| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Production from CD3intIL-2Rß+ T Cells1
*
Department of Immunology and Medical Zoology, and
Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, which in
combination with Fas ligand, inhibits IgE production from the B cells.
Our present results indicate that Mac-1+ cells and
double-negative CD3intIL-2Rß+ T cells,
uniquely abundant in the spleens of SJL mice, inhibit IgE production,
indicating their new role in IgE response. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Another mechanism for defective IgE production in SJL mice appears to
be linked to the absence of CD4+NK1.1+ T cells
that are assumed to produce initial IL-4 required for priming naive T
cells to develop into IL-4-producing cells in vivo (5, 6).
CD4+NK1.1+ T cells, expressing a limited set of
TCR-
ß V14, and Vß8 or Vß7, are specific for MHC class
I-like molecule CD1 and require CD1 for their development (7, 8, 9, 10, 11).
Indeed, CD1-deficient mice are lacking this lymphocyte population (12).
SJL mice have been reported to be defective in the expression of CD1.2
isoform of CD1 (13), providing us with the possibility of the genetic
basis for the marked diminution in the number of
CD4+NK1.1+ T cells in the spleens of SJL mice.
Alternatively, this diminution in the number of
CD4+NK1.1+ T cells might be due to excessive
production of IL-12 by macrophages in SJL mice. We have shown recently
that injection of IL-12 or a mixture of IL-12 and IL-18 to C57BL/6 mice
strikingly diminishes the proportion and number of
CD4+NK1.1+ T cells in the liver and splenic
lymphocytes, but induces CD4-CD8-
double-negative (DN)3
CD3intIL-2Rß+ T cells to develop into
IFN--producing cells in the liver (14). Furthermore, as we and
others have shown, injection of IL-12 or IL-12 and IL-18 into
Nippostrongylus brasiliensis-infected or
anti-IgD-treated mice inhibited polyclonal IL-4-dependent IgE
production by induction of IFN--producing T and B cells in these
mice (15, 16). Thus, we assumed that excessively produced IL-12 and
IL-18 might be responsible for diminishing
CD4+NK1.1+ T cells in the liver and spleen, but
inducing IFN--producing cells that inhibit IgE production in SJL
mice.
IL-12 is mainly produced by macrophages and dendritic cells and induces
naive CD4+ T cells to develop into Th1 cells (17), whereas
IL-18 has no such capacity (14). IL-18, originally called
IFN--inducing factor, is also produced by activated macrophages such
as Kupffer cells (18). The major activity associated with IL-18 is
induction of IFN- production from T cells and NK cells (18),
particularly in the presence of IL-12, and enhancement of their
cytotoxicity through Fas ligand (FasL)-mediated mechanism (19, 20).
Furthermore, IL-18 together with IL-12 induces IFN- production from
activated B cells (16). Therefore, it is important to determine whether
macrophages in SJL mice have the capacity to produce IL-12 and IL-18
that inhibit IgE production by induction of IFN--producing cells.
In this study, we demonstrated that T cell-depleted splenic B cells (standard B cells) from 6-wk-old SJL mice failed to proliferate and to produce IgE and IgG1 in response to LPS plus IL-4 in vitro. This diminished IgE and IgG1 production by SJL standard B cells could be restored by addition of anti-IL-12 Ab and enhanced by further addition of anti-IL-18 Ab. SJL standard B cells were heavily contaminated with B220- cells that mainly comprised Mac-1+ cells and DN CD3intIL-2Rß+ T cells. Addition of B220- cells inhibited IgE production by LPS plus IL-4-stimulated B cells in vitro in a dose-dependent and MHC-nonrestricted manner. In this work, we discuss the relative role of macrophages and DN CD3+IL-2Rß+ T cells in defective IgE/IgG1 production and cell growth seen with SJL standard B cells stimulated with LPS and IL-4.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Virus-free female BALB/c mice, 6 wk of age, were obtained from Japan SLC (Shizuoka, Japan). SJL mice, originally provided by Dr. N. Watanabe (Jikei Medical College, Tokyo, Japan), were bred at the animal facility of Hyogo College of Medicine (Nishinomiya, Japan) and were used at 6 wk of age.
Culture medium
RPMI 1640 supplemented with 10% FBS (HyClone, Logan, UT), 2-ME (50 µM), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and sodium pyruvate (1 mM) was used as culture medium.
Recombinant cytokines
Mouse rIL-4 was obtained and purified from a recombinant
baculovirus (AcMNPV.IL-4) prepared in our laboratory. Recombinant mouse
IFN-, IL-12, and IL-18 were kindly provided by Hayashibara
Biochemical Laboratories (Okayama, Japan).
Abs and reagents
Rat anti-mouse IgE (23G3) and biotin-conjugated rat
anti-mouse IgE (R35-118) were purchased from Southern Biotechnology
(Birmingham, AL) and PharMingen (San Diego, CA), respectively.
Affinity-purified goat Abs against mouse IgG1 and IgM, and
biotin-conjugated affinity-purified goat Abs against mouse IgG1 and IgM
were purchased from Southern Biotechnology. Rat Abs against mouse
IL-12p40 (C17.8, rat IgG2a) (21) and mouse IFN- (R4-6A2, rat IgG1)
were purified in our laboratory. Purified rat IgG1 (R3-34) and IgG2a
were purchased from PharMingen as control Abs for anti-IFN- and
anti-IL-12p40 Abs, respectively. We used affinity-purified hamster
anti-mouse FasL (MFL1) (22) and its control hamster IgG mAb (HH16)
(6). We also used rabbit-neutralizing anti-IL-18 Ab and its control
normal rabbit IgG fraction purified by protein G column. FITC rat
anti-mouse B220 (RA3-6B2), FITC goat anti-mouse IgM, FITC rat
anti-mouse Mac-1 (M1/70), FITC rat anti-mouse IFN- (XMG1.2),
FITC rat anti-mouse CD4 (RM4-5), PE rat anti-mouse CD8
(53-6.7), and Cy-Chrom anti-mouse CD3 (2C11) were purchased from
PharMingen. PE-labeled streptavidin was purchased from Becton Dickinson
(San Jose, CA). FITC anti-mouse CD3 (2C11) and biotin
anti-mouse IL-2Rß (TMß1) were prepared in our laboratory.
Cell preparation
Splenic B cells were prepared from BALB/c or SJL mice, pretreated with anti-asialo GM1 that was used to eliminate NK cells, by two rounds of complement-mediated lysis of T cells with anti-Thy-1.2 and anti-Lyt-1.2 mAbs (23). However, resultant cells that we termed standard B cells still retained macrophages and residual cells. Therefore, we depleted macrophages and residual cells and used the following two B cell populations; 1) Sephadex G-10 column (G10)-passed B cells (G10-B cells); 2) positively enriched B cells by a panning method (panned B cells) on anti-mouse IgM (Bet-2)-coated dishes (24).
For the preparation of B220- cells, SJL standard B cells were suspended at a concentration of 2 x 107/ml in RPMI 1640 containing 5 mM EDTA and 5% FBS. The cell suspension was incubated with 10 µg/ml of FITC anti-B220 for 30 min at 4°C on a turning wheel. The cells were then washed twice and resuspended with magnetic beads coated with sheep anti-FITC Abs (Advanced Magnetics, Cambridge, MA). Cells that had bound magnetic beads were depleted by two rounds of exposure to magnetic field. The residual cells were collected and washed twice, yielding B220- cells.
Assay for secreted Igs
Fractionated B cells (105/0.2 ml/well) were cultured
with LPS (20 µg/ml) in the presence or absence of anti-IL-12
and/or anti-IL-18 or anti-IFN- and/or anti-FasL Abs for
24 h, then provided with medium or serially diluted IL-4 for 7
days. Supernatants in triplicate cultures were collected at day 8 after
the initiation of the culture, and quantitative immunoassay for
secreted IgE, IgG1, and IgM was performed by using ELISA, as described
previously (4).
Lymphokine assays
IL-12, p40 and/or p70 was assayed with a specific two-site ELISA, with reference standard curves prepared using known amounts of IL-12 (Bio Source International, Camarillo, CA). IL-18 was determined by ELISA using protein G-purified rabbit polyclonal Ab to murine IL-18, with reference standard curves prepared using known amounts of IL-18 (Hayashibara Biochemical Laboratories) (14).
Fluorescence analysis
Fluorescence staining was performed at 4°C in 50 µl containing 106 each fractioned B cells. Staining was conducted with various combinations of FITC- and PE-conjugated Abs in PBS containing 0.1% BSA and 0.5% NaN3. For the detection of CD3intIL-2Rß+ T cells, FITC anti-CD3 and the combination of biotinylated anti-IL-2Rß and streptavidin PE were used. Fluorescence analysis was conducted with a FACScan flow cytometer (Becton Dickinson).
Propidium iodide staining and FACS analysis
B cell apoptosis was quantified by flow-cytometric determination of the proportion of cells with hypodiploid DNA by a procedure previously described (25). Briefly, collected cultured cells were centrifuged at 200 x g for 10 min and washed twice with PBS. A cell pellet was gently resuspended in 1.5 ml hypotonic fluorochrome solution (propidium iodide (PI), 50 µg/ml, in 0.1% sodium citrate plus 0.1% Triton X-100). The suspended cells were incubated at 4°C in the dark box before the flow-cytometric analysis. Apoptotic nuclei stained with PI were distinguished by their hypodiploid DNA content compared with the diploid DNA content in normal nuclei. The PI fluorescence of individual nuclei was measured using a FACScan. Region gates are drawn around the population of cells containing <2N DNA, apoptotic cells, and around the population of cells containing 2N–4N, nonapoptotic cells.
Intracellular cytokine staining
For analysis of intracellular IFN--positive cells, we
followed the modified protocol of immunofluorescent staining of
intracellular cytokines for the flow-cytometric analysis described in
our previous study (16). Briefly, prepared B220- cells
(1 x 106/ml/well) were cultured with or without LPS
(20 µg/ml) for 84 h with a pulse of 3 µg/ml of monensin during
the final 12 h to inhibit IFN- secretion (26). Such treated
B220- cells were first stained with Cy-Chrom anti-CD3
and a combination of biotin anti-IL-2Rß and PE-conjugated avidin,
and followed by fixation with 4% (w/v) paraformaldehyde in PBS and
permeabilization of cell membrane with ice-cold PBS containing 1% FCS
plus 0.1% saponin. Resultant cells were further stained with 0.5 µg
of FITC rat anti-mouse IFN- Ab in the presence or absence of
excess IFN- (10 µg/ml) and analyzed for their proportion of
cytoplasmic IFN--positive cells by three-color flow-cytometric
analysis by FACScan.
Analysis of expression of IL-12p40 and IL-18 mRNA
Total RNA from each fractionated B cell was prepared using the guanidium method, as described previously (6). As positive controls for IL-12p40 and IL-18, mRNAs extracted from the Propionibacterium acnes-elicited LPS-stimulated Kupffer cells were used (18). For analysis of expression of IL-12p40 and IL-18 mRNA, mRNAs were amplified by a modified standard reverse-transcription PCR (RT-PCR) amplification procedure, as described in our previous study (6). Primer sequences were as follows: IL-12p40, sense, CGT GCT CAT GGC TGG TGC AAA G, and antisense, GAA CAC ATG CCC ACT TGC TG; IL-18, sense, ACT GTA CAA CCG CAG TAA TAC, and antisense, AGT GAA CAT TAC AGA TTT ATC CC; and ß-actin, sense, GAT GAC GAT ATC GCT GCG CTG, and antisense, GTA CGA CCA GAG GCA TAC AGG. cDNAs were amplified for 30 cycles, each composed of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min (IL-18), or 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min (IL-12, ß- actin). At the end of 30 cycles, samples were stored at 4°C until analyzed. After amplification, PCR products were separated by electrophoresis in 8% acrylamide gels and visualized by UV light illumination after ethidium bromide staining.
Analysis of expression of IFN- and FasL mRNA in SJL
B220- cells
SJL B220- cells (2 x 106/ml) were
stimulated with medium alone or LPS (20 µg/ml) in the presence or
absence of anti-IL-12 (10 µg/ml) plus anti-IL-18 (10 µg/ml)
for 96 h. mRNAs were extracted from cultured cells and were
analyzed for IFN-, FasL, and ß-actin mRNA expression with RT-PCR.
As positive controls for IFN- and FasL mRNAs, mRNAs extracted from
total spleen cells from BALB/c mice treated with anti-CD3 Ab 90 min
before (6) and from cloned NK cell (LNK5E6) (19) were used,
respectively. Primer sequences were as follows: IFN-, sense, AAC GCT
TAC ACA CTG CAT CTT GG, and antisense, GAC TTC AAA GAG TCT GAG G; FasL,
sense, CTG GAA TGG GAA GAC ACA TA, and antisense, AAA GGT CTT AGA TTC
CTC AA. cDNAs were amplified for 30 cycles, each composed of 94°C for
15 s, 55°C for 15 s, and 72°C for 1 min (IFN-), or
95°C for 1 min, 55°C for 1 min, and 72°C for 1 min (FasL).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Splenic B cells prepared by complement-mediated T cell lysis were
designated as standard B cells. We first compared the capacities of
standard B cells from BALB/c and SJL mice to proliferate and to produce
IgE and IgG1 in response to LPS (20 µg/ml) and IL-4 (104
U/ml). B cells were cultured with LPS for 24 h, then additionally
stimulated with serially diluted IL-4 for the following 7 days. We
looked at their proliferative responses on a microscope every day and
measured the number of living cells at days 4, 5, 6, 7, and 8 after
initiation of the culture (Fig. 1
, A and B). Measurement of the concentrations of IgM,
IgG1, and IgE in the culture supernatants of standard B cells
stimulated with LPS and IL-4 was performed at day 8 (Fig. 1C).
|
A). Consistent with this observation, BALB/c
standard B cells gave markedly enhanced cell recovery at days 5
and 6, while similarly stimulated SJL standard B cells failed
(p < 0.01) (Fig. 1B). Beyond day 7,
standard B cells from both strains gave poor cell yields (Fig. 1B), although these poor cell yields seemed to occur in a
different manner. BALB/c standard B cells died and yielded much cell
debris at day 8, while SJL standard B cells died and disappeared
without yielding such cell debris (Fig. 1A), suggesting the
possibility that SJL dead cells might have been processed by phagocytic
cells before they became cell debris.
BALB/c standard B cells, used as a positive control, produced
high levels of IgM, IgG1, and IgE in response to LPS and IL-4 (Fig. 1C). In contrast, parallel stimulated SJL standard B cells
produced 6-fold less IgE (p < 0.01) and 2.5-fold
less IgG1 (p < 0.01), although they produced higher
level of IgM (5, 000 U/mL IL-4, p < 0.05; 10,000 U/mL IL-4,
p < 0.01) (Fig. 1C). To determine whether this
diminished IgG1 and IgE production can be accounted for solely on the
basis of a reduction in cell number of SJL B cells, we normalized the
levels of Igs produced to the cell numbers of each group. Since
stimulation of B cells with LPS and IL-4 results in the expression of
surface IgG1 and IgE on large fractions of the total B cell population
on days 4 and 5 of culture, respectively (27, 28), we normalized
the levels of IgM/IgG1 and IgE produced to the number of cells measured
at days 4 and 5 after initiation of culture, respectively. As
illustrated in Figure 1C, diminished IgG1 and IgE production
was not due solely to reduced cell yield, but also due to diminished
heavy chain isotype switching that might be responsible for inducing an
increase in IgM production.
Inhibition of B cell IgE production by the action of SJL B220- cells
There are at least two possibilities that account for this
poor cell recovery and low IgG1 and IgE production by SJL standard B
cells. First, SJL B cells are low responder to LPS and IL-4. Second, in
SJL standard B cells, there are regulatory cells that inhibit B cells
to proliferate and/or to produce IgE and IgG1 in response to LPS and
IL-4. We prepared standard B cells by complement-mediated lysis of T
cells. We removed NK cells in the spleens by a prior injection of the
mice with rabbit anti-asialo GM1. However, we did not remove
macrophages in the spleens of BALB/c or SJL mice, suggesting their
involvement in IgE inhibition. Therefore, we examined these
possibilities by using two B cell populations (Fig. 2A): 1) panned B cells (99%
surface IgM+), obtained by a positive selection panning
method; 2) G10-B cells (93% surface IgM+), obtained by
passing standard B cells through G10 column.
|
B). Furthermore, like BALB/c standard B cells, they
gave good cell yields (data not shown). These results strongly
suggested that there are regulatory cells in SJL standard B cells.
Passing SJL standard B cells through G10 column markedly diminished the
proportion of B220- cells in SJL standard B cells (Fig. 2A). Furthermore, this decrease was associated with an
increase in the capacity of SJL B cells to produce IgE and IgG1 (Fig. 2B). Therefore, we examined whether addition of
B220- cells into BALB/c or SJL G10-B cells inhibits their
capacity to produce IgE and IgG1. As shown in Figure 2C,
addition of B220- cells dose dependently and MHC
independently inhibited IgE production without affecting IgM production
(data not shown). These results indicated that there are IgE
class-specific suppressor cells in the fraction of B220-
cells in SJL standard B cells. Anti-IL-12 plus anti-IL-18 restored IgE/IgG1 production and cell yield
Since these suppressor cells were adherent to G10 column, we
assumed that they are macrophages or macrophage-like cells that produce
various cytokines, including IL-12 and IL-18, in response to LPS (17, 18). Because IL-12 or a mixture of IL-12 and IL-18 strongly inhibits
IL-4-dependent IgE production by induction of IFN--producing cells
in vitro and in vivo (15, 16), we suspected that IL-12 or IL-12 and
IL-18 from LPS-activated macrophages contaminated in SJL standard B
cells might have played a critical role in inhibition of B cell
proliferation and differentiation. To test this possibility, we
examined the capacity of Abs against IL-12p40 and/or IL-18 to restore
IgG1/IgE production and cell yield (Fig. 3, A and B).
Addition of anti-IL-12 Ab, but not control Ab (data not shown) or
rabbit anti-IL-18, strikingly restored both IgG1/IgE production and
cell yield in SJL standard B cells, although addition of a mixture of
anti-IL-12 and anti-IL-18 Abs most strikingly restored these
responses (Fig. 3, A and B). In contrast,
addition of a mixture of these Abs only modestly enhanced IgE
production from BALB/c standard B cells without affecting cell
yield (Fig. 3, A and B).
|
A), we examined
the possibility that LPS stimulation induces apoptotic cell death in
SJL standard B cells by induction of IL-12 and IL-18 production.
Because a method examining cell apoptosis by PI staining and flow
cytometry is quantitative (25), we used this method to measure the
percentage of apoptotic cells. Stimulation of SJL and BALB/c standard B
with LPS for 72 h induced 10.9 and 2.8% of cells with hypodiploid
DNA, respectively (Fig. 3C), indicating that LPS stimulation
induced cell apoptosis in SJL standard B cells. We also found that
costimulation with IL-4 failed to restore this LPS-induced cell
apoptosis (data not shown). However, as expected from the result of
Figure 3B, addition of a mixture of anti-IL-12 and
anti-IL-18 Abs, but not of control Abs (data not shown), markedly
reduced the percentage of apoptotic cells and rather induced an
increase in the proportion of proliferating cells. Furthermore,
addition of IL-12 and IL-18 markedly increased the proportion of
apoptotic cells in LPS-stimulated SJL standard B cells. These results
indicate that IL-12 and IL-18 are involved in LPS-induced B cell
apoptosis in SJL standard B cells. IL-12 production from LPS-stimulated G10-adhesive cells
To determine whether IL-12 and IL-18 were indeed produced by
G10-adhesive cells in SJL standard B cells, we examined the changes in
the spontaneous expression of mRNAs for IL-12p40 and IL-18 in standard
B cells before and after passing them through G10 column. As shown in
Figure 4A, freshly prepared
SJL, but not BALB/c standard B cells clearly expressed IL-12p40 mRNA,
whereas SJL G10-B cells had no such expression, indicating that there
are G10-adhesive cells that spontaneously express IL-12p40 mRNA in SJL
standard B cells. LPS stimulation of BALB/c and SJL standard B cells
induced or enhanced IL-12p40 mRNA expression, while LPS stimulation of
BALB/c or SJL G10-B cells did not (data not shown), indicating that
IL-12-producing cells were completely depleted by G10 treatment. We
also examined the expression of IL-18 mRNA. As shown in Figure 4A, freshly prepared standard B cells from SJL and BALB/c
mice equally expressed IL-18 mRNA. Depletion of G10-adhesive cells in
SJL and BALB/c standard B cells diminished this expression, suggesting
that G10-adhesive cells strongly expressed IL-18 mRNA. We performed
quantification of PCR products by serial dilution of RNA and by
quantitative densitometric analysis (6). These analyses revealed that
G10 treatment caused threefold diminution in the expression of IL-18
mRNA. Nevertheless, G10-B cells expressed IL-18 mRNA. Since G10-B cells
comprised high proportion of B cells (SJL G10-B cells, 93%
IgM+; BALB/c G10-B cells, 98% IgM+), we
suspected that IL-18 mRNA was constitutively expressed in SJL and
BALB/c B cells.
|
B, SJL standard B cells produced more
IL-12 (p40 and/or p70) than BALB/c standard B cells in response to LPS.
However, G10-B cells from both strains of mice failed to produce IL-12
(p40 and/or p70) in response to LPS, suggesting that G10-adhesive cells
produce IL-12. We also examined the capacity of standard B cells and
G10-B cells from SJL and BALB/c mice to produce IL-18 in response to
LPS. Although they constitutively express IL-18 mRNA, they failed to
produce detectable level of IL-18 by ELISA, indicating that expression
of IL-18 in mRNA does not always mean the production of functional
IL-18 in protein level (29). However, we could not exclude the
possibility that a very small amount of IL-18 was produced by
LPS-stimulated SJL standard B cells, because a mixture of
anti-IL-12 and anti-IL-18 restored IgG1/IgE production more
strongly than anti-IL-12 alone did (Fig. 3).
Anti-IFN- plus anti-FasL restored IgE/IgG1 production and
cell yield
We next examined the mechanism of how IL-12 and IL-18 from
LPS-stimulated B220- cells inhibit B cell growth and IgE
response in SJL standard B cells following stimulation with LPS and
IL-4. Since IgG1 and IgE responses are negatively regulated by the
action of Th1 cells, and treatment with anti-CD40 (CD40 ligand) and
anti-Fas (FasL) induces B cell apoptosis (30, 31, 32), we examined
whether IL-12 and IL-18 inhibit B cell IgE response by induction of Th1
cells that produce IFN- and FasL. Therefore, we examined the
capacity of anti-IFN- and/or anti-FasL Abs to restore this
diminished IgE production and cell yield. As shown in Figure 5A, BALB/c standard B cells
produced high level of IgE at day 8 and gave good cell yield at day 7
after initiation of culture, while SJL standard B cells gave poor IgE
production and cell yield. Addition of anti-IFN- or
anti-FasL Ab, but not control rat or hamster Ab (data not shown),
partially, but significantly, restored this diminished IgE production
and cell yield. Addition of a mixture of these Abs more strongly
restored these responses, although addition of anti-IL-12 Ab, but
not control rat IgG2a (data not shown), most strongly restored cell
yield and IgE response. This restoration of Ig production by treatment
with Abs to IFN- and FasL was also seen in the SJL G10-B cells
cultured with B220- cells (Fig. 5B), although
again addition of anti-IL-12 Ab most strongly restored IgE and IgG1
production. Thus, like anti-IL-12 Ab treatment, treatment with a
mixture of anti-IFN- and anti-FasL restored diminished
IgE/IgG1 production and cell yield that were induced by the action of
SJL B220- cells. Treatment with anti-IFN- Ab only
modestly enhanced IgE production, suggesting that level of IL-12 and
IL-18 produced was relatively low to induce IFN- that inhibits IgE
production by itself.
|
in response to IL-12 and IL-18
To determine the cells that produce IFN- in SJL
B220- cells, we tried to characterize B220-
cells. For this purpose, we first examined the composition of
Mac-1+ and CD3+ cells in SJL standard B cells.
Fractions of Mac-1+ cells and CD3+ cells in SJL
standard B cells were relatively high and 8.9 and 7.5%, respectively,
but these fractions became low after treatment with G10 column (Fig. 6). Characterization of CD3+
T cells revealed that they were DN
CD3intIL-2Rß+ T cells (data not shown) (Fig. 6). Therefore, this unique T cell population that we identified in the
spleens of SJL mice is very similar to the DN T cells that develop and
exit in the liver in their surface phenotype (33, 34). Total
splenocytes from BALB/c and SJL mice contained 3 to 4% and 12 to 14%
CD3intIL-2Rß+ T cells, respectively (data not
shown), whereas only standard B cells prepared from SJL mice contained
6 to 9% DN CD3intIL-2Rß+ T cells (deviation
of five mice) (Fig. 6), suggesting that
CD3intIL-2Rß+ T cells in BALB/c mice
disappeared after complement-mediated lysis of T cells with
anti-Thy-1 and anti-Lyt-1. We negatively enriched
B220- cells from SJL standard B cells by magnetic beads
and characterized them by examining their Mac-1, CD3, CD4, CD8, or
IL-2Rß expression. We found that they comprised 50.8%
Mac-1+ cells and 26% DN
CD3intIL-2Rß+ T cells (Fig. 6).
|
-producing cells in SJL
B220- cells stimulated with LPS for 72 h, we stained
them for cytoplasmic IFN- and analyzed them by FACS. As shown in
Figure 7A, 56.1% of
CD3intIL-2Rß+ T cells became positive for
IFN- after LPS stimulation. However, addition of Abs to IL-12p40 and
IL-18, but not combination of control Abs (data not shown), completely
abolished such action of LPS to induce IFN--producing cells,
substantiating further that IFN--producing cells were induced by
IL-12 and IL-18 from LPS-stimulated Mac-1+ cells (Fig. 7B). We simultaneously examined whether B220-
cells express FasL. We found that B220- cells
constitutively express FasL mRNA (Fig. 7B). We also found
that DN CD3intIL-2Rß+ T cells enriched by
cell sorting express FasL mRNA (data not shown). Because addition of
Abs to IL-12 and IL-18 diminished FasL mRNA expression, the expression
of FasL mRNA in B220- cells appeared to be up-regulated by
IL-12 and IL-18 from LPS-stimulated macrophages (Fig. 7B).
Again, we performed quantification of PCR products by serial dilution
of RNA and by quantitative densitometric analysis. These analyses
revealed that treatment with a mixture of anti-IL-12 and
anti-IL-18 Abs caused twofold diminution in the expression of FasL
mRNA in SJL B220- cells cultured with LPS. These results
taken together indicate that IL-12 and IL-18 from LPS-stimulated
Mac-1+ cells induced
CD3intIL-2Rß+ T cells to produce IFN- that
in combination with FasL strongly inhibited B cell proliferation and
IgE production.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(37) or IL-2 (28). CD4+ T cells
differ in the patterns of cytokines they express (31). Th1 cells
secrete IFN- and IL-2. Th2 cells secrete IL-4, IL-5, and IL-10.
Therefore, it has been suggested that the balance of Th2 and Th1
largely determines the levels of IgE produced during an immune
response. SJL mice are known to produce limited amounts of IgE in
response to a variety of stimulants in vivo (1, 2, 3). Watanabe et al.
demonstrated that T cells that express Lyt-1 inhibit IgE response in
SJL mice (3). An alternative possibility was proposed by Yoshimoto and
Paul (5). SJL mice have a striking defect in both IgE and IL-4
responses to polyclonal stimulation with anti-IgD Ab, and these
defects are associated with the absence of
CD4+NK1.1+ T cell population (5). Since it has
been proposed that this T cell population produces IL-4 for priming of
conventional T cell for IL-4 production in vivo (38), the absence of
this population might lead to Th1 dominance in the balance of Th2 and
Th1.
The defect in SJL mice in IgE production in response to helminth
infection or certain Ags has been studied extensively using an in vivo
system (1, 2, 3). Nevertheless, the nature of the IgE defect in SJL mice
remains to be enigmatic. In this study, we used an in vitro system to
reveal the mechanism underlying IgE defect in SJL mice. Taking
advantage of in vitro system, we prepared various types of splenic B
cells and stimulated them with LPS and IL-4. As shown in Figure 1C, standard B cells produced a limited amount of IgE.
Furthermore, they failed to give enhanced cell yield (Fig. 1B) and disappeared when they died without yielding cell
debris (Fig. 1A). We used standard B cells free from
contamination with Lyt-1.2+ T cells, which have been
proposed to be suppressor cells in vivo by Watanabe et al. (2).
Consistent with the previous report (4), panned B cells (99% surface
IgM+) and G10-B cells (95% surface IgM+)
proliferated and developed into IgE-producing cells following
stimulation with LPS and IL-4. We demonstrated that standard B cells
are heavily contaminated with B220- cells (Fig. 2), and
addition of B220- cells to the G10-B cells dose
dependently inhibited their capacity to produce IgE in response to LPS
and IL-4 (Fig. 2C), suggesting that the defect in SJL
standard B cells in IgE production is induced by active suppressive
mechanism.
In this study, we investigated this active suppressive mechanism by
B220- cells that principally comprise DN
CD3intIL-2Rß+ T cells and Mac-1+
cells (Fig. 6). This unique subpopulation of T cells is distinct from
thymic T cells in terms of their expression of or expressed level of
CD3, CD4, and CD8. It is recently established that there are
extrathymic pathways of T cell differentiation in the liver and other
organs of thymic mice and nonthymic mice (33, 34). Abo et al.
demonstrated that these hepatic T cells have unique properties as
primitive lymphocytes and contain DN
CD3intIL-2Rß+ cells (33, 34). Although the
central roles of these unique T cells remain uncertain, this population
increases in number at the target organ in autoimmune diseases (39) and
at tumor sites in malignancies (40). Thus, it would appear that these
primitive T cells are involved in many immunologic phenomena because of
their autoreactivity (33, 34).
We recently found that administration of P. acnes into
thymic or nonthymic mice stimulates Kupffer cells to produce IL-12 and
IL-18, which synergistically induce IFN- production from DN
CD3intIL-2Rß+ T in the liver (14, our
unpublished observation). Thus, it is intriguing to speculate that
IL-12 and IL-18 produced by SJL macrophages following helminth
infection stimulate a population of FasL-positive DN
CD3intIL-2Rß+ T cells to secrete IFN-
that, in combination with FasL, inhibits IgE production in vivo. To
understand the mechanism by which these unique T cells inhibit IgE
production and induce apoptotic cell death, we examined the capacity of
anti-IL-12 and/or anti-IL-18 or of anti-FasL and/or
anti-IFN- to restore these poor IgE production and poor cell
yield (Fig. 5). We found that a mixture of anti-IL-12 and
anti-IL-18 most strikingly restored these B cell responses. We also
found that a mixture of anti-IFN- and anti-FasL strongly
restored these B cell responses (Fig. 5).
In our previous study, we demonstrated that resting B cells do not
express Fas, but they express Fas when they are stimulated with
anti-CD40 or LPS and become highly susceptible to
anti-Fas-mediated apoptotic cell death (30). Others also
demonstrated that Th1 T cells induce B cell apoptosis in Fas-dependent
manner (32). LPS stimulation induces Fas mRNA as well as its protein in
SJL B cells (data not shown). DN
CD3intIL-2Rß+ T cells express FasL and begin
to produce IFN- after being stimulated with IL-12 and possibly IL-18
from LPS-stimulated Mac-1+ cells (Fig. 7). As shown in
Figure 4B, BALB/c standard B cells stimulated with LPS also
produce IL-12 (p40 and/or p70). Nevertheless, BALB/c standard B cells
produce IgE in response to LPS and IL-4, suggesting that IL-12 from
LPS-stimulated Mac-1+ cells could not inhibit IgE
production in the absence of DN CD3intIL-2Rß+
T cells and/or that produced IL-12 is nonfunctional p40 homodimer.
Therefore, IL-12 and possibly IL-18 from LPS-stimulated
Mac-1+ cells in SJL mice are cytokines that induce these
unique DN T cells to develop into IFN--producing Th1-type T cells.
We examined the level of FasL on DN
CD3intIL-2Rß+ T cells by staining with FITC
anti-FasL. Due to low level expression of FasL on these T cells, we
could not reveal the expression of FasL on the cell surface, although
we could detect FasL mRNA in B220- cells (Fig. 7) and
CD3intIL-2Rß+ T cells (data not shown).
We have reported recently that the defect in SJL mice may lie in an inability to produce sufficient IL-4 for priming of conventional T cells for IL-4-producing Th2 cells. The initial IL-4-producing cells appear to present a specialized population of CD4+ T cells since, in addition to markers associated with effector or memory cells (CD44high, LECAM-1negative, CD45RBdull), they express NK1.1. The mechanism for diminution in the proportion of CD4+NK1.1+ T cells in the spleens of SJL mice remains unclear at the present time. However, since CD1-deficient mice are lacking this population (12) and SJL mice have decreased expression of CD1.2 isoform of CD1 (13), recognition of CD1 may be essential for inducing and activating NK1.1+ T cells.
We have found recently that CD4+NK1.1+ T cells
in the liver are also strikingly low in SJL mice, constituting 0.3% of
liver lymphocytes in contrast to frequency of 19.2% in C57BL/6 mice
(unpublished observation). These CD4+NK1.1+
liver lymphocytes in C57BL/6 mice promptly produce IL-4 in response to
stimulation with anti-CD3 Ab in vitro and in vivo (14). We and
others have reported that CD4+NK1.1+ T cells in
the liver are down-regulated by IL-12 or IL-12 and IL-18 (14, 41). We
have also reported that treatment with IL-12 plus IL-18 induces an
increase in the number of DN CD3intIL-2Rß+ T
cells that produce IFN- in the liver of C57BL/6 mice (14). As noted
in this study, proportion of Mac-1+ cells in SJL standard B
cells is 8.9% (Fig. 6), which is higher than that (2%) in BALB/c
standard B cells (data not shown). Furthermore, these G10-adhesive
Mac-1+ cells constitutively express IL-12p40 mRNA and
produce IL-12 (p40 and/or p70) in response to LPS (Fig. 4). Therefore,
it is intriguing to speculate that IL-12 and possibly IL-18 from
Kupffer cells in the livers and activated macrophages in the spleens
may diminish the proportion of CD4+NK1.1+ T
cells and increase the proportion of DN
CD3intIL-2Rß+ T in the livers and spleens of
SJL mice.
In this study, we demonstrated that T cell-depleted splenic B cells
from SJL mice proliferated and produced IgE poorly in response to LPS
plus IL-4. This diminished IgE production and cell yield could be
markedly restored by addition of a mixture of anti-IL-12 and
anti-IL-18 Abs. Treatment with a mixture of anti-IFN- and
anti-FasL also restored defective IgE production and cell yield. In
this study, we also demonstrated the presence of cooperative
suppression of IgE production and B cell proliferation by the actions
of DN CD3intIL-2Rß+ T cells and
Mac-1+ cells. We showed that Mac-1+ cells and
DN CD3intIL-2Rß+ T cells are uniquely
abundant in the spleens of SJL mice, whereas, as we showed previously
(5), CD4+NK1.1+ T cells are uniquely poor in
the spleens and livers of SJL mice, suggesting that IL-12 and IL-18
affect these populations and regulate IgE production in vivo and in
vitro by stimulation of DN CD3intIL-2Rß+ T
cells to increase expression of FasL and to produce IFN-.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Kenji Nakanishi, Department of Immunology and Medical Zoology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663-8501 Japan. E-mail address:
3 Abbreviations used in this paper: DN, double-negative; FasL, Fas ligand; int, intermediate; PE, phycoerythrin; PI, propidium iodide; G10, Sephadex G-10.
Received for publication November 25, 1997. Accepted for publication April 2, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Immunol. 136:949.[Abstract]
chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8- T cells in mice and humans. J. Exp. Med. 180:1097. production from activated B cells. Proc. Natl. Acad. Sci. USA 94:3948. production by T cells. Nature 378:88.[Medline]
-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine NK cell clones. J. Immunol. 157:3967.[Abstract]
-inducing factor, a novel cytokine, enhances Fas ligand mediated cytotoxicity of murine T helper 1 cells. Cell. Immunol. 173:230.[Medline]
production and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672.[Medline]
inducing factor mediated by interleukin-1ß converting enzyme. Science 275:206. and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944.ß- cells in the liver is a common feature of autoimmune mice. J. Immunol. 147:2907.[Abstract]
/ß intermediate liver lymphocytes are down-regulated by Listeria monocytogenes. Eur. J. Immunol. 25:3321.[Medline]
This article has been cited by other articles:
|
|
 |
|
  E. A. Haddad, L. K. Senger, and F. Takei An Accessory Role for B Cells in the IL-12-Induced Activation of Resting Mouse NK Cells J. Immunol., September 15, 2009; 183(6): 3608 - 3615. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Suzukawa, M. Iikura, R. Koketsu, H. Nagase, C. Tamura, A. Komiya, S. Nakae, K. Matsushima, K. Ohta, K. Yamamoto, et al. An IL-1 Cytokine Member, IL-33, Induces Human Basophil Activation via Its ST2 Receptor J. Immunol., November 1, 2008; 181(9): 5981 - 5989. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. A. Korshunov, T. A. Nikonenko, V. A. Tkachuk, A. Brooks, and B. C. Berk Interleukin-18 and Macrophage Migration Inhibitory Factor Are Associated With Increased Carotid Intima-Media Thickening Arterioscler Thromb Vasc Biol, February 1, 2006; 26(2): 295 - 300. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Sasaki, T. Yoshimoto, H. Maruyama, T. Tegoshi, N. Ohta, N. Arizono, and K. Nakanishi IL-18 with IL-2 protects against Strongyloides venezuelensis infection by activating mucosal mast cell-dependent type 2 innate immunity J. Exp. Med., September 6, 2005; 202(5): 607 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Liu and D. I. Beller Distinct Pathways for NF-{kappa}B Regulation Are Associated with Aberrant Macrophage IL-12 Production in Lupus- and Diabetes-Prone Mouse Strains J. Immunol., May 1, 2003; 170(9): 4489 - 4496. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Taneichi, S. Naito, H. Kato, Y. Tanaka, M. Mori, Y. Nakano, H. Yamamura, H. Ishida, K. Komuro, and T. Uchida T Cell-Independent Regulation of IgE Antibody Production Induced by Surface-Linked Liposomal Antigen J. Immunol., October 15, 2002; 169(8): 4246 - 4252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Liu and D. Beller Aberrant Production of IL-12 by Macrophages from Several Autoimmune-Prone Mouse Strains Is Characterized by Intrinsic and Unique Patterns of NF-{kappa}B Expression and Binding to the IL-12 p40 Promoter J. Immunol., July 1, 2002; 169(1): 581 - 586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Walter, C. P. Wong, R. H. DeKruyff, G. J. Berry, S. Levy, and D. T. Umetsu IL-18 Gene Transfer by Adenovirus Prevents the Development of and Reverses Established Allergen-Induced Airway Hyperreactivity J. Immunol., May 15, 2001; 166(10): 6392 - 6398. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.B. Kay Allergy and Allergic Diseases- First of Two Parts N. Engl. J. Med., January 4, 2001; 344(1): 30 - 37. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ohkusu, T. Yoshimoto, K. Takeda, T. Ogura, S.-i. Kashiwamura, Y. Iwakura, S. Akira, H. Okamura, and K. Nakanishi Potentiality of Interleukin-18 as a Useful Reagent for Treatment and Prevention of Leishmania major Infection Infect. Immun., May 1, 2000; 68(5): 2449 - 2456. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Wild, A. Sigounas, N. Sur, M. S. Siddiqui, R. Alam, M. Kurimoto, and S. Sur IFN-{gamma}-Inducing Factor (IL-18) Increases Allergic Sensitization, Serum IgE, Th2 Cytokines, and Airway Eosinophilia in a Mouse Model of Allergic Asthma J. Immunol., March 1, 2000; 164(5): 2701 - 2710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Elhofy and K. L. Bost Limited Interleukin-18 Response in Salmonella-Infected Murine Macrophages and in Salmonella-Infected Mice Infect. Immun., October 1, 1999; 67(10): 5021 - 5026. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Fassbender, O. Mielke, T. Bertsch, F. Muehlhauser, M. Hennerici, M. Kurimoto, and S. Rossol Interferon-{gamma}-inducing factor (IL-18) and interferon-{gamma} in inflammatory CNS diseases Neurology, September 1, 1999; 53(5): 1104 - 1104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Baldini, I. Carla Lohman, M. Halonen, R. P. Erickson, P. G. Holt, and F. D. Martinez A Polymorphism* in the 5' Flanking Region of the CD14 Gene Is Associated with Circulating Soluble CD14 Levels and with Total Serum Immunoglobulin E Am. J. Respir. Cell Mol. Biol., May 1, 1999; 20(5): 976 - 983. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, SJL standard B cells;
SSB •, SJL panned B cells; SPB 
, SJL G10-B cells; SGB 
) was
cultured with LPS (20 µg/ml) for 24 h, then provided with medium
or serially diluted IL-4 (0–104 U/ml) for 7 days.
C, G10-B cells (105/0.2 ml/well) from BALB/c
and SJL mice were cultured with LPS (20 µg/ml) in the presence or
absence of SJL B220- cells (0–20%) for 24 h, then
provided with 104 U/ml IL-4 for 7 days. Concentrations of
IgE in triplicate cultures were measured on day 8 after initiation of
culture by ELISA. No significant difference in the levels of IgE, IgG1,
and IgM produced by BALB/c standard B cells, SJL panned B cells, and
SJL G10-B cells. Levels of IgE and IgG1 produced by SJL standard B
cells are significantly lower than those produced by other types of B
cells at IL-4 (200, 5,000, 10,000 U/ml, p < 0.01).
Levels of IgM produced by SJL standard B cells are significantly higher
than those produced by other types of B cells at IL-4 (1,000, 10,000
U/ml, p < 0.05). Data are mean ± SD.
