| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*
Department of Neurology, University of Maryland, Baltimore, MD 21201;
Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201;
Neuroimmunology Branch, National Institute of Neurologic Disease and Stroke, National Institutes of Health, Bethesda, MD 20892; and
§
Berlex Laboratories, Richmond, CA 94806
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in T cells and NK cells, promotion of Th1
responses, and enhancement of cytotoxic T cells, NK cells, and delayed
hypersensitivity responses (4, 5, 6). IL-12 production is
under positive and negative control by Th1 and Th2 cytokines,
respectively. IFN- enhances, whereas IL-10, IL-4, TGF-ß, and
IFN-
/ß suppress IL-12 production. Experimental evidence indicates
that IL-12 production is normally kept under tight inhibitory control
by Th-2 cytokines (reviewed in Ref. 3).
There is increasing evidence that implicates IL-12 in the pathogenesis
of a number of autoimmune diseases including multiple sclerosis
(MS),3
insulin-dependent diabetes mellitus, and rheumatoid arthritis (reviewed
in Ref. 3). In MS (reviewed in Ref.
7), it has been demonstrated that PBMC from patients with progressive disease secrete elevated levels of IL-12 (>8),
and PBMC production of IL-12 in response to IFN- plus bacterial
stimuli (9) or CD40-CD40 ligand interaction (10, 11) is increased in patients with MS. Elevated serum IL-12
levels have been reported in patients with progressive MS
(12). In addition, IL-12 is expressed in MS lesions
(13), and the degree of inflammation correlates with IL-12
release (14, 15). Studies in experimental allergic
encephalomyelitis (EAE), an animal model of MS, indicate that IL-12
plays an important pathogenic role in this model. For example,
neutralization of IL-12 prevents the development of EAE, and
administration of IL-12 to EAE animals in remission evokes relapses
(16, 17, 18, 19). Because IFN- has been shown to induce
relapses in MS (20), and because IL-12 is a potent inducer
of IFN-, it is plausible that IL-12 could have an important role in
disease progression in MS.
IFN-ß-1b (Betaseron) was the first immunomodulatory drug to be
approved for the treatment of MS (21). This was followed
by the approval of another form of IFN-ß: IFN-ß-1a (Avonex)
(22). These drugs have been shown to reduce the number of
relapses and to slow down disease progression in MS. There is evidence
both in vitro and in vivo that IFN-ß treatment inhibits many of the
activities of IFN- in MS (23). Because of the existence
of a positive regulatory feedback circuit between IFN- and IL-12
(2), we examined the effect of IFN-ß on IL-12 production
in PBMC and myelin basic protein (MBP)-specific T cell lines from
healthy individuals and MS patients. We demonstrate that IFN-ß-1b
inhibits IL-12 production, and that this inhibition is IL-10
dependent.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PBMC were obtained from healthy donors by leukapheresis performed at the Red Cross (Baltimore, MD), and from MS patients by venipuncture. An informed consent approved by the institutional review board was signed by the donors. PBMC were purified using lymphocyte separation medium (ICN, Aurora, OH) and were frozen in liquid nitrogen until used. PBMC were cultured in RPMI 1640 supplemented with 2% human AB serum (Sigma, St. Louis, MO), HEPES buffer, glutamine, gentamicin, and penicillin/streptomycin (Life Technologies, Gaithersburg, MD) in 24-well culture plates at 2 x 106 cells/well in 2 ml of medium.
MBP-specific T cell lines and clone
MBP-specific T cell lines were generated from two patients with MS as previously described (24). The MBP peptide 87–99-specific T cell clone 3A6 (HLA restriction DRB5*0101) was generated from PBMC of a MS patient and was described in an earlier publication (24). Its clonality was confirmed by TCR analysis.
Reagents
Recombinant human IL-12 p40, p70, and human IL-10 were purchased from R&D Systems (Minneapolis, MN) to be used as standards in the ELISA. Recombinant human IFN-ß-1b (Betaseron; sp. act., 32 x 106 IU/mg) was supplied by Berlex Laboratories (Richmond, CA). Anti-human IL-10 and IL-4 neutralizing Abs (both polyclonal goat IgG); capture mAbs for human IL-12 p40, p70, and IL-10; and detection Abs of biotinylated anti-human IL-12 and IL-10 polyclonal Abs were purchased from R&D Systems. Staphylococcus aureus Cowan (SAC) strain (Pansorbin) was purchased from Calbiochem (La Jolla, CA) as a 10% (w/v) suspension and was diluted 1/10 in RPMI and used at a final concentration of 20 µg/ml.
ELISA
IL-12 p40, p70, and IL-10 protein production were quantitated by a four-layer Ab sandwich ELISA. Ninety-six-well, high binding microtiter polystyrene base immunoassay plates (Dynex Technologies, Chantilly, VA), were coated with 0.4 µg/well of mouse anti-human IL-12 p40, IL-12 p70, or IL-10 mAb diluted in 100 µl of PBS at 4°C overnight. The plates were washed four times with PBS containing 0.1% Tween 20 and blocked with 200 µl of 10% nonfat milk/well for 2 h at room temperature. Standard concentrations of human recombinant IL-12 p40, p70, and IL-10 were diluted in 1% nonfat milk in PBS/0.1% Tween 20 to concentrations of 3200, 1600, 800, 400, 200, 100, 50, and 25 pg/ml. One hundred microliters of each diluted standard and test samples were added to duplicate wells and incubated at 4°C overnight. After washing four times with PBS/0.1% Tween 20, 100 µl of goat biotinylated anti-human IL-12 or IL-10 polyclonal Abs were added to each well at 300 ng/ml. After 1-h incubation at 37°C the plate was washed four times. One hundred microliters of streptavidin HRP conjugate (Southern Biotechnology Associates, Birmingham, AL) at 1/4000 dilution was added to each well and incubated for 30 min at 37°C. After rinsing four times, the color reaction was performed with TMB peroxidase enzyme immunoassay substrate kit from Bio-Rad (Hercules, CA) according to the manufacturer’s protocol. After 5 min, the plate was read in a microplate reader (Bio-Rad) at 450 nm, and the data were analyzed using Microplate Manager III Macintosh Data Analysis Software for Bio-Rad Microplate Readers. The detection range and sensitivity of the ELISAs were as follows: IL-12 p40, range of 31.25–2000 pg/ml and sensitivity of 7 pg/ml; IL-12 p70, range of 7.8–500 pg/ml and sensitivity of 2 pg/ml; and IL-10 range of 31.25–2000 pg/ml and sensitivity of 6.3 pg/ml.
Quantitative RT-PCR
Total RNA was isolated from cell cultures with an acid
guanidinium thiocyanate solution and phenol-chloroform extraction using
the Trizol reagent (Life Technologies, Gaithersburg, MD). RNA was
reverse transcribed to cDNA with random hexamer primers in a total
volume of 50 µl using the TaqMan RT reagents according to the
manufacturer’s instructions (Perkin-Elmer, Foster City, CA).
Quantitative real-time RT-PCR was performed on an ABI Prism-7700
Sequence Detection System (Perkin-Elmer). Briefly, this method monitors
the degradation of a dual-labeled fluorescent probe (TaqMan probe) in
real-time concomitant with PCR amplification (25). Input
target RNA levels are correlated with the time (measured in PCR cycles)
at which the reporter fluorescent emission increases beyond a threshold
level. For the detection of IL-10 and IL-12p40 transcripts,
predeveloped TaqMan assay reagents (Perkin-Elmer) containing cytokine
primers and probe were used according to the manufacturer’s
instructions. Samples were run in duplicate, and plasmids containing
the respective cytokine cDNA were included as positive controls. For
sample normalization, 18S ribosomal RNA was amplified using TaqMan
ribosomal RNA control reagents (Perkin-Elmer). Quantification of gene
expression relative to 18S ribosomal RNA was calculated by the
protocol’s 
CT method. This calculation is
based on equal amplification efficiencies of the target and reference
and correlates well with standard curve methods (26).
Statistical analysis
The significance of the effect of IFN-ß-1b treatment on cytokine production was analyzed by calculating means, SD, SEs, and p values. Paired Student’s t test and repeated measures ANOVA were performed using Macintosh PRISM 2.0 software. p < 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To examine the effect of IFN-ß-1b on IL-12 p40 production, PBMC
were cultured at a concentration of 2 x 106
cells in 2 ml of complete RPMI containing 2% human AB serum. PBMC were
stimulated with either LPS (1 µg/ml) or LPS (1 µg/ml) plus IFN-
(100 IU/ml) in the presence of serial doses of IFN-ß (10, 100, 1000,
and 5000 IU/ml). The stimulation protocol was as follows. One hour
after plating, cells were prestimulated with IFN-ß-1b for 4 h
before adding LPS and IFN-. The cytokines and LPS were left in the
culture medium until the cells were harvested. Control conditions
consisted of untreated cells, cells treated with IFN-ß-1b, LPS,
IFN-, or LPS plus IFN- 4 h after plating. After 24 h in
culture, supernatants were harvested and assayed for IL-12 p40 and p70
secretion by ELISA. Results obtained from six healthy donors are
presented in Table I
. IL-12 p40 was
undetectable in supernatants from unstimulated cells or those
stimulated with doses of IFN-ß-1b ranging from 10–1000 IU/ml.
However, levels of p40 in the range of 24–483 pg/ml were detected in
cultures stimulated with 5000 IU/ml of IFN-ß-1b. IFN-ß-1b either
had no significant effect (donors RC595 and RC696) or had a modest
inhibitory effect (donors RC898 and RC798) on LPS-induced IL-12 p40.
LPS-induced IL-12 p40 production was enhanced by cotreatment with
IFN- in cells from all donors examined. In each of the six donors,
IFN-ß-1b inhibited IL-12 p40 induced by the combination of LPS and
IFN-. This inhibitory effect was observed at IFN-ß-1b doses as low
as 10 IU/ml and increased at an IFN-ß-1b dose of 100 IU/ml, but
peaked at higher doses. The IL-12 p40 levels as well as the percent
inhibition are presented in Table I. This inhibitory effect ranged from
18–76% among the six donors. The mean levels of IL-12 p40 and SDs
from the six donors are presented in Fig. 1. The inhibitory effect of IFN-ß on
LPS- plus IFN--induced IL-12 p40 was statistically significant at
each dose, and the overall repeated measure ANOVA p value
was <0.0001. This inhibitory effect was also observed when IFN-ß was
added 4 h after stimulation with LPS plus IFN-, but the
magnitude of this inhibitory effect was slightly lower than that
obtained with pretreatment with IFN-ß (data not shown).
|
|
among the six donors. Therefore, we elected
to examine the effect of IFN-ß-1b treatment on IL-12 p40 and p70 in
response to SAC plus IFN- stimulation which is known to induce IL-12
p70 in PBMC (3).
IFN-ß-1b inhibits IL-12 production in PBMC induced by SAC and
IFN-
Examination of the effect of IFN-ß-1b on IL-12 p40 and p70
production in response to SAC plus IFN- followed the same
experimental design used for stimulation with LPS plus IFN-. SAC was
used at a final concentration of 20 µg/ml, and IFN- at the dose of
100 IU/ml. After 24-h treatment, supernatants were harvested and
assayed for p40 and p70. The effects of IFN-ß-1b on SAC-induced as
well as SAC- plus IFN--induced IL-12 p40 in six donors are presented
in Table II. IFN-ß-1b inhibited
SAC-induced IL-12 p40 production in PBMC from each of the six donors
examined, at doses ranging from 10–1000 IU/ml. However, at IFN-ß-1b
dose of 5000 IU/ml, inhibition was observed in four of the six donors,
and in the other two donors either no effect (RC 694) or a mild
stimulatory effect (RC 898) was obtained. The inhibitory effect of
IFN-ß-1b on SAC-induced p40 ranged from 6–51%, and the mean
inhibitory effect in the five donors was statistically significant at a
dose of IFN-ß-1b of 100 IU/ml. The inhibitory effect of IFN-ß-1b on
SAC- plus IFN--induced p40 was more prominent and ranged from
27–83% (Table II). This inhibitory effect was IFN-ß dose dependent
in the range of 10–1000 IU/ml, but no further inhibition was observed
at IFN-ß-1b dose of 5000 IU/ml. The mean level of IL-12 p40, SDs, and
p values are presented in (Fig. 2A). The inhibitory effect of
IFN-ß on SAC- plus IFN--induced IL-12 p40 was statistically
significant at all IFN-ß-1b doses tested, and the overall repeated
measures ANOVA p value was <0.0001.
|
|
(Table III and Fig. 2
B). In contrast to p40, the effect of IFN-ß-1b on
SAC-induced IL-12 p70 was additive in some donors, but the mean effect
was not statistically significant (Table III). However, IFN-ß-1b had
an inhibitory effect on IL-12 p70 induced by SAC plus IFN- in five
of the six donors examined, and this inhibitory effect was dose
dependent in the range of 10–1000 IU/ml. The mean levels for IL-12
p70, SDs, and p values are presented in Fig. 2B.
The inhibitory effect of IFN-ß-1b on SAC- plus IFN--induced IL-12
p70 was statistically significant at IFN-ß-1b doses of 100-5000
IU/ml, and the overall repeated measures ANOVA p value was
<0.0001.
|
Because IL-10 is a negative regulator of IL-12 production
(27, 28), and because IFN-ß has been reported to enhance
IL-10 production (29, 30), supernatants from the preceding
experiments were assayed for IL-10 levels to determine whether
IFN-ß-1b enhances IL-10 production in cells stimulated with LPS plus
IFN- or SAC plus IFN-. The mean effect of IFN-ß-1b on LPS- plus
IFN--induced IL-10 levels in the six donors is shown in Fig. 3A. Neither IFN- nor
IFN-ß individually had an effect on basal IL-10 production. LPS plus
IFN- induced higher levels of IL-10 in the presence of IFN-ß-1b
compared with those cultures stimulated with LPS plus IFN- alone
(Fig. 3A). The mean IL-10 levels induced in the presence of
IFN-ß-1b were significant as determined by repeated measures ANOVA
(p = 0.0422). Similarly, SAC- plus
IFN--induced IL-10 levels were significantly increased by IFN-ß
treatment (Fig. 3B; by repeated measures ANOVA,
p = 0.0084).
|
To determine whether the IFN-ß-1b inhibition of IL-12 observed
in PBMC from normal donors also occurs under pathological conditions,
we examined this inhibitory effect in PBMC from six MS patients with
relapsing-remitting disease. IL-12 p40, IL-12 p70, and IL-10 levels
were measured by ELISA. Because of the variation in the basal levels
for these cytokines and in the magnitude of IL-12 inhibition and IL-10
up-regulation, we analyzed the data as both absolute levels as well
percent change from baseline (Fig. 4). As
with the normal donors, in vitro treatment with IFN-ß-1b inhibited
SAC- plus IFN--induced IL-12 production in each of the six MS
patients. The mean SAC- plus IFN--induced IL-12 p40 levels (Fig. 4A) were significantly lower (p <
0.001) in the presence of IFN-ß-1b doses of 10, 100, and 1000 IU/ml
(by overall repeated measures ANOVA, p = 0.0006). The
mean percent change in p40 levels (Fig. 4b) was also
statistically significant (by overall repeated measures ANOVA,
p = 0.0001). A similar IFN-ß-1b inhibitory effect on
SAC- plus IFN--induced p70 was observed (Fig. 4C) and was
statistically significant when this effect was expressed as the percent
change from baseline (by overall repeated measures ANOVA,
p = 0.0004; Fig. 4D). IFN-ß treatment also
resulted in enhanced IL-10 production in PBMC from the six MS patients.
The overall increase in mean IL-10 levels and the percent change from
baseline were statistically significant (by repeated measures ANOVA,
p = 0.049 and 0.0366, respectively; Fig. 4, E and F).
|
Because IL-10 levels may not necessarily correlate with function,
we examined the contribution of IL-10 to the inhibitory effect of
IFN-ß-1b on IL-12 more directly by neutralizing IL-10 in our tissue
culture system. PBMC were stimulated with LPS plus IFN- or LPS plus
IFN- plus IFN-ß-1b in the presence of either a control Ab
(nonimmune goat IgG) or a neutralizing polyclonal anti-IL-10 Ab.
The dose of the anti-IL-10 Ab used (10 µg/ml) was sufficient to
neutralize 500 ng/ml of recombinant human IL-10. IL-10 neutralization
experiments were performed at serial IFN-ß-1b doses of 10, 100, 1000,
and 5000 IU/ml. The results obtained from four donors are shown in
Table IV. First, we observed that
addition of anti-IL-10 Ab to the control cultures (i.e., LPS plus
IFN- in the absence of IFN-ß) resulted in superinduction of IL-12.
This is consistent with an endogenous role for IL-10 in suppressing
IL-12 production. Second, in each of the four donors, the inhibitory
effect of IFN-ß-1b on IL-12 p40 production was markedly abolished by
the anti-IL-10 Ab, as IL-12 p40 levels approached the superinduced
levels (i.e., levels obtained in response to LPS plus IFN- in the
presence of anti-IL-10 Ab). This effect was consistently seen in
all four donors and at each of the IFN-ß-1b doses examined (Table IV). The mean levels of IL-12 p40 induced by LPS plus IFN- in the
presence of the anti-IL-10 or the control Ab in the four donors are
presented in Fig. 5. The mean inhibitory
effect of IFN-ß on IL-12 p40 was reduced from 57% in the presence of
the control Ab to 14% in the presence of the anti-IL-10 Ab. This
inhibitory effect was substantially reduced and was insignificant in
the presence of the anti IL-10 Ab (overall repeated measures ANOVA,
p = 0.204), whereas it was significant in the presence
of control Ab (p < 0.0001) or without Ab
(p < 0.0001). Next we determined whether IL-10
is involved in IFN-ß-1b inhibition of IL-12 induced by SAC plus
IFN-. The results were similar to those obtained in response to LPS
plus IFN-. A representative experiment is shown in Fig. 6. The inhibitory effect of IFN-ß-1b on
IL-12 p40 was significantly reduced in the presence of anti-IL-10,
but not the control Ab. The maximum inhibitory effect in the presence
of anti-IL-10 Ab was 22% compared with 90% without Ab or in the
presence of control Ab.
|
|
|
. Fig. 7 is a representative experiment that
shows titration of the ability of anti-IL-10 (Fig. 7, A
and C), but not the control Ab (Fig. 7, B and
D), to block the inhibitory effect of IFN-ß-1b on IL-12
induction by SAC plus IFN-. The IFN-ß-1b inhibitory effect on p70
appeared to be more readily IL-10 dependent than that on p40, as
blockade of this inhibitory effect was observed at lower anti-IL-10
Ab doses for p70 than for p40. In contrast to anti-IL-10 Ab,
polyclonal anti-IL-4 Ab (5 µg/ml) failed to abrogate the
inhibitory effect of IFN-ß-1b on inducible IL-12 (data not
shown).
|
To determine whether IFN-ß-1b inhibits IL-12 production in an
Ag-specific system, we generated several MBP-specific T cell lines
(TCL), and clones (TCC). We chose MBP because of the potential
relevance of this Ag to MS. We identified two TCL (MDPF3 and RSMD2) and
one TCC (3A6) that produce IL-12 p40 in which we examined the effect of
IFN-ß on IL-12 production. IFN-ß was added to the cultures at the
same time TCL were stimulated with Ag and autologous PBMC feeders. We
specifically did not irradiate the feeders, because they are the major
source of IL-12. IFN-ß-1b (100 IU/ml) produced marked inhibition of
IL-12 p40 in one of the two TCL (RSMD2; Fig. 8). IL-12 p70 levels were not detectable
in supernatants from these TCL, as p70 levels are generally 100-fold
lower than those of p40. In the MBP-specific TCC, IFN-ß markedly
inhibited IL-12 p40 at doses of 100 and 1000 IU/ml (Fig. 9A). This was associated with
a rise in IL-10 levels (Fig. 9B). Furthermore, the
inhibitory effect of IFN-ß-1b on IL-12 p40 was abolished in the
presence of anti-IL-10, but not control Ab (Fig. 9C),
indicating that the inhibitory effect of IFN-ß on IL-12 is IL-10
dependent.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It is unlikely that the reduced IL-12 levels in IFN-ß-1b-pretreated
PBMC are due to cell death or exhaustion induced by the latter
cytokine, for several reasons. First, IL-10 levels increased as a
result of IFN-ß pretreatment in the same supernatants in which IL-12
was inhibited. Second, the IFN-ß inhibitory effect on IL-12 was not
universal, as it was not observed in donor RC 599 (Table III) or one
MBP TCL (Fig. 8). Third, our previous studies indicate that IFN-ß is
toxic to human PBMC at doses exceeding 10,000 IU/ml
(32).
Our findings of IFN-ß-1b inhibition of inducible IL-12 are consistent
with those reported by Cousens et al. (33, 34),
demonstrating that IFN-/ß negatively regulates IL-12 production in
mouse splenic lymphocytes stimulated with SAC or infected with
lymphocytic choriomeningitis virus. Our study confirms these findings
in human PBMC and, furthermore, demonstrates that the inhibitory effect
of IFN-ß-1b on IL-12 is IL-10 dependent.
Although IFN-ß-1b inhibited IL-12 p40 induction in response to either
SAC or LPS in some donors, the magnitude of this inhibition was less
compared with the inhibition of IL-12 p40 levels induced by a
combination of LPS plus IFN- or SAC plus IFN-. Furthermore, while
the effect of IFN-ß on SAC-induced p70 was additive, the effect on
SAC- plus IFN--induced p70 was inhibitory (Table III). This suggests
a significant inhibitory effect of IFN-ß-1b on the priming effect of
IFN- in the induction of IL-12. This is also consistent with the
fact that IFN-ß-1b antagonizes many of the immunological effects of
IFN- (23). The mechanism for this antagonism is not
completely understood, but could involve competition for transcription
factors shared by both cytokines. Transcriptional regulation of the
IL-12 gene in response to IFN- priming, mitogen stimulation, and
infectious agents has been examined in a number of recent studies
(35, 36, 37, 38, 39). Several DNA binding sites have been identified
that could potentially contribute to regulation of the IL-12 promoter
activity, including Ets, NF-
B, AP-1, Sp-1, NF-IL-6, and IFN response
factor family binding sites. The Ets and the NF-B sites contribute
to the priming effect with IFN- on mitogen and pathogen-induced
IL-12 in murine macrophage cell lines (36, 37). In
addition, IFN consensus sequence binding protein (ICSBP) a member of
the IFN response factor family of transcription factors, contributes to
the regulation of IL-12 production during parasitic infection as
ICSBP-deficient mice display impaired resistance to intracellular
infection and defective IL-12 p40 production (38, 39).
ICSBP is inducible primarily by IFN-/ß and is generally believed
to mediate negative regulation (40). Therefore, it is
possible to speculate that IFN-ß-1b may inhibit the priming effect of
IFN- on IL-12 production by interfering with the activation of one
of the transcription factors, including Ets, NF-B, or ICSBP.
Although our studies were primarily conducted in an Ag-nonspecific
system, the findings are relevant to etiologic factors that can trigger
autoimmune disease. Microbial Ags alone or in combination with
IFN- induce IL-12 and are potential triggers of autoimmune diseases,
including MS (20, 41). Therefore, our findings of
IFN-ß-1b inhibition of LPS/SAC- plus IFN--induced IL-12 are of
potential pathogenic and therapeutic relevance. This is further
supported by the demonstration that IFN-ß-1b inhibits IL-12 in
Ag-specific T cells directed against the autoantigen MBP.
Furthermore, this study demonstrates that the inhibitory effect of
IFN-ß-1b on IL-12 production is IL-10 dependent. A number of factors
have been reported to negatively regulate IL-12 production (3, 42, 43, 44, 45, 46), notably Th2 cytokines such as IL-10, TGF-ß, and IL-4
(27, 28, 42, 43); soluble TNF receptor (44);
and PGE2 (3). It is clear from our
data that the inhibitory effect of IFN-ß-1b on IL-12 induction can be
substantially reduced, and in some donors completely abolished, in the
presence of anti-IL-10 Ab. This was observed at increasing doses of
the Ab as well as with serial doses of IFN-ß-1b. On the other hand,
unlike anti-IL-10, anti-IL-4 Ab repeatedly failed to reverse
the inhibitory effect of IFN-ß on IL-12 production. In addition,
PGE2 inhibition with indomethicin in doses of
1–100 µM did not reverse this inhibitory effect (data not shown).
Treatment with anti-TGF-ß Ab had a minimal effect in blocking the
inhibitory effect of IFN-ß on IL-12 (<10%) in cells from two donors
(data not shown). We propose that IL-10 has an important role in
IFN-ß-1b inhibition of inducible IL-12. This mechanism could involve
enhancement of IL-10 production by IFN-ß-1b, which, in turn, could
inhibit IL-12 production (18, 23, 28). This is based on
the finding that IL-10 levels increased in response to IFN-ß in
LPS/SAC- plus IFN--stimulated PBMC. The overall increase in mean
IL-10 levels was statistically significant as determined by repeated
measures ANOVA (Fig. 3). In addition, a statistically significant
increase in IL-10 levels was observed in cells from the six MS patients
(Fig. 4C) and in the MBP-T cell clone (Fig. 9B)
in response to IFN-ß.
There is increasing evidence that IL-10 negatively regulates IL-12
production under physiologic and pathologic conditions (18, 43). This, in fact, is supported by the data presented in Fig. 5
and Table IV. This data show that LPS- plus IFN--inducible IL-12
levels are higher in the presence of anti-IL-10 Ab than in cultures
without Ab or in the presence of the control Ab. These findings support
the fact that IL-10 has a tonic suppressive effect on IL-12 production,
and that if this effect is neutralized, IFN-ß would not be adequate
to suppress IL-12 production. Therefore, IL-10 is a critical and
necessary cofactor for the inhibitory effect of IFN-ß on inducible
IL-12 to occur.
Our findings could have important implications on the treatment of
autoimmune diseases, specifically MS, for which IFN-ß is an approved
therapy (21, 22). As indicated earlier, there is
increasing evidence that IL-12 may be involved in disease progression
in MS (). It is also well established that IFN-
provokes disease activity in MS (20). Because a positive
feedback loop between IFN- and IL-12 exists, inhibition of this
circuit by IFN-ß could be beneficial for the treatment of disease
progression in MS.
Reciprocal inhibition between IL-12 and IL-10 is likely to play an important role in autoimmune diseases such as MS and EAE. Segal et al. (18) found that anti-IL-12-treated unimmunized mice as well as those challenged with MBP/CFA consistently produce high IL-10 levels, but no IL-4. IL-10 seems to be produced in an Ag-independent manner by a CD4+ T cell different from the conventional Th2 cell. They concluded that there is tonic inhibition of IL-10 by the constitutive presence of IL-12. A recent study in patients with MS demonstrated that a decrease in IL-10 and an increase in IL-12 p40 mRNA are associated with disease activity in MS, whereas an increase in IL-10 correlates with recovery (47). Consistent with our in vitro findings, the IFN-ß-1b efficacy in MS may be related to an inhibitory effect on IL-12 mediated in part by IL-10 or dependent on IL-10. Alternatively, inhibition of IL-12 by IFN-ß may enhance endogenous IL-10 production and therefore confer protection. Furthermore, dependence of the inhibitory effect of IFN-ß on IL-10 could have implications on response to IFN-ß therapy in MS. It is possible to speculate that patients with high basal IL-10 levels are more likely to respond to IFN-ß.
In summary, this study demonstrates that IFN-ß-1b has a significant inhibitory effect on IL-12 production in human PBMC and in MBP-specific T cells, and that this inhibitory effect is IL-10 dependent. These findings suggest a mechanism of action for IFN-ß in the treatment of MS consistent with the Th1/Th2 cytokine paradigm changes in this disease.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Suhayl Dhib-Jalbut, Department of Neurology, University of Maryland Hospital, 22 South Greene Street, #N4w46, Baltimore, MD 21201.
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental allergic encephalomyelitis; MBP, myelin basic protein; SAC, Staphylococcus aureus Cowan; TCL, T cell line; TCC, T cell clone; ICSBP, IFN consensus sequence binding protein.
Received for publication September 3, 1999. Accepted for publication April 13, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Curr. Opin. Immunol. 9:17.[Medline]
(IFN-) production during differentiation of human T helper (Th) cells and transient IFN- production in established Th2 cell clones. J. Exp. Med. 179:1273. interferon. Lancet 1:893.[Medline]
, and interferon- in human monocytes infected with mycobacterium tuberculosis H37Ra. J. Infect. Dis. 178:1105.[Medline]
2a, and interferon-ß1b on HLA expression and lymphoproliferation: a preclinical model for treatment of multiple sclerosis. J. Interferon Cytokine 16:195.
/ß inhibition of interleukin 12 and interferon- production in vitro and endogenously during viral infection. Proc. Natl. Acad. Sci. USA 94:634./ß- and interleukin 12-mediated pathways in promoting T cell interferon responses during viral infection. J. Exp. Med. 189:1315. in monocytic cells. J. Exp. Med. 83:147.
B half-site. Mol. Cell. Biol. 15:5258.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  H. Qin, S. A. Niyongere, S. J. Lee, B. J. Baker, and E. N. Benveniste Expression and Functional Significance of SOCS-1 and SOCS-3 in Astrocytes J. Immunol., September 1, 2008; 181(5): 3167 - 3176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Bochtler, A. Kroger, R. Schirmbeck, and J. Reimann Type I IFN-Induced, NKT Cell-Mediated Negative Control of CD8 T Cell Priming by Dendritic Cells J. Immunol., August 1, 2008; 181(3): 1633 - 1643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Krakauer, P Sorensen, M Khademi, T Olsson, and F Sellebjerg Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-{beta} treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression Multiple Sclerosis, June 1, 2008; 14(5): 622 - 630. [Abstract] [PDF]
|
|
|
|
|
|
E. Wiesemann, M. Deb, C. Trebst, B. Hemmer, M. Stangel, and A. Windhagen Effects of interferon-{beta} on co-signaling molecules: upregulation of CD40, CD86 and PD-L2 on monocytes in relation to clinical response to interferon-{beta} treatment in patients with multiple sclerosis Multiple Sclerosis, March 1, 2008; 14(2): 166 - 176. [Abstract] [PDF]
|
|
|
|
|
|
F. Blanchette Clinical significance of glatiramer acetate antibodies Multiple Sclerosis, May 1, 2007; 13(1_suppl): 28 - 35. [Abstract] [PDF]
|
|
|
|
|
|
H. Qin, C. A. Wilson, K. L. Roberts, B. J. Baker, X. Zhao, and E. N. Benveniste IL-10 Inhibits Lipopolysaccharide-Induced CD40 Gene Expression through Induction of Suppressor of Cytokine Signaling-3 J. Immunol., December 1, 2006; 177(11): 7761 - 7771. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Teige, Y. Liu, and S. Issazadeh-Navikas IFN-beta Inhibits T Cell Activation Capacity of Central Nervous System APCs J. Immunol., September 15, 2006; 177(6): 3542 - 3553. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Qin, C. A. Wilson, S. J. Lee, and E. N. Benveniste IFN-{beta}-induced SOCS-1 negatively regulates CD40 gene expression in macrophages and microglia FASEB J, May 1, 2006; 20(7): 985 - 987. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
B. A. t Hart, H. P. M. Brok, E. Remarque, J. Benson, G. Treacy, S. Amor, R. Q. Hintzen, J. D. Laman, J. Bauer, and E. L. A. Blezer Suppression of Ongoing Disease in a Nonhuman Primate Model of Multiple Sclerosis by a Human-Anti-Human IL-12p40 Antibody J. Immunol., October 1, 2005; 175(7): 4761 - 4768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Dikopoulos, A. Bertoletti, A. Kroger, H. Hauser, R. Schirmbeck, and J. Reimann Type I IFN Negatively Regulates CD8+ T Cell Responses through IL-10-Producing CD4+ T Regulatory 1 Cells J. Immunol., January 1, 2005; 174(1): 99 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sturzebecher, K. P. Wandinger, A. Rosenwald, M. Sathyamoorthy, A. Tzou, P. Mattar, J. A. Frank, L. Staudt, R. Martin, and H. F. McFarland Expression profiling identifies responder and non-responder phenotypes to interferon-{beta} in multiple sclerosis Brain, June 1, 2003; 126(6): 1419 - 1429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Teige, A. Treschow, A. Teige, R. Mattsson, V. Navikas, T. Leanderson, R. Holmdahl, and S. Issazadeh-Navikas IFN-{beta} Gene Deletion Leads to Augmented and Chronic Demyelinating Experimental Autoimmune Encephalomyelitis J. Immunol., May 1, 2003; 170(9): 4776 - 4784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. P. M. Brok, M. van Meurs, E. Blezer, A. Schantz, D. Peritt, G. Treacy, J. D. Laman, J. Bauer, and B. A. 't Hart Prevention of Experimental Autoimmune Encephalomyelitis in Common Marmosets Using an Anti-IL-12p40 Monoclonal Antibody J. Immunol., December 1, 2002; 169(11): 6554 - 6563. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Nardelli, L. Zaritskaya, M. Semenuk, Y. H. Cho, D. W. LaFleur, D. Shah, S. Ullrich, G. Girolomoni, C. Albanesi, and P. A. Moore Regulatory Effect of IFN-{kappa}, A Novel Type I IFN, On Cytokine Production by Cells of the Innate Immune System J. Immunol., November 1, 2002; 169(9): 4822 - 4830. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Dhib-Jalbut Mechanisms of action of interferons and glatiramer acetate in multiple sclerosis Neurology, April 23, 2002; 58(90084): S3 - 9. [Abstract] [Full Text]
|
|
|
|
|
|
Z. Trobonjaca, A. Kroger, D. Stober, F. Leithauser, P. Moller, H. Hauser, R. Schirmbeck, and J. Reimann Activating Immunity in the Liver. II. IFN-{beta} Attenuates NK Cell-Dependent Liver Injury Triggered by Liver NKT Cell Activation J. Immunol., April 15, 2002; 168(8): 3763 - 3770. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G.-X. Zhang, H. Xu, M. Kishi, D. Calida, and A. Rostami The Role of IL-12 in the Induction of Intravenous Tolerance in Experimental Autoimmune Encephalomyelitis J. Immunol., March 1, 2002; 168(5): 2501 - 2507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Neuhaus, C. Farina, H. Wekerle, and R. Hohlfeld Mechanisms of action of glatiramer acetate in multiple sclerosis Neurology, March 27, 2001; 56(6): 702 - 708. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G Monteleone, S L F Pender, E Alstead, A C Hauer, P Lionetti, and T T MacDonald Role of interferon {alpha} in promoting T helper cell type 1 responses in the small intestine in coeliac disease Gut, March 1, 2001; 48(3): 425 - 429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ito, R. Amakawa, M. Inaba, S. Ikehara, K. Inaba, and S. Fukuhara Differential Regulation of Human Blood Dendritic Cell Subsets by IFNs J. Immunol., March 1, 2001; 166(5): 2961 - 2969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-G. Fischer and G. Reichmann Brain Dendritic Cells and Macrophages/Microglia in Central Nervous System Inflammation J. Immunol., February 15, 2001; 166(4): 2717 - 2726. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
