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*
The Wistar Institute, Philadelphia, PA 19104; and
Division of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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also plays a key role in orchestrating
inflammation and immunity. The reciprocal influence of these two
inflammatory mediators on each other may have significant impact on the
cytokine balance that shapes the type and extent of immune responses.
To investigate the relationship between TNF- and IL-12 production,
we analyzed the effects of exposure of human monocyte-derived
macrophages to TNF- on LPS- or Staphylococcus
aureus-induced IL-12 production in the presence or absence of
IFN-
. TNF- is a potent inhibitor of IL-12 p40 and p70 secretion
from human macrophages induced by LPS or S. aureus.
IL-10 is not responsible for the TNF--mediated inhibition of IL-12.
TNF- selectively inhibits IL-12 p40 steady-state mRNA, but not those
of IL-12 p35, IL-1, IL-1ß, or IL-6. Nuclear run-on analysis
identified this specific inhibitory effect at the transcriptional level
for IL-12 p40 without down-regulation of the IL-12 p35 gene. The major
transcriptional factors identified to be involved in the regulation of
IL-12 p40 gene expression by LPS and IFN-, i.e., c-Rel, NF-
B p50
and p65, IFN regulatory factor-1, and ets-2, were not affected by
TNF- when examined by nuclear translocation and DNA binding. These
data demonstrate a selective negative regulation on IL-12 by TNF-,
identifying a direct negative feedback mechanism for
inflammation-induced suppression of IL-12 gene
expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 production during an immune response is tightly modulated by both
positive and negative feedback signals. The positive feedback
regulation is exemplified by IL-12-induced IFN- secretion from NK
and T cells which in turn potently enhances the ability of monocytes
and polymorphonuclear cells to produce IL-12 (4). The
priming effect of IFN- for augmented IL-12 production may represent
a mechanism by which IL-12-induced Th1 responses are maintained in
vivo. The positive feedback amplification of IL-12 production mediated
by IFN- can also be a potentially dangerous mechanism leading to
uncontrolled inflammation and possibly shock. There are also effective
mechanisms that down-regulate IL-12 production and the responsiveness
of T and NK cells to IL-12, such as those mediated by cytokines IL-10
and TGF-ß (5). The understanding of the molecular
mechanisms governing the expression of IL-12 p40 and p35 genes in the
context of interactions between pathogens and the immune system is
essential to efforts in designing novel therapeutic strategies for
ameliorating infectious and malignant diseases.
TNF- is one of the principle mediators of inflammatory responses in
mammals (6). In addition to its well-known role in septic
shock, it has been implicated in the pathogenesis of chronic processes
such as autoimmunity (7), graft-vs-host disease
(8), rheumatoid arthritis (9), Crohn’s
disease (10), and the cachexia (11)
accompanying cancer and AIDS (12). Unexpectedly, it was
recently shown that IL-12 production in thioglycolate-elicited mouse
macrophages could be suppressed by TNF- and that TNF--deficient
mice developed a delayed but vigorous inflammatory response to
heat-killed Corynebacterium parvum with very high levels of
serum IL-12 production resulting in death (13, 14). The
prompt resolution of C. parvum-injected wild-type mice with
lower IL-12 production suggested TNF- may play a role in limiting
the extent and duration of murine inflammatory responses. The role of
TNF- in human proinflammatory diseases and the pivotal importance of
IL-12 in innate and adaptive cell-mediated responses underscores the
importance of understanding TNF- mechanisms of action in inhibiting
pathogen-induced IL-12 production by macrophages. We show here that in
human monocyte-derived macrophages
(MDM),3 TNF-
selectively inhibits IL-12 p40 transcription, but not p35, identifying
a novel regulatory mechanism of action for TNF--induced suppression
of IL-12 production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human TNF- was obtained from Genentech (San Francisco, CA);
murine TNF- and anti-murine TNF- (XT22) were purchased from
Genzyme (Cambridge, MA); human IFN- was obtained from Endogen
(Woburn, MA); fixed Staphylococcus aureus Cowan strain I
(binding capacity to human IgG: 2.0 ± 0.1 mg/ml suspension) and
actinomycin D were obtained from Calbiochem-Behring (La Jolla, CA); LPS
and polymixin B were obtained from Sigma (St. Louis, MO).
Preparation of MDM
Human PBMC were isolated from healthy donors by the procedure of Ficoll/Hypaque gradient centrifugation (Pharmacia, Uppsala, Sweden). Mononuclear cells were incubated for an hour in polystyrene tissue culture flasks (Falcon, Becton Dickinson, Franklin Lakes, NJ). Adherent cells, typically >94% CD14+ by FACS analysis, were cultivated in 10% pooled AB+ human serum (Sigma, St. Louis, MO) for 20 h before harvesting for experimental treatments. In some experiments, human monocytes were prepared by countercurrent elutriation. No difference was observed between elutriated and adherence-purified monocytes with regard to responses analyzed in this study.
Abs and cytokine assays
All Abs used for supershift and Western blot experiments were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). RIA for human
TNF- were performed using mAb pairs B154.9/B154.7 (15).
Human IL-12 p70 and p40 were measured in cell-free supernatants by RIA
as described in (16) using the mAb pairs 12H4/C8.6 and
C11.79/C8.6, respectively. The neutralizing anti-IL-10 mAb 19F1 was
kindly provided by Dr. K. Moore of DNAX (Palo Alto, CA).
RNase protection assay
RNase protection assays were performed using the PharMingen Multiprobe system hCK-2 (PharMingen, San Diego, CA) following the manufacturer’s instructions.
Nuclear run-on
Isolation of nuclei and in vitro transcription in the presence
of [32P]UTP (3000 Ci/mmol; DuPont, Wilmington,
DE) were performed essentially as described (17, 18).
Nuclear RNA was isolated after DNase I (Boehringer Mannheim,
Indianapolis, IN) and proteinase K (Boehringer Mannheim) treatment
followed by three phenol/chloroform/isoamyl alcohol extractions and
ethanol precipitation. Unincorporated [32P]UTP
was removed using Sephadex G-50 columns (Boehringer Mannheim). RNA was
partially degraded by treatment with 0.2 N NaOH for 10 min at 4°C.
32P-labeled nuclear RNA was hybridized for 2 days
at 60°C to prehybridized nylon filters (Schleicher & Schuell, Keene,
NH), on which 250 ng of denatured PCR-amplified cDNAs corresponding to
the coding regions of the IL-12 p40 and p35, TNF-, and ß-actin
genes had been immobilized using a slot-blot apparatus (Hoeffer
Scientific, San Francisco, CA). After hybridization, filters were
washed at room temperature with 2x SSC, and single-stranded RNA was
digested with the same solution containing 10 µg/ml RNase A at 37°C
for 30 min. Filters were then washed twice in 2x SSC, 0.1% SDS
for 15 min at 50°C and once in 0.1x SSC, 0.1% SDS for 30 min at
50°C. The extent of hybridization was quantified using the ImageQuant
software on a PhosphorImager 445SI (Molecular Dynamics, Sunnyvale,
CA).
Nuclear extract preparation
Nuclear proteins were isolated as described (19).
EMSA
End-labeled DNA probes (50,000 cpm/sample) were mixed with 1–2 µg of crude nuclear extracts and incubated at room temperature for 20–30 min in the presence of 1 µg of polydI-dC (Boehringer Mannheim) in a volume of 10 µl of 0.5x dialysis (D) buffer (10 mM HEPES, pH 7.9, 10% glycerol, 50 mM KCl, 0.1 mM EDTA, 0.25 mM DTT, 0.25 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin A). The mix was then fractionated through a 4% polyacrylamide gel in buffer containing 6.7 mM Tris-HCl, pH 7.5, 3.3 mM sodium acetate, 0.1 mM EDTA for 1 h at 200 V. The gel was dried and exposed in a PhosphorImager storage screen (Molecular Dynamics) and scanned. Supershift experiments were conducted by preincubating the nuclear extract with 1–2 µg of affinity-purified polyclonal Abs for 30 min at 4°C before the probe was added.
Western blot
Reducing SDS-PAGE (15%) was performed according to Laemmli (20) with 6–8 µg of nuclear proteins in each sample. The gel was then electroblotted in Tris-glycine buffer containing 40% methanol onto a nitrocellulose membrane (Trans-blot; Bio-Rad, Hercules, CA). After blocking the membrane with TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5% milk powder for 1 h at room temperature, primary Abs (rabbit anti-mouse IgG) were added at 1:1000 dilution (1 µg/ml) in TBST containing 1% milk powder for 1 h at room temperature. The membrane was washed three times for 7 min each with TBST, and incubated with secondary Ab (anti-rabbit or anti-goat IgG conjugated to HRP; Amersham, Arlington Heights, IL) at 1:5–10,000 dilution for 1 h at room temperature. After washing three times in TBST for 5 min each, and once in TBS for 5 min, the membrane was drained briefly and subjected to the enhanced chemiluminescence detection procedure (Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a potent and specific inhibitor of IL-12 p40 and p70
secretion from human macrophages
MDM were pretreated with TNF- (20 ng/ml) for 20 h followed
by IFN- (100 ng/ml) for 16 h before being stimulated with
S. aureus (1:10,000) for 24 h. Cell-free supernatants
were assayed by RIA for hIL-12 p40 and p70 secretion. Fig. 1
A shows that pre-exposure of
MDM to TNF- inhibited the production of IL-12 p40 and p70 induced by
IFN- and S. aureus in a dose-dependent manner. TNF-
also inhibited IFN- and LPS-stimulated IL-12 p40 and p70 production
similarly to S. aureus (data not shown). In all experiments
performed, regulation of IL-12 p70 secretion by TNF- is also
observed if measuring the secretion of IL-12 p40 chain. To determine
the specificity of the inhibitory effect of TNF-, MDM were incubated
with an anti-TNF- Ab B154.9 for 24 h or an isotype control
Ab (C20.8) before IFN- treatment. All cultures were kept in the
presence of 10 µg/ml of polymixin B to eliminate possible
contaminating endotoxin in our Ab preparations, which may complicate
the interpretation of the data (21). Importantly,
polymixin B did not restrict MDM IL-12 secretion following S.
aureus stimulation. Fig. 1B shows that addition of
anti-TNF- Ab B154.9 completely abolished the inhibitory effect
of the exogenous TNF- on IFN-/S. aureus-induced IL-12
production. The selectivity of the suppressive effect of TNF- on
IL-12 production was tested in parallel against proinflammatory
cytokine IL-6 as shown in Fig. 1C. Although IL-6 has been
reported to inhibit IL-12 secretion (22), our data showed
a selective effect by TNF- in suppressing IL-12 p40 secretion.
Finally, TNF- exposure of MDM had a positive effect on viability as
previously reported (23), ruling out a decrease of IL-12
p70 and p40 secretion due to an increase in programmed cell death.
Taken together, these results show that TNF- is a potent and
specific inhibitor of IL-12 production in macrophages.
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-mediated inhibition of IL-12 production
To determine the temporal characteristics of the TNF- action,
MDM were exposed to TNF- at different times during the stimulation
procedure, i.e., TNF- was added a day before IFN- priming, at the
time of IFN- priming, or at the time of stimulation with S.
aureus. In addition, a parallel culture treated equally was washed
at 20 h following TNF- treatment to determine the temporal
requirement for the presence or absence of TNF- to exert its
inhibitory effect. Fig. 2a
shows that the IFN-/S. aureus-stimulated IL-12 p70
production was inhibited strongly when TNF- was added before or at
the time of IFN- treatment (samples 3 and 4). However, the
inhibition was less pronounced when TNF- was added together with
IFN-. No inhibition was observed when TNF- was added at the same
time as S. aureus (sample 5). Washing the cells after an
overnight exposure to TNF- resulted in higher induction of IL-12 p70
production, but failed to reverse the inhibition (Fig. 2, a
and b, samples 2 and 3), suggesting that a constant presence
of TNF- is not required for its inhibitory effect.
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pretreatment interferes with the signaling of both IFN-
and S. aureus
To determine whether TNF- inhibitory effects extended to
S. aureus-induced or only IFN--enhanced IL-12 production
as observed in the murine system (13), MDM stimulation and
IL-12 secretion was measured in the presence of neutralizing Ab to
eliminate the endogenously produced IFN- in PBMC cultures with 10
µg/ml polymixin B. As expected, Fig. 3
shows that S. aureus-induced IL-12 p40 secretion by PBMC was
strongly inhibited by TNF- (sample 2 vs 3). Anti-IFN- treatment
resulted in a significant loss of S. aureus-induced IL-12
p40 production (sample 4), indicating that the endogenous IFN-
contributed to the production of IL-12 p40. However, the
IFN--independent IL-12 p40 production was significantly inhibited by
TNF- treatment. These results show that TNF- inhibits S.
aureus-induced IL-12 p40 production as well as the
IFN--enhanced production shown in Fig. 1
A.
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on IL-12 production is not
mediated by IL-10
IL-10 is one of the most potent inhibitors of macrophage
activation including the suppression of IL-12 production
(24). IL-10 exerts its repressive effect on IL-12
production by inhibiting directly the transcription of both the IL-12
p40 and the p35 genes (24). We tested whether
TNF--induced inhibition of IL-12 production in human MDM was
mediated by IL-10 because TNF- induces IL-10 expression
(25). MDM cultured in media containing 10 µg/ml
polymixin B were stimulated with S. aureus alone or with
IFN- and S. aureus after the pretreatment with TNF-,
in the absence or presence of the neutralizing anti-IL-10 Ab 12G8,
or an isotype control Ab (C20.8). Fig. 4
shows that the production of IL-12 p40 was stimulated with S.
aureus or IFN- priming plus S. aureus. Anti-IL-10
treatment substantially enhanced this production, while TNF-
pretreatment greatly inhibited it in the absence or presence of
anti-IL-10. These results show that TNF- inhibits IL-12 p40
production in human MDM independently of IL-10.
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-mediated inhibition is selective for IL-12 p40 gene
expression
RNase protection assay using the PharMingen multiprobe system was
performed on RNA samples derived from human MDM exposed to TNF-
before IFN-/LPS stimulation. Fig. 5
shows that IFN- priming before LPS stimulation significantly
enhanced the accumulation of mRNA for IL-12 p35, IL-12 p40, IL-10,
IL-1, IL-1ß, IL-1Ra, and IL-6. TNF- alone induced IL-10,
IL-1ß, IL-1Ra, and IL-6 mRNA expression. Pretreatment with TNF-
strongly inhibited IL-12 p40 mRNA induced by either LPS alone or
IFN- plus LPS while having only a modest and statistically
nonsignificant effect on the p35 message and an enhancing effect on
IL-1Ra. These results indicate that the inhibitory effect of TNF- is
selective for IL-12 p40 steady-state mRNA.
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inhibits IL-12 p40 gene expression at the transcriptional
level
To determine the molecular pathway through which TNF- affects
IL-12 gene expression, nuclear run-on assays were performed with
isolated nuclei derived from human MDM exposed to TNF- before
IFN-/LPS stimulation. Fig. 6 shows
that TNF- strongly inhibited the IFN-/LPS-stimulated IL-12 p40
transcription, and to a lesser degree that of the TNF- gene itself.
Interestingly, transcription of the IL-12 p35 gene was not affected by
TNF- priming. The stability of the IFN-/LPS-induced IL-12 p35 and
p40 mRNA was assessed following the blockade of de novo transcription
with actinomycin D. TNF- did not affect the half-life of the mRNA of
these two genes (data not shown). These results show that TNF-
inhibits IL-12 p40 gene expression in MDM at the transcriptional
level.
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treatment does not alter the nuclear translocation and/or
DNA binding of the major transcription factors involved in IL-12
p40 gene promoter activation
We analyzed by EMSA the binding and/or nuclear translocation of
all major transcription factors that are known to interact with the
IL-12 p40 promoter. We and others have shown previously that NF-B is
a critical factor involved in the induction of IL-12 p40 transcription
by LPS and IFN- (26, 27). Fig. 7 shows that pre-exposure of MDM to
TNF- alone for 40 h induced marginal NF-B binding to the
B "half site" element at -107/-117 (TGAAATTCCCC) of the IL-12
p40 promoter (26), whereas stimulation with LPS and
IFN- caused a strong increase in the binding activity to this
element, which was not inhibited by the pretreatment with TNF-. Ab
supershift experiments demonstrated that the NF-B complex binding to
the IL-12 p40 promoter in TNF--treated or untreated MDM is similarly
composed of p65 and p50 heterodimers. These results suggest that the
inhibitory effects of TNF- on IL-12 gene expression are not mediated
through NF-B.
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in the murine
macrophage cell line RAW264.7 and that contains ets-2, IFN regulatory
factor-1 (IRF-1), NF-B c-Rel, and a novel, ets-2-related protein
with a molecular mass of 109 kDa (28). However, the intact
F1 binding activity is difficult to analyze in primary human MDM due to
the differences in the nuclear extraction procedure necessarily applied
to the two cell types, which result in the disintegration of the F1
complex in primary human cells. Thus, we analyzed several previously
identified components of the F1 complex, including c-Rel, IRF-1, and
ets-2, with respect to their nuclear translocation in MDM exposed or
not to TNF- before IFN-/LPS stimulation. Fig. 8 shows a representative Western blot
analysis of two of these transcription factors, c-Rel and IRF-1, which
indicated that their nuclear translocation after IFN-/LPS
stimulation was not reversed by TNF-. The analysis of the nuclear
expression of ets-2 in three donors’ MDM also did not yield
significant effects following TNF- treatment (data not shown). These
results indicate that the mechanism of IL-12 p40 suppression by TNF-
is distinct in that its transcriptional effect is not mediated by
inhibition of the major factors identified to mediate IL-12 p40
transcription.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this report, we observed that TNF- is a potent and selective
inhibitor of IL-12 p40 and p70 production from human MDM (Fig. 1). This
inhibition is observed when MDM were pre-exposed to TNF- before, but
not at, the time of stimulation with LPS or S. aureus (Fig. 2). The fact that the inhibitory effects of TNF- on macrophages take
a considerable preincubation period (
16 h) to attain suggests that
TNF- may be inducing de novo protein syntheses required for its
inhibitory activities. Interestingly, inhibition of IL-12 can result
after transient exposure to TNF-, i.e., the contact between and its
receptors needs not to be continuous. In addition, the sustained
inhibitory effect of TNF- of S. aureus-induced IL-12 in
the presence of neutralizing Ab against endogenously produced IFN-
suggests that TNF- mechanism of action is independent of IFN-
signaling (Fig. 3). The TNF--mediated inhibition is also highly
selective against IL-12 p40 gene expression because it does not affect
IL-12 p35 or other inflammatory cytokines such as IL-1, IL-1ß, and
IL-6 expressed in macrophages in response to inflammatory stimuli
(Fig. 5).
TNF- has been shown to induce the production of IL-10 by resting and
LPS-stimulated macrophages in a potential mechanism of negative
feedback regulation of the immune response (25). However,
the TNF- action in the regulation of human IL-12 production is
independent of IL-10, as we showed that the inhibition of IL-12 by
TNF- was unabated in the presence of an effective neutralizing IL-10
Ab (Fig. 4). Interestingly, our observation of an IL-10 independence of
the TNF- action in human MDM is different from the report by
Hodge-Dufour et al. (13) on TNF- pretreatment of
thioglycolate-elicited murine macrophages resulting in suppression of
IL-12 p40 and p70 production following LPS or low m.w. hyaluronan
stimulation. The latter observations from comparing IL-10 null and
wild-type mice indicated that the inhibition of IL-12 by TNF- can be
mediated by both IL-10-dependent and IL-10-independent mechanisms,
although the IL-10-dependent mechanism appears dominant and the other
redundant and compensatory in the absence of the endogenous IL-10
production.
In contrast to the mechanism of IL-10-mediated suppression of both
IL-12 p40 and p35 gene transcription, nuclear run-on analysis indicated
that TNF- induced transcriptional inhibition of the IL-12 p40 gene
only, not of the p35 gene (Fig. 6). Moreover, mRNA stability analysis
in MDM after TNF- treatment showed that the half-life of
IFN-/LPS-induced IL-12 p40 mRNA was not affected by TNF-
treatment (data not shown). The latter shows the mechanism of
inhibition of IL-12 p40 by TNF- is different to that described for
other macrophage-derived mRNAs inhibited by TNF- (41).
Taken together, our data strongly suggest a selective inhibition by
TNF- of IL-12 p40 gene transcription induced by LPS/S.
aureus alone or the combination of IFN- and LPS/S.
aureus.
The binding of LPS/IFN--induced NFB to the B "half site"
at -107/-117 of the human IL-12 p40 promoter was not inhibited by the
pretreatment with TNF- (Fig. 7). Nor did this treatment alter the
composition of the NF-B complex, e.g., from the transactivating
p65/p50 heterodimer to an inhibitory homodimer such as p50/p50
(42). However, we cannot rule out the possibility that
there might be a difference in the transactivating potential of the
p65/p50 heterodimer, which may be adversely affected by TNF-, as has
been shown in the case of LPS induction of NF-B in THP-1 cells
(43). Another site of critical importance for the human
IL-12 p40 transcription in macrophages is the ets element located at
–206 to –211 of the p40 promoter. Cotransfection experiments with
various combinations of expression vectors for NF-B p65, p50, c-Rel,
and ets-2 demonstrated that ets-2 and c-Rel synergistically activate
the transfected p40 promoter in the IL-12 p40-expressing cell lines
(RPMI 8866 and RAW264.7), and nonexpressing cells such as Bjab
(EBV- B cell line) and Jurkat (T cell line),
strongly suggesting that c-Rel and ets-2 functionally cooperate as the
major transcription factors necessary and sufficient to direct the cell
type-specific expression of the p40 gene (27). Analysis of
several known components of the F1 complex, including c-Rel, IRF-1, and
ets-2, by Western blot indicated that the nuclear translocation of
these three transcription factors induced by IFN- and LPS is not
affected by pretreatment with TNF- (Fig. 8). The nuclear
translocation of IRF-1 in the presence of TNF- is consistent with an
inhibitory mechanism by TNF- of IL-12 production following
IFN-/LPS/S. aureus stimulation that is independent of
upstream IFN- signaling events. Overall, the lack of evidence for a
defect in the known IL-12 p40 transcription machinery induced by
LPS/S. aureus or IFN-/LPS/S. aureus in the
presence of TNF- pretreatment indicate that the molecular target(s)
of TNF- involved in the suppression of IL-12 p40 transcription may
be novel. Interestingly, this novel regulation of transcription appears
to be specific to the IL-12 p40 gene without affecting any of the
upstream pathways.
Another candidate mediator of the inhibition of IL-12 by TNF- in the
presence of IFN- is NO (44). Several reports
demonstrated that inducible nitric oxide synthase (NOS2)-deficient mice
developed enhanced Th1 cell responses following infections and
antigenic stimulation, producing more IFN- and less IL-4 compared
with similarly treated wild-type mice (45, 46). The same
group subsequently showed that peritoneal macrophages from
NOS2-deficient mice infected with Leishmania major in vivo
or stimulated with IFN- or LPS in vitro produced significantly
higher levels of IL-12 than those from heterozygous and wild-type mice.
IL-12 production from the macrophage cell line J774 activated with LPS
or LPS plus IFN- could be markedly enhanced by the NOS2 inhibitor
L-NG-monomethyl
arginine (L-NMMA) and profoundly inhibited by the
NO-generating compound
S-nitroso-N-acetyl-penicillamine
(47). Interestingly, the effect of NO on IL-12 p40
production is also transcriptional and is activation dependent, as is
the effect of TNF- on IL-12 p40 expression shown in our study.
However, another group recently reported the opposite observation that
at day 1 of L. major infection of mice, genetic deletion or
functional inactivation of NOS2 abolished IFN- and NK cell response
with a correlative down-modulation of IL-12 p40 mRNA expression in the
lymph node of infected mice. In contrast, peritoneal macrophages from
both NOS2+/+ and NOS2-/-
mice produced similar amounts of IL-12 p70 in response to IFN- plus
LPS within 24 h of stimulation (48). The reasons
underlying the differences in the data generated by the two groups are
presently unclear. In our own experiment where we used a neutralizing
mAb to eliminate endogenous and exogenous IFN-, hence the generation
of NO, TNF- was still able to inhibit S. aureus-induced
IL-12 p40 production. In addition, our preliminary data (not shown)
indicated that the use of
L-NG-monomethyl
arginine in IFN-/LPS-treated human MDM failed to reverse
TNF--induced inhibition of IL-12 p40 production.
TNF- was originally defined on the basis of its ability to induce
hemorrhagic necrosis of transplanted mouse tumors and by its selective
cytotoxicity for transformed cells (49). Since then,
TNF- has been found to play a key role in orchestrating a wide
spectrum of complex events involved in inflammation and immunity.
Recent studies (14, 50, 51) in TNF-- or TNFR
p55-deficient mice subjected to various pathological challenges suggest
that TNF- has an essential homeostatic role in limiting the extent
and duration of an inflammatory process in vivo (14). For
example, TNF--deficient mice showed little initial response and a
late but vigorous and disorganized inflammatory response to heat-killed
C. parvum leading to death, in contrast to the prompt
response (granuloma formation and hepatosplenomegaly) and subsequent
resolution phase in C. parvum-injected wild-type mice
(14). The ability of TNF- to inhibit IL-12 production
by human macrophages demonstrated here provides direct evidence that in
addition to the cascade of inflammatory and antiinflammatory cytokines
induced during inflammation, TNF- signaling can selectively inhibit
IL-12 gene expression upon macrophage activation as part of the scheme
of cytokine feedback and self-limiting modulations. This may benefit
the host in controlling inflammatory responses as exemplified in murine
models above while at the same time providing a potential
immune-evasion mechanism to intracellular pathogens whose infection is
associated with chronic TNF- expression. Indeed, ongoing experiments
are directed at assessing the role of TNF- in inducing a decreased
IL-12 response in HIV-1 infection where sustained immune activation and
expression of TNF- is associated with a decrease in IL-12 and
cell-mediated responses. It remains to be tested whether this mechanism
can provide a novel target for reversing immune defense suppression
during chronic inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Luis J. Montaner, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: MDM, monocyte-derived macrophages; IRF-1, IFN regulatory factor 1; NOS, NO synthase.
Received for publication August 5, 1999. Accepted for publication December 1, 1999.
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 S. Sinha, L. J. Kaler, T. M. Proctor, C. Teuscher, A. A. Vandenbark, and H. Offner IL-13-Mediated Gender Difference in Susceptibility to Autoimmune Encephalomyelitis J. Immunol., February 15, 2008; 180(4): 2679 - 2685. [Abstract] [Full Text] [PDF]
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 C. C. Keller, O. Yamo, C. Ouma, J. M. Ong'echa, D. Ounah, J. B. Hittner, J. M. Vulule, and D. J. Perkins Acquisition of Hemozoin by Monocytes Down-Regulates Interleukin-12 p40 (IL-12p40) Transcripts and Circulating IL-12p70 through an IL-10-Dependent Mechanism: In Vivo and In Vitro Findings in Severe Malarial Anemia Infect. Immun., September 1, 2006; 74(9): 5249 - 5260. [Abstract] [Full Text] [PDF]
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L. Buonaguro, M. L. Tornesello, M. Tagliamonte, R. C. Gallo, L. X. Wang, R. Kamin-Lewis, S. Abdelwahab, G. K. Lewis, and F. M. Buonaguro Baculovirus-derived human immunodeficiency virus type 1 virus-like particles activate dendritic cells and induce ex vivo T-cell responses. J. Virol., September 1, 2006; 80(18): 9134 - 9143. [Abstract] [Full Text] [PDF]
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 N. Khan, S. S. Rahim, C. S. Boddupalli, S. Ghousunnissa, S. Padma, N. Pathak, D. Thiagarajan, S. E. Hasnain, and S. Mukhopadhyay Hydrogen peroxide inhibits IL-12 p40 induction in macrophages by inhibiting c-rel translocation to the nucleus through activation of calmodulin protein Blood, February 15, 2006; 107(4): 1513 - 1520. [Abstract] [Full Text] [PDF]
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 Y. Ito, I. Kawamura, C. Kohda, K. Tsuchiya, T. Nomura, and M. Mitsuyama Seeligeriolysin O, a protein toxin of Listeria seeligeri, stimulates macrophage cytokine production via Toll-like receptors in a profile different from that induced by other bacterial ligands Int. Immunol., December 1, 2005; 17(12): 1597 - 1606. [Abstract] [Full Text] [PDF]
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M. Zakharova and H. K. Ziegler Paradoxical Anti-Inflammatory Actions of TNF-{alpha}: Inhibition of IL-12 and IL-23 via TNF Receptor 1 in Macrophages and Dendritic Cells J. Immunol., October 15, 2005; 175(8): 5024 - 5033. [Abstract] [Full Text] [PDF]
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E. Papasavvas, J. Sun, Q. Luo, E. C. Moore, B. Thiel, R. R. MacGregor, A. Minty, K. Mounzer, J. R. Kostman, and L. J. Montaner IL-13 Acutely Augments HIV-Specific and Recall Responses from HIV-1-Infected Subjects In Vitro by Modulating Monocytes J. Immunol., October 15, 2005; 175(8): 5532 - 5540. [Abstract] [Full Text] [PDF]
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 M. Dorsch, Y. Qiu, D. Soler, N. Frank, T. Duong, A. Goodearl, S. O'Neil, J. Lora, and C. C. Fraser PK1/EG-VEGF induces monocyte differentiation and activation J. Leukoc. Biol., August 1, 2005; 78(2): 426 - 434. [Abstract] [Full Text] [PDF]
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M. M. Freeman and H. K. Ziegler Simultaneous Th1-Type Cytokine Expression Is a Signature of Peritoneal CD4+ Lymphocytes Responding to Infection with Listeria monocytogenes J. Immunol., July 1, 2005; 175(1): 394 - 403. [Abstract] [Full Text] [PDF]
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H. Cho, T. M. Lasco, S. S. Allen, T. Yoshimura, and D. N. McMurray Recombinant Guinea Pig Tumor Necrosis Factor Alpha Stimulates the Expression of Interleukin-12 and the Inhibition of Mycobacterium tuberculosis Growth in Macrophages Infect. Immun., March 1, 2005; 73(3): 1367 - 1376. [Abstract] [Full Text] [PDF]
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 Q. Luo, X. Ma, S. M. Wahl, J. J. Bieker, M. Crossley, and L. J. Montaner Activation and Repression of Interleukin-12 p40 Transcription by Erythroid Kruppel-like Factor in Macrophages J. Biol. Chem., April 30, 2004; 279(18): 18451 - 18456. [Abstract] [Full Text] [PDF]
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M. A. P. Oliveira, G. M. A. C. Lima, M. T. Shio, P. J. M. Leenen, and I. A. Abrahamsohn Immature macrophages derived from mouse bone marrow produce large amounts of IL-12p40 after LPS stimulation J. Leukoc. Biol., November 1, 2003; 74(5): 857 - 867. [Abstract] [Full Text] [PDF]
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J. Liu, S. Cao, L. M. Herman, and X. Ma Differential Regulation of Interleukin (IL)-12 p35 and p40 Gene Expression and Interferon (IFN)-{gamma}-primed IL-12 Production by IFN Regulatory Factor 1 J. Exp. Med., October 20, 2003; 198(8): 1265 - 1276. [Abstract] [Full Text] [PDF]
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A. Smith, F. Santoro, G. Di Lullo, L. Dagna, A. Verani, and P. Lusso Selective suppression of IL-12 production by human herpesvirus 6 Blood, October 15, 2003; 102(8): 2877 - 2884. [Abstract] [Full Text] [PDF]
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G. J. Nau, A. Schlesinger, J. F. L. Richmond, and R. A. Young Cumulative Toll-Like Receptor Activation in Human Macrophages Treated with Whole Bacteria J. Immunol., May 15, 2003; 170(10): 5203 - 5209. [Abstract] [Full Text] [PDF]
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J.-N. Tournier, A. Q. Hellmann, G. Lesca, A. Jouan, E. Drouet, and J. Mathieu Fever-like thermal conditions regulate the activation of maturing dendritic cells J. Leukoc. Biol., April 1, 2003; 73(4): 493 - 501. [Abstract] [Full Text] [PDF]
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M. Rosenzwajg, F. Jourquin, L. Tailleux, and J. C. Gluckman CD40 ligation and phagocytosis differently affect the differentiation of monocytes into dendritic cells J. Leukoc. Biol., December 1, 2002; 72(6): 1180 - 1189. [Abstract] [Full Text] [PDF]
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Y. Drechsler, S. Chavan, D. Catalano, P. Mandrekar, and G. Szabo Fc{gamma}R cross-linking mediates NF-{kappa}B activation, reduced antigen presentation capacity, and decreased IL-12 production in monocytes without modulation of myeloid dendritic cell development J. Leukoc. Biol., October 1, 2002; 72(4): 657 - 667. [Abstract] [Full Text] [PDF]
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L. Zhao, C. A. Cuff, E. Moss, U. Wille, T. Cyrus, E. A. Klein, D. Pratico, D. J. Rader, C. A. Hunter, E. Pure, et al. Selective Interleukin-12 Synthesis Defect in 12/15-Lipoxygenase-deficient Macrophages Associated with Reduced Atherosclerosis in a Mouse Model of Familial Hypercholesterolemia J. Biol. Chem., September 13, 2002; 277(38): 35350 - 35356. [Abstract] [Full Text] [PDF]
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Y.-C. Chen and S.-Y. Wang Activation of Terminally Differentiated Human Monocytes/Macrophages by Dengue Virus: Productive Infection, Hierarchical Production of Innate Cytokines and Chemokines, and the Synergistic Effect of Lipopolysaccharide J. Virol., August 28, 2002; 76(19): 9877 - 9887. [Abstract] [Full Text] [PDF]
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M. Wittmann, P. Kienlin, S. Mommert, A. Kapp, and T. Werfel Suppression of IL-12 Production by Soluble CD40 Ligand: Evidence for Involvement of the p44/42 Mitogen-Activated Protein Kinase Pathway J. Immunol., April 15, 2002; 168(8): 3793 - 3800. [Abstract] [Full Text] [PDF]
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S. B. Flohe, J. Bruggemann, C. Herder, C. Goebel, and H. Kolb Enhanced proinflammatory response to endotoxin after priming of macrophages with lead ions J. Leukoc. Biol., March 1, 2002; 71(3): 417 - 424. [Abstract] [Full Text] [PDF]
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N. S. Goncalves, M. Ghaem-Maghami, G. Monteleone, G. Frankel, G. Dougan, D. J. M. Lewis, C. P. Simmons, and T. T. MacDonald Critical Role for Tumor Necrosis Factor Alpha in Controlling the Number of Lumenal Pathogenic Bacteria and Immunopathology in Infectious Colitis Infect. Immun., November 1, 2001; 69(11): 6651 - 6659. [Abstract] [Full Text] [PDF]
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 L. Steinman Blockade of gamma interferon might be beneficial in MS Multiple Sclerosis, October 1, 2001; 7(5): 275 - 276. [PDF]
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E. Bryl, A. N. Vallejo, C. M. Weyand, and J. J. Goronzy Down-Regulation of CD28 Expression by TNF-{alpha} J. Immunol., September 15, 2001; 167(6): 3231 - 3238. [Abstract] [Full Text] [PDF]
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X. Xu, K. Sumita, C. Feng, X. Xiong, H. Shen, S. Maruyama, M. Kanoh, and Y. Asano Down-Regulation of IL-12 p40 Gene in Plasmodium berghei-Infected Mice J. Immunol., July 1, 2001; 167(1): 235 - 241. [Abstract] [Full Text] [PDF]
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M. Wysocka, S. Robertson, H. Riemann, J. Caamano, C. Hunter, A. Mackiewicz, L. J. Montaner, G. Trinchieri, and C. L. Karp IL-12 Suppression During Experimental Endotoxin Tolerance: Dendritic Cell Loss and Macrophage Hyporesponsiveness J. Immunol., June 15, 2001; 166(12): 7504 - 7513. [Abstract] [Full Text] [PDF]
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M. J. Walter, N. Kajiwara, P. Karanja, M. Castro, and M. J. Holtzman Interleukin 12 p40 Production by Barrier Epithelial Cells during Airway Inflammation J. Exp. Med., January 29, 2001; 193(3): 339 - 352. [Abstract] [Full Text] [PDF]
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X. Ma and L. J. Montaner Proinflammatory response and IL-12 expression in HIV-1 infection J. Leukoc. Biol., September 1, 2000; 68(3): 383 - 390. [Abstract] [Full Text] [PDF]
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