©
International Immunology, Vol. 8, No. 5, pp. 791-798
1996 Oxford University Press
IL-4 and anti-CD40 protect against Fasmediated B cell apoptosis and induce B cell
growth and differentiation
Kenji Nakanishi, Kiyoshi Matsui1, Shin-ichiro Kashiwamura, Yasuhiro Nishioka2,
Jun Nomura3, Yoshiko Nishimura4, Nobuo Sakaguchi3, Shin Yonehara4,
Kazuya Higashino1 and Sohei Shinka
Department of Immunology and Medical Zoology and 1Third and 2Fifth Departments of Internal Medicine,
Hyogo College of Medicine, Nishinomiya, Hyogo 663, Japan
3
Department of Immunology, Kumamoto University School of Medicine, 211 Honjo, Kumamoto 860, Japan
institute for Virus Research, Kyoto University, Kyoto, Kyoto 606-01, Japan
Keywords: antibody-production, B cell apoptosis, CD40, Fas, IL-4, Th1/Th2
Abstract
Most Th2 clones, when activated, produce IL-4 and express CD40 ligand (CD40L) on their cell
surface. Therefore, they can induce growth and differentiation of B cells by cognate help. In
contrast, activated Th1 clones, which produce IFN-y and express both CD40L and Fas ligand (FasL)
on their cell surface, often induce B cell apoptotic cell death. To understand the mechanism by
which Th2 cells can induce B cell growth and differentiation in the presence of FasL-positive cells,
we stimulated B cells with IL-4, anti-IgM and/or anti-CD40 in the presence of anti-Fas. We report
here that addition of anti-Fas strongly inhibited anti-CD40-induced B cell proliferation without
affecting anti-lgM-induced B cell proliferation. Furthermore we showed that stimulation of B cells
with anti-CD40 induced the expression of Fas molecules on the B cells (~30%) and rendered them
highly sensitive to anti-Fas-mediated apoptotic cell death. Indeed, over 23% of anti-CD40stimulated B cells showed hypodiploid DNA after being incubated with anti-Fas, while <2% of antiCD40-stimulated B cells showed hypodiploid DNA after being incubated with medium alone. We
also showed that IL-4 enhanced expression of Fas on anti-CD40-induced B cells (~50%), although
co-stimulation with anti-CD40 and IL-4 protected B cells from anti-Fas-mediated apoptotic cell
death and induced their growth and differentiation. Our present result might suggest that Th2 cells
could dominate over FasL-positive Th1 cells by production of CD40L and IL-4, which in
combination induce antibody production and inhibit the Th1 cell-mediated immune response.
Introduction
Previous studies with use of mAb had suggested a role for
CD40 in B cell activation (1-9). A novel T cell surface molecule
that binds to CD40 has been cloned and designated as CD40
ligand (CD40L) (10-14). CD40L stimulates B cells much as
contact with helper T cells (10-14). Fas is a cell surface
glycoprotein that transduces an apoptotic cell death signal
(15-18). Although CD40 and Fas display opposite effects on
cell growth, they belong to the tumor necrosis factor (TNF)
receptor superfamily (17). A recently cloned novel Fas ligand
(FasL) is a type II transmembrane glycoprotein belonging to
the TNF and CD40L family and expressed on cytotoxic T
cells (18-26). Murine T helper cell clones are classified into
two distinct subsets, Th1 and Th2, on the basis of cytokine
secretion patterns. Th1 clones produce IL-2, TNF-p and IFNy, while Th2 clones produce IL-4, IL-5, IL-6, IL-10 and IL13 (27). These subsets differentially promote delayed-type
hypersensitivity or antibody responses respectively (27).
Activation of Th1 or Th2 clones with anti-CD3 induced CD40L
mRNA (14). CD40L-activated B cells have been reported to
express Fas and are highly sensitive to the killing action of
Th1 cells (24). In contrast, incubation of CD40L-activated B
cells with IL-4-producing Th2 cells induces B cell growth and
differentiation instead of inducing B cell apoptosis (14). Thus,
different relative expression of FasL and IL-4 by T h 1- and
Correspondence to: K. Nakanishi
Transmitting editor: M. M. Davis
Received 4 September 1995, accepted 7 February 1996
792
IL-4 protects against Fas-mediated B cell apoptosis
Th2-type T cells may help explain the reciprocal relationship
between delayed-type hypersensitivity and antibody production. To reveal the mechanism by which Th2-type T cells
dominate over Th1-type T cells and induce antibody production and inhibit delayed-type hypersensitivity, here we
assessed the ability of IL-4, anti-IgM antibody and/or antiC D ^ antibody to induce B cells to grow and differentiate
into antibody-producing cells in the presence of anti-Fas
antibody. Furthermore we examined the regulation of B cell
Fas expression by IL-4, anti-IgM and/or anti-CD40. Here we
provide evidence that anti-CD40 induces Fas on resting B
cells and renders them highly sensitive to anti-Fas-mediated
apoptotic cell death, and IL-4 protects against Fas-mediated
B cell apoptosis and induces B cell growth and differentiation.
Methods
Reagents and antibodies
Affinity purified goat anti-mouse IgM antibody, rat monoclonal
anti-mouse CD40 antibody (LB429, lgG2a) (28) and hamster
monoclonal anti-mouse Fas antibody (RK8) (29) were used.
Phycoerythrin (PE)-conjugated goat anti-hamster IgG antibody was obtained from Southern Biotechnology Associates
(Birmingham, AL).
Culture medium
RPMI-1640 supplemented with 10% FCS (HyClone, Logan,
UT), L-glutamine (2 mM), 2-mercaptoethanol (5X10~ 5 M),
penicillin (100 U/ml) and streptomycin (100 ng/ml) was used.
ml anti-Fas antibody. After incubation, cells were washed and
subsequently treated with PE-conjugated goat anti-hamster
antibody. The specificity of anti-Fas binding was established
by the inhibition of the binding of the labeled anti-Fas by
preincubation with the homologous unlabeled mAb (data not
shown). Stained cells were analyzed using a FACScan (Becton
Dickinson, Mountain View, CA). Ten thousand cells were
analyzed for each histogram and data were processed by
Lysis II (Becton Dickinson).
Propidium iodide (PI) 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 (33). Briefly, after culture the
cells were centrifuged at 200 g for 10 min and washed twice
with PBS. A cell pellet was gently resuspended in 1.5 ml
hypotonic fluorochrome solution (50 ng/ml PI in 0.1% sodium
citrate plus 0.1% TritonX-100). The suspended cells were
incubated at 4°C in the dark overnight before the flow
cytometric analysis. Apoptotic nuclei stained with PI were
distinguished by their hypodiploid DNA content compared
with the diploid DNA content of normal nuclei. The PI fluoresence of individual nuclei was measured using a FACScan
flow cytometry. All the data were recorded and processed by
Lysys II.
DNA preparation and electrophoresis
Recombinant murine IL-4 was obtained from a baculovirus
expression system (30). IL-4 activity was determined as
described previously (31).
Small-sized DNA was prepared from B cells (6x10 6 ) as
described by Laird et al. (34). DNA pellet was dried and
resuspended in 20 |il of TE (pH 8.0). Aliquots of 10 nl of each
DNA sample were electrophoresed in a 1.4% agarose gel
containing ethidium bromide. DNA was visualized by a UV
transilluminator and gels were photographed by a Polaroid
camera.
Factors
Mice and preparation of B cells
Assay for secreted Ig
Specific pathogen-free female BALB/c and MRL Ipr/lpr (Ipr/
Ipr) mice obtained from Shizuoka Laboratory Animal Center
(Shizuoka, Japan) were used at 7-9 weeks of age. Splenic B
cells were prepared by a procedure described in our previous
report (32), which is able to deplete CD5 + B cells by antiCD5 antibody plus C-mediated CD5 + B cell lysis, T cells by
treatment with anti-CD5 antibody/anti-Thy-1.2 antibody plus
C-mediated T cell lysis and activated lymphocytes/macrophages by passing through a Sephadex G-10 column. This
procedure routinely yields cells that are >98% surface IgM
and < 1 % CD3 + .
Purified B cells (105) were cultured in flat-bottom, 96-well
microtiter plates in 0.2 ml of medium containing anti-CD40
(2 |xg/ml) for 24 h and then incubated with medium or serially
diluted IL-4 in the presence or absence of anti-Fas (100-400
pg/ml). Culture supernatants were collected at 8 days after
the initiation of the culture, and quantitative immunoassays
for secreted lgG1 and IgE were performed by using an ELISA
method (32).
B cell proliferation assay
IL-4 blocks the capacity of anti-Fas antibody to inhibit antiCD40 antibody-induced B cell proliferation
B cells were cultured in flat-bottomed 96-well microtiter plates
(No. 3596; Costar, Cambridge, MA) at 105 per well with antiIgM antibody (-100 ng/ml), anti-CD40 antibody (-10 ng/ml)
and anti-Fas antibody (-400 pg/ml), either individually or in
various combinations, with or without serially diluted IL-4 for
3 days, with a pulse of 1 nCi [3H]thymidine during the final
16 h.
Cytofluorometric analysis of Fas expression
B cells were incubated for 30 min on ice in 50 nl of staining
buffer (PBS with 2% FCS and 0.05% NaN3) containing 20 \ig/
Results
Highly purified B cells proliferated in response to stimulation
with anti-IgM antibody (1, 10 and 100 ^g/ml) (Fig. 1a) or antiCD40 antibody (0.1, 1 and 10 ng/ml) (Fig. 1a). Costimulation
with IL-4 dose-dependently enhanced the proliferation of B
cells induced by stimulation with anti-IgM (10 ng/ml) and/or
anti-CD40 (2 |ig/ml) (Fig. 1b-d). We first investigated whether
anti-Fas antibody could inhibit these B cell proliferative
responses. Addition of anti-Fas antibody strongly inhibited
anti-CD40-dependent B cell proliferation without affecting
anti-lgM-dependent or anti-IgM plus IL-4-dependent B cell
IL-4 protects against Fas-mediated B cell apoptosis
a.
anti-IgM
or
b.
anti-CD40
anli-CD40
anti-IgM +
793
IL-4
SOOO-i
«OOO-
3000-
2000 •
1000
IL-4
c.
anti-CD40 +
d.
IL-4
anti-IgM
+
anti-CD40
(U/ml)
+
IL-4
30000 -|
20000n
10000
IL-4
(U/ml)
IL-4
(U/ml)
Fig. 1. Anti-Fas, antibody inhibited anti-CD40-dependent but not anti-lgM-dependent B cell proliferative responses, (a) The proliferative
response of B cells (105/0.2 ml/well) to anti-IgM antibody or anti-CD40 antibody in the presence or absence of anti-Fas antibody. Cultures
were incubated for 72 h, with a pulse of 1 |iCi [3H]thymidine during the final 16 h. (b-d) The proliferative response of B cells (105/0.2 ml/well)
to anti-IgM antibody (10 ng/ml) and/or anti-CD40 antibody (2 |ig/ml) in the presence or absence of anti-Fas antibody with various concentrations
of IL-4. B cells were cultured for 3 days with a pulse of 1 nCi [3H]thymidine during the final 16 h.
proliferation (Fig. 1a and b). Furthermore, such anti-Fas
treatment significantly inhibited anti-CD40 plus IL-4-dependent or anti-IgM, anti-CD40 plus IL-4-dependent B cell proliferation, especially when the B cells were stimulated with lower
concentrations of IL-4 (Fig. 1c and d). However, stimulation
with higher concentrations of IL-4 (1000-10,000 U/ml) induced
the proliferation of B cells even in the presence of anti-Fas
antibody (Fig. 1c and d). In accord with the findings by
Rothstein et al. (24), although partially, addition of anti-IgM
(10 ng/ml) blocked anti-Fas-mediated inhibition of anti-CD40induced B cell proliferation (Fig. 1c versus d).
Expression of Fas is regulated by anti-CD40 antibody and IL-4
We next examined the expression of Fas on the B cells which
had been incubated with medium alone or with anti-IgM
antibody (10 \ig/m\) and/or anti-CD40 antibody (2 ng/ml) in
the presence or absence of IL-4 (5000 U/ml) for 48 h (Fig. 2).
Freshly purified B cells did not express Fas (data not shown).
B cells cultured by themselves or with anti-IgM antibody and/
or IL-4 (5000 U/ml) expressed Fas meagerly (Fig. 2a-d). In
contrast, culture with anti-CD40 antibody markedly induced
Fas expression on B cells (-30%) (Fig. 2e). Furthermore,
IL-4 treatment enhanced Fas expression on anti-CD40-stimulated B cells (-50%) (Fig. 2f). Clearly, augmentation in the
expression of Fas on anti-CD40-stimulated B cells was dosedependently induced by IL-4; 1000-10,000 U/ml of IL-4
caused greater enhancement than 10-100 U/ml of IL-4 did
(data not shown). However, stimulation of B cells with antiIgM antibody modestly diminished the expression of Fas as
well as background fluorescence intensity (Fig. 2c, d, g and
h), suggesting that goat anti-IgM antibody used for stimulation
of B cells competed with PE-goat anti-hamster IgG used for
cell staining for FcyR on B cells. Kinetic study indicated that
B cells stimulated with anti-CD40 antibody and IL-4 1 day
previously showed marked enhancement and B cells stimulated 2-4 days previously showed maximal enhancement
(data not shown).
The specificity of binding of mAb against Fas was deter-
794
IL-4 protects against Fas-mediated B cell apoptosis
a Fas
Fas Expression (Log 1 0 )
Fig. 2. Stimulation with anti-CD40 induces Fas expression on resting
B cells and IL-4 enhances Fas expression on anti-CD40-stimulated
B cells. B cells (10 6 ml/well) were cultured alone or with IL-4 (5000
U/ml), anti-IgM antibody (10 ng/ml) and anti-CD40 antibody (2 \ig/
ml), either individually or in various combinations. After 2 days of
culture, each culture group was harvested, washed and then stained
with hamster anti-Fas antibody and PE-conjugated goat anti-hamster
IgG. One-color immunofluorescence diagrams of cells in the
lymphocyte gate for Fas. Background staining of cells provided by
PE-conjugated goat anti-hamster IgG treatment is shown by the open
histogram.
3.
Medium
D.
anti-CD40
C
anti-CD40
1078
872
603
310
M
1
Fig. 4. Induction of DNA fragmentation in anti-CD40-activated B cells
by anti-Fas treatment. B cells incubated with anti-CD40 antibody
(2 ng/ml) for 48 h were washed and subsequently incubated at 10°/
ml with anti-Fas antibody (400 pg/ml) in the presence of anti-CD40
antibody (2 ng/ml) anti/or IL-4 (5000 U/ml) for 8 h. Small-sized DNA
was prepared and was electrophoresed in a 1.4% gel containing
ethidium bromide.
mice (Fig. 3b and c) but not from Ipr/lpr mice (Fig. 3e and f),
establishing further the specificity of staining used here for
Fas expression.
Blocking of anti-Fas antibody-mediated B cell apoptosis by
IL-4
Fas Expression ( Log 10 )
Fig. 3. Stimulation with anti-CD40 or anti-CD40 plus IL-4 induces Fas
expression on resting B cells from BALB/c but not from MRL Ipr/lpr
mice. B cells (106 ml/well) were cultured alone or with anti-CD40
antibody (2 ng/ml) in the presence or absence of IL-4 (5000 U/ml).
After 2 days of culture, each culture group was harvested, washed
and then stained with hamster anti-Fas antibody and PE-conjugated
goat anti-hamster IgG. One-color immunofluorescence diagrams of
cells in the lymphocyte gate for Fas. Background staining of cells
provided by PE-conjugated goat anti-hamster IgG treatment is shown
by the open histogram.
mined by the capacity of unlabeled anti-Fas antibody to
compete with binding of labeled anti-Fas antibody to Faspositive cells (data not shown). To substantiate further the
specificity of staining for Fas expression, we stained B cells
from BALB/c and Fas-deficient Ipr/lpr mice after stimulation
of B cells with anti-CD40 or anti-CD40 plus IL-4 for 48 h
(Fig. 3). A combination of hamster anti-Fas antibody and PEgoat anti-hamster antibody stained both anti-CD40-stimulated- and anti-CD40 plus IL-4-stimulated B cells from BALB/c
We next investigated the mechanism how anti-Fas antibody
inhibited B cell proliferation and how IL-4 blocked such ability
of anti-Fas antibody. We prepared anti-CD40-stimulated B
cells by incubation of B cells with anti-CD40 antibody (2 \igl
ml) for 48 h. We separated viable cells on a Ficoll-Hypaque
gradient and then incubated them with medium alone or with
anti-Fas antibody (400 pg/ml) in the presence and absence
of anti-CD40 antibody (2 ng/ml) and/or IL-4 (5000 U/ml) for
8 h. Treatment with anti-Fas antibody clearly induced DNA
fragmentation in anti-CD40-stimulated B cells (Fig. 4, lane 2).
In contrast, induction of such B cell apoptosis was markedly
blocked by incubation with IL-4 (Fig. 4, lane 3), and almost
completely blocked by incubation with anti-CD40 antibody
plus IL-4 (Fig. 4, lane 5).
As shown in Fig. 4, cell apoptosis can be qualitatively
evaluated by electrophoresis. However, this technique is
unable to determine the percentage of apoptotic cells in a
heterogenous cell population. Since a method for measuring
cell apoptosis by PI staining and flow cytometry is more
accurate and quantitative (33), we used this method for
measuring the percentage of apoptotic cells (Table 1). Cultures of anti-CD40-stimulated B cells with anti-Fas antibody
in the presence and absence of anti-CD40 antibody for 24 h
induced 21.3 and 23.5% of apoptotic cells respectively. In
IL-4 protects against Fas-mediated B cell apoptosis
30-
Table 1. IL-4 blocks Fas-mediated BALB/c B cell apoptosis
anti-Fas (ng/ml)
Condition of stimulation
Anti-Fas
Anti-CD40
795
IgG 1
Percentage of apoptotic cells
IL-4
BALB/c
MRL Iprllpr
1.4
23.5
0.6
0.7
2.8
2.5
1.6
1.6
1.9
1.7
2.0
1.7
21.3
4.9
0.7
4.1
6
B cells (2x 10 cells/ml) from BALB/c or MRL Ipr/lprwere stimulated
with anti-CD40 antibody (2 ng/ml) for 48 h. Viable cells (106 cells/ml/
well) separated on a Ficoll-Hypaque gradient were incubated with
anti-CD40 antibody (2 ng/ml) or anti-Fas antibody (500 pg/ml) in the
presence or absence of IL-4 (5000 U/ml) for 24 h; then the cells were
permeabilized and their DNA was stained with PI, as described in
Methods. Region gates are drawn around the population of cells
containing <2/v DNA, apoptotic cells and around the population of
cells containing 2N-4N, nonapoptotic cells.
contrast, cultures of anti-CD40-stimulated B cells with antiFas and IL-4 in the presence and absence of anti-CD40 for
24 h reduced these percentages of apoptotic cells to 4.1
and 4.9% respectively, indicating that IL-4 is principally
responsible for protecting against Fas-mediated B cell
apoptosis (Table 1). As expected from the result of Fig. 3,
treatment with anti-Fas antibody did not induce apoptosis in
anti-CD40-stimulated B cells from Iprllpr mice. These results
taken together indicated that, although IL-4 augmented Fas
expression on anti-CD40-activated B cells, IL-4 protected
against Fas-mediated B cell apoptosis and in conjunction
with anti-CD40 antibody induced the proliferation of B cells.
Anti-CD40 antibody and IL-4 induced lgG1 and IgE production
in the presence of anti-Fas antibody
We finally examined whether anti-CD40 (2 ng/ml) and IL-4
could induce lgG1 and IgE production from B cells in the
presence of anti-Fas antibody (Fig. 5). Like human B cells
stimulated with anti-CD40 antibody and IL-4 (7,8), murine B
cells activated with anti-CD40 antibody produced lgG1 and
IgE in response to IL-4. Addition of anti-Fas antibody inhibited
these Ig responses, when B cells were stimulated with antiCD40 antibody and 1000-5000 U/ml IL-4. However, marked
lgG1 and IgE responses were observed when B cells were
stimulated with anti-CD40 antibody and 10,000 U/ml of IL-4.
Although the IL-4-induced increase in B cell Ig production
required a higher concentration of IL-4 than the IL-4-induced
increase in B cell proliferation did (Fig. 1c), a combination of
anti-CD40 antibody and high concentration of IL-4 strongly
overcame the inhibitory action of anti-Fas antibody and
induced B cells to develop into lgG1 and IgE producing cells.
Both the activation and the functions of CD4 + T cells
mediated through intimate interaction with activated B cells.
The cytokines produced by the T cells are concentrated in
the small space (immunological synapse) between the two
interacting cells (35). Thus B cells might be stimulated with
high concentration of IL-4 (10,000 U/ml) in vivo. Alternatively,
100
1000
10000
IL - 4 ( U/ml )
Fig. 5. Stimulation of lgG1 and IgE production from B cells by antiCD40 antibody and IL-4 with or without anti-Fas antibody. Splenic B
cells cultured at 105/0.2 ml/well with anti-CD40 antibody (2 ng/ml) for
24 h were additionally stimulated with serially diluted IL-4 in the
presence or absence of anti-Fas antibody. Concentrations of Igs in
triplicate cultures were measured on day 7 of culture after addition
of IL-4 by ELISA.
some other cytokines derived from CD4 + cells might synergize
with IL-4 and/or lower threshold of IL-4 signaling.
Discussion
Studies with a variety of Ig transgenic mice elucidated the
mechanisms involved in induction of B cell tolerance at early
B cell development: clonal anergy and clonal deletion (3638). B cell anergy is induced by secreted antigens that can
only cause limited cross-linking of slg receptor, while B cells
developing in the marrow are deleted by contact with those
self antigens that are able to extensively cross-link slg receptors, suggesting that self antigen structure itself plays a key
role in determining the cellular mechanism of tolerance (36).
Several studies using immature B cells and transformed cell
lines also demonstrated that ligation of the slg receptor at an
early stage of B cell development results in deletion by
apoptosis in vitro (39-42).
It was demonstrated in vivo that interaction of anti-lgD with
slgD receptor on B cells in the absence of T cell help leads
to B cell apoptosis (43), suggesting that T cells also play a
key role in determining the cellular response to antigen
binding. Indeed, treatment with anti-CD40 antibody prevented
anti-lgM-induced apoptosis in the transformed cell line WEHI231 (42). Treatment with IL-4 abrogated the capacity of antiIgM to induce a striking reduction in in vitro growth of cloned
murine B lymphoma line (BCL r CL-3) (44). This anti-lgMinduced reduction in in vitro growth of this B lymphoma cells
was due to cell apoptosis (our unpublished observation).
Furthermore, hypercross-linking of slgM or slgD receptors on
mature B cells in vitro induced apoptosis that is reversible
by co-stimulation with anti-CD40 antibody and IL-4 (45),
796
IL-4 protects against Fas-mediated B cell apoptosis
suggesting that T cells prevent slg cross-linked B cells from
B cell apoptosis by stimulation with CD40L and IL-4.
Rothstein era/, have shown another type of B cell apoptosis,
i.e. Fas-dependent killing of CD40L-activated B cells by T h 1type T cells (24). TCR engagement induces both CD40L and
FasL on Th1 cells (19,46,47). Thus, activated Th1 cells could
induce Fas by stimulation of B cells with CD40L and induce
B cell apoptosis by further ligation of this with FasL. B cells
stimulated with lipopolysaccharide also express Fas and are
sensitive to the killing action of CD4 + T cells (24,26). In
contrast, there is no evidence that activated Th2 cells which
express CD40L and IL-4 induce B cell apoptosis.
There are several possible mechanisms through which Th2
cells induce activated B cells to grow and differentiate without
inducing cell apoptosis. First, activated Th2 cells express
CD40L but not FasL. Indeed, Th2 cells were shown to express
no or low levels of FasL (19,47), although some activated Th2
cells clearly express FasL (19). Second, Th2 cells produce
some cytokines that inhibit the action of FasL on CD40Lactivated B cells. Our present results support this possibility,
because IL-4 abrogates the capacity of anti-Fas to induce
DNA fragmentation in anti-CD40-stimulated B cells (Fig. 5
and Table 1). Third, cytokines from Th2 cells diminish Fas
expression on activated B cells. To our surprise, however, IL4 rather enhanced Fas expression on anti-CD40-stimulated
B cells (Figs 3 and 4). We might need further study to examine
such a possibility of other cytokines such as IL-10.
This novel mechanism for protection of CD40-activated B
cells from Fas-mediated death by IL-4 appears unique among
the mechanisms of determination of immune response. Infections with intracellular microbes tend to induce the differentiation of naive T cells into the T h 1, which promotes phagocytic
elimination of these microbes by IFNy-dependent activation
of phagocytic cells (27,35). Th2 cells, on the other hand,
produce IL-4, IL-5, IL-10 and IL-13, which together with IL-4
suppress cell-mediated immunity (27,35). Since Th1 cells
induce tissue damage by production of IFNyand possibly by
expression of FasL, Th2 cells may also act to regulate the
potential tissue damaging effects of responses induced by
FasL. Thus, the present result may help explain the reciprocal
relationship between cell-mediated immunity and humoral
immunity.
A recent study has revealed that clonal deletion of autoreactive B cells is a Fas-dependent elimination of anergic B cells
upon interaction with CD4 + T cells (48), suggesting that clonal
deletion is determined by the ability of anergic B cells to
express Fas and to induce cytotoxic CD4 + T cells. Anergic
B cells might lack molecules that trigger T cell cytokines such
as IL-4. Alternatively these B cells might produce IL-12 that
induces Th1 cells. Another intriguing possibility is that properly
activated B cells produce IL-10, while anergic B cells cannot
produce IL-10 which inhibits IL-12 production from macrophages (49). We also studied the action of anti-IgM on Fasmediated B cell apoptosis. Consistent with the result of
Rothstein et al. (24), we could demonstrate that B cells
stimulated with anti-IgM expressed Fas meagerly (Fig. 2c)
and such stimulated B cells could proliferate even in the
presence of anti-Fas antibody (Fig. 1 a). Furthermore, we could
demonstrate that treatment with anti-IgM partially protected
against anti-Fas-mediated B cell growth inhibition (Fig. 1c
versus d). However, B cells that had been chronically exposed
to autoantigen carried desensitized slg (48). Thus, their
antigen receptor engagement failed to transduce slg signaling
that may protect against Fas-dependent Th1-mediated
apoptosis. Therefore, both slg signal and IL-4 are required
for expansion and differentiation of antigen-stimulated B cells
in the presence of Th1 cells.
It has been shown recently that Fas ligation induced DNA
fragmentation in CD40-activated human B lymphocytes even
in the presence of IL-4 (50 U/ml) (50). Furthermore, Fas
ligation inhibits cytokine (IL-4 or IL-10 plus IL-2)-dependent
Ig secretion of CD40-activated B cells (50). However, here we
have demonstrated that IL-4 protects against Fas-mediated B
cell apoptosis. Furthermore, we have shown that IL-4 and
anti-CD40 induce B cells to develop into lgG1 and IgE
producing cells in the presence of anti-Fas. Therefore their
result and ours appear to be entirely opposite. We used
murine B cells stimulated with anti-CD40. In contrast, they
used human B cells stimulated with CD40L. We stimulated B
cells with a high concentration of IL-4 (5000-10,000 U/ml),
while they stimulated with a low concentration of IL-4 (50 U/
ml). Thus, at the present time we can only suggest that the
major explanation that accounts for this discordance is that
we used a high concentration of IL-4 and murine B cells.
Thus, in conclusion, we have identified IL-4 as a factor that
protects against Fas-mediated B cell apoptosis. This effect
of IL-4 may help explain the reciprocal relationship between
antibody production and cell-mediated immunity. Furthermore, this effect of IL-4 may explain one possible mechanism
of autoimmunity by inhibiting Fas-dependent elimination of
anergic B cells upon interaction with Th1 cells.
Acknowledgement
This work was supported in part by the Osaka Foundation for
Promotion of Clinical Immunology.
Abbreviations
CD40L
FasL
Ipr/lpr
PE
PI
TNF
CD40 ligand
Fas ligand
MRL Iprllpr
phycoerythrin
propidium iodide
tumor necrosis factor
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