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Laboratories of
*
Immunology and
Parasitic Diseases, National Institute of Allergy and Infectious Diseases, 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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. Because T cells from these mice could
produce normal amounts of IFN- when activated by
IL-12/IL-18-independent stimuli, the defect appeared to be a failure to
respond to IL-12. This defect extended to NK cells, which also failed
to produce IFN- when stimulated by IL-12. The capacity of NK cells,
but not activated T cells, to produce IFN- in response to IL-12
could be partially restored by IL-18. The expression of the IL-12R
1- and 2-chains on T cells and NK cells from B10.Q/J mice was
normal. However, activated T cells from B10.Q/J mice did not signal
normally through the IL-12R and manifested a defect in their capacity
to phosphorylate Stat4. This defect was partial in that it could be
overcome by increasing both the concentration of IL-12 and the
incubation times in the Stat4 phosphorylation assays. Because Stat4
function is apparently intact in B10.Q/J mice, the defect in IL-12
signaling can be localized between the IL-12R complex and Stat4. This
subtle abnormality in IL-12 responsiveness results in a profound defect
in the generation of Th1 cells and the development of autoimmune
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by NK cells and is the
major cytokine responsible for driving Th1 development in response to
protein Ags. The effects of IL-12 are mediated through a heterodimeric
receptor complex consisting of the 1 and 2 receptor subunits
(1). The 1 subunit is expressed on resting T cells,
whereas expression of the 2 subunit is up-regulated during the
process of T cell activation (4). Upon high affinity
binding of IL-12 by its receptor, the associated kinases, Jak2 and
Tyk2, become phosphorylated and in turn phosphorylate critical tyrosine
residues within the cytoplasmic domains of both the 1 and 2
receptor subunits (5). Tyrosine phosphorylation of the
receptor tails recruits Stat4. Stat4 becomes phosphorylated by the
associated JAK kinases and subsequently forms homodimers that
translocate to the nucleus and function as transcriptional activators
by binding to specific DNA response elements in the promoter regions of
IL-12 inducible genes (6). Stat1, Stat3, and Stat5 may
also be capable of mediating some responses induced by IL-12
(7).
The critical role of IL-12 in Th1 development has been demonstrated by
studies of IL-12 deficient (-/-),
IL-12R1-/-, and
Stat4-/- mice (8, 9, 10). Mutations
in cytokine signaling pathways in humans have provided important
additional insights into the role played by specific signaling proteins
in the activation of lymphocytes (11). Children with
spontaneous mutations in the IL-12 p40 subunit that result in the
inability to secrete the biologically active p70 heterodimer have
impaired Ag-driven IFN- production that could be reconstituted by
the addition of exogenous recombinant IL-12 (12, 13).
Children with mutations in the IL-12R1 gene that results in the
inability to express the receptor on the cell surface also have
defective in vitro Ag-driven IFN- production, but production could
not be augmented by exogenous IL-12. Both of these deficiencies are
characterized clinically by recurrent disseminated BCG infection after
neonatal immunization, infections with nontuberculous mycobacteria, and
invasive Salmonella infections, the majority due to nontyphi
Salmonella species (14).
In this report and the accompanying paper (15), we characterize a defect in the IL-12-signaling pathway in both NK cells and activated T cells that was detected in the subline of B10.Q (B10.Q-H2q/SgJ, which will be referred to as B10.Q/J) mice maintained at The Jackson Laboratory (Bar Harbor, ME). Although the expression of both IL-12R subunits on activated T cells was normal, stimulation with IL-12 failed to induce phosphorylation of Stat4. Because Stat4 gene function in these mice appears to be normal (15), B10.Q/J mice appear to have a novel defect in the IL-12 signal transduction cascade that is localized between receptor binding and activation of Stat4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.Q-H2q/SgJ (B10.Q/J) and C57BL10/J mice were obtained from The Jackson Laboratory. B10.Q/Ai mice were obtained from Taconic Farms (Tarrytown, NY). All mice were housed under specific-pathogen-free conditions. Males between the ages of 8 and 12 wk were used exclusively.
Reagents
Bovine type II collagen was obtained from Elastin Products (Evansville, MO). IFA and desiccated Mycobacterium H37Ra were purchased from Difco (Detroit, MI). Recombinant murine IL-2, IL-12, and IL-18 and human IL-12 were from PeproTech (Rocky Hill, NJ). Con A, PMA, and chicken OVA were purchased from Sigma (St. Louis, MO). Anti-mouse CD3, anti-mouse CD4, anti-mouse CD8, and anti-mouse NK1.1 were purchased from PharMingen (San Diego, CA).
Abs to the 1- and 2-chains of murine IL-12R were the kind gift of
Drs. Chang-you Wu (National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD) and Rolf Erhardt
(Bioseek, Burlingame, CA), respectively. Isotype controls were obtained
from PharMingen. Rabbit anti-mouse Stat4 was purchased from Santa
Cruz Biotechnologies (Santa Cruz, CA) and rabbit
anti-phospho-specific Stat4 was from Zymed (South San Francisco,
CA). Peroxidase-conjugated anti-rabbit Ab was from Boehringer
Mannheim (Indianapolis, IN).
FACS analysis
Cells (1–2 x 106) were resuspended
in PBS and placed in individual wells of a 96-well V-bottom Costar
plate. Cells were then pelleted by a brief spin at 1400 rpm and 4°C
for 2 min. Cells were then incubated with either 1 µg normal hamster
IgG or 1 µg hamster anti-murine IL-12R1 or hamster
anti-mouse IL-12R2 diluted in PBS containing 5% BSA. Cells were
incubated with the respective Ab for 20 min at 4°C and then washed
twice with PBS. The cells were then incubated in PBS-normal mouse serum
for 10 min before the addition of 2 µg streptavidin-PE-biotinylated
goat anti-hamster IgG (Jackson ImmunoResearch Laboratories, West
Grove, PA) diluted in PBS-BSA. Cells were subsequently washed twice in
PBS and resuspended in streptavidin-PE (PharMingen) diluted in PBS.
After a 10-min incubation at 4°C, FITC-labeled anti-CD4 or
anti-CD8 (PharMingen) was added directly to wells for an additional
20 min incubation at 4°C. Cells were again washed twice in PBS and
resuspended in PBS. 7-Amino actinomycin D was added immediately before
FACS analysis to exclude dead cells from analysis. Analysis was
performed using CellQuest software (Becton Dickinson, San Diego,
CA).
Immunization and disease induction
Mice were immunized with an emulsion of type II collagen in CFA as described previously (16), and animals were scored for disease beginning on day 7. Serum was obtained by retroorbital sinus puncture 21 days after immunization and isotype-specific ELISAs for type II collagen-specific Abs were performed as previously described (16).
Cell culture
For testing Ag-specific responses, draining lymph node
(LN)2 cells
(3) were harvested 10–14 days after immunization and
processed as previously described (16). In brief, cells
(4 x 106/ml) were cultured in RPMI 1640
containing 10% FCS and standard supplements (cRPMI) in the presence of
either purified protein derivative (PPD) (10 µg/ml) or medium alone.
To assess global T cell responsiveness, spleen cells from naive animals
were prepared and cultured as above, except soluble anti-CD3
(1
µg/ml) or the combination of PMA (5 ng/ml) and ionomycin (300 ng/ml)
were used. Supernatants were obtained at 48 h and frozen at
-70°C until used. To assess the immune response to exogenous
cytokine administration, spleen cells from naive animals were prepared
as above and cultured in the presence of IL-2 (10 ng/ml), IL-12 (10
ng/ml), IL-18 (10 ng/ml), various combinations of these cytokines, or
medium alone. Supernatants were harvested at 24, 48, and 72 h and
frozen at -70°C until used. In some experiments,
CD4+ T cells were purified from spleens by
positive selection with magnetic anti-CD4 Microbeads (Miltenyi
Biotec, Auburn, CA), and CD4+ T cells were
isolated using an autoMACS magnetic cell separator. After positive
selection of CD4+ T cells, the negative fraction
(CD4-) was irradiated (3000R) and used as APC.
CD4+ T cells (2 x
106/ml) were combined with the irradiated
negative fraction (2 x 106/ml) and cultured
with 2.5 µg/ml Con A for 72 h. The cells were then harvested and
cultured (2 x 105/well) with IL-12 or
IL-18. After 18 h, cells were pulsed with 1 µCi
[3H]TdR and incubated at 37°C. After an
additional 18 h of culture, [3H]TdR
incorporation was determined by liquid scintillation counting. Con
A-activated CD4+ cells were also tested for their
capacity to secrete IFN- as described above for unseparated spleen
cells.
ELISA
IFN- ELISA kits were purchased from PharMingen, and
supernatants from previously mentioned experiments were assessed for
IFN- according to the manufacturer’s recommendations.
EMSA
EMSA were performed essentially as reported previously
(6). Naive splenocytes were cultured for 48 h in the
presence of soluble anti-CD3 (1 µg/ml) with or without the
addition of exogenous recombinant IL-12 (10 ng/ml). Cells were
harvested, washed extensively with PBS, and cultured in serum-free
cRPMI 1640 for 4 h. Subsequently, 5 x
106 cells were cultured in 1 ml cRPMI in the
presence of IL-2 (100 ng/ml), IL-12 (50 ng/ml), or medium alone for 20
min. Each culture was then washed with 10 ml ice-cold PBS/100 µM
sodium vanadate. Cell pellets were then resuspended in 20–40 µl
lysis buffer (0.5% Nonidet P-40, 50 mM Tris (pH 8.0), 10% glycerol,
100 µM EDTA (pH 8.0), 50 mM NaF, 150 mM NaCl, 100 µM
Na3VO4, 1 mM DTT, 400 µM
PMSF, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 µg/ml aprotinin)
and incubated on ice for 60 min with frequent vortexing. Lysates were
centrifuged at 15,000 rpm for 15 min at 4°C. Supernatants were
harvested and stored at -70°C. Protein concentrations were
determined using the Bio-Rad protein assay (Bio-Rad Laboratories,
Hercules, CA) with BSA as standard. For assays, 25 µg of cell lysate
were incubated with 100 ng 32P-labeled
oligonucleotide in reaction buffer (40 mM KCl, 1 mM
MgCl2, 0.1 mM EDTA, O.5 mM DTT, 20 mM HEPES (pH
7.9), 6% glycerol, 1 mg/ml BSA, 0.1 mg/ml poly(dI-dC)) for 15 min at
room temperature. Reactants were separated by electrophoresis on a
4.5% polyacrylamide gel buffered with 0.22x Tris-buffered EDTA. Gels
were dried and exposed directly to Kodak Biomax MR film (Sigma). To
generate a Stat4-binding DNA element, a double-stranded oligonucleotide
corresponding to an IFN- activation site (GAS)-like element found in
the mouse FcR1 promoter
(5'-gatcGCATGTTTCAAGGATTTGAGATGTATTTCCACAGAAAAGG) was synthesized
with a 5'-GATC overhang on each end (denoted by lower case letters) and
labeled with [32P]dCTP using Klenow DNA
polymerase by standard techniques (17). For generation of
an IL-2-responsive Stat5-binding element, the GAS-like binding element
in the CD23 promoter region (5'-gatcAAGACCTTTCTAAGAACTTTAATCT) was
constructed using similar techniques.
Immunoblotting
Spleen cells were cultured in cRPMI and Con A, 2.5 µg/ml, for 48 h. As a positive control, the IL-12-responsive NK3.3 cells (a gift of Dr. J. Kornbluth, Arkansas Cancer Research Center, Little Rock, AR) was grown in RPMI with 15% FCS further supplemented with 10% Lymphocult-T (Biotest Diagnostics, Denville, NJ) and 10 ng/ml human IL-2. Cells were washed in acidified RPMI (pH 6.4) and rested overnight in RPMI 1640 medium with 1%FCS-1% BSA. Cells were then washed and stimulated (50 x 106 cells/ml) for 5 or 15 min in the presence of 20 ng/ml IL-12. For some experiments, cells were stimulated for up to 50 min in the presence of 50 ng/ml IL-12.
Cell lysis in 1% Triton X-100 buffer, SDS-PAGE resolution, and subsequent immunoblotting were performed essentially as described (6). For immunoblotting with anti-Stat Abs, membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dried milk and incubated sequentially with phospho-specific anti-Stat4 (1 µg/ml) followed by HRP-conjugated goat anti-rabbit IgG (1:8000). Detection was performed using enhanced chemiluminescence. Membranes were stripped using conventional methods and reprobed with anti-Stat4 to confirm equal loading of lanes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the course of experiments on cytokine-mediated regulation
of collagen-induced arthritis (CIA (16)), we compared the
susceptibility of two sublines of B10.Q mice that bear the permissive
H-2q MHC haplotype. Surprisingly, mice purchased
from The Jackson Laboratory (B10.Q/J) were resistant to the induction
of CIA when immunized with heterologous type II collagen, whereas
B10.Q/Ai mice, which had been bred at Taconic Farms, developed a severe
inflammatory polyarthritis (Fig. 1
A). B10.Q/J mice also had a
blunted humoral response to collagen with defects in the production of
both the IgG2a and IgG1 fraction of type II collagen-specific Abs (Fig. 1B).
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production, we next
assessed the ability of these mice to produce IFN- in response to
nominal Ag. Cells were obtained from draining LN 14 days after
immunization and cultured for 48 h in the presence of PPD. LN
cells from B10.Q/J mice failed to produce IFN- in response to PPD,
whereas cells from B10.Q/Ai strain produced significant amounts of
IFN- (Fig. 2A). Similar
defects in Ag-specific IFN- production were observed when the two
strains were immunized with OVA in CFA and restimulated in vitro with
OVA (data not shown). We have not specifically tested the responses of
cells from the two strains to collagen, because we have had great
difficulty measuring collagen-specific IFN- production even when
cells from mice with severe disease were tested. In any case, the
defect in generating an Ag-specific IFN- response in B10.Q/J mice
did not appear to be Ag specific. Because IFN--deficient mice have
an enhanced susceptibility to CIA (16), the failure to
generate disease and a Th1 response is mostly likely secondary to
defects in the ability to produce or respond to cytokines (IL-12,
IL-18) that play important roles in the generation of IFN--producing
Th1 cells. The latter explanation appears likely given that naive
splenocytes from both of these strains produced similar amounts of
IFN- when stimulated with a high concentration of soluble
anti-CD3 or the combination of PMA and ionomycin (Fig. 2B). The capacity of both of these stimuli to induce IFN-
production is IL-12/IL-18 independent under these conditions (R.
Orlmann and E. M. Shevach, unpublished observations).
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Because the experiments described above were designed to measure
IFN- production by T cells, it was also of interest to determine
whether NK cells from the B10.Q/J would be capable of producing IFN-
when stimulated with IL-12 alone. Single-cell suspensions of
splenocytes from naive animals of several different strains were
cultured with increasing concentrations of IL-12 for 24 h, and
IFN- production was evaluated by ELISA. As seen in Fig. 3, splenocytes from B10.Q/J
animals responded very poorly when compared with all other strains
tested. To further characterize the cell type producing IFN- in
response to IL-12, T cells were depleted from the spleen cell
preparation, and the resultant population similarly stimulated, with
nearly identical results (Fig. 3). This suggests that the NK cell
population in B10.Q/J mice, the primary source of IFN- in response
to IL-12 in naive animals, is defective in its capacity to respond
appropriately to stimulation with exogenous IL-12.
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response to IL-12 by NK cells from B10.Q/J
mice is partially corrected by the addition of exogenous IL-18
Because IL-18 alone has been reported to induce IFN- production
by NK cells (18), splenocytes from both of the
strains were stimulated with IL-2, IL-12, IL-18, and combinations of
these cytokines to determine whether the IFN- response to IL-12
could be normalized. Splenocytes from B10.Q/Ai mice produced low levels
of IFN- when stimulated with IL-12 or IL-18 alone, but manifested a
robust response to combinations of IL-2 and IL-12, IL-2 and IL-18, and
IL-12 and IL-18 at both the 24-h and 72-h time points. No IFN-
production could be detected when cells from the B10.Q/J mice were
stimulated with all the cytokine combinations at 24 h (Fig. 4A), but low levels of IFN-
were observed after 72 h stimulation with IL-2/IL-12, and
substantial levels of IFN- were seen in response to the combination
of IL-12 and IL-18 (Fig. 4B).
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Because the studies with naive splenocytes strongly suggested that
NK cells from B10.Q mice manifest a partial defect in IL-12
responsiveness, we next evaluated whether CD4+ T
cells from this strain had a similar defect. Because resting T cells do
not express the IL-12R2-chain and do not respond to IL-12 alone, we
activated CD4+ T cells from both strains with Con
A. The blast cells were extensively washed and then cultured in IL-2,
IL-12, IL-18, and IL-12/IL-18. Con A blasts from B10.Q/Ai mice produced
IFN- when stimulated with IL-12, IL-18, or the combination, whereas
blasts cells from the B10.Q/J mice were unresponsive (Fig. 5A). In addition to inducing
the production of IFN-, IL-12 is capable of inducing a proliferative
signal to activated T cells. CD4+ blasts obtained
from B10.Q/Ai mice demonstrated a strong proliferative response (Fig. 5B, bottom) to IL-12, but not IL-18. The addition of IL-18
did not lead to enhancement of the proliferative response induced by
IL-12. In contrast, blasts cells from B10.Q/J mice reproducibly
demonstrated a higher basal level of proliferation to medium alone, and
only slight enhancement of this response was seen when cytokines were
added (Fig. 5B, top).
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The failure of cells from B10.Q/J mice to respond to IL-12 could
be secondary to a defect in the expression of the IL-12R. The recent
production of mAbs to the 1- and 2-chains of the mouse IL-12R
complex has facilitated analysis of the cell surface expression of this
receptor (19). The 1-chain is expressed at low levels
on resting CD4+ T lymphocytes and is up-regulated
on T cell activation (1). The IL-12R1-chain was
expressed on a slightly lower percentage (marked area, 15% vs 24%)
and at a lower level on CD4+ T cells from B10.Q/J
mice than on CD4+ T cells from normal C57BL/10
mice (Fig. 6). After stimulation with Con
A, the levels of expression of the IL-12R1-chain were
indistinguishable on CD4+ and
CD8+ T cells from the two strains (Fig. 7). The IL-12R2-chain was not
expressed on resting CD4+ or
CD8+ T cells from either strain (data not shown)
but was up-regulated to the same extent after stimulation with Con A
(Fig. 7). Thus, both chains of the IL-12R on cells from B10.Q mice
appear to be transcribed normally and expressed on the cell surface of
CD4+ T cells.
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in response to
IL-12 in naive splenocyte populations, we examined the level of
IL-12R2-chain expression that is constitutively expressed on
unstimulated NK cells. NK cells from B10.Q and C57BL10/J mice expressed
similar levels of the IL-12R 1- and 2-chains (Fig. 6
). Thus, a
defect in the expression of the IL-12R does not appear to account for
the nonresponsiveness of either T or NK cells from the B10.Q/J mice to
IL-12. B10.Q/J mice do not signal normally through the IL-12R
Signaling through the IL-12R is thought to involve ligand binding
to the 1-chain, heterodimerization with the 2-chain, and
subsequent phosphorylation of two receptor-associated Janus kinases,
Tyk2 and Jak2 (5). These phosphorylated intermediates
recruit Stat4 to the complex (6). Stat4 is then itself
phosphorylated, homodimerizes, and then is transported to the nucleus
where it regulates transcription of a number of genes.
Because the IL-12R is expressed normally on activated T cells from
B10.Q/J mice, we next attempted to determine whether signal
transduction via the IL-12R complex was normal. Anti-CD3 induced blasts
were incubated with IL-12, and whole cell lysates were subsequently
incubated with a labeled GAS probe and analyzed by EMSA. Phosphorylated
Stat4 homodimers can bind to this probe and retard its migration
through a polyacrylamide gel. IL-12 induced a prominent band shift with
extracts of blast cells from B10.Q/Ai mice, but not with extracts
prepared from B10.Q/J mice, even when the extracts were from cells that
initially were stimulated with IL-12 in addition to anti-CD3 (Fig. 8A). This abnormality is not a
manifestation of global cytokine signaling dysfunction, as generation
of a CD23 promoter-binding element in response to IL-2 is completely
normal in these mice (Fig. 8B).
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One of the requirements for Stat4 homodimerization and subsequent
binding to DNA is that it be phosphorylated (6). To assess
the ability of IL-12 to induce Stat4 phosphorylation in cells from
B10.Q/J mice, Con A blasts were serum and cytokine starved for 18
h and then incubated with IL-12 for 5 or 15 min, which is generally
adequate time to induce phosphorylation of Stat4. Lysates from cells
thus treated were run out on SDS-PAGE and immunoblotted with an Ab
specific for the phosphorylated form of Stat4. As can be seen in Fig. 9A, Stat4 phosphorylation was
seen at 5 min in both the blots from the control human NK cell line,
NK3.3, and B10.Q/Ai cells. No Stat4 phosphorylation was noted, even
after 15 min, in the blots prepared from B10.Q/J cells, whereas lysates
from the control animal cells show increased phosphorylation at the
later time point. These differences are not due to different levels of
Stat4, as the reblot in Fig. 9A (bottom)
shows similar levels of protein expression.
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and in the accompanying article
(15), describing the in vivo study of resistance of
B10.Q/J mice to Toxoplasma gondii infection, under certain
circumstances cells from B10.Q/J mice appear to be capable of
responding to IL-12, suggesting that IL-12 can signal appropriately
through the IL-12R complex. To further address this issue, we increased
both the concentration of IL-12 and prolonged the incubation time in
the Stat4 phosphorylation assays. Surprisingly, when B10.Q/J blasts
were cultured with 50 ng/ml IL-12 for 50 min, Stat4 phosphorylation was
induced (Fig. 9B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing Th1 cells by immunization of this strain
with Ag in CFA. Furthermore, we were unable to induce CIA in this
strain which expresses the susceptible MHC haplotype,
H-2q, whereas a subline of B10.Q that has been
maintained separately at Taconic Farms is readily susceptible to CIA.
In many respects, the B10.Q/J mouse resembles
IL-12R1-/- mice (9). NK cells
and Con A-activated splenocytes from both strains fail to produce
IFN- when stimulated with IL-12, and activated T cells from both
strains fail to proliferate when cultured with IL-12. However,
T-depleted spleen cells from the B10.Q/J mice did produce low levels of
IFN- when stimulated with 50 ng/ml IL-12, whereas the
IL-12R1-/- mice did not respond to
concentrations of IL-12 as high as 5000 ng/ml.
Although the B10.Q/J and IL-12R1-/- mice
share many phenotypic features, our studies using mAbs to both the
IL-12R 1- and 2-chains clearly demonstrate that their expression
on activated T cells and NK cells is indistinguishable from that of the
B10.Q/Ai strain. Although mutations of the IFN-R have been described
that result in failure of IFN- to bind to its receptor in the
presence of normal receptor expression (20), similar
mutations of the IL-12R complex in humans have not been reported. In
any case, sequencing of both chains of IL-12R from the B10.Q/J and
B10.Q/Ai strains has not revealed any differences; however, there
appears to be significant polymorphism in the extracellular domain of
the IL-12R 1-chain among several of the strains examined (D. Frucht
and J. O’Shea, unpublished observations).
We also considered that the defect in the B10.Q/J strain was a mutation in the Stat4 that is responsible for IL-12-mediated gene activation. However, normal levels of Stat4 were present in B10.Q/J mice, and we were able to induce phosphorylation of Stat4 with high concentration of IL-12 and prolonged incubation times. In any case, the complementation studies in the accompanying paper (15) demonstrate that Stat4 function in the B10.Q/J strain is completely normal. Collectively, the lack of genetic defects in Stat4 or both chains of the IL-12R complex with major defects in Stat4 phosphorylation in response to IL-12 strongly point to defects in the only two known upstream components of this pathway, the Jak kinases, Tyk2 and Jak2. It is unlikely that the defect is at the level of Jak2 given that this kinase is critically important for signaling by other cytokine receptors including IFNs and hemopoietic growth factors (21, 22). Jak2-deficient mice also show embryonic lethality due to the absence of erythropoiesis. It remains possible that the mutation in the B10.Q/J mouse might be in an adapter protein that specifically couples the IL-12R, but not other cytokine receptors, to Jak2.
We have attempted to analyze Tyk2 phosphorylation in response to IL-12
and determine whether differences exist between the B10.Q/J and
B10.Q/Ai strains. However, we have been unable to detect specific
phosphorylation of Tyk2 in either strain using Con A T blasts and
the reagents that are presently commercially available. Very recently,
two groups have generated Tyk2-deficient mice (23, 24).
There are several features in common between the
Tyk2-/- mice and the B10.Q/J mice, but several
subtle differences are also present. Stat4 phosphorylation was
detectable in blast cells from Tyk2-/- mice at
15 min but was quantitatively lower than the level of phosphorylation
seen in the wild type. In contrast, B10.Q/J mice had absolutely no
detectable phosphorylation at these time points at the concentrations
of IL-12 tested but did manifest similar low levels of Stat4
phosphorylation after prolonged exposure to a higher concentration of
IL-12. It appears as if the loss of Tyk2 results in a dampening effect,
whereas the defect in the B10.Q/J strain seems to have a delaying
effect on Stat4 phosphorylation. T cell blasts from B10.Q mice failed
to specifically proliferate when stimulated with IL-12, whereas T cells
blasts from the Tyk2-/- demonstrated a normal
proliferative response. Although one group (24) reported a
profound decrease in the capacity of activated T cells from the
Tyk2-/- to produce IFN- in response to IL-12
resembling what we have observed with cells from B10.Q/J mice, the
studies by the other group (23) demonstrated a substantial
(50–80%), but incomplete, reduction in IL-12 responsiveness. Lastly,
in contrast to the Tyk2-/- mice, the capacity
of B10.Q/J mice to respond to LPS by activation of NO production was
normal (G. Yap, unpublished observations). Resolution of these
differences will require more detailed studies of Tyk2 function in the
B10.Q/J strain, sequencing of the Tyk2 genes in both strains, as well
as breeding Tyk2-/- mice to B10.Q/J
mice.
One important finding in our in vitro experiments was the ability of
IL-18 to partially reconstitute the capacity of T-depleted spleen cells
containing NK cells to respond to IL-12 by producing IFN- after
72 h of culture. In contrast, activated T cell blasts failed to
produce IFN- when similarly stimulated. One possibility is that at
the level of the NK cell, IL-18 modulates the expression of multiple
other signaling pathways that augment the low levels of IL-12-mediated
Stat4 signaling. As will be demonstrated in the accompanying report, in
vivo treatment of B10.Q mice with IL-18 had profound effects on
enhancing their resistance to infection with T. gondii by an
IL-12-dependent mechanism. If the defect in the B10.Q/J mice is at the
level of a subtle mutation in Jak2 or Tyk2, it is possible that the
defective function of these kinases may be overridden by excess
IL-18.
Studies of mice with selective deficiencies of components of the Th1
cytokine pathway have strongly supported the critical role of Stat4 in
IL-12 responsiveness and Th1 differentiation. However, recent
observations with lymphocytes from patients with increased
susceptibility to atypical mycobacteria and Salmonella
infections have raised the possibility that IL-12-mediated activation
of other Stats may also be needed to generate a Th1 response of
sufficient potency to eradicate intracellular infections. Thus, Gollob
et al. (7) have recently described a patient with
Mycobacterium avium infection and recurrent
Staphylococcus aureus sinusitis whose cells resembled the
B10.Q/J mouse, in that they expressed normal levels of the IL-12R 1-
and 2-chains but, in contrast to the B10.Q/J mouse, phosphorylated
Stat4 normally in response to IL-12. However, the activation of Stat1,
-3, and -5 by IL-12 was completely absent. These results emphasize the
complexity of the IL-12-mediated Th1 differentiation pathways. The
availability of an inbred strain of animals, such as the B10.Q/J mouse,
with a defect in IL-12 responsiveness should be a valuable resource in
analyzing the complexity of IL-12-mediated signal transduction.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: LN, lymph node; cRPMI, RPMI 1640 containing 10% FCS and standard supplements; PPD, purified protein derivative; GAS, IFN- activation site; CIA, collagen-induced arthritis.
Received for publication January 12, 2001. Accepted for publication March 1, 2001.
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by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115.2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185:817. production and type I cytokine responses. Immunity 4:471.[Medline]
1 chain (IL-12R1)-deficient mice. IL-12R1 is an essential component of the functional mouse IL-12 receptor. J. Immunol. 159:1658.[Abstract]
(IFN-) pathway. Clin. Exp. Immunol. 121:417.[Medline]
production is cytokine-dependent. Proc. Natl. Acad. Sci. USA 94:3189.2 chain expression and signaling. J. Exp. Med. 191:847. receptor 1 deficiency, cell surface receptors fail to bind IFN-. J. Clin. Invest. 105:1429.[Medline]
signaling, although it is required for IL-12-mediated T cell function. Immunity 13:561.[Medline]This article has been cited by other articles:
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