binding sites for IL-12 on the Con
A-activated splenocytes. This is in agreement with previously reported
results from studies of IL-12 binding to Ba/F3 cells expressing
IL-12R1 that mouse IL-12R1 appears to be predominantly
responsible for mediating both high and low affinity binding of IL-12
(6). Therefore, IL-12R2 does not contribute
significantly to mouse 125I-IL-12 binding.
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2-/- splenocytes are unresponsive to
IL-12 stimulation
Because both high and low affinity IL-12 binding sites are
detected on splenocytes from IL-12R2-/-
mice, we investigated whether IL-12 binding could mediate IL-12-induced
biological activity. First, we checked whether IL-12 was able to induce
IFN- production by Con A-activated splenocytes obtained from
IL-12R2-/- mice. Con A-activated splenocytes
from IL-12R2+/+ mice were used as a positive
control. Cells were cultured with increasing doses of IL-12 at 37°C
for 48 h, and the levels of IFN- in the cell-free culture
fluids were measured by ELISA. Results are shown in Fig. 3. IL-12 induced the production of high
levels of IFN- by Con A-activated splenocytes from
IL-12R2+/+ mice; however, no IFN- could be
detected in culture fluids from Con A-activated splenocytes of
IL-12R2-/- mice, even when the cells were
treated with very high concentrations of IL-12 (up to 5000 ng/ml). In
vivo induction of IFN- production by IL-12 was also deficient in
IL-12R2-/- mice. Administration of 1
µg/mouse of mouse rIL-12 for 5 days resulted in high levels of
IFN- production detected in the sera of
IL-12R2+/+ mice but not in sera of
IL-12R2-/- mice (Fig. 4).
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2+/+
mice proliferated in response to IL-12 in a dose-dependent manner (Fig. 5A). In contrast, no
proliferation was detected in Con A-activated splenocytes from
IL-12R2-/- mice even at concentrations of
IL-12 as high as 67 nM (Fig. 5A). In contrast, Con
A-activated splenocytes from both IL-12R2+/+
and IL-12R2-/- mice proliferated equally
well when stimulated with IL-2 (Fig. 5B). These results
indicate that IL-12R2-/- T cells have a
defective proliferative response to IL-12 but not to IL-2.
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2-/- splenocytes was
examined. Cytolytic assays using 51Cr-labeled
YAC-1 target cells were performed. Splenocytes from both
IL-12R2-/- and
IL-12R2+/+ mice were cultured in the presence
of the indicated concentrations of IL-12 or IL-2 for 4 days at 37°C.
Cells were then harvested and tested for lytic activity. NK lytic
activity of IL-12R2-/- splenocytes was
enhanced when incubated with IL-2 (Fig. 6B) but not when incubated
with IL-12 (Fig. 6A). In contrast, NK lytic activity of
IL-12R2+/+ splenocytes was enhanced in
response to both IL-12 (Fig. 6A) and IL-2 (Fig. 6B).
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2-/- mice
IL-12 has been found to play an important role in promoting Th1
responses both in vitro and in vivo (reviewed in Ref. 2).
Upon subsequent activation with mitogens or specific Ags, Th1 cells can
produce a large amount of IFN- (25, 26, 27). Therefore,
Ag-induced IFN- production in IL-12R2-/-
and IL-12R2+/+ mice treated with or
without IL-12 was compared. For this purpose, mice were immunized with
alum-precipitated KLH combined with heat-killed P. acnes as
adjuvant. Lymph nodes were harvested after 4 days, and IFN-
production by LNCs cultured with KLH was measured by ELISA. Results are
shown in Fig. 7. LNCs from KLH-immunized
IL-12R2-/- mice were unable to produce
IFN- when cultured with KLH, whereas
IL-12R2+/+ LNCs secreted large amounts of
IFN-.
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2-/- mice are
defective in producing IFN- in response to polyclonal stimulation in
vitro and in vivo
It has been found that mitogens like Con A and anti-CD3 mAb
are able to induce IFN- production by mouse splenocytes. To
investigate whether IL-12R2 plays a role in this process,
splenocytes from both IL-12R2+/+ and
IL-12R2-/- mice were stimulated with an
anti-CD3 mAb and Con A for 48 h, cell-free culture fluids were
collected, and levels of IFN- were measured by ELISA (Fig. 8). Splenocytes from
IL-12R2-/- mice are defective in secreting
IFN- in response to either anti-CD3 mAb or Con A stimulation.
These results are similar to those observed using
IL-12R1-/- mice (7).
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1 is involved in elevating IFN- production when mice are
treated with endotoxin LPS in vivo (7). To investigate
whether IL-12R2 plays a role in stimulating IFN- production in
vivo, IL-12R2+/+ and
IL-12R2-/- mice were injected with LPS i.p.,
serum samples were collected 6 h later, and levels of IFN- were
assayed by ELISA. Similar to previous results with
IL-12R1-/- mice (7), IFN-
production in IL-12R2-/- mice was markedly
reduced (0.27 ± 0.07 ng/ml) as compared with
IL-12R2+/+ mice (1.40 ± 0.32 ng/ml)
(Fig. 9).
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2-/- mice
To address whether the signal transduction pathways mediated by
IL-12 were also impaired, Con A-activated splenocytes were analyzed for
their ability to induce STAT-4 phosphorylation in response to
IL-12. In contrast to cells obtained from
IL-12R2+/+ mice, phosphorylation of STAT-4 was
not observed in response to IL-12 in splenocytes obtained from
IL-12R2-/- mice (Fig. 10). However, cells obtained from
IL-12R2+/+ and
IL-12R2-/- mice were equally responsive to
IL-2-mediated signal transduction as measured by phosphorylation of
STAT-5 following IL-2 treatment (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1
and IL-12R2 due to their sequence homology to other type
cytokine receptors like gp130 and leukemia-inhibitory factor receptor.
Previous studies have demonstrated the important role of IL-12R1 in
IL-12 responsiveness and the generation of Th1 cells (7).
To determine the role of IL-12R2 and elucidate its function in
IL-12-mediated events, we generated and characterized
IL-12R2-deficient mice. These mice are viable, fertile, of normal
size and weight, have no gross abnormalities, and appear to have normal
B and T cell development, indicating that IL-12R2 is not involved in
normal mouse development, including the development of immune cells.
However, consistent with the deficiency in
IL-12p40-/- and
IL-12R1-/- mice (10, 7),
defects in several immunological responses were observed in
IL-12R2-/- mice. These include
unresponsiveness to IL-12 as measured by multiple parameters as well as
severely impaired Th1 responses in vitro and in vivo.
Several lines of evidence suggest that responsiveness to IL-12 requires
a functional receptor consisting of both IL-12R1 and IL-12R2
subunits. Blocking of IL-12 binding to IL-12R1 by using an
anti-IL-12R1 Ab (28) or IL-12p40 homodimer, a known
IL-12 receptor antagonist, abrogates the biological activities induced
by IL-12 (29). Likewise, Con A-activated splenocytes from
IL-12R1-/- mice fail to proliferate, produce
IFN-, or generate Th1 cells in response to IL-12 (7).
It has been recently reported that PBMCs from IL-12R1-deficient
patients produce much lower amounts of IFN- than PBMCs from normal
donors when cells are stimulated with Ags and mitogens (30, 31). IL-12 could not induce proliferation or IFN- production
by PBMCs from these patients. Importantly, patients with IL-12R1
deficiency were extremely susceptible to mycobacterial and salmonella
infections, suggesting that IL-12-induced IFN- production and Th1
generation are essential for controlling resistance to these pathogens
in humans (30, 31). Data presented in this paper
demonstrates the additional requirement for IL-12R2 in the mouse
system.
Recent studies suggest that IL-12R2 expression in both humans and
mice may be confined to Th1 cells and suppressed on Th2 cells.
Following differentiation of CD4+ T cells in
vitro, Th2 cells expressing IL-12R1, but not IL-12R2, fail to
respond to IL-12. Expression of IL-12R2 appears to be regulated by
cytokines. IFN-
in humans, IFN- in mice, and IL-12 itself
enhanced, whereas IL-4 inhibited IL-12R2 expression
(32, 33, 34). It was shown in this study that Con A-activated
splenocytes from IL-12R2-/- mice, which
displayed both high and low affinity binding sites for IL-12, were
still deficient in the IL-12-induced biological activities tested.
These included IL-12-induced proliferation, IFN- secretion in vitro
and in vivo, and enhancement of NK lytic activity. In agreement with
previous findings in IL-12p40- and p35-deficient mice and in
IL-12R1-deficient mice, IL-12R2-/- mice
had a severe defect in their ability to generate Th1 responses, as
measured by production of IFN-. The lack of IL-12 responsiveness was
further substantiated by the observation that in contrast to the
wild-type control cells, STAT-4 phosphorylation is not observed after
treatment of IL-12R2-deficient splenocytes with IL-12. STAT-4
phosphorylation has been previously shown to be an important downstream
event in IL-12-mediated signal transduction (reviewed in Ref.
35). In contrast, treatment of splenocytes from both
wild-type and IL-1212R2-deficient mice with IL-2 resulted in
phosphorylation of STAT-5 as expected (36, 37). Taken
together, the control of IL-12R2 expression may constitute an
important mechanism for regulating IL-12 responsiveness.
Previous studies have identified two IL-12 receptor subunits to date in
both mice and humans (3, 4, 5) that have differing affinities
for IL-12. The relative contribution of the IL-12 receptor subunits to
IL-12 binding differs significantly between the human and mouse
systems. This was demonstrated using mouse pro-B cells (Ba/F3). Ba/F3
cells transfected with only human IL-12R1 or IL-12R2 bind human
IL-12 with only low affinity. Coexpression of both human subunits
resulted in the appearance of both high
(Kd = 50 pM) and low affinity
(Kd = 5 nM) binding sites, which
correspond to the high and low affinity binding sites detected on human
PHA lymphoblast cells (17). In contrast to human IL-12
receptor subunits, Ba/F3 cell lines expressing mouse IL-12R1 alone
displayed both high (Kd = 50–100 pM)
and low (Kd = 0.5–2 nM) affinity
IL-12 binding sites (6), whereas Ba/F3 cells expressing
mouse IL-12R2 alone bound IL-12 very poorly. Thus, in the mouse
system, IL-12R1 is the primary binding component, and IL-12R2
adds very little additional binding capacity.
IL-12 appears to interact with IL-12R1 via domains on the IL-12p40
subunit and with IL-12R2 via a heterodimer-specific region of IL-12
to which the IL-12p40 and p35 subunits may both contribute
(38). In vitro studies using blocking Abs or purified
mouse IL-12p40 homodimer indicate that IL-12p40 interacts primarily
with IL-12R1 because Ba/F3 cells expressing mouse IL-12R1 alone
bound mouse IL-12p40 homodimer with both high and low affinity binding
sites (39). Multiple interactions between IL-12
heterodimer and IL-12R complex can also be observed with human receptor
subunits transfected into COS-7 cells. Two classes of IL-12 inhibitors
were identified based on their ability to interfere with the binding of
125I-IL-12 to these receptor subunits expressed
on COS-7 cells. Anti-IL-12R1 Ab (2B10) and mouse Il-12p40 homodimer
blocked the binding of IL-12 to IL-12R1 but not to IL-12R2
(38). In contrast, anti-human IL-12
heterodimer-specific mAb (20C2) selectively inhibits the binding of
IL-12 to IL-12R2-transfected COS-7 cells. These two classes of IL-12
inhibitors have a synergistic effect on blocking IL-12-mediated
proliferation and IFN- production (38).
Activated splenocytes from IL-12R2-/- mice
bind IL-12 with both high and low affinity, corresponding to that
observed in lymphoblasts from wild-type mice. This study as well as our
previous results suggest that IL-12R1 is primarily responsible for
binding IL-12 in the mouse system, and IL-12R2 is further required
for IL-12 responsiveness. Disruption of either IL-12R1 or IL-12R2
activity abrogates IL-12-mediated biological functions.
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Footnotes |
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2 C.-y.W., and X.W. contributed equally to this work.
3 Current address: OSI Pharmaceuticals, 777 Old Saw Mill River Road, Tarrytown, NY 10591.
4 Address correspondence and reprint requests to Dr. Jeanne Magram, OSI Pharmaceuticals, 777 Old Saw Mill River Road, Tarrytown, NY 10591.
5 Abbreviations used in this paper: ES, embryonic stem; TCM, tissue culture medium; LNCs, lymph node cells; KLH, keyhole limpet hemocyanin;
Received for publication February 10, 2000. Accepted for publication September 1, 2000.
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