Recombinant interleukin 12 (IL-12) can profoundly suppress cellular immune responses in
mice. To define the underlying mechanism, recombinant murine (rm)IL-12 was given to
C57BL/6 mice undergoing alloimmunization and found to transiently but profoundly suppress in vivo and in vitro allogeneic responses and in vitro splenocyte mitogenic responses. Use of
neutralizing antibodies and genetically deficient mice showed that IFN-
(but not TNF-
)
mediated rmIL-12-induced immune suppression. Splenocyte fractionation studies revealed that
adherent cells from rmIL-12-treated mice suppressed the mitogenic response of normal nonadherent cells to concanavalin A and IL-2. Addition of an inhibitor of nitric oxide synthase
(NOS) restored mitogenic responses, and inducible (i)NOS
/ mice were not immunosuppressed by rmIL-12. These results support the view that suppression of T cell responses is due
to NO produced by macrophages responding to the high levels of IFN- induced by rmIL-12.
When a NOS inhibitor was given with rmIL-12 during vaccination of A/J mice with irradiated SCK tumor cells, immunosuppression was averted and the extent of rmIL-12's ability to
enhance induction of protective antitumor immunity was revealed. This demonstrates that
rmIL-12 is an effective vaccine adjuvant whose efficacy may be masked by its transient immunosuppressive effect.
Key words:
 |
Introduction |
Interleukin (IL)-12 is an immunoregulatory cytokine with
potent antitumor, antiviral, and antimicrobial effects (1, 2). Many of its activities are attributable to its ability to induce Th1 CD4+ T cell differentiation, CD8+ T cell cytotoxicity,
and NK cell activation. IL-12 is proinflammatory through its
ability to induce production of IFN-, TNF-, GM-CSF, and other cytokines by T and NK cells. A particularly important mediator of IL-12 effect is IFN-, which, among
other actions, activates macrophages and induces the production of nitric oxide (NO).1 IFN- also acts on many
other types of cells, including tumor cells, and its ability to
upregulate MHC expression, slow cell proliferation, and
inhibit angiogenesis (3) may contribute to IL-12's antitumor effects.
Therapeutic use of rIL-12 can be accompanied by severe
toxicities. Dose- and schedule-dependent toxicities have
been seen during clinical trials (6, 7) and in mice (8). Administration of recombinant murine (rm)IL-12 during lymphocytic choriomeningitis virus (LCMV) infection in mice
has been associated with adverse immunological effects
manifest by higher viral loads, decreased antiviral CTL activity, and poorer outcome (9). While studying the effects
of rmIL-12 during vaccination with genetically modified tumor cells, we identified a dose-dependent transient suppression of the immune response that was accompanied by
suppressed in vitro splenocyte responses to T cell mitogens
(10). These effects of high-dose rmIL-12 were generalized,
affecting responses to allogeneic vaccination and splenocyte
mitogenic responses in naive mice of many strains, and
appeared to result from impairment of immune effector
function rather than failure to induce immunity. Studies
described in this manuscript examine the mechanism of rmIL-12 immune suppression that appears to result from
inhibition of T cell proliferation by NO generated by macrophages activated by the IFN- produced in response to
rmIL-12. When inhibitors of NO generation were given
with rmIL-12 during vaccination, it prevented immune
suppression and allowed the potent vaccine adjuvant effect
of rmIL-12 to be fully revealed.
| |
Materials and Methods |
Mice and Cell Lines.
5-8-wk-old female C57BL/6 (H-2b)
mice were purchased from Harlan-Sprague-Dawley (Indianapolis, IN). IFN-/ and iNOS/ C57BL/6 mice and wild-type
controls were purchased from The Jackson Laboratory (Bar Harbor, ME). IFN-R1/ C57BL/6 × SV129 mice and controls
stemmed from breeding pairs that were gifts from Dr. Michel
Aguet (University of Zurich, Zurich, Switzerland; reference 11).
TNF- p55 and p75 receptor/ C57BL/6 × SV129 mice and
controls were provided by Dr. Philip Scott and Michelle Nashleanas (University of Pennsylvania, Philadelphia, PA) with permission from Genentech (South San Francisco, CA) and Dr.
Horst Bluethmann of Roche Pharmaceuticals (Basel, Switzerland; references 12, 13). 5-8-wk-old female A/J (H-2a) mice were purchased from The Jackson Laboratory. HKB cells were established
from a spontaneous tumor that arose in an aged, unmanipulated female A/J mouse and are maintained in RPMI with 10% FCS
and penicillin/streptomycin. They are MHC class I+ and are nontumorigenic in A/J mice when 106 cells are injected subcutaneously. SCK murine mammary carcinoma cells and SCK tumor
cells engineered to secrete GM-CSF (SCK.GM cells) were maintained as previously described (10).
Immunization and Challenge.
Immunization of C57BL/6 mice
with HKB cells or A/J mice with SCK or SCK.GM cells was
performed with cells suspended in PBS at 107 trypan blue-
excluding cells/ml. Cells were irradiated with 6,000 rads from a
137Cs source, and mice were vaccinated with 106 cells subcutaneously (day 0). Mice given rmIL-12 (Genetics Institute, Andover,
MA) were injected intraperitoneally with 500 ng/day (all strains
except A/J) or 250 ng/day (A/J strain) on days 0-4 and 7-11 (10 injections) unless otherwise noted, whereas control mice received
PBS injections. Where indicated, vaccinated mice received 1 mg
of anti-IFN- (XMG6) or anti-TNF- (XT22) mAbs on days
1, 3, and 7. Where indicated, mice received 0.2 mg N-nitro-
L-arginine methyl ester (L-NAME) or N-nitro-D-arginine methyl
ester (D-NAME) on days 0-4 and 7-11. Vaccinated and naive A/J
mice were challenged with 2.5 × 104 trypan blue-excluding
SCK cells to assay for the presence of tumor immunity.
Mitogen and Alloantigen Stimulation of Splenocytes.
In vitro mitogenic stimulation of splenocytes with 2.5 µg/ml Con A or 100 U/ml rmIL-2 was performed as previously described (10). Proliferative responses to allogeneic antigens (MLR) were measured
when splenocytes were stimulated with 105 mitomycin C-treated
A/J splenocytes. Splenocyte fractionation was performed by allowing 105 splenocytes to adhere for 90 min in 96-well plates after which the nonadherent cells were removed and cocultured
with adherent cells from different wells for assay. When added,
antibodies (XMG6 for IFN-, XT22 for TNF-, AE5 for IL-10,
and C17.8 for IL-12) were used at 10 µg/ml final concentration
and N-methyl-L-arginine (L-NMMA) and N-methyl-D-arginine
(D-NMMA) were used at 500 µM final concentration (Sigma
Chemical Co., St. Louis, MO). After 72 h of exposure to mitogen, cultures were pulsed with 1 µCi [3H]thymidine for 16 h,
cells were harvested and 3H incorporation was measured by scintillation counting. Supernatants from cultures assayed for IFN-
by RIA (using antibodies AN18 and XMG6; reference 14) were
harvested 24 or 72 h after stimulation.
Delayed Type Hypersensitivity Assessment.
For assessment of allogeneic delayed type hypersensitivity (DTH) responses, mice
were injected with 50 µl PBS containing 106 irradiated SCK cells
in the right footpad and with 50 µl PBS in the left footpad. Footpad thickness measurements were taken just before injection and
24 h later using a Starrett pocket gauge (Athol, MA). Data are
presented as the difference in footpad swelling induced by SCK
cells and by PBS.
Measurement of Nitrite Production.
NO production measured as
nitrite concentration in stimulated cell culture supernatants was
measured by the Greiss assay (15). Supernatant (100 µl) was
added to 96-well plates; 100 µl of a 1:1 mixture of 1% sulfanilamide dihydrochloride in 2.5% H3PO4 and 0.1% naphthylethylenediamine dihydrochloride in 2.5% H3PO4 was then added to
samples. Plates were incubated at room temperature for 10 min
and A540 was determined using a microplate reader with reference to sodium nitrite standard curves. SNAP (S-nitroso- N-acetyl-penicillamine; Sigma Chemical Co.) was used as an acellular source of NO in cultures.
| |
Results |
IFN- Mediates rmIL-12-induced Immunosuppression.
In
studies of rmIL-12 effects in A/J mice on vaccination with
irradiated SCK tumor cells, we previously had found that
high doses of the cytokine transiently suppressed tumor
protection in vivo and proliferative responses of splenocytes
to T cell mitogens in vitro (10). We also had found that
rmIL-12 exerted similar effects (suppressed in vivo DTH
and in vitro mitogenic and alloproliferative responses) in
C57BL/6 (H-2b) mice vaccinated with irradiated allogeneic HKB (H-2a) cells. Since most effects of IL-12, both
beneficial and inhibitory, are mediated by IFN-, and because TNF- is implicated in rmIL-12-suppressive effects
during LCMV infection (16), we examined the roles of these two cytokines in rmIL-12-induced suppression of
responses to alloimmunization. Studies using neutralizing
mAbs in C57BL/6 mice given irradiated HKB cells and a
course of rmIL-12 on days 0-4 and 7-11 showed that
XMG6 (anti-IFN-) completely restored DTH responses,
XT22 (anti-TNF-) only partially restored responses, and
XMG6 + XT22 restored responses no better than XMG6
alone (data not shown). Although these results suggested
that IFN- is crucial for rmIL-12 suppression of immune
responses and that the role of TNF- is less certain, we examined the role of these cytokines more definitively by
testing the effect of rmIL-12 on alloimmunization in mice
genetically deficient for these cytokines or their receptors. After vaccination with HKB cells, a course of rmIL-12-
suppressed DTH responses to background levels in wild-type C57BL/6 mice but had no suppressive effect in IFN-/ C57BL/6 mice (Fig. 1 A). Similarly, rmIL-12 suppressed DTH responses in wild-type but not in IFN-R1/
C57BL/6 × SV129 mice vaccinated with HKB cells (data
not shown). From these results, we conclude that IFN- is
crucial for rmIL-12-induced immunosuppression. To examine the role of TNF-, we studied C57BL/6 × SV129
mice deficient for both the p55 and p75 TNF- receptors. HKB-vaccinated TNFR/ mice treated with rmIL-12
had depressed DTH responses like wild-type C57BL/6 × SV129 mice (Fig. 1 B), indicating that TNF- responses were dispensable for rmIL-12 immune suppression. However, DTH responses without rmIL-12 were lower in the
TNFR/ mice, suggesting that TNF- responses might
be necessary for maximal responses.
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
IFN- but not TNF- is required for rmIL-12-induced immunosuppression. (A) C57BL/6 mice genetically deficient for IFN- and
wild-type controls were vaccinated with 106 irradiated HKB cells and
given 500 ng rmIL-12 or PBS on days 0-4 and 7-11 or left unvaccinated.
They were challenged with 106 irradiated SCK cells in the right foot,
with PBS control injection in the left foot on day 12. Mean footpad
swelling (+ SE) is presented for unvaccinated mice (white bars; two mice)
and vaccinated mice given PBS (black bars; four mice) or rmIL-12 (gray
bars; four mice). (B) C57BL/6 × SV129 mice deficient for both the p55
and p75 TNF- receptors and control mice were vaccinated with 106 irradiated HKB cells and given 500 ng rmIL-12 (gray bars) or PBS (black
bars) on days 0-4 and 7-11. DTH assessment was performed on day 12 as
described and mean footpad swelling (+ SE, three mice per group) is presented.
|
|
In our earlier studies, suppression of in vitro splenocyte
mitogenic responses correlated well with suppression of in
vivo immune responses. This correlation held up in studies
of IFN-/ and TNFR/ mice: Con A, IL-2, and allogeneic stimulation of splenocytes from rmIL-12-treated
IFN-/ mice resulted in normal proliferative responses,
whereas responses of splenocytes from rmIL-12-treated
TNFR/ mice were suppressed (data not shown).
Adherent Cells Mediate IL-12-induced Suppression of Splenocyte Mitogenesis.
To identify the cell population responsible for the suppressed splenocyte mitogenic responses,
splenocytes from C57BL/6 mice given rmIL-12 or PBS
were fractionated by adherence to plastic and reconstituted
in various combinations of adherent and nonadherent cells
before stimulation with Con A or IL-2. As expected, cultures of adherent and nonadherent splenocytes from rmIL-12-treated mice had suppressed mitogenic responses compared with cultures of adherent and nonadherent cells from
PBS-treated mice (Fig. 2). Nonadherent cells from rmIL-12-treated mice cocultured with adherent cells from PBS-treated mice had normal mitogenic responses, indicating that mitogenesis of T cells from rmIL-12-treated mice is
not intrinsically or irreversibly defective. When nonadherent cells from control mice were mixed with adherent cells
from rmIL-12-treated mice, proliferative responses were
severely impaired whether the stimulus was Con A, IL-2,
or alloantigen (Fig. 2). These results indicate that T cells
from rmIL-12-treated mice can respond to mitogens and
generate an antigen-specific mitogenic response in the
presence of adherent cells from normal mice, and that adherent cells are largely responsible for the defect after rmIL-12 therapy.
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2.
Adherent cells from rmIL-12-treated mice confer immunosuppression. Splenocytes from rmIL-12- and PBS-treated C57BL/6 mice
were allowed to adhere to 96-well plates for 90 min. Nonadherent cells
were overlaid on adherent cells and cocultures were stimulated. Data are
from one of five experiments that produced similar results and are shown
as the percentage (+ SE) of stimulation elicited in cultures of adherent
and nonadherent cells from spleens of PBS-treated mice. Cocultures were
stimulated with either 2.5 µg/ml Con A (black bars), 100 U/ml IL-2-
treated (hatched bars) or 105 mitomycin C-treated A/J (H-2a) splenocytes
(stippled bars). Data are from triplicate determinations and are significantly
different from control mixtures (adherent and nonadherent cells from
PBS treated mice, P < 0.05) where indicated (*).
|
|
IFN- was readily detected by RIA in cocultures of adherent cells from rmIL-12-treated mice and nonadherent
cells from PBS-treated mice at both 24 and 72 h after stimulation with Con A, IL-2, or alloantigen (data not shown).
Addition of anti-IFN- antibody to these cocultures restored mitogenic responses, whereas addition of antibodies
to IL-12, IL-10, or TNF- had little effect (Fig. 3 A).
These antibodies did not suppress mitogenic responses in cocultures containing adherent and nonadherent splenocytes from PBS-treated mice (data not shown), indicating
that they had no intrinsic suppressive effects that could
have shrouded beneficial effects of cytokine neutralization.
These data support the results of in vivo experiments showing
a critical role for IFN- in rmIL-12 immune suppression.
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
IFN--induced NO mediates rmIL-12-induced immunosuppression. Cocultures were established from splenic adherent cells and nonadherent cells of PBS-treated mice or from the adherent cells of rmIL-12-treated mice and nonadherent cells from PBS-treated mice. (A) Antibodies
XMG6 (to IFN-), XT22 (to TNF-), AE5 (to IL-10), and C17.8 (to IL-12) were added to a final concentration of 10 mg/ml in cocultures containing
adherent cells from spleens of rmIL-12-treated mice. Data from Con A- (black bars) and IL-2-stimulated (hatched bars) cultures are from triplicate determinations and are significantly different from control cocultures (P < 0.05) where indicated (*). (B) L-NMMA and D-NMMA were added to the coculture
containing adherent cells from rmIL-12-treated mice at a final concentration of 500 mM. Data from Con A- (black bars) and IL-2-stimulated (hatched bars)
cultures are from triplicate determinations and are significantly different from control cocultures (P < 0.05) where indicated (*).
|
|
Adherent Cell-derived NO Inhibits Proliferative and Immune
Responses.
Knowing that adherent cells are important for
rmIL-12 suppression of in vitro mitogenic and immunological responses and that IFN- is necessary for this effect,
we considered that NO from activated macrophages might
mediate suppression. To examine this possibility, we added
an inhibitor of iNOS, L-NMMA, to cocultures of adherent
cells from rmIL-12-treated mice and nonadherent cells from control mice. We found that it reduced NO levels in
the culture supernatant by 58 and 94% in two independent
measurements and restored mitogenesis (Fig. 3 B) when
compared with addition of D-NMMA, a noninhibitory
isoform. If secreted NO is responsible for suppression of
mitogenesis, an acellular source of NO should have a similar effect. We added SNAP (S-nitroso-N-acetyl-penicillamine), an NO donor, to splenocytes from HKB-vaccinated C57BL/6 mice and found that cultures with NO
levels as low as 2.6-3.5 mM inhibited mitogenic responses
83-98%. Together, these data suggest that adherent splenocytes (probably macrophages) activated by rmIL-12 treatment to secrete NO are responsible for impaired T cell mitogenic responses.
The ability of an iNOS inhibitor to reverse rmIL-12-
induced suppression of mitogenesis in vitro suggested that
mice lacking iNOS might be resistant to the immunosuppressive effects of rmIL-12. iNOS/ and wild-type
C57BL/6 mice were vaccinated with irradiated HKB cells
and given a course of rmIL-12 or PBS. iNOS/ mice receiving rmIL-12 had DTH responses that were at least as great as those of PBS-treated iNOS/ and wild-type mice
and that were substantially higher than those of wild-type
mice given rmIL-12 (Fig. 4 A). Although rmIL-12 induced splenomegaly in iNOS/ mice as in wild-type mice (17),
their splenocytes had proliferative responses like those of
splenocytes from control mice after in vitro stimulation
with mitogens or alloantigens (Fig. 4 B). Together, these data
show that macrophage-derived NO is essential for rmIL-12- induced immunosuppression, whereas rmIL-12-induced
splenomegaly and associated pathological changes are not.
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
iNOS/ mice do not experience rmIL-12-induced immunosuppression. iNOS/ and wild-type C57BL/6 mice were vaccinated
with irradiated HKB cells and received rmIL-12 (gray bars) or PBS (black
bars) injections. (A) Footpad injections for DTH assessment were performed on day 12, and swelling 24 h later is presented as the mean (+ SE)
from three mice in each treatment group. (B) Mitogenic (Con A, black
bars; IL-2, hatched bars) and allogeneic stimulation (stippled bars) of splenocytes was performed as described in the legends to Figs. 2 and 3.
|
|
An Inhibitor of NO Generation Prevents rmIL-12 Suppression of Vaccine Efficacy and Reveals the Extent of rmIL-12 Adjuvant Activity.
Finding that rmIL-12 does not suppress
allogeneic responses in iNOS/ mice, we investigated
whether iNOS inhibitors would prevent immunosuppression in mice given rmIL-12 during tumor cell vaccination.
Previously, we showed that vaccinating A/J mice with irradiated SCK.GM cells was highly protective, but that administration of rmIL-12 abrogated protection 2 wk after
vaccination (but had no deleterious effect 4 wk after vaccination) (18). We gave A/J mice undergoing SCK.GM vaccination and rmIL-12 treatment either L-NAME, an
inhibitor of iNOS that acts similarly to L-NMMA, or
D-NAME, the inactive isoform. As expected, SCK.GM
vaccination protected the great majority of mice from
tumor cell challenge 2 wk after vaccination (19% developed
tumors), and rmIL-12 severely impaired this protection (94% developed tumors). L-NAME, but not D-NAME,
prevented this impairment (Fig. 5; the difference in tumorigenesis between rmIL-12-treated mice given L-NAME
versus either D-NAME or nothing is significant at P < 0.05). In mice not treated with rmIL-12, L-NAME, and
D-NAME had no effect on SCK.GM-induced protection
(data not shown), showing that L-NAME acts by preventing rmIL-12 suppression of SCK.GM vaccine efficacy.
rmIL-12 also impairs tumor protection in A/J mice with established SCK immunity if it is given just before tumor
cell rechallenge (18). We found that L-NAME but not
D-NAME given with the rmIL-12 prevented this impairment of immune rejection: only 25% of rmIL-12-treated
mice given L-NAME developed tumors, whereas 75% of
rmIL-12-treated mice given D-NAME developed tumors (data not shown). Thus, L-NAME prevents rmIL-12 suppression of established antitumor immune responses. In
these studies, levels of NO were not consistently measurable in mice given rmIL-12 (at or below the sensitivity
limits of the assay), so lower levels in mice also given
L-NAME could not be demonstrated.
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibition of iNOS function reverses rmIL-12 suppression of
immunologic protection. Female A/J mice were vaccinated with 106 irradiated SCK.GM cells and received either PBS (gray lines), rmIL-12 (solid
black lines), rmIL-12 + L-NAME (hatched black lines), or rmIL-12 and
D-NAME (double dashed black lines) on days 0-4 and 7-11. Mice were
challenged 14 d after vaccination with 2.5 × 104 SCK cells in the opposite flank. Tumorigenesis was scored daily. The * designates statistical differences at P < 0.05 for rmIL-12- and L-NAME-treated mice versus
rmIL-12- and D-NAME-treated mice and rmIL-12- and L-NAME-
treated mice versus rmIL-12-treated mice. Data are compiled from two
separate experiments that produced consistent results (15-17 mice per
group total).
|
|
Previously, we had shown that vaccination of A/J mice
with irradiated wild-type SCK cells protected only ~10%
of mice from a tumor cell challenge, i.e., SCK cells are intrinsically poorly immunogenic (18). Giving rmIL-12 with
vaccination did not improve protection when mice were
challenged 14 d after vaccination but did improve protection when they were challenged at 28 d. Since an iNOS
inhibitor prevented transient immunosuppression by rmIL-12, we asked whether its use might reveal rmIL-12's effectiveness as a vaccine adjuvant at the earlier time point. As
shown in Fig. 6, only 38% of mice given L-NAME with
irradiated SCK cells and rmIL-12 developed tumors when
they were challenged on day 14, whereas 75% of mice
given D-NAME developed tumors. This indicated that
rmIL-12 improves SCK cell vaccine efficacy markedly and
rapidly but that the improvement at day 14 was obscured
by rmIL-12's immunosuppressive effect. The level of protection with L-NAME at 14 d (62%) was similar to the
level of protection seen at 28 d in SCK-vaccinated mice
given rmIL-12 alone (75%) or rmIL-12 with L-NAME
(50%) or D-NAME (50%), indicating that use of L-NAME
did not impair long-term protection afforded by rmIL-12
and SCK vaccination.
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
Inhibition of iNOS function reveals rmIL-12 adjuvant effects. Female A/J mice (8 mice per group) were vaccinated with 106 irradiated SCK cells and received either PBS (gray lines), rmIL-12 (solid black
lines), rmIL-12 + L-NAME (hatched black lines), or rmIL-12 and D-NAME
(double dashed black lines) on days 0-4 and 7-11. Mice were challenged 14 d
after vaccination with 2.5 × 104 SCK cells in the opposite flank. Tumorigenesis was scored daily.
|
|
Use of an iNOS inhibitor to alleviate rmIL-12 immune
suppression would be problematic if it reduces the antitumor efficacy of the cytokine, an important consideration
since the antitumor effects of rmIL-12 are diverse and some
are not immunological (3, 4). Thus, we tested the effect of
L-NAME on the antitumor activity of rmIL-12 against
SCK tumors. Similar to what was seen in the past (19),
rmIL-12 injections started on the day of SCK cell injection
delayed tumor appearance by about 5 d (Fig. 7; medians of
9 d to tumor appearance without rmIL-12 and 14 d to tumor appearance with rmIL-12). Mice given L-NAME with
their rmIL-12 developed tumors after a median of 17 d
which was 8 d later than in untreated mice and 3 d later
than in mice given rmIL-12 alone or with D-NAME.
L-NAME given without rmIL-12 had no effect on SCK
tumorigenesis (data not shown). Together, these data indicate that iNOS inhibitors can prevent rmIL-12 immune
suppression and reveal its effectiveness as a vaccine adjuvant
without adversely affecting rmIL-12 antitumor activity.
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 7.
Inhibition of iNOS function enhances rmIL-12-induced delay of SCK tumorigenesis. Female A/J mice were injected with 2.5 × 104
SCK cells and either PBS (gray lines), rmIL-12 (solid black lines), rmIL-12 + L-NAME (hatched black lines), or rmIL-12 and D-NAME (double dashed
black lines) on days 0-4 and 7-11. Tumorigenesis was scored daily. Data
are compiled from two separate experiments that produced consistent results (11-12 mice per group total).
|
|
| |
Discussion |
rIL-12 is a proinflammatory cytokine with potentially
useful effects against tumors and infectious agents. However, recently it has been shown to suppress cellular immune responses in mice (10). To understand the mechanisms underlying this suppression without the confounding
influence of tumor burden or host infection with pathogens, we studied the effects of rmIL-12 on allogeneic immune responses. In vivo and in vitro evidence indicates
that alloimmunization is transiently but profoundly suppressed by high-dose rmIL-12. It seems to impair immune
effector mechanisms because responses in mice with established immunity are also suppressed, and may not impair
induction of immunity because rmIL-12 given during tumor cell vaccination provides enhanced protective antitumor immunity after the period of immunosuppression.
rmIL-12 impairment of cellular immune responses is consistently associated with and likely due to impaired T cell
mitogenic responses. In this study, we provide in vivo and
in vitro evidence that anti-IFN-, but not antibodies to
other cytokines, prevents rmIL-12-induced suppression. The importance of IFN- was confirmed by studies in
IFN-/ and IFN-R1/ mice that are not immunosuppressed by rmIL-12. Studies using splenocyte fractionation,
an inhibitor of NO generation, and iNOS/ mice revealed that adherent cells of the spleen, through stimulated production of NO by iNOS, are responsible for suppressing T cell mitogenesis in vitro and cellular immune responses in vivo. Having identified the probable mechanism, we used an iNOS inhibitor with rmIL-12 during
tumor cell vaccination that prevented immune suppression and allowed the full extent of rmIL-12 adjuvant activity to
be revealed.
These observations led us to propose that events leading
to immune suppression by high-dose rmIL-12 administration are initiated by its induction of IFN- production
by host lymphocytes. Levels of IFN- production high
enough to activate macrophages and induce iNOS activity
generate levels of NO that impair the proliferation of T
cells in response to mitogens. That adherent cells rather than T cells are primarily responsible for the pathogenesis
of rmIL-12 immune suppression is supported by the fact
that T cells from the spleens of rmIL-12-treated mice are
normally mitogenic when cocultured with adherent cells
from normal mice or from rmIL-12-treated iNOS/ mice.
Impairment is transient presumably because T cell proliferative responses recover as IFN- production and consequent macrophage activation wanes after completion of
rmIL-12 therapy. How NO impairs T cell proliferative responses is uncertain, but a recent study indicates that NO
allows initial T cell activation up to and including IL-2
receptor expression and IL-2 production but impedes effective signal transduction by reversibly inhibiting JAK3
and STAT5 phosphorylation (20). Other studies have suggested that NO may induce apoptosis in conjunction with
drug therapy or infection (21, 22).
Our identification of NO as a mediator of rmIL-12-
induced immunosuppression is consistent with its known
activities. NO, a key component of host defense mechanisms against invading pathogens, is produced by iNOS
in macrophages activated by IFN- and other cytokines.
Although other NOS (nNOS or NOS1 and eNOS or
NOS3) also generate NO, iNOS (or NOS2) is the high-output source of NO, and phagocytic cells are the major
source of iNOS during inflammation (23). Impaired splenocyte mitogenesis during Salmonella typhimurium, Trypanosoma cruzi, Toxoplasma gondii, and Listeria monocytogenes infections is due to NO production associated with high
levels of IFN-, endogenous IL-12, and/or other proinflammatory cytokines (24). When splenocytes from
these mice are fractionated, the adherent population containing macrophages and producing NO appears to suppress splenic T cell mitogenic responses, and this suppression is reversed by inhibitors of iNOS (28). Thus, diverse
processes that induce NO production by macrophages (24,
25, 29) can impair T cell proliferative responses. To this list
can be added high-dose rmIL-12 that, through induction
of high levels of IFN- and in the absence of additional inflammatory stimuli, sufficiently activates macrophages to
engender immunosuppression.
Although NO is a mediator of impaired T cell mitogenesis, the cytokines that impair mitogenesis or induce NO
production may vary with the circumstance. In studies of
parasite infection, IFN- appears to play a significant role,
and IL-10 plays a minor role in impaired mitogenesis (30).
In studies of rmIL-12-induced immunosuppression during
LCMV infection, TNF- has been implicated as a mediator (16). Although endogenously produced IL-12 is important for controlling Toxoplasma gondii infection, it also suppresses splenocyte mitogenic responses seen during infection (24). IL-12-directed pathways are also implicated in
immune suppression after vaccination with attenuated Salmonella typhimurium (26). These studies raise the point that
agents that naturally induce strong inflammatory responses
may engender immunosuppression through endogenous
production of proinflammatory cytokines. The specific or
spectrum of cytokines involved may vary with the initiating agent, but suppression of antigen-specific immune responses is likely to develop if the result is vigorous activation of phagocytic cells and their production of NO. In our
studies of rmIL-12-induced immune suppression, we were
only able to identify IFN- as an essential mediator, which
may reflect the simplicity or paucity of pathways activated
by rmIL-12 compared with infectious agents. The importance of other proinflammatory cytokines for suppressing T
cell responses associated with infection and giving rmIL-12 during LCMV infection probably reflects a contribution of
these cytokines to the inflammatory response and macrophage activation under these circumstances.
Therapeutic applications of rmIL-12 may benefit from reduction or elimination of its transient immunosuppressive
side effects, and the mechanism elucidated here suggests several potential approaches. Reducing or eliminating rmIL-12
immunosuppression by using lower or fewer doses of the
cytokine is one option. Although this approach may be
beneficial (31), finding the "proper" regimen of rmIL-12 may be involved, and the results may be idiosyncratic, e.g.,
we have not found a lower dose of rmIL-12 that improves
its adjuvant effect during tumor cell vaccination (data not
shown). Inhibiting IFN- action is another alternative for
avoiding rmIL-12 immunosuppression but is an approach
that would be difficult to implement (other than in mice)
and that may be self-defeating because IFN- may be an
essential mediator of rmIL-12 therapeutic effects. The
promising approach tested here, inhibition of NO generation, alleviates rmIL-12 immune suppression and allows its
adjuvant effects to be fully revealed. This finding suggests
that some studies testing rmIL-12 as an immunological adjuvant should be reevaluated since studies performed during the period of immune suppression are unlikely to reflect its true adjuvant potential. Of course, use of inhibitors
of NO generation in conjunction with rmIL-12 must be
examined for its independent effect on desired therapeutic endpoints. As use of the iNOS inhibitor, L-NAME, enhanced and did not impair rmIL-12's limited efficacy against
SCK tumors, our studies do not reveal any problems with its
use. Clearly, it will be important to determine whether recombinant human IL-12 produces similar immune suppressive effects in patients and, if so, whether inhibitors of iNOS
function might be beneficial in this setting.
Address correspondence to William M.F. Lee, 663 Clinical Research Building, 415 Curie Blvd., University
of Pennsylvania, Philadelphia, PA 19104-6140. Phone: 215-898-0258; Fax: 215-573-7912; E-mail: leemingf
@
We thank Genetics Institute for generously supplying the rmIL-12. We thank Dr. Cameron Koch for use of
the cell irradiator, Dr. Laurence Turka for helpful discussions and generous gifts of reagents, and Dr. Philip
Scott and Michelle Nashleanas (University of Pennsylvania School of Veterinary Medicine), Dr. Mark Moore (Genentech), and Dr. Horst Bluethmann (Roche Pharmaceuticals) for p55/p75 knockout mice. We thank Dr.
Denise LaTemple for critical reading of the manuscript.
Supported by funds from the Department of the Army Predoctoral Training Grant DAMD17-94-J-4027 (to H.K.
Koblish) and National Institutes of Health grants AI-42334-01 TMP (to C.A. Hunter), AI-34412, CA 10815, CA
20833, and CA 32898 (to G. Trinchieri), and U01 CA-65805 and R01 CA-77851 (to W.M.F. Lee).
| 1.
|
Brunda, M.J..
1995.
Role of IL-12 as an anti-tumor agent:
current status and future directions.
Res. Immunol.
146:
622-628
[Medline].
|
| 2.
|
Trinchieri, G..
1995.
Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate
resistance and antigen-specific adaptive immunity.
Annu.
Rev. Immunol.
13:
251-276
[Medline].
|
| 3.
|
Voest, E.E.,
B.M. Kenyon,
M.S. O'Reilly,
G. Truitt,
R.J. D'Amato, and
J. Folkman.
1995.
Inhibition of angiogenesis
in vivo by interleukin-12.
J. Natl. Cancer Inst.
87:
581-586
[Abstract/Free Full Text].
|
| 4.
|
Sgadari, C.,
A.L. Angiolillo, and
G. Tosata.
1996.
Inhibition
of angiogenesis by interleukin-12 is mediated by the interferon-inducible protein-10.
Blood.
87:
3877-3882
[Abstract/Free Full Text].
|
| 5.
|
Coughlin, C.M.,
K.E. Salhany,
M. Wysocka,
E. Aruga,
H. Kurzawa,
A.E. Chang,
C.A. Hunter,
J.C. Fox,
G. Trinchieri, and
W.M.F. Lee.
1998.
Interleukin-12 and interleukin-18
synergistically induce murine tumor regression which involves inhibition of angiogenesis.
J. Clin. Invest.
101:
1441-1452
[Medline].
|
| 6.
|
Atkins, M.B.,
M.J. Robertson,
M. Gordon,
M.T. Lotze,
M. DeCoste,
J.S. DuBois,
J. Ritz,
A.B. Sandler,
H.D. Edington,
P.D. Garzone, et al
.
1997.
Phase I evaluation of intravenous
recombinant human interleukin 12 in patients with advanced
malignancies.
Clin. Cancer Res.
3:
409-417
[Abstract].
|
| 7.
|
Cohen, J..
1995.
IL-12 Deaths: explanation and a puzzle.
Science.
270:
908-910
.
|
| 8.
|
Coughlin, C.M.,
M. Wysocka,
G. Trinchieri, and
W.M.F. Lee.
1997.
The effect of interleukin-12 desensitization on the
antitumor efficacy of recombinant interleukin-12.
Cancer Res.
57:
2460-2467
[Abstract/Free Full Text].
|
| 9.
|
Orange, J.S.,
S.F. Wolf, and
C.A. Biron.
1994.
Effects of IL-12 on the response and susceptibility to experimental viral infections.
J. Immunol.
152:
1253-1264
[Abstract].
|
| 10.
|
Kurzawa, H.,
M. Wysocka,
E. Aruga,
A.E. Chang,
G. Trinchieri, and
W.M.F. Lee.
1998.
Interleukin 12 enhances
cellular immune responses to vaccination only after a period
of suppression.
Cancer Res.
58:
491-499
[Abstract/Free Full Text].
|
| 11.
|
Huang, S.,
W. Hendriks,
A. Althage,
S. Hemmi,
S. Bluethmann,
R. Kamijo,
J. Vilhachekcek,
R.M. Zinkernagel, and
M. Aguet.
1993.
Immune response in mice that lack the interferon-gamma receptor.
Science.
259:
1742-1745
[Abstract/Free Full Text].
|
| 12.
|
Kalb, A.,
H. Bluethmann,
M. Moore, and
W. Lesslauer.
1996.
Tumor necrosis factor receptors (Tnfr) in mouse fibroblasts deficient in Tnfr1 or Tnfr2 are signaling competent and
activate the mitogen-activated protein kinase pathway with
differential kinetics.
J. Biol. Chem.
271:
28097-28104
[Abstract/Free Full Text].
|
| 13.
|
Rothe, J.,
W. Lesslauer,
H. Lotscher,
Y. Lang,
P. Koebel,
F. Kontgen,
R. Althage,
R. Zinkernagel, and
H. Bluethmann.
1993.
Mice lacking the tumor necrosis factor receptor I are
resistant to TNF-mediated cytotoxicity but highly susceptible
to infection by Listeria monocytogenes.
Nature.
364:
798-802
[Medline].
|
| 14.
|
Wysocka, M.,
M. Kubin,
L.Q. Vieira,
L. Ozmen,
G. Garotta,
P. Scott, and
G. Trinchieri.
1995.
Interleukin-12 is required
for interferon-gamma production and lethality in lipopolysaccharide-induced shock in mice.
Eur. J. Immunol.
25:
672-676
[Medline].
|
| 15.
|
Green, L.C.,
D.D.A. Wagner,
J. Glogowski,
P.L. Skepper,
J.S. Wishnok, and
S.R. Tannenbaum.
1982.
Analysis of nitrate, nitrite and [15N]nitrate in biological fluids.
Anal. Biochem.
126:
131-138
[Medline].
|
| 16.
|
Orange, J.S.,
T.P. Salazar-Mather,
S.M. Opal,
R.L. Spencer,
A.H. Miller,
B.S. McEwen, and
C.A. Biron.
1995.
Mechanism of interleukin 12-mediated toxicities during experimental viral infections: role of tumor necrosis factor and glucocorticoids.
J. Exp. Med.
181:
901-914
[Abstract/Free Full Text].
|
| 17.
|
Car, B.D.,
V.M. Eng,
B. Schnyder,
M. LeHir,
A.N. Shakov,
G. Woerly,
S. Huang,
M. Aguet,
T.D. Anderson, and
B. Ryffel.
1995.
Role of interferon-gamma in interleukin-12-
induced pathology in mice.
Am. J. Pathol.
147:
1693-1707
[Abstract].
|
| 18.
|
Rakhmilevich, A.L.,
J. Turner,
M.J. Ford,
D. McCabe,
W.H. Sun,
P.M. Sondel,
K. Grota, and
N.S. Yang.
1996.
Gene gun
mediated skin transfection with interleukin 12 genes results in
regression of established primary and metastatic murine tumors.
Proc. Natl. Acad. Sci. USA.
93:
6291-6296
[Abstract/Free Full Text].
|
| 19.
|
Coughlin, C.M.,
M. Wysocka,
H. Kurzawa,
W.M.F. Lee,
G. Trinchieri, and
S.L. Eck.
1995.
B7-1 and interleukin 12 synergistically induce effective antitumor immunity.
Cancer Res.
55:
4980-4987
[Abstract/Free Full Text].
|
| 20.
|
Bingisser, R.M.,
P.A. Tilbrook,
P.G. Holt, and
U.R. Kees.
1998.
Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling
pathway.
J. Immunol.
160:
5729-5734
[Abstract/Free Full Text].
|
| 21.
|
Singhal, P.C.,
P. Sharma,
A.A. Kapasi,
K. Reddy,
N. Franki, and
N. Gibbons.
1998.
Morphine enhances macrophage apoptosis.
J. Immunol.
160:
1886-1893
[Abstract/Free Full Text].
|
| 22.
| Lopes, M.F., V.F. daVeiga, A.R. Santos, M.E.F. Fonseca, and
G.A. DosReis. 1995. Activation-induced CD4+ T cell death
by apoptosis in experimental Chagas' disease. J. Immunol. 154:
744-752.
|
| 23.
|
MacMicking, J.,
Q.W. Xie, and
C. Nathan.
1997.
Nitric oxide and macrophage function.
Annu. Rev. Immunol.
15:
323-350
[Medline].
|
| 24.
|
Hunter, C.A.,
E. Candolfi,
C. Subauste,
V. Van Cleave, and
J.S. Remington.
1995.
Studies on the role of interleukin-12
in acute murine toxoplasmosis.
Immunology.
84:
16-20
[Medline].
|
| 25.
|
Gregory, S.H.,
E.J. Wing,
R.A. Hoffman, and
R.L. Simmons.
1993.
Reactive nitrogen intermediates suppress the
primary immunological response to Listeria.
J. Immunol.
150:
2901-2909
[Abstract].
|
| 26.
|
Schwacha, M.G., and
T.K. Eisenstein.
1997.
Interleukin-12
is critical for induction of nitric oxide-mediated immunosuppression following vaccination of mice with attenuated Salmonella typhimurium.
Infect. Immun.
65:
4897-4903
[Abstract].
|
| 27.
|
Fernandez-Gomez, R.,
S. Esteban,
R. Gomez-Corvera,
K. Zoulika, and
A. Ouaissi.
1998.
Trypanasoma cruzi: Tc52 released protein-induced increased expression of nitric oxide
synthase and nitric oxide production by macrophages.
J. Immunol.
160:
3471-3479
[Abstract/Free Full Text].
|
| 28.
|
Candolfi, E.,
C.A. Hunter,
J.S. Remington, and
R.C. Mulligan.
1994.
Mitogen- and antigen-specific proliferation of T
cells in murine toxoplasmosis is inhibited by reactive nitrogen
intermediates.
Infect. Immun.
62:
1995-2001
[Abstract/Free Full Text].
|
| 29.
|
Fecho, K.,
K.A. Maslonek,
M.E. Coussens-Read,
L.A. Dykstra, and
D.T. Lysle.
1994.
Macrophage-derived nitric oxide
is involved in the depressed concanavalin A responsiveness of
splenic lymphocytes from rats administered morphine in
vivo.
J. Immunol.
152:
5845-5852
[Abstract].
|
| 30.
|
Candolfi, E.,
C.A. Hunter, and
J.S. Remington.
1995.
Roles
of gamma interferon and other cytokines in suppression of
the spleen cell proliferative response to concanavalin A and
toxoplasma antigen during acute toxoplasmosis.
Infect. Immun.
63:
751-756
[Abstract].
|
| 31.
|
Noguchi, Y.,
E.C. Richards,
Y.-T. Chen, and
L.J. Old.
1995.
Influence of interleukin 12 on p53 peptide vaccination
against established Meth A sarcoma.
Proc. Natl. Acad. Sci.
USA.
92:
2219-2223
[Abstract/Free Full Text].
|
Induction of Nitric Oxide
Synthase 2 (iNOS) Activity: Inhibitors of NO Generation
Reveal the Extent of rIL-12 Vaccine Adjuvant Effect
Top
Abstract
Introduction
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
