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Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this factor.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, for in
IL-12-/- mice egg injection fails to stimulate increased
production of either of these cytokines. Furthermore, IL-6 also
up-regulates IL-10 production which, together with IL-6, negatively
regulates IL-12 and IFN- production. Finally, IL-10 down-regulates
the production of its inducer, IL-6. These data indicate that the
anti-inflammatory role of IL-6 may be effected through negative
regulation of type 1 (IFN-) and type 1-associated (IL-12) cytokines
either directly (by IL-6) or indirectly (through the induction of
IL-10) and suggest that one mechanism by which eggs may support the
development of Th2 responses is through the negative regulation of the
type 1 response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The eggs of the parasitic worm Schistosoma mansoni induce
the development of a strong Th2 response during natural infection
(12) or when injected i.p. (13), i.v.
(14), or s.c. (15) into a naive animal.
Previous studies have shown that after injection of eggs into the
footpads of naive mice a transient Th0 response develops that is
characterized by increased production of IFN- (15) and
also IL-6 (5). Despite the initial increase, IFN- is
down-regulated and a strong type 2 response develops (15, 16). The factors involved that drive the development of the Th2
response to eggs are still unclear, but recent studies have shown that,
although IL-6 may be involved in Th2 differentiation under certain
conditions (11), its absence does not affect the
development of Th2 responses to schistosome eggs (5).
In addition to its role in promoting IL-4 production by precursor T
helper cells (11), current work has demonstrated a role
for IL-6 in regulating Th1-associated cytokines (17, 18, 19, 20, 21).
Because IL-6 is up-regulated in response to
schistosome egg injection (5, 22), it may play a role not
in directing Th2 development but in controlling the initial IFN-
production and associated Th0 response. To understand the role of IL-6
in the development of the response to schistosome eggs, we investigated
both the factors involved in its up-regulation as well as the factors
that were regulated by it. To this end, we found that egg-induced IL-12
led to the up-regulation of both IFN- and IL-6 in response to T
cell-dependent and -independent stimuli. IL-6 in turn up-regulated
IL-10 production and down-regulated IL-12 and IFN-. These studies
indicate that IL-6 is playing a role in controlling type 1 responses
after exposure to schistosome eggs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 x SV129 F1 hybrids, IL-6-/- (C57BL/6 x SV129), and IL-12-/-p35 (C57BL/6) mice (The Jackson Laboratory, Bar Harbor, ME) were bred and used at 6–12 wk of age. Female C57BL/6 mice were purchased from Taconic (Germantown, NY). S. mansoni (Puerto Rican strain NMRI) eggs were isolated from the livers of infected mice, washed extensively, resuspended at 50,000 eggs/ml in low endotoxin PBS (Sigma, St. Louis, MO), and stored at -70°C until use, as previously described (23, 24). Mice were injected with 50 µl of egg suspension or with an equal volume of PBS per hind footpad. For infection, mice were exposed percutaneously to 70 cercariae.
Abs, Ags, cytokines, and reagents
Rat anti-IL-6 mAb 20F3 (F. Finkelman, Division of
Immunology, University of Cincinnati College of Medicine, Cincinnati,
OH), rat anti-IFN- mAb XMG 1.2 (PharMingen, San Diego, CA), rat
anti-IL-12p 40 mAb C17.8 (kind gift of Giorgio Trinchieri, Wistar
Institute, Philadelphia, PA), rat anti-IL-10 mAb JES5-2A5
(PharMingen), normal rat Ig (Accurate Chemicals, Westbury, NY),
purified hamster anti-CD3 mAb (PharMingen), and purified hamster
anti-CD40 ligand (anti-CD40L)3 mAb
(Phar-Mingen) were used in in vitro culture. FITC-, PE-, and/or
CyChrome-conjugated mAbs specific for mouse CD4, CD8, B220, IFN-,
IL-10, and IL-6 were purchased from PharMingen. Schistosome egg Ags
were prepared as described (23, 24). PMA, ionomycin, and
LPS were purchased from Sigma. Recombinant murine IL-6 and recombinant
murine IL-10 were purchased from Intergen (Purchase, NY). Recombinant
IFN- was obtained from Genzyme (Cambridge, MA) and recombinant IL-12
was from R&D Systems (Minneapolis, MN).
Lymph node (LN) and splenocyte isolation and culture
Popliteal LN were harvested from egg-injected mice and popliteal and axillary LN were isolated from PBS-injected mice. Single-cell suspensions were prepared using sterile 70-µm cell strainers (Falcon, Franklin Lakes,NJ) as previously described (25). LN cells were resuspended at 5 x 106 cells/ml in complete T cell medium containing DMEM (Sigma), 10% FCS (HyClone, Logan, UT), 100 U/ml penicillin plus 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD), 10 mM HEPES (Life Technologies), L-glutamine (Life Technologies), and 5 x 10-5 M 2-ME (Sigma). Cells (106) were cultured in 96-well flat-bottom plates (Falcon) at 37°C and 5% CO2. Culture supernatants were harvested at 24 or 72 h for cytokine analysis. Spleens were harvested and single-cell suspensions were prepared as previously described (26). Splenocytes were resuspended to 107 cells/ml in complete T cell medium and were cultured in 96-well flat-bottom plates at 37°C and 5% CO2. Culture supernatants were harvested at 72 h for cytokine analysis.
Cytokine ELISAs
Sandwich ELISAs were used to measure IL-6, IL-10, and IFN- as
previously described (5, 27). Rat anti-IL-12p40/70
C15.6 mAb (PharMingen) was used for the IL-12 p40 capture Ab, and
biotinylated rat anti-IL-12p40 mAb C17.8 (gift of Phil Scott,
Department of Pathobiology, University of Pennsylvania, Philadelphia,
PA) and then peroxidase-labeled streptavidin (Jackson ImmunoResearch,
West Grove, PA) were used for detection. Recombinant IL-12 (R&D
Systems) was used for the IL-12 ELISA standard.
Intracellular cytokine staining and flow cytometry
Cells were stained ex vivo on ice for 20 min with FITC-, PE-, or cytochrome c-conjugated Abs against surface markers. After washing twice with 1% FCS, 0.08% NaN3 (Sigma) in PBS (Sigma), cells were resuspended in fixative (1% formaldehyde (Sigma) in PBS). For intracellular cytokine staining, LN cells were cultured in vitro for 72 h before a 6-h incubation with 250 ng/ml ionomycin (Sigma), 50 ng/ml PMA (Sigma), and 1 µg/ml brefeldin A (PharMingen). After a 15-min incubation with Fc block, LN cells were incubated with FITC- or CyChrome-labeled Abs to surface markers, fixed, permeabilized, and stained with PE-conjugated anti-cytokine Abs using reagents from the Cytofix/cytoperm kit (PharMingen). All cells were analyzed using a FacsCaliber flow cytometer (Becton Dickinson, Franklin Lakes, NJ) with the CellQuest program (Becton Dickinson).
Statistical analysis
Data were analyzed using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the relationship between IL-12 and IL-6 in the
initial response to schistosome eggs, we assessed cytokine production
by popliteal LN cells harvested from mice 24 h after footpad
injection of eggs. Popliteal LN cells from mice injected with
schistosome eggs 24 h previously are primed to make IL-12 in
response to anti-CD3 stimulation (Fig. 1
a) or, more strongly, in
response to LPS (Fig. 1b). The production of IL-12 in LN
cultures stimulated with anti-CD3 is in part dependent on
CD40-CD40L interactions for addition of neutralizing anti-CD40L mAb
to LN cultures from egg-injected mice significantly reduced IL-12
production after anti-CD3 but not LPS stimulation (Fig. 1, a and b). The production of this early IL-12 is
important for the initial priming of the immune response because LN
cells isolated from egg-injected IL-12-/- mice
made less IFN- and IL-6 in response to LPS than did LN cells from
egg-injected wild-type (WT) mice (Fig. 2a). Similar results were
found using anti-CD3 stimulation (Fig. 2b). Furthermore,
the addition of rIL-12 to LN cultures from PBS and egg-injected WT mice
promoted IL-6 (Fig. 2c). CD4 T cells were found to be the
source of IL-6 in in vitro cultures stimulated with anti-CD3,
whereas both CD4 T cells and a nonlymphocyte,
Mac-1+ population were the principle IL-6
producers after LPS stimulation (data not shown). This result further
supports the idea that IL-12 is one of the stimuli promoting IL-6
production by CD4 T cells and macrophages early after egg-injection.
Because T cell-dependent responses are the focus of this study, further
work will primarily present data from LN cells stimulated with
anti-CD3. IFN- production was also increased after the addition
of rIL-12 to anti-CD3-stimulated LN cultures from PBS and
egg-injected mice, and production by both CD4 and CD8 T cells was
affected (data not shown). Finally, the addition of anti-IL-12 mAb
decreased IL-6 and IFN- production after anti-CD3 stimulation
(data not shown). These results indicate that IL-12 is produced after
egg injection and that this IL-12 is important in priming the immune
response to produce optimal levels of IL-6 and IFN-.
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production
In contrast to the positive effect that IL-12 has on IL-6
production, IL-6 was found to negatively regulate IL-12 production
after egg injection. IL-12 production by LN cells was greater in
egg-injected IL-6-/- mice stimulated with
anti-CD3 or LPS compared with those stimulated with LN cells from
WT mice (Fig. 3a). Addition of
rIL-6 to IL-6-/- LN cultures or of
anti-IL-6 mAb to WT LN cultures stimulated with anti-CD3
resulted in suppression or enhancement of IL-12 production,
respectively (Fig. 3b), although in vitro treatment
(anti-IL-6 or rIL-6) did not result in the levels seen with
IL-6-/- or WT LN cells alone. This difference
is presumably due to the additional priming that occurs in vivo. These
results suggest that whereas IL-12 increases the production of IL-6
after egg injection, once IL-6 is produced the production of IL-12 is
suppressed.
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. Addition of neutralizing anti-IL-6 mAb
enhanced IFN- production in cultures from egg-injected WT mice, and
similarly the production of IFN- was reproducibly elevated in
cultures from egg-injected IL-6-/- mice
compared with WT mice. In further support, IFN- production could be
specifically inhibited by the addition of rIL-6 (Fig. 4, a and b).
Intracellular cytokine analysis revealed that CD8 T cells were the
primary IFN--producing cells in the WT and
IL-6-/- LN cultures and that rIL-6 reduced not
only the level of IFN- produced but also the number of cells
producing IFN- (Fig. 4b). Although CD4 T cells did not
produce IFN- in WT LN cultures, a small but significant population
of IFN--producing CD4 T cells (evident as
CD8- cells in Fig. 4b) was present in
IL-6-/- cultures, suggesting that IL-6 may be
inhibiting IFN- production by CD4 T cells in WT LN cultures.
Although IFN- levels are higher in cultures from egg-injected vs
PBS-injected mice 1 day after injection (Fig. 2
b), levels
are reduced within 3 days after egg injection (15) and
more dramatically at 7 days after egg injection (see below and Fig. 9),
further supporting the initial enhancement of IFN- production by
IL-12 and the subsequent down-regulation by IL-6. These results suggest
that one of the mechanisms by which schistosome eggs could enhance Th2
differentiation may be through the induction of IL-6, which then
negatively regulates both IL-12 and IFN- production.
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production
Egg injection led to the enhanced production of IL-10 in cultures
from egg-injected vs PBS-injected mice after 72 h of in vitro
stimulation with anti-CD3, and this increased production was absent
in cultures from IL-6-/- mice (Fig. 5a). Intracellular cytokine
staining revealed that T cells, both CD4 and CD8, were the primary
producers of IL-10 in vitro (data not shown). Macrophages, which
comprised less than 5% of the LN population, and B cells did not
produce significant levels of IL-10 (data not shown). Ab-mediated
neutralization of IL-6 in WT cultures reduced the IL-10 to levels seen
in IL-6-/- mice, and addition of exogenous IL-6
brought the levels of IL-10 in the supernatant of
IL-6-/- cultures to those measured in WT
cultures (Fig. 5b). The positive regulation of IL-10 by IL-6
suggests that the anti-inflammatory properties of IL-6 may be due
in part to IL-10.
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c, neutralization of IL-10 in vitro enhanced IL-12
production, whereas the addition of exogenous IL-10 substantially
reduced IL-12 levels in WT and IL-6-/- LN
cultures. Both IL-6 and IL-10 appear to manifest similar
down-regulatory effects on IL-12 production (Figs. 3b and
5c). However, IL-10 can act alone in regulating IL-12
production because the addition of IL-10 in the absence of IL-6
(IL-6-/- cultures) reduced IL-12 production
(Fig. 5c). The effect of IL-6 on IL-12 is in part
independent of IL-10 because the addition of exogenous IL-6 to WT
cultures treated with anti-IL-10 resulted in a reduced production
of IL-12 compared with the results from anti-IL-10 treatment alone
(data not shown). In addition, the neutralization of both IL-6 and
IL-10 in WT cultures led to greatly increased levels of IL-12
(2875 ± 156 pg/ml) compared with either anti-IL-10 alone
(1408 ± 130; Fig. 5c) or anti-IL-6 alone (562
± 14; Fig. 3b). These results suggest that although IL-10
and IL-6 can each independently inhibit IL-12 production, part of
the effect of IL-6 on IL-12 production is due to IL-6-induced IL-10
production.
IL-10 is also a strong inhibitor of IFN- production (29, 30), and therefore the reduced IFN- production seen after
addition of exogenous IL-6 could be a result of an increase in IL-10.
To determine whether this was the case, IL-6 and IL-10 were either
neutralized or the exogenous cytokine was added in vitro, and the
relative change in IFN- production was measured. As with IL-12
production, both IL-6 and IL-10 were capable of reducing IFN-
production (Fig. 6a); the
difference in the activities of the two cytokines lays in the kinetics
of the inhibition. Whereas rIL-6 reduced IFN- levels within the
first 24 h of culture, IL-10 was more effective after 72 h of
in vitro culture (data not shown). Additionally, neutralization of IL-6
in cultures from egg-injected WT mice was more effective than the
neutralization of IL-10 at increasing IFN- levels within the first
24 h of culture (data not shown) when IL-6 but not IL-10 levels
were high (Fig. 6b). As with IL-12 production,
neutralization of both IL-10 and IL-6 elevated IFN- levels above
those seen in cultures in which either cytokine was neutralized alone
(Fig. 6a). The different effects of IL-6 and IL-10 on
IFN- production argue that while part of the negative effect of IL-6
may be mediated through IL-10, IL-6 also has a direct effect on IFN-
(Fig. 6a). Furthermore, this direct negative regulation of
IFN- may be important at very early time points after priming when
IL-6 but not IL-10 has been up-regulated.
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Although IL-6 is an important inducer of IL-10 (31),
it is clearly not the sole agent responsible because IL-10 is produced
by IL-6-/- LN cells, albeit at lower levels
than those in WT cultures. To determine whether the increased
production of IL-12 or IFN- by cells from
IL-6-/- mice could be promoting the production
of IL-10, the effect of neutralizing IL-12 or IFN- on IL-10
production in vitro was studied. Whereas the neutralization of IFN-
had only a marginal effect on IL-10 production by
IL-6-/- LN cells from egg-injected mice (data
not shown), anti-IL-12 mAb significantly reduced the levels of
IL-10 detected in the cultures (Fig. 7).
Addition of rIL-12 further increased the levels of IL-10 in
IL-6-/- cultures (Fig. 7) to those detected in
WT cultures. These results give additional evidence that IL-12 in
addition to IL-6 may act to promote the resolution of an immune
response through the induction of the anti-inflammatory cytokine,
IL-10.
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IL-10 has been shown to be important in the resolution of the
inflammatory response by its ability to down-regulate the
Th1-associated cytokines IL-12 (18, 28) and IFN-
(29, 30) and also the Th2 cytokines IL-4 (32)
and IL-6 (33). To understand whether IL-10 could also be
important for the control of IL-6 production in our model, the effect
of IL-10 on IL-6 was investigated. After the addition of neutralizing
anti-IL-10 mAb, the production of IL-6 was elevated, whereas the
addition of rIL-10 decreased the levels detected in LN cultures from
egg-injected WT mice (Fig. 8). A similar
pattern was seen in LN cultures from PBS-injected mice, albeit at lower
levels (data not shown). Taken together these data suggest that in a
manner similar to the counterregulation of IL-12 and IL-6, IL-6
promotes the production of IL-10, which in turn down-regulates the
production of IL-6 and hence allows for the resolution of the innate
response.
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To discern whether the enhanced type 1 response in LN cells from
egg-injected IL-6-/- mice had any effect on Th
response development, cytokine production by LN cells isolated 7 days
after egg injection was examined. At this time point, previous studies
showed that the Ag-specific Th2 cytokine response was not abolished in
the absence of IL-6 (5). Subsequent studies focusing on
type 1 responses revealed that IFN- production was not
down-modulated in LN cultures from IL-6-/- mice
7 days after egg injection vs PBS injection, as was seen with WT mice
(Fig. 9a). Furthermore, spleen
cells from infected IL-6-/- mice also failed to
down-regulate IFN- production after anti-CD3 stimulation, in
contrast to the marked reduction in IFN- levels in cultures of
splenocytes from infected vs uninfected WT mice (Fig. 9b).
Taken together, these data indicate that while IL-6 is not necessary
for Th2 response development, it may be responsible for controlling
type 1 cytokine production (most notably IFN- production) during
schistosome infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-10 in T cell-dependent responses were
studied and particular emphasis was placed on the role of IL-6 in
directing the early immune response. Previous studies have shown that
the absence of IL-6 does not abolish the production of type 2 cytokines
(IL-4 and IL-5) nor does it ultimately affect the development of
Ag-specific Th2 cells (5, 36). This study extends the
previous findings by demonstrating that the absence of IL-6 instead
enhances the production of IL-12 and IFN- and leads to decreased
IL-10 production. These results suggest that although IL-6 does not
appear to be necessary for the development of Ag-specific Th2 cells,
one mechanism by which strong egg-specific Th2 effector responses may
be maintained is through IL-6-mediated suppression of the type 1
response.
Exposure to eggs results in the induction of IL-6 transcripts as well
as the increased production of IL-6 in anti-CD3 or LPS-stimulated
in vitro LN cultures (5). Increased levels also have been
found in the lungs of mice 1–3 days after the i.v. injection of eggs
(22). Although these studies have shown that IL-6 is
up-regulated in response to eggs, neither have identified the factor
mediating IL-6 production. This study found that IL-12 is an important
cytokine in mediating IL-6 up-regulation, for in the absence of IL-12,
reduced IL-6 levels were detected. When exogenous IL-12 was added to
the cultures, IL-6 production was enhanced. The induction of IL-6
production by IL-12 has been shown before in murine
CD5+ and CD5- B cells
(37); however, in this model CD4 T cells and macrophages
are the principle IL-6 producers. The up-regulation of IL-6 may be a
direct effect of IL-12 but also may be an indirect result of
IL-12-mediated TNF-
production (17), a known inducer of
IL-6 (1). In either case, these results indicate that the
optimal induction of IL-6 after egg injection is ultimately the result
of IL-12 up-regulation.
The finding of IL-12-producing cells in the draining LN 24 h after
egg injection suggests that one of the initial events that occurs after
the exposure to eggs is the induction of IL-12 production. Egg-specific
induction of IL-12 transcripts in the lungs has been shown before in a
study by Wynn et al. (22) in which NK-derived IFN-
played a major role in IL-12 induction in vivo. In contrast, we found
that optimal IFN- production in vitro was dependent on IL-12
production during in vivo priming. These differences may be due to
different cellular compositions, for NK cells were found to be a minor
population (<1%) in the popliteal LN (our unpublished observations),
or due to the use of different mouse strains (C3H/HeN vs C57BL/6 and
C57BL/6 x 129SV). This report shows enhanced IL-12 production
after in vitro stimulation of LN cells with anti-CD3 or LPS. This
production of IL-12 after T cell-dependent stimulation (anti-CD3)
but not after T cell-independent stimulation (LPS) required CD40-CD40L
interactions (18, 38) and therefore suggests that part of
the in vivo priming that occurs after egg injection may be the
induction of CD40L expression on T cells or the induction of CD40 on
APC. Because macrophages and/or dendritic cells, which are the most
likely cellular source of the IL-12 in LN cultures, comprise <5% of
the LN population (Ref. 5 and data not shown), the
moderately high levels of IL-12 detected in the LN cultures indicate
that on a per-cell basis egg injection strongly primes for IL-12
production.
After IL-12-induced IL-6 production, IL-12 is negatively regulated by
IL-6. The down-regulation of IL-12 by IL-6 has previously been
described by Takenaka et al. (18), who focused on the
independent effects of IL-4, IL-10, and IL-6 on T cell-dependent and
-independent IL-12 production. Takenaka et al. (18)
clearly showed that IL-6 could inhibit CD40-CD40L-dependent (T
cell-dependent) IL-12 production and that IL-10 could inhibit both T
cell-independent (LPS-stimulated) and -dependent IL-12 production. Our
results concur with this study and indicate that after egg injection
IL-6 may inhibit T-cell-dependent IL-12 production through several
possible pathways. First, IL-6 may directly inhibit IL-12 production as
suggested by Takenaka et al. (18). Second, IL-6 may work
indirectly through the promoting IL-10 or by directly inhibiting
IFN- at early time points when IFN- strongly enhances IL-12
production. Because the addition of rIL-10 reduces IL-12 levels in
cultures from IL-6-/- mice, our results argue
that IL-12 down-regulation is not solely a direct effect of IL-6.
Despite that, neutralization of both IL-10 and IL-6 results in IL-12
levels greater than those found with neutralization of either cytokine
alone, suggesting that IL-6 can inhibit IL-12 production independently
of IL-10, possibly through inhibiting IFN- production. Although it
is well established that IL-10 is a potent inhibitor of IL-12
production (17, 18, 28), this study proposes that IL-6 may
be an intermediary in that pathway as well as having a direct
inhibitory effect on T cell-dependent IL-12 production.
In conjunction with the increased levels of IL-12 produced in the
absence of IL-6, IFN- levels were also greatly increased. Despite
the established role for IL-10 in down-regulating IFN- (29, 30), this study points toward a role for IL-6 in negatively
affecting IFN- production at early time points before the induction
of IL-10. Several investigators using different model systems have also
shown enhanced IFN- production in the absence of IL-6
(19, 20, 21). In our studies, egg-induced IL-12 production led
to an early and transient increase in IFN- production, which is
consistent with the development of the early Th0 response as has been
described previously (15). Along with this increase in
IFN-, IL-12 induced IL-6, which in turn down-regulates IL-12 and
IFN-. The down-regulation of IFN- production is most evident 7
days after egg injection and, as at 1 day, does not occur in cultures
from IL-6-/- mice. Similar results were found
using splenocytes from infected WT and IL-6-/-
mice. Because reduced levels of IFN- are not seen after egg
injection or after natural schistosome infection in the absence of
IL-6, these data suggest that type 1 responses in part may be
controlled directly or indirectly through IL-6.
IL-6 has been classified as an anti-inflammatory cytokine (1, 21). The finding that IL-6 induces IL-10 production as well as
its role in inhibiting IFN- may account for some of its
anti-inflammatory properties. Nishimura et al. (31)
have shown that IL-6 promotes IL-10 production using a model of
UV-induced skin damage, and our results expand upon that finding by
showing that IL-6 can enhance IL-10 production by CD4 and CD8 T cells
after injection with schistosome eggs. The enhancement of IL-10 is
implicated in the down-regulation of IL-12 seen after the addition of
exogenous IL-6 to LN cultures from egg-injected WT mice. In
further support, in LN cultures from IL-6-/-
mice, which produce significantly reduced IL-10 levels, IL-12
production is increased. Addition of exogenous IL-6 or exogenous IL-10
are equally effective in reducing IL-12 levels in the
IL-6-/- cultures to those in WT cultures.
Despite the evidence that IL-10 is the major negative regulator of
IL-12 in this system, the possible contributions of IL-6 in reducing
IFN- and therefore IL-12 production must not discounted.
Along with the observation that IL-6 is a major inducer of IL-10 (31) is the finding that IL-12 can induce IL-10 independently of IL-6. Studies by Finkelman et al. (39) and Wynn et al. (40) have shown that during immune responses to parasitic worms or worm Ags, administration of exogenous IL-12 results in increased IL-10 mRNA in the mesenteric LN, Peyer’s patch, or lungs of mice. The induction of IL-10, specifically in T cells, by IL-12 has also been reported before but primarily in human T cells (41, 42). Our work supports these findings and additionally demonstrates that the induction of IL-10 by IL-12 is independent of IL-6, suggesting that IL-12 itself is important not only in the initial priming of the immune response to eggs but also in up-regulating factors that control the resolution of that response.
Previous work has shown that Th2 response development is not abolished
in the absence of IL-6 (5, 36). Although reduced levels of
IL-4 are produced by IL-6-/- CD4 T cells
isolated 1 day after egg injection compared with levels produced by WT
CD4 T cells, Ag-specific IL-4 and IL-5 production are equivalent by 7
days after egg injection (5). From this work we concluded
that IL-6 was not essential for the development of a Th2 response. The
results presented here suggest that instead of controlling Th2
responses, IL-6 appears to play a role in controlling type 1 responses.
Although an egg-specific Th1 response does not develop 7 days after egg
injection of IL-6-/- mice (5) or
after infection of IL-6-/- mice
(36), the reduced levels of IFN- are not found when
IL-6-/- cells are polyclonally stimulated
(i.e., anti-CD3), suggesting that the IFN--producing cells are
not inhibited by the presence of Ag-specific Th2 cells in the absence
of IL-6. In addition to the direct effect on controlling type 1
responses, IL-6 can mediate its effects through IL-10. In support of
this conclusion, we found that in the absence of IL-6, Ag-specific
IL-10 production by LN cells 7 days after egg injection was reduced
(5). Further studies are underway to assess the potential
effect of IL-6 on Th1 and Th2 effector functions (i.e., granuloma size
and fibrosis) in vivo during infection with S. mansoni.
Taken together these studies implicate IL-6 directly and indirectly
through IL-10 in regulating type 1 responses after exposure to
schistosome eggs.
The regulation of Th2 responses by IL-12 after injection of schistosome
eggs was previously investigated by Wynn et al. (22, 40, 43). In their system, the administration of exogenous IL-12 at
the time of egg injection led to decreased Th2 responses in WT mice.
However, in IFN--/- mice, Th2 responses were
not reduced (40). The authors concluded from these data
that IL-12 diminished Th2 responses indirectly by stimulating IFN-
production in vivo. Our results suggest that in addition to this
pathway, another pathway exists whereby IL-12 can indirectly control
Th2 responses after exposure to schistosome eggs. We have found that
IL-12-induced IL-6 leads to IL-10 production, which in turn regulates
both type 1 and Th2 responses. Therefore, although the absence of IL-6
does not lead to diminished Th2 responses despite increased IFN-
production, this finding can be explained by the significantly reduced
IL-10 production without IL-6. Together, these two counterregulating
pathways balance the type of immune response that develops after
exposure to schistosome eggs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Edward J. Pearce, Department of Microbiology and Immunology, C5-165 Veterinary Medical Center, Cornell University, Ithaca, NY 14853. E-mail address:
3 Abbreviations used in this paper: CD40L, CD40 ligand; LN, lymph node; WT, wild type.
Received for publication October 15, 1999. Accepted for publication December 22, 1999.
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References |
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in the pathogenesis of T-cell activation-associated hepatitis and regulatory mechanisms of interleukin-6 for the manifestations of hepatitis. Hepatology 23:1608.[Medline]
and TNF by antitumor T cells or macrophages in the tumor-bearing state. J. Immunol. 154:2281.[Abstract]
production following in vitro culture. Scand. J. Immunol. 39:12.[Medline]
-mediated cachexia and death during acute schistosomiasis. J. Immunol. 159:777.[Abstract]
synthesis and lymphokine-activated killer activity. Int. Immunol. 4:563.. J. Immunol. 154:3999.[Abstract]
and interleukin-10. J. Exp. Med. 183:2559.. J. Immunol. 157:1127.[Abstract]
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