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B Activation and IL-10 Production
* Department of Immunology, National Jewish Medical and Research Center, University of Colorado Health Sciences Center, Denver, CO 80206; and
Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, People’s Republic of China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B
activation and IL-10 production. Taken together, our findings suggest
that TRAF3 is a negative regulator of BAFF-R-mediated NF-B
activation and IL-10 production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-10 (3, 6). Like most members of the TNF family, the
extracellular domain of TALL-1 can be cleaved to form a soluble
cytokine (2). Crystal structure studies suggest that
soluble TALL-1
(sTALL-1)3 contains an
unique "flap" region that is important for its virus-like assembly,
receptor binding, and biological activities (7, 8, 9). TALL-1 signals through three receptors, including BCMA, TACI, and BAFF-R, which are members of the TNFR family (5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). All three receptors are mainly expressed by B lymphocytes, while TACI is also induced in a subset of T cells following their activation (17, 18, 19, 20, 21, 22, 23, 24). Early studies suggest that sTALL-1 can potently stimulate B lymphocyte proliferation in vitro, either alone or in synergy with anti-IgM (2, 3, 10, 11, 12, 13). It is now believed that sTALL-1 promotes either differentiation of B cells or survival of mature B cells (25, 26, 27, 28). Administration of recombinant sTALL-1 or overexpression of sTALL-1 in mice leads to increased numbers of mature B lymphocytes, splenomegaly, anti-DNA Abs, proteinuria, and glomerulonephritis, phenotypes that mimic those of systemic lupus erythemia (3, 5, 14, 29, 30). Conversely, it has been shown that recombinant soluble TACI-Ig fusion proteins can significantly inhibit progression of lupus-like autoimmune syndrome and collagen-induced arthritis in animal models (27, 31). Recently, gene knockout studies further confirmed that TALL-1 is required for normal B cell development (26). Surprisingly, gene inactivation studies also indicated that BAFF-R, but not TACI and BCMA, is required for TALL-1-triggered B cell development (26, 32, 33, 34). Taken together, these studies suggest that the TALL-1/BAFF-R signaling plays critical roles in regulation of B cell function and autoimmune diseases.
The signal transduction pathways triggered by BCMA, TACI, and BAFF-R
are poorly characterized. Like many other members of the TNFR family,
BCMA and TACI can bind to TNFR-associated factor (TRAF) proteins and
activate the transcription factor NF-B and the serine/threonine
protein kinase JNK (10, 11, 12, 13, 16, 35). In addition, it has
also been shown that TACI can activate the transcription factor NF-AT
(36).
The intracellular signaling pathways and downstream effectors triggered
by BAFF-R are not known. In this study, we identified TRAF3 as a
BAFF-R-associated protein. Our findings suggest that TRAF3 negatively
regulates BAFF-R-mediated NF-B activation and IL-10 production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mammalian-derived recombinant Flag-tagged human sTALL-1 (10) and Escherichia coli-derived recombinant His6-tagged human sTALL-1 (7) were previously described. The human embryonic kidney 293 (Dr. Z. Cao, Tularik, South San Francisco, CA), the B lymphoma Bjab (Dr. J. Hagman, National Jewish Medical and Research Center, Denver, CO) and RPMI 8226 (American Type Culture Collection (ATCC), Manassas, VA) cells, the mAbs against Flag (Sigma-Aldrich, St. Louis, MO) and hemagglutinin (HA) (Covance, Berkeley, CA) epitopes, and the rabbit polyclonal anti-TRAF3 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) were obtained from the indicated sources.
Bjab and RPMI 8226 cells were cultured in RPMI 1640 medium containing 10% FBS. 293 cells were maintained in high glucose DMEM containing 10% FCS.
Yeast two-hybrid screening
To construct a BAFF-R bait vector, a cDNA fragment encoding for aa 97–184 of BAFF-R was inserted in-frame into the Gal4 DNA-binding domain vector pGBT (Clontech Laboratories, Palo Alto, CA). The human B cell cDNA library (ATCC) was screened as described (37, 38).
Coimmunoprecipitation and Western blot
293 cells (5 x 106) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were lysed in 1 ml lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF). For each immunoprecipitation, 0.4 ml aliquot of lysates was incubated with 0.5 µg of the indicated mAb or control mouse IgG, and 25 µl of a 1:1 slurry of GammaBind G Plus Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) for at least 1 h. The Sepharose beads were washed three times with 1 ml lysis buffer containing 500 mM NaCl. The precipitates were fractionated on SDS-PAGE and subsequent Western blot analyses were performed as described (37, 38).
Vectors
To construct C-terminal HA-tagged expression plasmids for BCMA, TACI, BAFF-R, and BAFF-R mutants, cDNAs for these proteins were amplified by PCR from a B cell cDNA library and inserted into a C-terminal HA-tagged pcDNA3 plasmid (39).
To make retroviral vectors for TRAF3 and its TRAF domain (aa 258–568)
mutant (TRAF3-C), cDNAs encoding for full-length TRAF3 and aa 258–568
were amplified by PCR and inserted into the MSCV2.2IRES-GFP
retroviral vector (provided by Dr. P. Marrack, National Jewish Medical
and Research Center).
Expression plasmids for TRAF1, 2, 3, 5, 6 (Dr. D. Goeddel, Tularik) and
NF-B-luciferase reporter plasmid (Dr. G. Johnson, University of
Colorado Health Sciences Center, Denver, CO) were provided by
the indicated investigators.
Establishment of BCMA, TACI, and BAFF-R stable cell lines
Expression plasmids for C-terminal HA-tagged BCMA, TACI, and BAFF-R were linearized and transfected into RPMI 8226 cells or Bjab cells by electroporation. The transfected cells were selected by G418 (1 mg/ml) for 2 wk. Cells with high-level receptor expression were sorted with Flag-tagged sTALL-1 by flow cytometry. Overexpression of the receptors in the stable lines were confirmed by Western blot with anti-HA Ab.
Establishment of TRAF3 and TRAF3-C-terminal stable cell lines
Retroviral plasmid (15 µg) for wild-type TRAF3 or TRAF3-C was
transfected into the packaging cell line 293–10A1 (
2 x
106) by calcium phosphate precipitation. Eighteen
hours after transfection, the cells were washed with PBS and cultured
in 5 ml of fresh medium for 24 h. The recombinant
retrovirus-containing medium was collected and centrifuged. The
supernatant, supplemented with 4 µg/ml of polybrene, was used to
infect RPMI 8226 or Bjab cells (2 x
106). Two days after infection, green fluorescent
protein-positive cells were isolated using a cell sorter.
Flow cytometry analysis
RPMI 8226 or Bjab cells were incubated in staining buffer (PBS/2% FBS) in the absence or presence of Flag-sTALL-1 (100 ng/ml) for 40 min. Cell staining was performed by sequential incubation (each 40 min) with anti-Flag mAb (1 µg/ml) and R-PE-conjugated goat anti-mouse IgG (1/200 dilution) in staining buffer. Cells were washed two times with staining buffer following each incubation. Cells with high-level Flag-sTALL-1 binding were isolated by a cell sorter.
Reporter gene assays
Bjab stable cell lines (2 x 105)
were seeded on 6-well (35-mm) dishes and were transfected with 1.0 µg
of NF-B-luciferase reporter plasmid by Lipofectamine 2000
(Invitrogen, Carlsbad, CA). To normalize for transfection efficiency
and protein amount, 0.5 µg of RSV-
-galactosidase plasmid
was added to all transfections. Fourteen hours after transfection,
cells were treated with His-sTALL-1 (200 ng/ml) or untreated for 6
h. Luciferase reporter assays were performed using a luciferase assay
kit (BD PharMingen, San Diego, CA) and following the manufacturer’s
protocols. -Galactosidase activity was measured using the
Galacto-Light chemiluminescent kit (Tropix, Medford, MA). Luciferase
activities were normalized on the basis of -galactosidase expression
levels.
ELISA
Human IL-10 ELISA were performed using the human IL-10 ELISA Ready-Set-Go kit (eBioscience, San Diego, CA) by following procedures recommended by the manufacturer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The intracellular signaling pathways mediated by BAFF-R are
unknown. Similar to other members of the TNFR family, BAFF-R does not
have intrinsic enzymatic activity and is believed to transduce signals
through physical interaction with downstream signaling proteins. To
identify BAFF-R-associated signaling proteins, we used the yeast
two-hybrid system to screen a human B cell cDNA library with the
intracellular domain of BAFF-R as bait. We screened a total of 5
million independent clones and obtained 78 -galactosidase-positive
clones. Sequence analysis of 20 clones indicated that most clones
encode a poorly characterized protein p14.5 (40).
Subsequent transient transfection and coimmunoprecipitation experiments
indicated that BAFF-R did not interact with p14.5 in 293 cells (data
not shown). One of the sequenced clones encodes for TRAF3.
Because TRAF proteins are involved in signaling by many members of the
TNFR family (41, 42, 43), we decided to investigate a
potential role for TRAF3 in BAFF-R signaling. To determine whether
TRAF3 is associated with BAFF-R in mammalian cells, expression plasmids
for C-terminal HA-tagged BAFF-R and N-terminal Flag-tagged TRAF3 were
transfected into 293 cells. Coimmunoprecipitation experiments indicated
that BAFF-R interacted with TRAF3 (Fig. 1
A). In the same experiments,
BAFF-R did not interact with other cytoplasmic TRAF proteins, including
TRAF1, TRAF2, TRAF5, and TRAF6 (Fig. 1A). TRAF4 was not
included in these experiments because it is primarily localized in the
nucleus in mammalian cells (41, 42, 43). These data suggest
that BAFF-R specially interacts with TRAF3.
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TRAF proteins interact with members of the TNFR family through
their TRAF domains (41, 42, 43). We determined whether the
TRAF domain of TRAF3 (TRAF3-C) is sufficient to interact with BAFF-R.
As shown in Fig. 1
B, transient transfection and
coimmunoprecipitation experiments suggest that the TRAF domain of TRAF3
is sufficient for interaction with BAFF-R.
Previously, conserved TRAF-binding motifs, such as (P/S/A/T)x(Q/E)E and
PxQxxD, have been identified in the cytoplasmic domains of some TNFR
family members (41, 42, 43, 44). However, these conserved
TRAF-binding motifs are not easily recognizable in the cytoplasmic
domain of BAFF-R. To determine the regions of BAFF-R that are required
for interaction with TRAF3, we constructed a series of C-terminal
HA-tagged deletion mutants of the BAFF-R (Fig. 2A). Transient transfection
and coimmunoprecipitation experiments suggest that six aa (PDGDKD) at
the membrane proximal region (aa 117–122) and the C-terminal tail (aa
150–184) are both required for interaction with TRAF3 (Fig. 2, A and B).
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Under physiological conditions, BAFF-R is specifically expressed in B lymphocytes. We next determined whether BAFF-R interacts with TRAF3 in B lymphoma cells and whether this interaction is affected by TALL-1 stimulation.
To do this, the Bjab lymphoma cells were stably transfected with
C-terminal HA-tagged BAFF-R, BCMA, and TACI. To determine whether
BAFF-R is associated with endogenous TRAF3 in these cells, we performed
coimmunoprecipitation experiments with anti-HA Ab and the
immunoprecipitates were analyzed by Western blots with anti-TRAF3
Ab. These experiments indicated that BAFF-R could weakly interact with
endogenous TRAF3 in Bjab cells (Fig. 3).
Moreover, sTALL-1 stimulation significantly enhanced the interaction
between BAFF-R and TRAF3. The weak interaction between BAFF-R and TRAF3
in the absence of sTALL-1 is probably due to the fact that
overexpression of BAFF-R mimics TALL-1 stimulation of endogenous
BAFF-R. It is possible that the association of TRAF3 with BAFF-R is
dependent on TALL-1 stimulation in untransfected cells. In these
experiments, TRAF3 was also recruited to TACI, but not to BCMA, in a
ligand-dependent manner (Fig. 3). These data are consistent with
previous observations that TRAF3 interacts with TACI, but not BCMA, in
mammalian overexpression systems (10, 13).
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B activation by TRAF3
Many TNFR family members, including TALL-1 receptors BCMA and
TACI, can activate the transcription factor NF-B. Previously, we
have shown that TALL-1 can induce NF-B activation in the B lymphoma
RPMI 8226 cells (39). To determine whether BAFF-R can
activate NF-B, we transfected a NF-B-luciferase reporter plasmid
into Bjab cells overexpressing BAFF-R. Luciferase assays indicated that
overexpression of BAFF-R could significantly induce NF-B activation,
and this effect was enhanced by sTALL-1 stimulation (Fig. 4). Moreover, overexpression of TRAF3 and
TRAF3-C both inhibited BAFF-R-mediated NF-B activation (Fig. 4).
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Previously, we showed that TALL-1 stimulation or overexpression of
BAFF-R in RPMI 8226 cells induced IL-10 production (39).
To determine whether TRAF3 is involved in BAFF-R-induced IL-10
production, we transfected TRAF3 or its TRAF domain into
BAFF-R-overexpressing RPMI 8226 cells by retroviral-mediated gene
transfer and measured IL-10 levels by ELISA. The results indicated that
both wild-type TRAF3 and TRAF3-C inhibited BAFF-R-induced IL-10
production (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the three TALL-1 receptors, BCMA and TACI are not important for B cell development because normal B cell maturation is found in BCMA- and TACI-deficient mice (32, 33, 34). In contrast, inactivation of BAFF-R in mice causes loss of T2 and mature B cells (26). These studies suggest that BAFF-R signaling is essential for TALL-1-triggered B cell survival and maturation.
The downstream effector molecules responsible for
TALL-1/BAFF-R-triggered B cell survival and maturation are not clear.
It is possible that TALL-1 signaling initiates the anti-apoptotic
activity that is associated with the activation of NF-B. Previously,
it has been shown that disruption of NF-B activation pathways, such
as by gene knockout of IKK, Rel, and RelA, impairs B cell
maturation (45, 46, 47). In this context, we found in this
study that BAFF-R could also activate NF-B.
Both BCMA and TACI can activate NF-B but are not important for
TALL-1-triggered B cell survival, suggesting that activation of NF-B
is not sufficient for TALL-1-triggered B cell survival. Recently, we
identified multiple downstream genes transcriptionally induced by
TALL-1, including the cytokine IL-10, the chemokine LAG-1, and GCP-2,
the secreted protein pre-B cell colony enhancing factor, among others
(39). Among the genes induced by TALL-1, IL-10 is
particularly interesting. It has been shown that IL-10 can suppress
cytokine production and several accessory cell functions by Th1 cells,
macrophages, and NK cells and is regarded as a potent suppressor of the
effector functions of these cells (48). Conversely, IL-10
is a potent stimulator of B cell proliferation and differentiation and
is critically involved in regulating autoantibody-secreting B cell
activities in systemic lupus erythemia (49, 50). Our
previous studies suggest that BAFF-R, but not TACI and BCMA, can
dramatically up-regulate IL-10 expression in primary and transformed B
cells (39), pointing to the possibility that IL-10 is an
important effector molecule for TALL-1-triggered B cell survival and
maturation.
In an attempt to decipher the intracellular signaling pathways mediated
by BAFF-R, we identified TRAF3 as a cytoplasmic protein physically
binding to the cytoplasmic domain of BAFF-R. TRAF3 is a member of the
TRAF protein family, which is involved in signaling by many members of
the TNFR family (41, 42, 43). TRAF3 was first identified as a
molecule that binds to the cytoplasmic tails of CD40 and
EBV-transforming protein LMP1 (51, 52, 53, 54). Signaling through
CD40 in B cells causes rescue from apoptosis, proliferation,
differentiation, Ig production, class switching, and expression of
costimulatory molecules. Overexpression of TRAF3 inhibits CD40-mediated
Ab secretion (54). The TRAF domain of TRAF3 is sufficient
to bind to CD40 and also inhibits CD40-mediated Ab secretion when
overexpressed, suggesting that the physical association of TRAF3 with
CD40 mediates its negative regulatory function (55). TRAF3
is also recruited in a ligand-dependent manner to lymphotoxin-
receptor (LTR) and has an inhibitory effect on LTR-mediated
NF-B activation (56, 57). In this context, it is
interesting that both TRAF3 and its TRAF domain inhibit BAFF-R-mediated
downstream effects, such as NF-B activation and IL-10 production.
Taken together, these studies suggest that TRAF3 is involved in
negative regulation of signaling by several TNFR family members,
including BAFF-R, CD40, and LTR.
The cytoplasmic domain of BAFF-R is not conserved with those of other
TNFR family members. The major conserved TRAF-binding motif,
(P/S/A/T)x(Q/E)E (44), is not easily recognizable in the
cytoplasmic domain of BAFF-R. A 6-aa sequence at position 117 of
BAFF-R, PDGDKD, is weakly similar to the minor consensus TRAF-binding
motif PxQxxD found in the EBV LMP1 protein and the TRAF-interacting
protein I-TRAF/TANK (44). Previously, it has been shown
that TRAF3 binds to LTR through amino acids PEEGDPG at position 389
of LTR (57). This site is also not found in BAFF-R. We
made a series of deletion mutants of BAFF-R and examined their
interaction with TRAF3 in coimmunoprecipitation experiments. These
studies suggest that the PDGDKD motif at position 117 and the
C-terminal 35 aa are both required for BAFF-R’s association with
TRAF3. The simplest explanation for this observation is that these
amino acids form a novel spatial structure for BAFF-R binding to TRAF3.
In this context, it has previously been shown by crystal structure
studies that binding of TRAF3, but not TRAF2, to CD40 is influenced by
both the PXQXT motif and residues distal to this site
(58, 59, 60). Alternatively, it is also possible that TRAF3
binds only to one site in the cytoplasm of BAFF-R in vivo and the
results from our mapping experiments are due to altered folds caused by
deletion.
In conclusion, we have identified TRAF3 as a BAFF-R-associated
signaling protein. TRAF3 can inhibit BAFF-R-mediated NF-B activation
and IL-10 production, suggesting a negative regulatory role for TRAF3
in TALL-1-triggered B cell survival and maturation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hong-Bing Shu, Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, K516c, Denver, CO 80206. E-mail address: shuh{at}njc.org
3 Abbreviations used in this paper: sTALL-1, soluble TALL-1; T2, transitional type II; LTR, lymphotoxin- receptor; TRAF, TNFR-associated factor; HA, hemagglutinin.
Received for publication July 12, 2002. Accepted for publication October 11, 2002.
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B kinase is essential for mature B cell development and function. J. Exp. Med. 193:417.B and putative signal transducer for the lymphotoxin- receptor. J. Biol. Chem. 271:14661. receptor for tumor necrosis factor receptor-associated factor binding, subcellular localization, and activation of cell death and NF-B pathways. J. Biol. Chem. 275:11121.B and Jun N-terminal kinase activation. J. Biol. Chem. 274:22414.This article has been cited by other articles:
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) or left untreated (
) for
6 h. Luciferase activities were measured and normalized based on
