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*
Department of Veterinary Surgery, University of Osaka Prefecture College of Agriculture, Sakai, Osaka;
Noda Institute for Scientific Research, Noda, Noda-shi, Chiba-ken; and
Department of Veterinary Practice, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan;
§
College of Medicine, Jagiellonian University Krakow, Krakow, Poland; and
¶
Section of Allergy and Clinical Immunology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production
by CS effector T cells stimulated with hapten-conjugated APC, and we
also measured CS ear swelling in vivo. The in vitro responses of T
cells to hapten-APC became absent in tolerized mice, paralleling
impaired in vivo CS responses. Addition of IL-12 to cultures
manifesting this fully established in vitro tolerance completely
restored impaired responses of tolerized T cells. The reversing effects
of IL-12 were not blocked by anti-IFN- mAb, but were blocked by
mAbs against B7-1, more strongly by anti-B7-2, and by both Abs
together. Additional in vivo ear-swelling response experiments
confirmed the reversing effects of IL-12 on established tolerance. To
examine whether the IL-12 effect depended on stimulation of IFN-, we
directly injected IFN- into tolerized mice. This partially mimicked
but did not fully reconstitute the effects of IL-12. In summary, IL-12
abrogation of established tolerance of CS may have been partially due
to endogenous production of IFN-, but appeared mainly due to direct
activation of the tolerized T cells by affecting signaling through
costimulatory molecules B7-1 and B7-2. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 is a heterodimeric Th1-associated cytokine (5, 6) that
consists of disulfide-linked 35- and 40-kDa chains (7) and is produced
by macrophages, B cells, and possibly other cells (8, 9). IL-12
regulates the growth and function of T cells (10, 11) and especially
regulates the development of Th1 cells by stimulating the production of
IFN- (6, 12, 13). Prior results showed that IL-12 promoted
resistance to Leishmania (14, 15) and other parasites (16, 17) and inhibited airway hypersensitivity in an asthma model (18) by
directing immunity toward Th1 and away from Th2 responses, and in other
systems similarly promoted cell-mediated immunity against bacteria,
intracellular parasites, and tumors (19, 20, 21).
An important role of IL-12 also has been reported in induction and elicitation of CS (22, 23). In particular, neutralization of IL-12 by in vivo administration of mAb impaired generation of CS responses, switching the development of CS immunity to induction of tolerance (22). These results suggested that IL-12 also was a positive regulator of the development of CS responses. Furthermore, in vivo injection of IL-12 at the induction reversed UV-induced tolerance (23, 24). Tolerance induced by UV irradiation was reported to be due to IL-10 released from UV-irradiated local keratinocytes (25, 26). Accordingly, anti-IL-10 mAb treatment prevented UV tolerance (25, 27). Furthermore, tolerance of CS was induced by IL-10 administration at induction, suggesting a role for Th2 cytokines in CS tolerance (28). The above studies implied that a cytokine imbalance between Th1 vs Th2 cells was responsible for development of UV-induced tolerance in CS, and IL-12 could prevent the induction of tolerance by antagonizing Th2 cytokines. However, compared with the induction of tolerance, there is little evidence that these inhibitory Th2 cytokines also are involved in the maintenance of established tolerance. Accordingly, most of the effects of IL-12 in CS have been investigated at the time of immunization by injecting or neutralizing IL-12 during primary immune responses (i.e., the induction phase), but not at the stimulation of secondary immune responses (i.e., elicitation phase) of CS. Thus, the effects of IL-12 on this established tolerance that was induced by i.v. administration of high doses of reactive hapten, and the molecular mechanisms that might underlie the reversal of established tolerance by IL-12 were still unclear.
In the current study, we analyzed the underlying molecular mechanisms
in previously induced and established tolerance of CS, including the
effect of IL-12 on a form of established CS tolerance not studied
previously. Fully established CS tolerance was induced by i.v.
administration of high doses of reactive hapten, and then CS
immunization was attempted. Also, we employed newly developed in vitro
assays of T cell proliferation and IFN- production by CS effector T
cells responding to hapten-conjugated APC that paralleled in vivo
tolerance of CS ear-swelling responses. We found that in vitro addition
of IL-12 largely reversed established tolerance of in vitro responses,
and that these effects of IL-12 required both B7-1 and B7-2 molecules,
since Abs to both B7-1 and B7-2 blocked the ability of IL-12 to reverse
CS tolerance. Furthermore, administration of IL-12 in vivo also
reversed this established tolerance of CS. In contrast, IFN-
administration in vivo only partially mimicked the effects of
IL-12.
In summary, these results suggest that IL-12 may have reversed
established CS tolerance by direct activation of tolerized CS effector
T cells in which both B7-1 and B7-2 molecules are required for
production of IFN- and proliferation. Thus, one of the underlying
mechanisms that might be involved in the reversal of tolerance by IL-12
might be a synergistic action of this cytokine with B7-1 and B7-2
molecule-mediated signals in CS effector T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male 6- to 8-wk-old CBA/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME), kept in filter-topped microisolators, and rested 1 to 2 wk before use.
Reagents
Picryl chloride (PCl) (trinitrophenyl (TNP) chloride) was obtained from Chemica Alta (Edmonton, Alberta, Canada), recrystallized from methanol/H2O, and stored in a light-protected desiccator at room temperature. Oxazolone (4-ethoxymethylene-2-phenyloxazolone (OX)) and trinitro-benzene sulfonic acid (TNBSA) were purchased from Sigma Chemical Co. (St. Louis, MO) and Eastman Kodak Co. (Rochester, NY), respectively.
Abs and cytokines
Hybridoma cells producing anti-mouse IL-10 mAb (clone;
JES5–2A5.1.1, rat IgG1) were obtained from the American Type Culture
Collection (Rockville, MD) with the permission of DNAX Research
Institute (Palo Alto, CA) and were maintained in RPMI 1640 containing
10% heat-inactivated FCS (Gemini, Calabasas, CA), 100 U/ml penicillin,
100 µg/ml streptomycin, 2 mM L-glutamine, and 25 mM
HEPES. Purified mAb to IL-10 was prepared by passage of hybridoma
culture supernatants over a protein G-Sepharose column (Pharmacia,
Piscataway, NJ). mAbs to TGF-ß2 (clone; 1D11.16, mouse IgG1) (29) was
provided by Dr. Wendy Waegel at Celtrix Laboratories (Santa Clara, CA).
Neutralizing mAb to murine B7-1 (clone; 1G10, rat IgG2a) and B7-2
(clone; 2D10, rat IgG2b) were provided by Dr. Denise Faherty at
Hoffmann-La Roche (Nutley, NJ). Murine rIL-12 and sheep polyclonal Ab
to murine IL-12 were generous gifts from Dr. Stanley F. Wolf at
Genetics Institute (Cambridge, MA). Neutralizing Abs to murine IFN-
(clone; XMG1.2, rat IgG1) and murine IL-2 (clone; C300–1A12) were
provided by Dr. Robert Coffman at DNAX Research Institute. Murine
rIFN- (4.6 x 106 U/mg) was provided by Toray
Industry, Inc. (Kanagawa, Japan).
Hapten conjugation of splenic APC
Normal mouse spleen cells were treated with 100 µg/ml of mitomycin C (Sigma Chemical Co.) at 37°C for 30 min. Then, after washing with PBS, the cells were incubated with 10 mM TNBSA (pH 7.2) or 6 mM Oxazolone in HBSS (pH 7.2), at 37°C for 10 min. Oxazolone was first dissolved in 100 µl ethanol, heated to 37°C, diluted with warmed HBSS, and used immediately.
In vitro measurement of CS immune lymph node cell (LNC) proliferation
A single cell suspension of LNC (axial, brachial, and inguinal)
was prepared under aseptic conditions, and 4 x
105 LNC were incubated in flat-bottom 96-well
microplates (Falcon, Oxnard, CA) with various numbers of
hapten-conjugated syngeneic normal spleen cells as APC in 0.2 ml of
RPMI 1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2
mM L-glutamine, 25 mM HEPES, 5 x 10-5 M
2-ME, and 10% FCS for 72 h. [3H]thymidine (1
µCi/well; Amersham, Aylesbury, U.K.) was added for the last 6 of
72 h of culture incubation, and [3H]thymidine
incorporation was determined by beta liquid scintillation counting.
Ssupernatant was also collected at 48 h for assay of IFN-
production and was stored at -20°C until use.
ELISA detection of IFN- in culture supernatants
Quantitative ELISA of IFN- employed two different mAb
specific for mouse IFN-, according to the manufacturer (PharMingen,
San Diego, CA). Briefly, wells of 96-well microtiter plates (Corning,
Corning, NY) were coated with 1 µg/ml of capture mAb (clone R4-6A2)
in 0.1 M NaHCO3 (pH 8.3) at 4°C overnight. Following
blocking with 3% dry milk in PBS at room temperature for 2 h,
samples and standard recombinant mouse IFN- dilutions (Genzyme,
Cambridge, MA), were added and incubated overnight at 4°C. Then, 0.5
µg/ml of biotinylated detection mAb to mouse IFN- (clone XMG1.2)
and subsequently 1/3000 diluted horseradish peroxidase-conjugated
streptavidin (Vector Laboratories, Burlingame, CA) were added to probe
for IFN-, and then TMB (tetramethylbenzidine), the peroxidase
substrate, and TMB one-component stop solution (Kirkegaard and Perry,
Gaithersburg, MD) were used to obtain ODs determined at 450
nm.
Contact sensitization and i.v. tolerance induction
Positive control mice were contact sensitized by topical skin application of 5% PCl (TNP-chloride) or 3% OX (150 µl) in an ethanol-acetone mixture (3:1) to the shaved abdomen and four footpads. For prior TNBSA tolerization, separate mice initially received i.v. 0.35 ml of 1% TNBSA in PBS (pH adjusted to 7.2 with 1 M NaOH) on days 0 and 3. Then, to evaluate tolerance, these mice were contact sensitized by skin painting on day 7 with the same or a different hapten (TNP vs OX), used for tolerance. Since no water-soluble salt of OX existed for OX tolerization similar to TNBSA, OX hapten-conjugated syngeneic spleen cells were prepared (1). Briefly, 1 mg of OX initially dissolved in a small volume of ethanol was brought to 1 ml with HBSS (pH 7.2), and spleen cells were incubated at 25°C for 1 h in this solution at 107 cells/ml. After washing with PBS, 5 x 107 of the OX hapten-conjugated cells/mouse were injected i.v. on day 0. Then, to test tolerance, these OX-injected mice were contact sensitized on day 7 by applying 3% OX or 5% PCl to the shaved abdomen and four footpads. To determine the CS ear-swelling responses of these i.v. tolerized and then CS-immunized animals, the mice were challenged with 10 µl of 0.8% PCl or OX in olive oil onto each side of both ears, and subsequent increases in ear thickness were measured 24 h after challenge, using a dial thickness gauge (Langenmessgerate, Berlin, Germany).
In some experiments, murine rIL-12 (0.5 µg/mouse) or IFN- (100 or
500 U/mouse) was injected i.p. into various mice 24 and 1 h before
ear challenge.
Statistics
Data were analyzed using Student’s t test (two-tailed) for independent samples, and p < 0.05 was taken as the level of significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study established Ag-specific tolerance of CS in vitro, we
examined proliferative responses of LNC from mice that were tolerized
by i.v. injection of hapten in vivo and then were tested in vivo for
resulting tolerance by attempted CS induction via hapten skin painting.
The LNC from mice tolerized i.v. with TNBSA before TNP contact
sensitization showed complete in vitro unresponsiveness of T cells to
proliferation induced by various doses of TNP-APC compared with LNC of
mice that were just contact sensitized with TNP and not previously i.v.
tolerized (Fig. 1
A).
This T cell unresponsiveness in vitro was Ag specific, since mice
tolerized instead with OX, a non-cross-reacting hapten, and then
similarly contact sensitized with TNP, in contrast, had intact in vitro
proliferative responses to TNP-APC (Fig. 1A).
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B). Thus, LNC from OX-tolerized and
then OX contact-sensitized mice were unresponsive in vitro to OX-APC,
but, in contrast, LNC from TNBSA-tolerized and then similarly OX
contact-sensitized mice had intact proliferative responses to OX-APC in
vitro (Fig. 1B). Therefore, Ag-specific tolerance
induced in vivo by i.v. injection of high doses of hapten could be
demonstrated in vitro. These in vitro results of unresponsive T cell
proliferation were exactly analogous to previously reported tolerized
in vivo ear-swelling responses, in which decreased hapten-specific
ear-swelling responses were induced by similar i.v. administration of
high doses of hapten before subsequently testing the tolerance by
contact sensitization via skin painting with reactive hapten (1, 2, 30).
IL-12 added in vitro reverses the reduced T cell proliferation and
IFN- production of tolerized mice
Since IL-12 has a strong ability to stimulate Th1 cells (6, 12, 13) that usually mediate CS (31, 32, 33), we determined whether IL-12 could
reverse established high dose i.v. hapten Ag tolerance. In vitro T cell
proliferative responses to hapten-APC were determined in the presence
or the absence of IL-12 added in vitro. Addition of IL-12 in vitro over
3 days caused restoration of proliferative responses to increasing
doses of TNP-APC in previously tolerized T cells (Fig. 2). The reversal of tolerance by IL-12
was Ag specific, since these proliferative responses were not induced
to the inappropriate hapten OX-APC (Fig. 2). This ability of IL-12 to
reverse Ag-specific tolerance of T cell proliferative responses was
neutralized completely by addition of polyclonal anti-IL-12 Ab
(Fig. 3), indicating that the restoring
activity was due to IL-12 itself and not to LPS contamination.
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by CS effector T
cells in response to TNP-APC was decreased substantially in LNC from
TNBSA-tolerized mice (Fig. 4, group A vs
group B), suggesting that in vitro Ag-specific established tolerance
was also observed for T cell cytokine production in addition to T
cell-proliferative responses (Figs. 1 and 2). Again, IL-12 largely
restored diminished IFN- production (Fig. 4, group D). Thus,
although addition of IL-12 alone, without Ag stimulation, caused a very
small increase in the basal production of IFN- (Fig. 4, group C),
addition of IL-12 with appropriate TNP-APC produced a strong reversal
of T cell IFN- production to 52 ng/ml (Fig. 4, group D). This
reached 60% of the level of IFN- produced by positive CS effector
LNC from nontolerized mice (Fig. 4, group A). In contrast, in vitro
addition of IL-12 to the i.v. TNBSA cells tolerized in vivo and then
stimulation with the inappropriate hapten OX-APC in vitro led to the
production of only 7 ng/ml IFN- (Fig. 4, group E), similar to the
small amounts produced by tolerized LNC without Ag stimulation, even in
the presence of IL-12 (Fig. 4, group C). We also demonstrated that the
effect of IL-12 on IFN- production by tolerized T cells responding
to hapten-APC was dose dependent (Fig. 5,
groups A–E) and again was neutralized by anti-IL-12 (Fig. 5, group
E vs group F). These experiments suggested that IL-12 added in vitro
restored Ag-specific hyporesponsive T cell proliferation and IFN-
production by LNC from mice with established in vivo tolerance induced
by high doses of hapten i.v.
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and requires costimulatory molecules, B7-1 and B7-2
Next we tried to determine the mechanisms of IL-12 reversal of
tolerance of CS effector cells. Since IL-12 was known to have a strong
ability to promote IFN- production (5, 6, 34), which is an important
Th1 cytokine for mediating CS responses (4, 31, 32, 33), we investigated
whether the restoring effect of IL-12 was mediated by endogenously
produced IFN- by experiments in which a neutralizing Ab to IFN-
was added in vitro. As shown in Figure 6,
the restoring effect of IL-12 in proliferative responses was not
affected by the presence of anti-IFN- mAb and was only partially
inhibited to a minor degree by anti-IL-2 mAb.
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(34, 35), and that costimulatory molecule-mediated signals had an
important role in this direct action of IL-12 on T cells (33, 36).
Thus, we added neutralizing mAbs to B7-1 and B7-2 to cultures of the
previously tolerized cells in the presence of IL-12 and found that the
ability of IL-12 to induce reversal of tolerance and activation of T
cells was blocked in vitro by anti-B7-1 and B7-2 mAbs when we
measured production of IFN- (Fig. 7A, left)
and proliferation (Fig. 7A, right) of LNC.
Interestingly, both B7-1 and B7-2 molecules seemed to be necessary for
the restoring effects of IL-12. However, if we looked at
hapten-specific proliferative responses and IFN- production in
normally immune LNC from CS mice that were not tolerized (Fig. 7B), the B7-1 molecule appeared to contribute little
to hapten-specific proliferative responses in these assays (Fig. 7B, right, group B), but B7-2 was involved in
proliferative and IFN- responses of these CS immune mice (Fig. 7B, group C). These results suggested that selective usage
of B7-1-mediated signals was involved in the reversal of proliferative
responses of tolerized LNC by IL-12, and B7-2-mediated signals also
were required for the reversal effects on CS tolerance of IL-12 as well
as for hapten-specific activation of normally immune LNC.
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Since IL-12 reversed tolerance in vitro, we investigated whether
IL-12 given in vivo also could reverse tolerance of CS by measuring
alterations of impaired CS ear-swelling responses. IL-12 was injected
twice into the mice, 24 and 1 h before Ag challenge on the ears.
Tolerance was already established, having been induced by TNBSA i.v. on
days 0 and 3, and then CS immunization was attempted on day 7. IL-12
administration to contact-sensitized mice that had not been tolerized
did not affect CS responses (Fig. 8,
groups C and D). Also, background ear-swelling responses in nonimmune
mice were unaffected (Fig. 8, groups A and B). In contrast, IL-12
caused significant restoration of hyporeactive CS ear-swelling
responses in mice with established tolerance (Fig. 8, group E vs group
F). These in vivo results confirmed the in vitro results reported above
and clearly demonstrated that IL-12 also reversed established tolerance
of CS ear-swelling responses in vivo.
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partially reverses hyporeactive
CS ear-swelling responses in tolerized mice
The above results showed that IL-12 restoration in vivo of
hyporeactive CS ear-swelling responses correlated with in vitro
restoration of T cell proliferative responses and IFN- production in
tolerized mice. Since IL-12 is known to have a strong ability to
promote IFN- production (5, 6, 34), which is an important Th1
cytokine for mediating CS responses (4, 31, 32, 33), we examined whether
the in vivo restorative effect of IL-12 may have been due to its
ability to induce production of IFN-. Thus, to imitate this effect
we directly injected IFN- into mice in vivo with the same timing as
that used for IL-12 administration, i.e., 24 and 1 h before Ag ear
challenge.
We choose this approach rather than administration of
anti-IFN- mAb together with IL-12 because IFN- is known to be
a critical cytokine for elicitation of the CS effector responses we
studied (4, 31, 32, 33). Thus, we postulated that anti-IFN- mAb
administration would disturb elicitation of all the fundamental CS
effector responses we were measuring, and thus that these effects might
not necessarily be mediated by administration of IL-12. Accordingly,
IFN- administration (100 or 500 U) in vivo had little
effect on CS ear-swelling responses of mice that were immunized but
were not tolerized, or tended to very slightly decrease CS responses
(Fig. 9, group B vs group C or D). In
contrast, statistically significant but small and partial restoration
of CS ear-swelling responses was observed when the same doses of
IFN- were administrated to mice with established tolerance and prior
Ag ear challenge (Fig. 9, group E vs group F or G). However,
restoration of ear-swelling responses by IFN- (group F, 42% of
positive nontolerized controls; group G, 40% of positive nontolerized
controls) was much smaller than restoration by IL-12 (78% of positive
nontolerized controls) (Fig. 8, group F). These results suggested that
increased endogenous production of IFN- may at best have been only
partially responsible for the restoration of hyporeactive CS
ear-swelling responses observed in tolerized mice injected with
IL-12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since IL-12 is a strong inducer and promoter of Th1 responses, we
investigated whether IL-12 could influence this in vivo high dose
hapten-induced established tolerance of Th1 CS responses. Importantly,
we tried to understand the molecular mechanisms of this hapten-induced
tolerance by developing an in vitro assay of CS tolerance. Using this
in vitro system, we determined the state of activation of CS effector T
cells by measuring hapten-specific T cell proliferative responses and
also IFN- production. We showed that CS effector T cells proliferate
and produce IFN- in response to increasing doses of specific
hapten-conjugated APC. Also, we found that T cells from high dose i.v.
hapten-treated, tolerized animals showed strongly reduced proliferative
responses and reduced in vitro IFN- production to specific
hapten-APC (Fig. 1). This in vitro unresponsiveness correlated with
inhibited CS ear-swelling responses that follow identical tolerogenesis
in vivo (1, 2, 30). Importantly, we showed further that IL-12 restored
impaired in vitro Ag-specific proliferative responses and IFN-
production of LNC from mice with high dose hapten tolerance (
Figs. 2–6), and these IL-12 effects required costimulatory molecule-mediated
pathways (Fig. 7). Further, we confirmed these in vitro results by
showing that in vivo administration of IL-12 restored this same
tolerance of CS itself (Fig. 8). Reversal by IL-12 clearly showed that
this tolerance was not due to T cell deletion and further suggested
that tolerance may not have simply been due to anergy (38).
Since development of tolerance in a different system that was induced
by UV irradiation was reported to be mediated by yet another mechanism,
namely down-regulatory Th2 cytokines such as IL-10 (25, 26), and recent
studies on CS due to CD8 effector T cells (39) also demonstrated
down-regulatory CD4 Th2 cells (40), it was possible that the mechanism
of IL-12 reversal of established tolerance involved the induction of
Th1 cytokines such as IFN- to antagonize the well-known
down-regulatory effects of Th2 cytokines. However, in contrast to
UV-induced tolerance (25, 26) or inhibition of CD8 CS (40), IL-10 was
not involved in the induction and maintenance of tolerance that we
induced in CD4 CS (37), since in vivo administration of neutralizing
mAb against IL-10 during induction of this tolerance did not prevent
the development of tolerance (our unpublished observations). In
addition, treatment of in vitro cultures of established tolerized cells
from our system with neutralizing mAbs against IL-4 and IL-10 did not
reverse tolerance (our unpublished observations), indicating that the
established high dose i.v. hapten-induced tolerance that we have
employed is different from UV-induced tolerance, and once tolerance has
been established, Th2 cytokines such as IL-4 and/or IL-10 might not be
involved in the maintenance of tolerance. Thus, the mechanisms of
reversal of established tolerance by IL-12 that we observed here seemed
to be completely different from that of prevention of UV-induced
tolerance by IL-12, i.e., direct activation of CS effector cells via
costimulatory molecules in our system vs antagonizing effects of IL-12
against suppressive Th2 cytokines.
In many in vivo experiments in which exogenous IL-12 has been used to
reverse Th2 responses, it has been shown that the effect is due to the
strong ability of IL-12 to stimulate the secretion of positive-acting
IFN- by T cells or NK cells (16, 41). We did not try to block the
effect of IL-12 in vivo by the administration of anti-IFN-
because of the obvious effect that anti-IFN- would have had on
elicitation of CS responses per se. However, and importantly, we
observed almost comparable IL-12 reversal of in vitro proliferative
responses of tolerized T cells in the presence of neutralizing Abs
against either IFN- or IL-2 (Fig. 6). This strongly suggested a
direct action of IL-12 on CS effector T cells in our established i.v.
hapten tolerance system, as suggested by another system, in which IL-12
acted directly on Th1 clones to induce IL-2-independent proliferation
(42). This direct effect of IL-12 on T cells probably synergizes with
signals due to costimulatory molecules, without effects due to
additional production of IFN- to induce T cell activation (36, 43).
In fact, we observed that the reversing effects of IL-12 were blocked
in vitro by anti-B7-1, more strongly than by anti-B7-2 mAb, and
by both Abs (Fig. 7A). Since in contrast to
anti-B7-2 mAb, anti-B7-1 mAb had little effect on proliferative
responses of nontolerized LNC (normally CS immunized) to haptenized APC
(Fig. 7B), there appeared to be some differences in
responsiveness to these costimulatory molecule-mediated pathways
between normal CS immune cells and tolerized CS effector T cells.
We do not know yet from our results how IL-12 synergizes with
costimulatory molecule mediated signals to T cells, and how these
pathways are different between CS immune cells and tolerized cells.
IL-12 has been reported to be produced by monocytes and macrophages,
and these cells function as important APC during normal immune
responses. B7 molecules on these APC interact with CD28 and CTLA-4 on
activated T cells, and these interactions are implicated in the priming
of naive T cells, but not in the activation of memory T cells (43).
However, some systems suggest that costimulation via B7 is required for
functional activation of memory effector T cells (44), and IL-12 is
also an important cytokine for activating memory T cells with
costimulation via these molecules (43). These results are well
correlated with our findings that costimulatory molecule-mediated
pathways are required not only for full activation of CS effector cells
(Fig. 7B), but also, more importantly, for
reactivation of tolerized cells by IL-12 (Fig. 7A).
As important roles for expression of B7-1 molecules and IL-12 in
breaking peripheral tolerance have been implicated in other systems,
such as in the pathogenesis of autoimmune diseases (45, 46) and in the
augmentation of tumor immunity (47), the same mechanisms may be
operative in our system to reverse tolerance by IL-12.
We also observed almost comparable reversal of impaired CS in
established tolerance in vivo by administrating IL-12 systemically to
mice at the time of elicitation of CS ear-swelling responses (Fig. 8).
This effect was mimicked partially by in vivo administration of
IFN-, although the magnitude of reversal of tolerance for CS
ear-swelling responses was significantly greater for IL-12 (Fig. 8 vs
Fig. 9). Moreover, we could not obtain further enhancement of CS
ear-swelling responses by increasing the dose of IFN- (Fig. 9).
These results again correlated well with our in vitro data, showing
that IL-12 has an additional mechanism, beyond induction of IFN-, to
reverse established CS tolerance by synergizing with B7-1 and B7-2
costimulatory molecule signal pathways.
In summary, these results taken together suggest that IL-12 probably
acts by two possible pathways to produce a reversal of the established
tolerance of CS that was induced previously by high i.v. doses of
hapten. One pathway may have been the reversal of tolerance due to the
production of a small amount of endogenous IFN- that is essential
for elicitation of CS responses (4, 31, 32, 33). Our data indicate that it
is unlikely that this small increase in IFN- occurred by IL-12
inhibiting counterbalancing Th2 responses. The other and probably major
pathway of IL-12 reversal of established CS tolerance is proposed to
have been due to a direct action of IL-12 on CS effector T cells to
strengthen their responses, probably by increasing T cell costimulation
mediated by signaling mechanisms acting via B7-1 and B7-2 (36, 43).
Finally, it has been suggested that IL-12 might be used clinically to augment impaired immune T cell function in clinical immunodeficiencies such as in HIV patients (48) or to augment tumor immunity (21, 47), in which reversing Ag-specific tolerance probably plays an important role. Thus, these current results may have important implications for the use of IL-12 to augment a broad range of impaired clinical immune responses, some of which may involve established tolerance.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Philip W. Askenase, Section of Allergy and Clinical Immunology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520. E-mail address:
3 Abbreviations used in this paper: CS, contact sensitivity; PCl, picryl chloride (trinitrophenyl chloride); TNP, trintrophenyl; OX, oxazolone; TNBSA, trinitro-benzene sulfonic acid; LNC, lymph node cell.
Received for publication January 13, 1997. Accepted for publication November 5, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or OX tolerized (
), and
then to test this tolerance, they were contact sensitized by skin
painting with active TNP (PCl). Subsequently, LNC were harvested and
then cultured in microwells with various numbers of TNP
hapten-conjugated normal spleen cells as APC. B, LNC (4
x 105 per well) were from mice that were not tolerized
(
), or i.v. TNBSA-tolerized (
), or i.v.
OX-tolerized (
). Then to test this tolerance by subsequent contact
sensitization with OX, LNC were cultured subsequently with various
numbers of OX hapten-conjugated normal spleen cells as APC. In
A and B, LNC proliferative responses were
measured 72 h after beginning coincubation of LNC and hapten-APC,
using [3H]thymidine incorporation. Data were expressed as
the mean counts per minute ± SE of two experiments. Triplicate
wells at each of the indicated Ag-APC doses were employed in each
experiment. * indicates p < 0.001 compared with
responses of mice that were not tolerized and responses of OX- or
TNBSA-tolerized and then contact-sensitized mice. ** indicates
p < 0.02 compared with responses of nontolerized mice
in A. NS, not significant vs non-tolerized mice in
B.
