| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Allergology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
)3 and dendritic cells (DC)
(3, 4). IL-12 is a unique heterodimeric cytokine composed of p35 and
p40, and the heterodimeric structure was shown to be essential for its
biological activity (5). p40, especially homodimeric p40, was reported
to inhibit biological activity of the heterodimer both in vitro (6) and
in vivo (7). Both p35 and p40 subunits have to be produced in the same
cell to be secreted as an active dimer (5). Production of these
subunits could be regulated cooperatively by mechanisms activated by
different stimulations. Promoter regions of the p35 and p40 genes have
different NF binding sites (8). In cell lines, the expression of p40
transcripts was shown to correlate with the ability of the cells to
produce IL-12, whereas p35 mRNA was reported to be ubiquitously
expressed in almost all cell lines of either hematopoietic or
nonhematopoietic origin (9). These findings led to an assumption that
p40 production is representative of bioactive IL-12 production.
However, IL-12 production of LPS-stimulated human monocytes was shown
to be regulated by p35 subunit synthesis (10).
IL-12 production by the cells in a monocyte-M lineage was shown to
be mediated by the interaction of CD40 with CD40 ligand (CD40L) on
activated T cells in both human (11) and mouse (12). In our previous
experiments (12), an incubation of M with Th1 cells in the presence
of relevant Ag up-regulated the expression of both p35 and p40 mRNA,
and the p40 mRNA accumulation was shown to be induced by the CD40-CD40L
interaction. However, the molecule on APC to induce p35 mRNA
accumulation has not been elucidated yet.
In the present experiments, we have analyzed the cell surface molecule mediating up-regulation of the p35 mRNA accumulation in APC, and results indicate that an interaction of MHC class II Ag peptide with TCR mediates the up-regulation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan) and used at 7–10 wk of age.
Culture media
RPMI 1640 (JRH Biosciences, Lenexa, KS) supplemented with 10% FCS (Summit Biotechnology, Fort Collins, CO), 5 x 10-5 M 2-ME, and 100 µg/ml kanamycin was used. MEM (JRH Biosciences) was used for cell washing.
Reagents and Abs
OVA (grade VII) was purchased from Sigma (St. Louis, MO). The
OVA preparation contained 2.4 ng LPS/mg OVA, and it was confirmed in
preliminary experiments not to induce either IL-12 p35 or p40 mRNA
accumulation in spleen cells at 1 mg/ml. Purchased from PharMingen (San
Diego, CA) were the following: purified anti-mouse CD40L (MR1,
hamster IgG) (13), anti-B220 (RA3-6B2, rat IgG2a), biotinylated
anti-mouse CD40L (MR1), biotinylated normal hamster IgG,
PE-anti-CD4 (RM4-5, hamster IgG), FITC-anti-Mac-1 (M1/70, rat
IgG2b), PE-anti-NK1.1 (PK136, mouse IgG2a), biotinylated
anti-IgM (Bet 2, rat IgG1), and avidin-Cy-chrome.
PE-anti-hamster IgG (goat IgG) was purchased from Caltag
(Burlingame, CA). FITC-streptavidin was purchased from Life
Technologies (Gaithersburg, MD). Culture supernatant of hybridoma N418
was used as a source of anti-CD11c (hamster IgG). Ascites
containing anti-Thy1.2 (HO13.4, IgM) was used for T cell depletion
with baby rabbit complement. Anti-CD4 (GK1.5, rat IgG2b), anti-CD8
(53-6.72, rat IgG2a), anti-Thy1.2 (30-H12, rat IgG2b),
anti-Fc
RII/III (2.4G2, rat IgG2b), and anti-I-Ab
(M5/114, rat IgG2b) (14) used for blocking a MHC class II/TCR
interaction were purified from ascites on a protein G column (Pharmacia
Fine Chemicals, Piscataway, NJ). Anti-CD3 (145-2C11, hamster IgG) and
normal hamster IgG were purified on a protein A column (Pharmacia) from
ascites and serum, respectively. Anti-I-Ab (M5/114) was
confirmed in preliminary experiments not to induce either p35 or p40
mRNA expression in splenic adherent cells even at 30 µg/ml.
Anti-I-Ab (28-16-8S, IgM) previously shown to stimulate B
cell activation (15) was purified from ascites on an Immunoassist MG-PP
column (Kanto Chemical, Tokyo, Japan). Purified anti-Mac-1 (M1/70,
rat IgG2b) and anti-H-2Db (28-11-5S, IgM) were used as
Ig subclass- and class-matched controls for M5/114 and 28-16-8S mAb,
respectively.
Cells
An OVA-specific Th1 clone, 35-9D, was established from C57BL/6
mouse lymph node cells and maintained as described (16). 35-9D cells
cultivated for at least 3 wk after the last Ag stimulation were used
for experiments as the clone under resting conditions. They were
confirmed not to grow in the medium with exogenous IL-2 and to be
CD40L-. For some experiments, 35-9D cells stimulated for
6 h with plate-bound anti-CD3 (100 ng/well of a 24-well plate)
were fixed with 1% paraformaldehyde in PBS (pH 7.2) and used as
activated Th1 cells after they were confirmed to be CD40L+.
T cell-depleted (T-d) spleen cells were prepared by the treatment of
spleen cells with anti-Thy1.2 (HO13.4) and baby rabbit complement.
The resultant cells contained 85.4–90.1% of B220+ B
cells, 5.9–8.7% of Mac-1+NK1.1- M/DC,
2.1–3.1% of CD11c+NK1.1- DC, and 2.7–5.1%
of NK1.1+ cells, and the remaining CD3+ T cells
constituted <0.6%. B cells were purified from T-d spleen cells by
passing through Sephadex G-10 columns twice. The preparations contained
>95% of B220+µ+ B cells. When the B cells
were used as APC for IL-12 activity assay, they were irradiated with 8
Gy. For some experiments, splenic M/DC were enriched. To enrich
M/DC populations, spleen cells were treated with 1 mg/ml collagenase
from Clostridium histolyticum (Wako Pure Chemical, Osaka,
Japan) in HBSS, and the resultant single-cell suspension was incubated
with a mixture of 1 µg/ml of anti-B220, anti-Thy1.2 (30-H12),
anti-CD4, and anti-CD8, followed by the negative selection
using Dynabeads (M-450) coated with anti-rat IgG and magnetic
particle concentrators (Dynal, Oslo, Norway). After dead cells were
depleted by centrifugation over Ficoll-Hypaque, these cells were
incubated with FITC-anti-Mac-1 and PE-anti-NK1.1 in the
presence of anti-FcRII/III, and Mac-1+
NK1.1- cells were sorted in a FACS Vantage (Becton
Dickinson, Mountain View, CA). The sorted cells contained 19.0–20.1%
of CD11c+ DC and 77.1–77.8% of
CD11c-Mac-1+ M and NK1.1+,
CD3+, or surface µ+ cells in these
preparations were <1%. They were used as M/DC in the present
experiments. Chinese hamster ovary (CHO) cells transfected with CD40L
cDNA (CD40L-CHO) (17) were a generous gift from Drs. H. Yagita and K.
Okumura (Juntendo University, Tokyo, Japan) and were used after
fixation with 1% paraformaldehyde in PBS as described (18).
Stimulation of T-d spleen cells or M/DC
Three million T-d spleen cells or 5 x 105
M/DC were incubated with indicated numbers of resting or activated
35-9D clone cells in 0.6 ml/well culture medium in a 48-well plate in
the presence or absence of OVA. In some experiments, T-d spleen cells
or M/DC were stimulated with agonistic anti-I-Ab
(28-16-8S) plus CD40L-CHO. Supernatants of these cultures were assayed
for IL-12 activity, and the cells were assayed for IL-12 p40 and p35
mRNA expression.
Bioassay for IL-12 activity
IL-12 activity was assayed by the proliferation of Th1 clone
35-9D stimulated with OVA on B cell APC as described (19). When culture
supernatants of M/DC stimulated in the presence of
anti-I-Ab (28-16-8S or M5/114) were assayed for IL-12
activity, 35-9D cells were stimulated overnight with OVA on B cell APC
and then used for assay after depletion of the B cells by a
centrifugation over Ficoll-Hypaque. The proliferation of 35-9D cells
was evaluated by pulsing with [3H]TdR for the last 8
h of the culture. The [3H]TdR incorporation was counted
in a Matrix 96 system (Packard Instrument, Meriden, CT) under a gas
phase according to the manufacturer’s instructions.
Assay for CD40L expression
CD40L expression on 35-9D clone stimulated with OVA on T-d
spleen cells was assayed as described previously (12). Briefly, 2
x 105 35-9D clone cells per culture were incubated with
3 x 106 T-d spleen cells per culture in the presence
of 1 µg/ml biotinylated anti-CD40L to prevent down-regulation of
CD40L expressed; incubated with anti-FcRII/III; and then stained
with biotinylated anti-CD40L, FITC-streptavidin, and
PE-anti-CD4. The cells positively stained with anti-CD4 were
analyzed for CD40L expression on a FACScan using Lysis II software
(Becton Dickinson, San Jose, CA). As a control for biotinylated
anti-CD40L, biotinylated normal hamster IgG was used. Results are
expressed as ratio of mean fluorescence intensity of cells stained with
anti-CD40L to that of cells stained with normal hamster IgG.
Assay for IL-12 p35 and p40 mRNA accumulation
IL-12 p35 and p40 mRNA accumulation was assayed by competitive RT-PCR. Total RNA was isolated by the acid guanidinium-thiocyanate-phenol-chloroform method (20). One microgram of the total RNA was reverse transcribed into cDNA in a 50-µl reaction mixture containing 1x reverse transcriptase buffer, 0.2 mM each dNTP, 0.5 ng of oligo-(dT) primer, and 20 U Moloney murine leukemia virus reverse transcriptase (all from Life Technologies), and 20 U ribonuclease inhibitor (Wako Pure Chemical).
Competitive RT-PCR was conducted as described previously (21). In
brief, a competitive DNA fragment containing the IL-12 p35 primer
sequence was constructed using a PCR MIMIC construction kit (Clontech,
Palo Alto, CA) according to the manufacturer’s instructions. The
multiple competitor PQRS (22), kindly provided by Dr. R. M.
Locksley (University of California, San Francisco, CA), was used for
IL-12 p40 and hypoxanthine phosphoribosyltransferase (HPRT) cDNA. A
fixed amount of competitor was added in a PCR amplification of the
target cDNA in a reaction mixture containing
[
-32P]dCTP. The amplification was performed in a DNA
thermal cycler (ASTEC, Tokyo, Japan). The conditions for PCR were the
following: 60 s at 94°C, 60 s at 60°C, and 120 s at
72°C for 35–40 cycles. The optimal competitor concentrations
were determined by amplifying target cDNA in the presence of 2-fold
serial dilutions of competitor. After PCR amplification, the RT-PCR
mixture was electrophoresed and the radioactivity of the specific bands
was measured. The results are presented as the ratio of target to
competitor PCR products normalized with that of HPRT.
In some experiments, simple RT-PCR was conducted to amplify the cDNA
preparations for TNF- and HPRT using 1 U Taq polymerase
(Toyobo, Tokyo, Japan) and 0.5 µM primers as described previously
(12). The conditions for PCR were as follows: TNF-, 60 s at
94°C, 60 s at 55°C, and 120 s at 72°C for 26 cycles,
and HPRT, 40 s at 94°C, 20 s at 60°C, and 40 s at
72°C for 26 cycles. RT-PCR signals for TNF- and HPRT were
confirmed to be proportional to the amplification cycle from 22 to 30
cycles. The PCR products were size fractionated on an agarose gel,
blotted onto a nylon membrane (Bio-Rad Laboratories, Richmond, CA), and
hybridized with appropriate internal probes to verify the specificity
of the amplification.
The following primers were used for cDNA amplification: IL-12 p40
sense, 5'-ATG GCC ATG TGG GAG CTG GAG-3', and antisense, 5'-TTT GGT GCT
TCA CAC TTC AGG-3'; IL-12 p35 sense, 5'-ATG ATG ACC CTG TGC CTT GG-3',
and antisense, 5'-CCT TTG GGG AGA TGA GAT GT-3'; TNF- sense, 5'-GAT
CTC AAA GAC AAC CAA CTA GTG-3', and anti-sense, 5'-CTC CAG CTG GAA
GAC TCC TCC CAG-3'; and HPRT sense, 5'-GTT GGA TAC AGG CCA GAC TTT GTT
G-3', and antisense, 5'-GAG GGT AGG CTG GCC TAT AGG CT-3'. The internal
probes for hybridization were as follows: TNF-, 5'-CCC GAC TAC GTG
CTC CTC ACC-3', and HPRT, 5'-GGA AAA GCC AAA TAC AAA GCC-3'.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the first experiment, 3 x 106 T-d spleen
cells per culture were incubated for 6 h with various
concentrations of OVA in the presence or absence of 2 x
105 35-9D cells per culture and assayed for IL-12 p35 and
p40 mRNA accumulation. When T-d spleen cells were incubated with 35-9D
cells in the presence of 100 µg/ml or more OVA, both p35 and p40 mRNA
were accumulated (Fig. 1
, A
and B). Both p35 and p40 mRNA accumulations were confirmed
to peak at 6 h in separate experiments (data not shown). However,
neither p35 nor p40 mRNA was accumulated in T-d spleen cells or 35-9D
clone when they were separately incubated with OVA. Although a marginal
expression of p35 mRNA was seen in T-d spleen cells on several
occasions of repeated experiments even in the absence of OVA, the
expression was not increased by the inclusion of OVA in cultures.
Anti-CD3 stimulation did not induce p35 mRNA accumulation in 35-9D
cells either (data not shown). These results suggest that both p35 and
p40 mRNA accumulations observed by the cultivation of T-d spleen cells
with 35-9D clone were induced by interaction of 35-9D cells with APC.
|
).
|
To examine further the role of TCR/MHC class II-Ag peptide interaction
in p40 and p35 mRNA accumulation, T-d spleen cells were incubated with
resting 35-9D clone and 0–1000 µg/ml OVA in the presence or absence
of 10 µg/ml anti-I-Ab (M5/114). However, neither p40
nor p35 mRNA was accumulated in the cells incubated in the presence of
anti-I-Ab (M5/114) (data not shown), although both
mRNAs were accumulated in the absence of anti-I-A, as shown in Fig. 1
.
When 35-9D cells were stimulated with OVA on T-d spleen cells, they
expressed CD40L on their surface in an OVA dose-dependent manner. The
expression was detected 90 min after the stimulation, peaked at 6
h, and decreased thereafter. The inclusion of 10 µg/ml
anti-I-Ab (M5/114) in cultures abrogated the CD40L
expression even at the maximum expression at 6 h (Fig. 3). Therefore, T-d spleen cells were
incubated with activated CD40L+ 35-9D cells and 0–1000
µg/ml OVA in the absence or presence of 10 µg/ml
anti-I-Ab (M5/114) or control Ab and assayed for p40
and p35 mRNA accumulation to examine the effect of TCR/MHC class II-Ag
peptide interaction on p40 mRNA accumulation stimulated with CD40L. OVA
dose-dependent accumulations of p35 mRNA observed in the absence of the
anti-I-Ab were all abrogated by the addition of the
anti-I-Ab (Fig. 4,
A and B), suggesting that the
anti-I-Ab effectively blocked the TCR/MHC class II-Ag
peptide interaction. In contrast to p35 mRNA, the accumulation of p40
mRNA was not affected by the dose of OVA included in cultures, and the
addition of anti-I-Ab (M5/114) did not affect the p40
mRNA accumulation either (Fig. 4, A and B).
|
|
Inhibition with anti-I-A (M5/114) of p35 mRNA accumulation induced by the interaction of T-d spleen cells with fixed-resting 35-9D
In the above experiment, there remains a possibility that
Ag-stimulated 35-9D clone expressed some molecule to stimulate T-d
spleen cells to induce p35 mRNA accumulation. In the next experiment,
therefore, 35-9D cells were fixed with paraformaldehyde in their
resting conditions, and the fixed 35-9D cells (1 x
106 cells/culture) were incubated with T-d spleen cells
(3 x 106 cells/culture) in the presence of 500
µg/ml OVA for 6 h and assayed for p40 and p35 mRNA accumulation.
35-9D cells were used after it was confirmed that they did not express
CD40L. In this experiment, the effect of anti-I-Ab
(M5/114) on p35 and p40 mRNA accumulation was also examined. Although
p40 mRNA was not accumulated by the incubation, p35 mRNA accumulation
was increased to reach the maximum at 6 h and declined thereafter,
and the accumulation was suppressed by the inclusion of
anti-I-Ab (M5/114) in a dose-dependent manner, but not
by control Ab. The results at 6 h of a representative experiment
are shown in Fig. 5, A and
B. The control Ab did not inhibit the p35 mRNA accumulation
for 24 h even at 10 µg/ml (data not shown). p35 mRNA was
accumulated in T-d spleen cells in the presence of OVA even when the
fixed 35-9D included in culture was decreased in number to 3 x
105 cells/culture, but not in the absence of OVA (data not
shown). p40 mRNA was not accumulated for 24 h even when 3 x
106 fixed 35-9D cells per culture were included in culture
(data not shown).
|
p35 mRNA accumulation in APC by agonistic anti-I-A stimulation
To examine whether MHC class II molecule stimulation induces p35
mRNA accumulation, T-d spleen cells were stimulated with 10 µg/ml
anti-I-Ab (28-16-8S) and assayed for p35 and p40 mRNA
accumulation. p35 mRNA accumulation was seen 3 h after
stimulation, peaked at 6 h, and declined thereafter; however, a
control Ab, anti-H-2Db, did not affect the accumulation
(Fig. 6, A and B).
The p35 mRNA accumulation increased depending on the dose (1–10
µg/ml) of anti-I-Ab (28-16-8S) included in cultures
(data not shown). On the other hand, p40 mRNA accumulation was not
affected for 12 h after the stimulation with
anti-I-Ab (28-16-8S) (Fig. 6, A and
B). The accumulation was confirmed not to be affected for
72 h (data not shown). Because human monocytes were shown to
express TNF- mRNA by the stimulation with anti-MHC class II (24, 25), we also examined the agonistic effect of anti-I-Ab
(28-16-8S) on TNF- mRNA expression in T-d spleen cells and confirmed
the accumulation of TNF- mRNA (Fig. 6C). On the other
hand, anti-I-Ab (M5/114) used as a blocking Ab in the
above experiment was confirmed not to induce TNF- mRNA accumulation
(data not shown).
|
/DC and B cell preparations were obtained
from spleen cells, and both cell preparations were stimulated with 10
µg/ml anti-I-Ab (28-16-8S) for 6 h and assayed
for p35 and p40 mRNA accumulation by competitive RT-PCR. p40 mRNA
accumulation was not affected by the anti-I-Ab
stimulation either in M/DC or B cells. In contrast, p35 mRNA
accumulation was apparently increased in M/DC by the
anti-I-Ab stimulation; however, p35 mRNA accumulation
in B cells was not increased (Fig. 7,
A and B), although p35 mRNA accumulation in B
cells was relatively high without stimulation, indicating that the
cells in which p35 mRNA was accumulated by MHC class II stimulation in
the above experiments are M/DC, but not B cells.
|
/DC by stimulation with CD40L-CHO and
anti-I-A (28-16-8S)
In the next experiment, we examined whether the p35 mRNA
accumulation induced by the agonistic anti-I-Ab
stimulation affects the bioactive IL-12 production of M/DC incubated
with CD40L-CHO. M/DC were incubated for 9 h with various
numbers of paraformaldehyde-fixed CD40L-CHO cells in the presence of 10
µg/ml anti-I-Ab (28-16-8S) or control Ab, and
supernatants were assayed for IL-12 activity. M/DC stimulated with
1 x 104 cells/culture or less CD40L-CHO cells
produced bioactive IL-12, depending upon the dose of CD40L-CHO cells,
only when anti-I-Ab (28-16-8S) was in the cultures.
When M/DC were stimulated with 3 x 104 CD40L-CHO
cells per culture, a low IL-12 activity was detected in the
supernatants without inclusion of anti-I-Ab (28-16-8S)
in cultures, and the IL-12 production was enhanced by adding 10 µg/ml
anti-I-Ab (28-16-8S) (Fig. 8). M/DC did not produce IL-12 when
they were stimulated with 10 µg/ml anti-I-Ab
(28-16-8S) in the presence of CHO. In separate experiments, the
anti-I-Ab was confirmed not to affect the assay for
IL-12 activity. These results indicate that ligations of both CD40 and
the MHC class II molecule are required for efficient IL-12 production
of M/DC, although intensive CD40 stimulation itself was indicated to
induce a small amount of IL-12 production.
|
/DC depending on the dose of CD40L-CHO, even when
anti-I-Ab (28-16-8S) was not included in cultures. p35
mRNA appeared to be accumulated a little in M/DC stimulated with
3 x 104 fixed CD40L-CHO cells per culture, but not
with 1 x 104 or fewer CD40L-CHO cells per culture.
However, fixed CHO cells did not affect p35 mRNA accumulation even at
3 x 104 cells/culture in the absence of
anti-I-Ab (28-16-8S). On the other hand, p35 mRNA was
accumulated in M/DC stimulated with anti-I-Ab
(28-16-8S) in either the presence or the absence of CD40L-CHO cells.
These results indicate that the I-A molecule mediates an important
signal for p35 mRNA accumulation in M/DC, although intensive
stimulation to CD40 may also induce a weak accumulation of p35 mRNA.
These results, shown in Fig. 9,
A and B, are consistent with the findings in Fig. 8.
|
/DC were incubated in the presence
or absence of 500 µg/ml OVA for 9 h with activated 35-9D cells
fixed with paraformaldehyde, and the effect of blocking
anti-I-Ab (M5/114) on the IL-12 production was
examined. M/DC produced IL-12 by the incubation with the activated
CD40L+ 35-9D cells in the presence of OVA, but not in the
absence of OVA. The addition of >1 µg/ml anti-I-Ab
(M5/114) significantly (p < 0.01) suppressed
the IL-12 production, and the production was abrogated by the inclusion
of 10 µg/ml anti-I-Ab (M5/114) (Fig. 10A). When aliquots of
M/DC used for this experiment were assayed for p40 and p35 mRNA
accumulation, p40 mRNA was accumulated 6 h after cultivation with
activated 35-9D cells, and the accumulation was not affected by the
addition of either OVA or anti-I-Ab (M5/114); however,
p35 mRNA was accumulated in M/DC only in the presence of OVA.
Moreover, the p35 mRNA accumulation was inhibited by the addition of
anti-I-Ab (M5/114) in a dose-dependent manner (Fig. 10, B and C).
|
/DC by the stimulation of MHC class II, and the accumulation plays
a role in bioactive IL-12 production. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/DC is up-regulated by the
adherence to plastic surface. This is why we prepared M/DC without
using the adherence technique.
Our present results indicate that stimulation of MHC class II molecule
via interaction with TCR plays an important role in the accumulation of
p35 mRNA in M/DC. For bioactive IL-12 production, both p35 and p40
subunits have to be produced in the same cell (5). In our previous
report (12), a CD40-CD40L interaction induced the accumulation of p40
mRNA, but not p35 mRNA. Taken together, these results indicate a
possible mechanism for IL-12 production in a Th cell-APC interaction;
i.e., MHC class II-Ag peptide complex interaction with TCR induces an
activation signal for p35 production in APC and, at the same time, the
interaction induces CD40L expression on Th cells. The CD40-CD40L
interaction stimulates p40 production in APC. p35 and p40 subunits
produced in the same cell form biologically active dimer p70 to be
secreted. Consistent with our results, bioactive IL-12 production in DC
stimulated with anti-CD40 was reported to be enhanced by
anti-I-A stimulation (29). In the experiments of Koch et al. (29),
stimulations with CD40L and anti-I-A independently up-regulated
IL-12 production, and DC stimulated only with anti-I-A produced a
low amount of p70 IL-12, although they did not assay p35 mRNA
expression. In our experiments, T-d spleen cells stimulated with a high
dose of anti-I-A (28-16-8S), such as 30 µg/ml, produced a very
low amount of bioactive IL-12 as almost the lowest limit in our assay
(data not shown). As shown in Fig. 8 and Fig. 9, A and
B, splenic M/DC seemed to accumulate a small amount of
p35 mRNA and produced a small amount of bioactive IL-12 when they were
stimulated with 3 x 104 CD40L-CHO cells per culture
but not with CHO cells. When M/DC were stimulated with 3 x
104 to 1 x 106 CD40L-CHO cells per
culture, p35 mRNA accumulation and IL-12 production were increased a
little, but not with CHO cells, in the absence of
anti-I-Ab (28-16-8S) (data not shown). The increase in
p35 mRNA accumulation was not so remarkable as that observed in the
cells stimulated with anti-I-Ab (28-16-8S), and the
IL-12 production was significantly enhanced by the presence of
anti-I-Ab (28-16-8S) (data not shown). When T-d spleen
cells from CD40-deficient mice were incubated with activated 35-9D
cells, they accumulated p35 mRNA, not p40 mRNA, only in the presence of
relevant Ag (data not shown). Our present results indicate that p35 and
p40 mRNA expressions were up-regulated mostly by the ligations of MHC
class II and CD40, respectively, in Th cell-APC interaction, although a
weak accumulation of p35 mRNA could be induced by a CD40-CD40L
interaction. Cells stimulated for bioactive IL-12 production were
reported to secrete a free p40 protein, although free p35 has not been
detected (30, 31). The recombinant p40 was shown to inhibit biological
activity of p70 heterodimer (6). Th1 responses were shown to be reduced
in p40 transgenic mice (7). Therefore, free p40 may act as a
physiologic antagonist of IL-12. If this is the case, up-regulation of
p35 production may reduce the production of free p40, and regulate the
physiologic role of free p40. In our present results, the ligation of
MHC class II molecule was shown to increase the production of
biologically active IL-12. Free p35 produced by the I-A stimulation
without CD40 stimulation might play a role in the regulation of a
function(s) of MHC class II+ cells, although the
function(s) is not known.
The production of bioactive IL-12 is well documented to be regulated by
various cytokines such as IL-10, IFN-, and IL-4 (28, 32). In these
cytokines, IL-10 is one of the potent inhibitors of IL-12 production of
monocyte-M lineage cells stimulated with CD40L (33) or LPS (32).
IL-10 was shown to suppress the accumulation of both p35 and p40 mRNAs
in APC incubated with alloreactive T cells (33) or in LPS-stimulated
monocytes (32). Prostaglandin E2 was also reported to
inhibit IL-12 production (34). In contrast to these substances, IFN-
was proven to enhance IL-12 production. The enhancement is considered
to be due, at least partly, to the inhibition of IL-10 production (35).
These soluble factors regulating IL-12 production are products of Th
cells and/or cells in a monocyte-M lineage, indicating that IL-12
production is regulated directly or indirectly in an interplay between
Th cells and APC or accessory cells.
IL-12 production was also shown to be induced by the infection with
microorganisms such as Staphylococcus aureus, Listeria
monocytogenes, and Leishmania major. In CD40-deficient
mice, IL-12 p40 mRNA accumulation in the draining lymph nodes of
L. major-infected sites was shown to be significantly lower
than that in wild-type mice (36), suggesting that CD40-CD40L
interaction plays a role in IL-12 production in L. major
infection. However, IL-12 was also produced by L.
monocytogenes-activated spleen M in SCID mice (37). The results
indicate that there may be some other mechanisms for IL-12 production
than APC-T cell interaction or LPS stimulation. IL-12 is considered to
play a pivotal role in the development of Th1 cells to regulate a
Th1/Th2 balance. Therefore, understanding of the mechanisms of IL-12
production is important for the analysis of immune responses and may
provide a means to manipulate Th1 and Th2 responses.
In the present paper, we have provided evidence to show that TCR/MHC class II-Ag peptide interaction regulates IL-12 production through the stimulation of IL-12 p35 mRNA accumulation.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Hideo Nariuchi, Department of Allergology, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, 108-8639 Tokyo, Japan. E-mail address:
3 Abbreviations used in this paper: M, macrophage; DC, dendritic cell; CD40L, CD40 ligand; T-d, T cell depleted; CHO, Chinese hamster ovary; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication June 16, 1998. Accepted for publication March 16, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Immunol. 152:1883.[Abstract]
genes by MHC class II ligands. J. Immunol. 146:2310.[Abstract]
gene expression in human monocytes by major histocompatibility complex class II ligands. Eur. J. Immunol. 26:2417.[Medline]
of lipopolysaccharide-inducible p35 and p40 genes. Blood 86:646., and IL-1ß production in human myeloid leukemia cell lines and peripheral blood mononuclear cells. Blood 83:1847.-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041. inhibits interleukin-10 production by monocytes. J. Exp. Med. 177:523. are costimulators of interferon production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin-10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90:3725.This article has been cited by other articles:
|
|
 |
|
  R. Sporri and C. Reis e Sousa Newly Activated T Cells Promote Maturation of Bystander Dendritic Cells but Not IL-12 Production J. Immunol., December 15, 2003; 171(12): 6406 - 6413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Lemos, L. Fan, D. Lo, and T. M. Laufer CD8{alpha}+ and CD11b+ Dendritic Cell-Restricted MHC Class II Controls Th1 CD4+ T Cell Immunity J. Immunol., November 15, 2003; 171(10): 5077 - 5084. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Morelli, A. F. Zahorchak, A. T. Larregina, B. L. Colvin, A. J. Logar, T. Takayama, L. D. Falo, and A. W. Thomson Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation Blood, September 1, 2001; 98(5): 1512 - 1523. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
