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in Th1 Differentiation: IFN- Regulates IL-18R
Expression by Preventing the Negative Effects of IL-4 and by Inducing/Maintaining IL-12 Receptor 
2 Expression
* Laboratory of Immunology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
Howard Hughes Medical Institute, National Institutes of Health Research Scholars Program, Bethesda, MD 20814; and
Bioseek, Burlingame, CA 94010
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting Th1 cells: up-regulation of IL-12R
2 and
IL-12-driven up-regulation of IL-18R
. We previously demonstrated
that IL-12-driven up-regulation of IL-18R expression is severely
impaired in IFN--/- mice. However, it was unclear from
these studies how IFN- influenced IL-18R since IFN- alone had
no direct effect on IL-18R expression. In the absence of IL-4,
IL-12-dependent up-regulation of IL-18R/IL-12R2 was independent
of IFN-. However, in the presence of IL-4, IFN- functions to
limit the negative effects of IL-4 on both IL-18R and IL-12R2.
Neutralization of IL-4 restored IL-12-driven up-regulation of
IL-18R/IL-12R2 in an IFN--independent fashion. In the absence
of both IL-12 and IL-4, IFN- up-regulates IL-122 expression and
primes IFN--producing Th1 cells. When T cells were primed in the
presence of IL-4, no correlation was found between the levels of
expression of the IL-18R or the IL-12R2 and the capacity of these
cells to produce IFN-, suggesting that IL-4 may also negatively
affect IL-12-mediated signal transduction and thus Th1 differentiation.
These data clarify the role of IFN- in regulation of
IL-18R/IL-12R2 during both IL-12-dependent and IL-12-independent
Th1 differentiation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing Th1 cells (1). Ligation of the TCR
induces IL-12R2 expression rendering T cells responsive to IL-12.
Binding of IL-12 leads to IFN- production and further enhancement of
IL-12R2 expression (2, 3, 4, 5, 6). In addition to IL-12R2,
IL-12 up-regulates IL-18R expression (7, 8, 9). Although
IL-18 alone does not appear to be sufficient for Th1 development
(10, 11), T cells primed in the presence of IL-12 will
subsequently respond to both IL-12 and IL-18 in a paracrine fashion by
secreting large amounts of IFN- (12, 13, 14). High levels
of IL-18R expression correlate with the ability of IL-18 to induce,
as well as synergize with IL-12 for significant IFN- production
(7). This synergistic effect of IL-12 and IL-18 occurs in
the absence of TCR ligation and may represent an alternative pathway
for production of IFN- during ongoing inflammatory responses.
Previously we reported that IL-18R is expressed on a majority of
peripheral CD4+ T cells (7). The
combination of TCR ligation and IL-12 leads to significant enhancement
of IL-18R expression during Th1 differentiation. We and others have
demonstrated that IFN- itself did not directly affect
IL-18R expression, but that IFN- was required for IL-12-driven
up-regulation of IL-18R expression (7, 15); however,
the mechanism by which IFN- influenced IL-18R expression and thus
IL-18 responsiveness was not elucidated.
The goal of the present study was to further define the role of IFN-
in regulation of IL-18R expression and thus Th1 differentiation in
general. Since IL-12 plays an important role in both processes, we also
included an analysis of IL-12R2 expression. As IFN- has been
reported to be capable of inducing Th1 differentiation in the absence
of IL-12, we extended our studies to examine the role of IFN- in the
regulation of IL-18R/IL-12R2 expression and function on Th1 cells
primed in the absence of IL-12. Although previous studies have
frequently examined expression of these receptors at the mRNA level or
in binding assays, we made use of a polyclonal Ab to the IL-18R and
a newly described mAb to the mouse IL-12R2 chain, thereby
facilitating analysis of receptor expression at the single-cell level.
In addition to surface expression of each receptor, we used
intracellular cytokine staining to measure their functionality by the
ability of IL-12 and IL-18 to induce IFN- production. We show that
IFN- is required for preventing the negative effects of IL-4 on
IL-18R and subsequently Th1 differentiation. In the presence of
IL-4, IFN- is absolutely required for IL-12-induced up-regulation of
both IL-18R and IL-12R2 expression. Neutralization of IFN- led
to down-regulation of both IL-18R and IL-12R2, as well as
IL-12/IL-18 responsiveness. Conversely, in the absence of IL-4,
IL-12-dependent up-regulation of IL-12R2/IL-18R was independent
of IFN-. No correlation was found between the levels of
IL-12R2/IL-18R and the degree of Th1 differentiation. Although
IL-12 is sufficient for Th1 differentiation when IL-4 is limiting,
IFN- is also capable of inducing Th1 differentiation independent of
IL-12. IFN--primed T cells secreted IFN- both when
restimulated through their TCR and when challenged with the
combination of IL-12 and IL-18 in the absence of TCR ligation. These
studies illustrate the pleiotropic effects of IFN- in the regulation
of IL-12R2/IL-18R expression and function, and thus control of
IL-12-dependent and IL-12-independent Th1 differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female B10.A, 5CC7 B10.A/Ai, and 5CC7 B10.A/Ai recombination activation gene (RAG)2 2-deficient (RAG-2-/-) mice expressing a TCR transgene specific for cytochrome c, DO11.10 BALB/c and DO11.10 BALB/c RAG-2-/- expressing a TCR transgene specific for OVA, were obtained from Taconic Farms (Germantown, NY) and used at 6–10 wk of age. Female BALB/c mice were obtained from the National Cancer Institute (Frederick, MD) and used at 6–8 wk of age. All animals were housed under specific pathogen-free conditions and provided food and water ad libitum. Animals were maintained according to National Institutes of Health Animal Care Guidelines.
Priming conditions
CD4+ T cells were isolated from spleens of
mice by positive selection using auto-MACS magnetic separation
(Miltenyi Biotec, Auburn, CA), and purity was confirmed by FACS
analysis. CD4+ T cells (2 x
105/ml) were stimulated in vitro for 7 days with
irradiated spleen cells as APC (106/ml) and 1
µM of the appropriate peptide in IL-2-supplemented media containing
10% FBS, L-glutamine, antibiotics, and 2-ME. Cells were
primed under the following conditions: 1) IL-12 (10 ng/ml; R&D Systems,
Minneapolis, MN); 2) IL-12 and anti-IFN- (XMG 1.2, 20 µg/ml);
3) IL-12, anti-IFN-, and anti-IL-4 (11B11, 10 µg/ml); 4)
IFN- (10 ng/ml) and anti-IL-12 (C17.8, 10 µg/ml); 5) IFN-,
anti-IL-12, and anti-IL-4; 6) anti-IFN- and
anti-IL-12; 7) anti-IFN-, anti-IL-12, and anti-IL-4;
8) IL-4, anti-IL-12, and anti-IFN- (Th2 polarizing
conditions); 9) no additional cytokine (neutral conditions); and 10)
anti-IL-12. An equal volume of fresh IL-2 medium was added on day 4
for 7-day cultures.
FACS analysis of IL-12R2 expression
Cells were harvested and washed twice in cold PBS, resuspended
in cold PBS, and plated (1–2 x 106) in a
96-well V-bottom Costar plate (Corning Glass, Corning, NY). All
incubations were done at 4°C. Cells were first incubated with either
purified hamster IgG (BD PharMingen, San Diego, CA) or hamster
anti-mouse IL-12R2 diluted in PBS containing 5% BSA (PBS-BSA)
for 30 min. Cells were washed twice with cold PBS and then incubated in
goat serum/PBS-BSA for 10 min before staining with long
spacer-biotinylated goat anti-hamster IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA)/PBS-BSA for 20 min. Cells
were subsequently washed twice in cold PBS and resuspended in
streptavidin-PE (BD PharMingen) diluted in PBS. After a 10-min
incubation, FITC-labeled anti-mouse CD4 (BD PharMingen) was added
directly to wells for an additional 10-min incubation. Cells were
washed once in PBS and resuspended in PBS. 7-Amino actinomycin
D (BD PharMingen) was added immediately before FACS analysis to
assay for viability. Analysis was performed using CellQuest software
(BD Biosciences, San Diego, CA). Data are presented as the difference
(
) between the mean fluorescence intensity (MFI) of the positive
stain (anti-IL-12R2) and the MFI of the negative control
(goat Ig).
FACS analysis of IL-18R expression
Analysis of IL-18R expression was performed as previously
described (7). Briefly, cells were first incubated for 10
min with 1 µg of rat anti-mouse CD16/CD32 (Fc block; BD
PharMingen) to block nonspecific binding of goat Ig/anti-IL-18R
to Fc receptors. Cells were then incubated for 30 min with either 1
µg of biotinylated goat IgG or 1 µg of biotinylated goat
anti-mouse IL-18R (R&D Systems) diluted in PBS-BSA. Cells were
subsequently washed twice in PBS and then resuspended in
streptavidin-PE as above. The remainder of the staining protocol is as
described above for IL-12R2 expression.
Intracellular cytokine staining
Primed T cells were harvested on day 6, washed, then resuspended
in fresh IL-2 medium. Cells (1–2 x 106)
were stimulated overnight in 12-well plates with plate-bound
anti-CD3 (5 µg/ml), and 2 µM monensin was added directly to
each well 4 h before intracellular staining. Alternatively, primed
T cells were stimulated overnight (1–2 x
106) in 24-well round-bottom plates as follows:
1) no additional cytokine (IL-2 only), 2) IL-12 (10 ng/ml), 3) IL-18
(30 ng/ml), and 4) IL-12 and IL-18. Monensin was added to each well as
above. Both anti-CD3 and cytokine-stimulated cells were harvested
and washed twice in PBS before being transferred (1–2 x
106) to a 96-well V-bottom plate. Cells were
first incubated with 0.5 µg of Fc block and 0.5 µg of FITC-labeled
anti-CD4 for 10 min at room temperature. Cells were then washed
twice in PBS and incubated with 50 µl of FACS fix (PBS with 1% BSA,
4% paraformaldehyde, and 0.01% NaN3) for
5 min at 37°C. One hundred fifty microliters of Perm buffer (0.1%
saponin in PBS) was added to each well before spinning the cells down
and resuspending in 2 µg of allophycocyanin-conjugated rat
anti-mouse IFN- (BD PharMingen) and 2 µg of PE-rat
anti-mouse IL-4 (BD PharMingen) for 15 min at room temperature.
Finally, cells were washed once in PBS and analysis was performed using
CellQuest software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in IL-12-dependent differentiation of Th1 cells
We and others (7, 15) have previously shown that the
ability of IL-12 to up-regulate IL-18R expression is severely
impaired in cultures of T cells from IFN--/-
mice. Although IFN- itself had no effect on IL-18R expression,
the combination of both IL-12 and IFN- restored up-regulation of
IL-18R. To clearly define the roles of endogenous IFN- and IL-4
in regulation of both IL-18R/IL-12R2 expression and function, we
primed T cells from TCR-transgenic mice with Ag, APC, and IL-12. We
took advantage of two transgenic models, 5CC7 (B10.A) and DO11.10
(BALB/c) mice, because of their genetic predisposition toward either a
Th1 or Th2 response (16, 17). When primed under neutral
conditions (cytochrome c and APC only), 5CC7-transgenic T
cells become predominantly IFN- producers with very little IL-4
production (data not shown). In contrast, DO11.10 mice primed with
OVA/APC develop into predominantly IL-4 producers (data not shown).
CD4+ T cells from 5CC7 and DO11.10 mice were
primed in vitro with Ag, APC, exogenous IL-12, and neutralizing Ab to
IFN- or IL-4. Cells were collected after 1 wk in culture and
IL-18R and IL-12R2 expression were evaluated by FACS
analysis.
The addition of anti-IFN- had no effect on the ability of IL-12
to up-regulate IL-18R expression on CD4+ T
cells from 5CC7 mice (Fig. 1
, a and b). The absence of IFN- also had no effect on
IL-12R2 expression (Fig. 1, g and h).
Endogenous IL-4 did not appear to play any role in priming 5CC7 T cells
as addition of anti-IL-4 had no noticeable effect on either
IL-18R or IL-12R2 (Fig. 1, c and i). In
contrast to 5CC7 cultures, neutralization of IFN- in DO11.10
cultures resulted in a significant decrease in IL-18R expression
(Fig. 1, d and e) and also resulted in
undetectable levels of IL-12R2 expression (Fig. 1, j and
k). Neutralization of IL-4 restored the ability of IL-12 to
up-regulate IL-18R on T cells from DO11.10 mice in the absence of
IFN- (Fig. 1, d–f), resulting in higher levels of
IL-18R expression than those induced on T cells primed with IL-12
alone (Fig. 1, d and f). Addition of
anti-IL-4 also partially restored IL-12-induced IL-12R2
expression in the absence of IFN- (Fig. 1, k and
l). These data confirm that one major role of IFN- in
IL-12-induced Th1 differentiation is to prevent the negative effects of
IL-4 on up-regulation of the IL-18R and the IL-12R2.
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/IL-12R2 expression with Th1
differentiation
To determine whether the levels of IL-18R/IL-12R2
expression are correlated with Th1 differentiation, we restimulated T
cells 7 days after priming with immobilized anti-CD3 as well as
with IL-12 and IL-18 separately and together and quantitated IFN-
production by intracellular staining. T cells from 5CC7-transgenic mice
primed with IL-12 produced IFN- (Fig. 2a, 91%), but not IL-4, when
restimulated with immobilized anti-CD3. These cells also produced
IFN- when stimulated with IL-12 alone, IL-18 alone, and the
combination of IL-12 and IL-18 (Fig. 2, b–e).
Neutralization of IFN- or both IL-4 and IFN- in 5CC7 cultures had
minimal effects on IFN- production in response to anti-CD3 or
IL-12 and IL-18 (Fig. 2, f–o). This result correlates with
the expression of high levels of IL-18R/IL-12R2 on these cells
and the lack of effects of IFN- or IL-4 on their expression
(Fig. 1
).
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-producing cells (Fig. 3a, 24% IL-4; 20% IFN-).
Although these cells expressed levels of IL-18R/IL-12R2
comparable to those expressed by similarly stimulated 5CC7 T cells
(Fig. 1), they did not respond to IL-12 or IL-18 and responded only
modestly to the combination of IL-12 and IL-18 (cf Figs. 2, b–e, and 3, b–e). Neutralization of IFN- in
DO11.10 cultures led to a marked reduction in the percentage of
IFN--producing cells (Fig. 3, a and f, 20 vs
9%), enhanced the development of IL-4-secreting cells (Fig. 3, a and f, 24 vs 42%) in response to anti-CD3
stimulation, and resulted in almost a complete loss of responsiveness
to IL-12/IL-18 (Fig. 3, e and j, 26 vs 5%); this
failure to respond to IL-12/IL-18 is consistent with the very low
levels of IL-18R and IL-12R2 expressed by these cells (Fig. 1, e and k).
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almost completely restored expression of both
IL-18R and IL-12R2 (Fig. 1, d–f and j–l),
we next determined whether restoration of IL-18R/IL-12R2
expression in anti-IL-4-treated cultures was accompanied by an
increase in IL-18/IL-12 responsiveness. Addition of anti-IL-4 to
DO11.10 cultures primed with IL-12/anti-IFN- resulted in an
increase in IL-12- and IL-18-induced IFN- production (Fig. 3, h and m, 0 vs 9%, and i and
n, 2 vs 11%, respectively) as well as significant
enhancement of responsiveness to the combination of IL-12 and IL-18
(Fig. 3, j and o, 5 vs 84%). The ability to
secrete IFN- in response to cytokines was also paralleled by an
increase in IFN- production after restimulation with anti-CD3
(Fig. 3k). Taken together, these studies with D011.10 T
cells demonstrate discordance between the levels of
IL-18R/IL-12R2 expression and commitment of the cells to Th1
differentiation. Thus, while the level of expression of the
IL-18R/IL-12R2 is comparable on 5CC7 and DO11.10 cells stimulated
in the presence of IL-12 (Fig. 1, a and d),
DO11.10 cells have only partially committed to Th1 differentiation as
measured by their responses to anti-CD3 or IL-12/IL-18.
Furthermore, neutralization of endogenous IL-4 production does not
completely abolish the differences between the two strains (cf Figs. 3, k–o, and 2, k–o).
Comparison of DO11.10 with DO11.10 x RAG-2-/-
mice reveal IL-4-independent regulation of IL-12R2
Because the experiments performed in
Figs. 1–3 utilized
CD4+ T cells from conventional mice, it is likely
that memory T cells present in these cultures could influence the
subsequent priming of naive cells. Therefore, we performed identical
experiments using transgenic T cells from either 5CC7 or DO11.10 mice
on a Rag-2-/- background. The results obtained
with cells from 5CC7 mice on a RAG-2-/- background were
identical to those obtained with cells from 5CC7 mice on a conventional
background (data not shown). However, T cells from DO11.10 x
RAG-2-/- developed exclusively into
IFN--producing cells, which is in contrast to conventional DO11.10 T
cells, which are mostly IL-4 producers (data not shown). This suggests
that IL-4 derived from memory T cells in DO11.10 mice on a conventional
background can influence the priming of naive T cells.
The addition of IL-12 to DO11.10 x
RAG-2-/- cultures led to a significant
enhancement of IL-18R (Fig. 4a), IL-12R2 (Fig. 4b), as well as both TCR (Fig. 4c) and IL-12-
plus IL-18-induced IFN- production (Fig. 4, d and
e). The amount of IFN- produced in response to TCR or
cytokine stimulation was very similar to that of 5CC7 cultures primed
with IL-12 (Fig. 2, a–e). Neutralization of IFN- led to
a partial decrease in IL-12R2 (Fig. 4g, MFI 41 to MFI
24), but not IL-18R (Fig. 4f) and a decrease in IFN-
production by approximately one-third following stimulation with
anti-CD3 or IL-12 plus 18 (Fig. 4, h–j). Interestingly,
neutralization of IL-4 in DO11.10 x
RAG-2-/- cultures did not restore the full
effects of IL-12 as T cells expressed less IL-12R2 and produced less
IFN- (Fig. 4, k–o). Similar to DO11.10 cultures,
neutralization of endogenous IL-4 production in DO11.10 x
RAG-2-/- cultures does not completely abolish
the differences between the two strains (i.e., B10.A and BALB/c) and
reveals an IL-4-independent, IFN--dependent component to the control
of IL-12R2 expression.
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and IL-4 in IL-12-independent Th1 differentiation
A number of studies have suggested that IFN- alone in the
absence of IL-12 is capable of inducing Th1 differentiation
(18, 19, 20, 21, 22, 23), but this issue remains controversial
(24). Although the studies described above have focused on
the effects of IFN- in preventing the negative effects of IL-4 on
Th1 differentiation in the presence of IL-12, the model systems used in
these studies also allow us to examine the IL-12-independent effects of
IFN- on expression of the IL-18R/IL-12R2 as well as TCR- and
cytokine-induced IFN- production. 5CC7 or DO11.10-transgenic T cells
were therefore primed in the presence of IFN- and neutralizing Ab to
IL-12. T cells from 5CC7 mice primed with IFN-/anti-IL-12
expressed levels of IL-18R similar to those primed with
anti-IL-12 alone (Fig. 5, a and b). This is consistent with our previous
data that IFN- alone does not directly up-regulate IL-18R
expression. T cells from DO11.10 mice primed with anti-IL-12
expressed lower levels of IL-18R when compared with 5CC7 T cells
(Fig. 5, a and d), but again the addition of
IFN- had no direct effect on IL-18R expression (Fig. 5e). In contrast to 5CC7 T cells, addition of anti-IL-4
to anti-IL-12/IFN--primed DO11.10 cultures resulted in marked
enhancement of IL-18R expression (Fig. 5, e and
f), consistent with the possibility that endogenous IL-4
production is responsible for lower levels of IL-18R expression seen
on DO11.10 cells compared with 5CC7 cells in anti-IL-12/IFN-
cultures. T cells from DO11xRAG-2-/- mice
primed with anti-IL-12/IFN- expressed much higher levels of
IL-18R when compared with DO11.10 cultures (data not shown), again
consistent with this hypothesis since primed cells from
DO11xRAG-2-/- mice made little IL-4. This
result demonstrates that the presence of IFN- is not sufficient to
prevent IL-4-mediated down-regulation of IL-18R in the absence of
IL-12.
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enhanced IL-12R2 expression in both 5CC7 (Fig. 5, g and h, MFI 3 vs 9) and DO11.10 cultures
(Fig. 5, j and k, MFI 6 vs 28) in the absence of
IL-12. It should be noted that the low levels of IL-12R2 expression
seen on cells cultured in the presence of anti-IL-12 were due to
endogenous IFN- production, since addition of both anti-IL-12
and anti-IFN- led to undetectable IL-12R2 expression on both
DO11.10 and 5CC7 cells (Fig. 5, m and n).
Addition of anti-IL-4 had no effect on the ability of IFN- to
enhance IL-12R2 expression on DO11.10 T cells (Fig. 5, k
and l).
Although the levels of both the IL-18R and IL-12R2 seen in Fig. 5
are much lower than those induced during Th1 differentiation in the
presence of IL-12 (Fig. 2), we next determined whether T cells primed
with IFN- in the absence of IL-12 had developed into Th1 cells as
determined by their capacity to produce IFN- after restimulation
through their TCR or with IL-12/IL-18. T cells from 5CC7 mice primed in
the presence of anti-12 produced low levels of IFN- after
stimulation with either anti-CD3 (Fig. 6a, 13%) or the combination
of IL-12 and IL-18 (Fig. 6e, 16.5%), but did not respond to
either IL-12 or IL-18 separately (Fig. 6, b–d). This
IL-12-independent IFN- production by 5CC7 T cells likely reflects
endogenous IFN- production, as inclusion of both anti-IL-12 and
anti-IFN- significantly reduced the number of IFN--positive
cells (data not shown). Surprisingly, priming of 5CC7 cells with
IFN- and anti-IL-12 led to a significant increase in the number
of IFN--secreting cells (Fig. 6f, 50%) in response to
anti-CD3. Although these T cells responded poorly to either IL-12
or IL-18 alone, they responded quite well to the combination of IL-12
and IL-18 (Fig. 6, g–j, 63%). Addition of anti-IL-4 to
these cultures led to only a modest increase in the ability of IFN-
to prime cells for further IFN- production (Fig. 6, k–o). Although the level of expression of the IL-18R on
the primed 5CC7 cells did not correlate with responsiveness to
IL-12/IL-18, the relatively small increase in the level of expression
of the IL-12R2 seen in the presence of exogenous IFN- did
correlate with enhanced responsiveness to the combination of
IL-12/IL-18. T cells primed with IFN- appear, in part, to be like
classical IL-12-primed Th1 cells in that they are capable of secreting
IFN- via both TCR and cytokine stimulation; however, they do not
respond to IL-12 or IL-18 alone presumably because the levels of
receptor expression are below some critical threshold.
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failed to produce IFN- after restimulation
with either anti-CD3 (Fig. 7, a and f) or the combination of IL-12 and IL-18
(Fig. 7, e and j), despite the expression
of higher levels of the IL-12R2 than those expressed on similarly
primed 5CC7 cells (Fig. 5, k and h). Although the
percentage of IL-4 producers was similar between the two groups (Fig. 7, a and f, 40 vs 53%), the addition of
anti-IL-4 to DO11.10 cultures primed with IFN- and
anti-IL-12 led to a striking increase in the number of IFN-
producers (Fig. 7, k and o). Although the level
of expression of the IL-18R was enhanced in the absence of IL-4, the
level of the IL-12R2 was not enhanced. These data demonstrate that
one critical factor that allows IFN- to induce Th1 differentiation
in the absence of IL-12 is the absence of IL-4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 and IL-18R, while these receptors are
absent from differentiated Th2 cells. We and others (7, 15) recently reported that the ability of IL-12 to increase
IL-18R expression was severely impaired in
IFN--/- mice. Although the addition of
IFN- alone had no effect on IL-18R expression, the addition of
both IL-12 and IFN- restored IL-12-enhanced IL-18R expression.
The purpose of this study was to determine the role of IFN- in
regulation of IL-18R and IL-12R2 expression during Th1
differentiation
We have clearly demonstrated that IFN- is not required for
IL-12-induced up-regulation of IL-18R or IL-12R2 expression.
Studies with T cells from 5CC7 TCR-transgenic mice demonstrated
that in the absence of IL-4, IL-12 alone was sufficient and IFN- was
dispensable for both IL-12R2 and IL-18R expression and Th1
differentiation. In the presence of IL-4, however, IFN- was required
for IL-12-induced Th1 differentiation and functioned to limit the
negative effects of IL-4 on both IL-18R and IL-12R2.
Neutralization of IFN- during priming of DO11.10 with IL-12 led to a
marked down-regulation of both IL-18R and IL-12R2, while
simultaneous neutralization of IL-4 and IFN- enhanced IL-18R
expression to levels higher than those seen with IL-12 alone and almost
completely restored IL-12R2 expression. The fact that anti-IL-4
did not completely restore IL-12R2 expression in DO11.10 cultures is
not surprising since there are several reports of IL-4-independent
down-regulation of IL-12R2 in BALB/c mice (25, 26, 27). As
IFN- does not enhance IL-18R expression, we conclude from this
study that its effects on IL-18R expression are to primarily limit
the negative effects of IL-4. Although IFN- also antagonizes the
negative effects of IL-4 on IL-12R2 expression, IFN- can also
directly enhance and/or maintain IL-12R2 expression.
Although high levels of expression of IL-18R and IL-12R2 have
been proposed as markers for Th1 cells (2, 4, 28), our
studies have also demonstrated that a rather poor correlation exists
between the levels of expression of these receptors and the Th1
differentiation status of CD4+ T cells. After 1
wk of priming with Ag and IL-12, T cells from 5CC7 and DO11.10 mice
expressed identical levels of IL-18R and IL-12R2 (cf Fig. 2, a and g with d and j). Yet,
5CC7 T cells had differentiated into Th1 cells as demonstrated by high
percentages of cells producing IFN- when stimulated with immobilized
anti-CD3 or with the combination of IL-12 and IL-18. Smaller, but
highly significant, numbers of cells produced IFN- when stimulated
with IL-12 or IL-18 alone. In contrast, DO11.10 T cells were a mixed
population that produced both IL-4 and IFN- in response to
anti-CD3, responded weakly to the combination of IL-12 and IL-18,
and failed to respond to IL-12 or IL-18 alone. It is therefore
impossible to correlate Th1 differentiation with the levels of
expression of the IL-18R and IL-12R2 as measured by FACS.
Although the high concentrations of IL-12 used in the priming cultures
can overcome most of the negative effects of IL-4 on expression of the
IL-18R and IL-12R2, the levels of endogenous IL-4 produced by the
DO11.10 T cells dominate over the added IL-12 in directing T cell
differentiation. It is possible that IL-4 may exert inhibitory effects
on IL-12 signaling that are distinct from its effects on IL-12R2
expression (29, 30).
We have also examined the effects of IFN- on expression of the
IL-18R and IL-12R2 and Th1 differentiation in the absence of
IL-12. The concept of IL-12-independent Th1 differentiation has
remained controversial. Although some reports have shown that IFN-
is capable of driving Th1 differentiation independent of IL-12
(18, 19, 20, 21, 22, 23, 31), others do not support such a role for
IFN- (24). Although IL-12-/-
mice exhibit a defect in their ability to mount Th1 responses
(1), polarized CD4+ Th1 cells
secreting high levels of IFN- can be detected in
IL-12-/- mice in response to certain viral
infections (32, 33). STAT-4-/-
mice exhibit a defect in their ability to mount a Th1 response as the
result of disruption of the major IL-12 signaling pathway. However,
simultaneous disruption of the STAT-6 gene
(STAT-4, STAT-6-/- mice) leads to
the development of IFN--producing T cells (31). TGF-
has also been shown to induce the development of IFN--producing T
cells independent of IL-12 (34), reportedly via inhibition
of STAT-6/GATA-3 (35). IFN- has been shown to induce
T-bet (T box expressed in T cells) expression, an important
transcription factor expressed early by Th1 cells and which regulates
IFN- production (19, 36, 37). The IL-12-independent,
IFN--dependent induction of t-bet may thus drive Th1
differentiation.
T cells from 5CC7 and DO11.10 mice up-regulated their expression of the
IL-12R2 chain, but not the IL-18R, in the absence of IL-12 and in
the presence of IFN-. Under these conditions, IFN- was acting
directly on the responder T cells to induce or maintain IL-12R2
expression as no further augmentation of IL-12R2 expression was seen
when anti-IL-4 was added. As was seen with T cells primed in the
presence of IL-12, neutralization of IL-4 led to enhancement of
IL-18R expression on DO11.10 T cells. Although the levels of
IL-18R and IL-12R2 expression were considerably lower than T
cells primed in the presence of IL-12, T cells from 5CC7 mice appeared
to have differentiated into Th1 cells as 50–60% of the primed cells
produced IFN- in response to anti-CD3 and the combination of
IL-12 and IL-18, but not either cytokine alone. It appears that very
small differences in the expression of the IL-12R2 can determine
IL-12 responsiveness. 5CC7 T cells primed in the absence of endogenous
or exogenous IFN- responded poorly (5 and 16.5%, respectively) to
IL-12/IL-18, whereas T cells primed in the presence of IFN-
responded vigorously (63%). The ability of IFN- to induce Th1
differentiation was markedly compromised by the presence of IL-4 and
this may account for the failure of some studies to see the
Th1-inductive effects of IFN-.
An important distinction must be made when comparing Th1 priming by
IFN- with priming by IL-12. IFN- can increase IL-12R2 on
DO11.10 cells in the presence of IL-4 to levels higher than those
expressed on 5CC7 cells, but it cannot prevent the negative effects of
IL-4 on IL-18R. Nevertheless, IFN--primed DO11.10 T cells
functionally resembled IL-12-primed DO11.10 cells as they failed to
produce IFN- in response to anti-CD3 or the combination of IL-12
and IL-18. In the absence of IL-4, IFN--induced Th1 priming of
DO11.10 was as efficient as IFN--induced priming of 5CC7 T cells.
The most likely explanation for this result is that the endogenous
levels of IL-4 produced by DO11.10 T cells dominate over the high
concentrations of exogenous IFN- used in these studies and
inhibit Th1 differentiation, but not IL-12R2 expression.
Our results also have a number of implications for the generation of
potent Th1 responses in vivo during vaccination. The use of adjuvants
such as CpG oligonucleotides that are potent IL-12 inducers has been
advocated for enhancement of the induction of a Th1 response
(38). Our findings suggest that concomitant inhibition of
IL-4 production and/or action might represent a very useful adjunct to
such an approach. Lastly, it remains possible that IFN- itself might
be used to enhance the priming of a Th1 response in situations where
the administration of IL-12-inducing adjuvants may have adverse
systemic effects.
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Footnotes |
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2 Abbreviations used in this paper: RAG, recombination activation gene; MFI, mean fluorescence intensity.
Received for publication March 5, 2002. Accepted for publication April 15, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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