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Harold C. Simmons Arthritis Research Center and Departments of Internal Medicine,
Pharmacology, and
Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B
activation, the outcomes of TRAF3-initiated signaling are less
characterized. To delineate CD40-induced TRAF3-dependent events, Ramos
B cells stably transfected with a dominant negative TRAF3 were
stimulated with membranes expressing recombinant CD154/CD40 ligand. In
the absence of TRAF3 signaling, activation of p38 and control of Ig
production were abrogated, whereas Jun N-terminal kinase activation and
secretion of IL-10, lymphotoxin-
, and TNF- were partially
blocked. By contrast, induction of apoptosis, activation of NF-B,
generation of granulocyte-macrophage CSF, and up-regulation of CD54,
MHC class II, and CD95 were unaffected by the TRAF3 dominant negative.
Together, these results indicate that TRAF3 initiates independent
signaling pathways via p38 and JNK that are associated with specific
functional outcomes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early biochemical events following CD40 engagement include activation
of src family tyrosine kinases as well as PLC-
2,
JAK3-STAT3, PI3K, and STAT6 (13, 14, 15, 16). Since the cytoplasmic domain of
CD40 lacks intrinsic enzymatic activity, it has been hypothesized that
these early biochemical signals as well as the subsequent activation of
kinases such as MKK13 (17),
ERK (18, 19), JNK (19, 20, 21), and p38 (22, 23), and the nuclear
translocation of transcription factors AP-1, NF-AT (24), and NF-B
(24, 25) induced by CD40 engagement may be mediated by adaptor
molecules. In this regard, a family of TNF receptor-associated factor
(TRAF) molecules that is induced to associate with members of the TNF
receptor superfamily, including CD40 (26), upon engagement has been
characterized. Whereas the C-terminal portions of TRAF2, -3, and -5
associate with overlapping sites contained in residues 226 to 249
(27, 28, 29, 30, 31) of the cytoplasmic tail of CD40, the C-terminal portion of
TRAF6 associates with the more membrane-proximal residues 210 to 225 of
CD40 (32, 33).
The signaling portion of TRAF2, -3, -5, and -6 has been found to be
contained in the N-terminal ring and zinc fingers. Whereas TRAF2 (27),
TRAF5 (28), and TRAF6 (32, 33) have been shown to mediate activation of
NF-B following engagement of CD40, the downstream transducers
involved in TRAF3 signaling are unknown (29, 30, 31). These considerations
prompted an examination of the functional consequences of
TRAF3-mediated signaling following engagement of CD40 on human B cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Ramos Burkitt lymphoma R-F6 and R-D4 cell lines were stably transfected with the control vector pEBVHis/lacZ or a DN mutant of TRAF3, pEBVHis/C26, respectively (29) (gifts from Dr. Seth Lederman, Columbia University, New York, NY). The TRAF3 DN (C26) contains C-terminal TRAF-C and TRAF-N domains, but lacks the N-terminal leucine zippers as well as the ring and zinc fingers. R-F6 and R-D4 cell lines were maintained in hygromycin (Calbiochem, La Jolla, CA)/DMEM (Life Technologies, Grand Island, NY) selection medium supplemented with penicillin G (200 U/ml), gentamicin (10 µg/ml), L-glutamine (0.3 mg/ml), and 10% FCS (Life Technologies).
Culture conditions
Ramos cells were cultured in U-bottom 96-well microtiter plates (1 x 105/well; Costar, Cambridge, MA). Where indicated, Ramos cells were stimulated with 400 µM sorbitol (Sigma, St. Louis, MO). In some cases, the cells were activated with 10 µg/ml mouse IgG1 anti-human CD40 mAb (G28.5, American Type Culture Collection, Manassas, VA; or 626.1, gift from Dr. Shu Man Fu, University of Virginia, Charlottesville, VA) or an isotype-matched control mAb (MOPC; American Type Culture Collection) in the presence or the absence of cross-linking with immobilized goat anti-mouse Ig (GaMIg). GaMIg (4 ng/well) was immobilized on plates by incubation in Tris buffer for 2 h at 37°C before washing extensively with PBS. Alternatively, Ramos cells were incubated with membranes from Sf9 cells infected with wt AcMNPV or recombinant baculovirus encoding mCD154/CD40L prepared as previously described (5) in the presence or the absence of 5 to 10 µg/ml of either MR1, a hamster anti-mouse CD154 mAb (gift from Dr. Randolph Noelle, Dartmouth Medical School, Lebanon, NH), or 2C11, a control hamster anti-murine CD3 mAb (American Type Culture Collection) that has no reactivity with human lymphocytes, to demonstrate specificity.
The degree of CD40 engagement on Ramos B cells by a given amount of
mCD154-expressing membranes was determined by a competitive binding
assay using a mCD154-CD8 construct (5) (gift from Peter Lane, Basel
Institute for Immunology, Basel, Switzerland). Specifically, 100
x 103 B cells were incubated with increasing volumes of
membranes from either wt or mCD40L-Sf9 cells for 2 h at 37°C.
Cells were washed to remove unbound membranes before incubation with a
nonsaturating amount of mCD40L-CD8 (5 µl of a 1/10 dilution) for
30 min at 4°C. After washing to remove unbound construct, cells were
incubated with a 1/20 dilution of anti-CD8-FITC (YTS 169.4;
American Type Culture Collection) for 30 min at 4°C. Before analysis
with the use of the FACScan system, cells were washed and resuspended
in PBS with 2% normal human serum. Live cells were gated using
propidium iodide staining (Sigma).
Inhibitors
SB203580 (10 µM; Calbiochem) was used to inhibit activation of
the and ß isoforms of p38 expressed in lymphocytes (34, 35, 36, 37, 38).
Cyclosporine (100 ng/ml; Sandoz, East Hanover, NJ), N-acetyl
cysteine (100 mM; Sigma Chemical Co.), and zVAD-fluoro methyl ketone
(20 µM; Enzyme Systems Products, Livermore, CA) were added to
cultures to inhibit activation of NF-AT, NF-B, and caspase,
respectively.
Analysis of TRAF3 protein
Control (R-F6) or TRAF3 DN (R-D4)-expressing Ramos cells were
washed once with PBS and lysed for 10 min in cold buffer (20 mM HEPES
(pH 8), 1 mM EDTA, 1 mM DTT, 10 mg/ml leupeptin, 10 mg/ml limabean
extract, and 16 µg/ml each of PMSF,
L-p-tosylamino-2-phenylethyl chloromethyl
ketone, and TN--p-tosyl-L-lysine
chloromethyl ketone) before homogenization using a 27.5-gauge needle.
Lysates were centrifuged at 3000 x g for 2 min to
remove nuclei before concentration using Microcon 10 columns (Amicon,
Beverly, MA) following the manufacturer’s instructions. The protein
concentration was determined using the DC protein assay (Bio-Rad,
Hercules, CA) before analysis by SDS-PAGE and transfer to
polyvinylidene difluoride membranes (Millipore, Bedford, MA). Blots
were blocked for 1 h at room temperature in Tris-buffered saline,
pH 7.6, with 0.5% Tween-20, 5% nonfat dried milk, and 5% BSA before
incubation for 2 h with 1 µg/ml of affinity-purified
biotinylated polyclonal rabbit anti-human TRAF3 (26) (gift from Dr.
Randolph Noelle) directed against the C-terminus or a 1/250 dilution of
a rabbit polyclonal anti-TRAF3 (gift from Dr. Marilyn Kehry,
Boehringer Ingelheim, Ridgefield, CT) directed against the C-terminus.
Following extensive washing in Tris-buffered saline with 0.2% Tween-20
and 0.2% BSA, blots were incubated for 2 h at room temperature
with either a 1/2000 dilution of strepavidin-horseradish peroxidase
(Vector, Burlingame, CA) or a 1/3000 dilution of goat anti-rabbit
horseradish peroxidase (Bio-Rad). After extensive washing as described
above, blots were developed using ECL reagents (Amersham, Arlington
Heights, IL) and analyzed by autoradiography. The densities of the
resulting bands were digitized, quantitated, and expressed as a fold
increase over background.
Analysis of p38 and JNK
Following stimulation, cytoplasmic lysates were analyzed for p38
and JNK by Western analysis using a mixture of anti-p38 polyclonal
rabbit antisera (P287, gift from Dr. Melanie Cobb, University of Texas
Southwestern Medical Center, Dallas, TX; C-20, Santa Cruz
Biotechnology, Santa Cruz, CA) or anti-JNK polyclonal rabbit
antisera (0977, gift from Dr. Cobb; C-17, Santa Cruz Biotechnology) and
for specific kinase activity using GST-ATF2
1–254 and
GST-c-jun1–221 (gifts from Dr. Cobb) as
respective substrates.
Specifically, cells were centrifuged, washed once with PBS, and lysed
in cold buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 0.2 mM
MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 100 mM
NaF, 0.2 mM sodium orthovanadate, and 1% PMSF). Lysates were
centrifuged for 10 min at 14,000 rpm at 4°C, supernatants were
transferred to fresh Eppendorf tubes, and protein content was
quantitated using the Bio-Rad protein assay. Following
immunoprecipitation for 2 h at 4°C with appropriate Ab and
protein A-Sepharose (Pharmacia, Piscataway, NJ), immunoprecipitates
were centrifuged for 5 min at 6000 rpm and washed twice with lysis
buffer and once with 50 mM Tris (pH 7.4), 1 mM DTT, and 10 mM
MgCl2. Kinase reactions were conducted for 40 min at 30°C
in this same buffer with the addition of 50 µM ATP, 10 µCi
[-32P]ATP/sample, and 0.3 mg/ml of the appropriate
substrate. Reactions were terminated by centrifugation, addition of
sample buffer, and boiling for 5 min. Proteins were resolved on a 10%
SDS-polyacrylamide gel and visualized by Coomassie blue staining.
Following destaining in MeOH/H2O/acetic acid, the gel was
dried and analyzed by autoradiography. Kinase activity was quantitated
by liquid scintillation spectroscopy of appropriate bands.
Analysis of B cell function
After activation for 4 h at 37°C and permeabilization with Triton X-100 (Sigma), cells were stained with propidium iodide to detect DNA strand breaks and were analyzed for percent apoptosis by flow cytometry using the FACScan with CellQuest Software (Becton Dickinson, San Jose, CA). Alternatively, apoptosis was detected by [3H]TdR release. Cells were labeled for 12 h at 37°C with 1 µCi of [3H]TdR (6.7 Ci/mM; ICN Biomedicals, Irvine, CA) in the presence of 50 µg of fluorouridine deoxyribose (Sigma) and then were washed, counted, and incubated at 37°C under various conditions. [3H]TdR release was determined by liquid scintillation spectroscopy.
Proliferation was analyzed by [3H]thymidine incorporation as previously described (39). Secreted Ig was analyzed by ELISA as previously described (39), and cytoplasmic IgM content was analyzed by intracellular staining. To analyze cytoplasmic IgM, cells were preactivated with CD154 for 1 h at 37°C, after which 500 ng/ml brefeldin A (Sigma) was added, and the cells were incubated for 5 h. Cells were harvested, washed with PBS, fixed with 4% paraformaldehyde, and washed again with 0.1% BSA/PBS before being resuspended in 10% DMSO/1% BSA/PBS and frozen for 12 h at -80°C. After incubation at 37°C for 5 min, cells were washed with PBS and incubated for 10 min in the dark with in the presence or the absence of permeabilization reagents (Becton Dickinson). Cells (2.6 x 105) were washed with 0.1% BSA/0.1% NaN3/PBS, blocked for 10 min with 10% mouse and rat sera in 0.1% BSA/0.1% NaN3/PBS at room temperature, and incubated with rabbit anti-human IgM-FITC (Dako, Carpinteria, CA). As a control, cells were incubated with rabbit anti-human IgA-FITC (Dako) that has no reactivity with IgM+ Ramos B cells (data not shown). Cells were washed and resuspended in 1% paraformaldehyde before analysis using the FACScan (Becton Dickinson).
Secretion of GM-CSF, LT-/TNF-ß, TGF-ß, IL-1, IL-4 (R&D Systems,
Minneapolis, MN), IL-6, IL-10, IL-12, IL-13, IFN-, and TNF-,
(Biosource International, Camarillo, CA) was analyzed by ELISA
according to the manufacturer’s instructions and normalized to the
number of cells present at supernatant harvesting.
EMSA analysis of NF-B activation
Nuclear extracts were incubated with a 32P-labeled
double-stranded DNA probe containing the sequence of the I
NF-B
binding site (5'-AGC TTC AGA GTG GGG TTC CCG AGA GG-3' and 5'-TC GAC
CTC TCG GGA ACC CCA CTC TGA-3') in the presence or the absence of a
10-fold excess of unlabeled competitor probe and analyzed for NF-B
binding activity following electrophoresis on a 4% native
polyacrylamide gel and autoradiography. The densities of the resulting
bands were digitized, quantitated, and expressed as the fold increase
over background.
Flow cytometric analysis
Cells were stained for surface Ags with mAb as previously
described (39). Anti-human IgM-FITC, IgG-FITC, IgA-FITC, and a control
FITC are mouse IgG1 F(ab')2 (Caltag Laboratories, South San
Francisco, CA). Anti-human CD19-FITC, CD20-FITC (Becton Dickinson, San
Jose, CA), CD30-FITC, CD10-FITC, CD38-FITC, and a control FITC (Caltag)
are mouse IgG1. Anti-CD21 (THB.5; American Type Culture Collection),
CD23 (MHM; Dako), CD39 (AC2; Biodesign, Kennebunk, ME), CD44 (A3D8;
American Type Culture Collection), CD70 (Dako), CD95 (DX2; PharMingen,
San Diego, CA), and HLA-DR/MHC class II (L243; American Type Culture
Collection) are mouse IgG1. Anti-CD11a/LFA1 (TS1/18.1; American Type
Culture Collection), CD18/LFA-1ß (TS1/22.1.1; American Type Culture
Collection), and CD54/ICAM-1 (R6.5; gift from Dr. Robert Rothlein,
Boehringer Ingelheim) are mouse IgG2a. Anti-CD77 (Biodesign) is rat
IgM. The isotype-matched control mAb were MOPC (IgG1; American Type
Culture Collection), P1.17 (IgG2a; American Type Culture Collection),
and rat IgM (Biodesign). Where required, the secondary stain was GaMIg
[F(ab')2]-FITC (Calbiochem).
Expression of LMP1 in control (R-F6) and TRAF3 DN (R-D4)-expressing Ramos cells was determined by intracellular staining. Cells (2 x 105) were washed with 1% BSA/0.1% NaN3/PBS at room temperature, blocked with 2% normal human serum for 15 min, and incubated for 10 min in the dark with FACS permeabilization solution (Becton Dickinson) before additional washing and blocking. Cells were stained with either 2 µg of mouse IgG1 anti-human LMP1 mAb (Dako) or the isotype-matched control MOPC, washed, and blocked before development with goat anti-mouse IgG1-FITC (The Binding Site, Birmingham, U.K.). Cells were washed and resuspended in 1% paraformaldehyde before analysis using the FACScan (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Furthermore, R-F6 and R-D4 express
equivalent levels of CD19, CD20, IgM, and HLA-DR and have comparable
surface phenotypes typical of Burkitt lymphoma cell lines (42), in that
they are positive for CD10, CD38, CD54, CD70, CD77, and CD95 (Fig. 1)
and are negative for IgG, IgA, CD11a, CD18, CD21, CD23, CD30, CD39, and
CD44 (data not shown). In addition, R-F6 and R-D4 constitutively
secrete similar amounts of Ig, GM-CSF, TNF-, and IL-6, but do not
secrete IL-1, IL-4, IL-10, IL-12, IL-13, IFN-, LT-, or TGF-ß1
(data not shown). Finally, Western analysis of TRAF3 protein
demonstrated equivalent expression of the 66-kDa wt protein in R-F6 and
R-D4 cell lines. Of importance, the 30-kDa TRAF3 DN protein was
expressed in R-D4 cells at a level approximately ninefold less than the
wt protein, but was not found in R-F6 cells (Fig. 2).
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B). In addition, the
DN mutant of TRAF3 selectively inhibited CD40-mediated p38 and JNK
activation as the pharmacologic agent, sorbitol, stimulated p38 and JNK
activity in both the control Ramos cells as well as those expressing
the DN version of TRAF3 (Fig. 3C).
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; data not shown).
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B (data not shown). Importantly,
CD40-induced apoptosis and inhibition of proliferation did not involve
a TRAF3-dependent signaling pathway, as they were unaffected by the
TRAF3 DN mutant (Figs. 4 and 5
). The increase in apoptosis as well as
the inhibition of DNA synthesis following CD40 engagement were similar
for both TRAF3 DN and control cells, even though the basal level of
proliferation differed.
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C). Of note, engagement of CD40 with anti-CD40 mAb also
decreased Ig secretion (Fig. 6B). In contrast to the effect
on apoptosis and proliferation, the decrease in Ig secretion induced by
engaging CD40 with mCD40L-expressing Sf9 membranes was dependent upon
TRAF3-mediated p38 activation, as it was abrogated by either the DN
TRAF3 construct or pharmacologic inhibitors of p38 (Fig. 6, A and C). Inhibition of Ig production caused by
an anti-CD40 mAb was also blunted by the DN TRAF3 (Fig. 6B). The dichotomy between the finding that
CD40-induced TRAF3-mediated signals regulated Ig secretion, but
not cellular growth, is supported by previous findings that B cell
growth and Ig production were independently regulated (4, 5, 12).
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, LT-, and
GM-CSF. Induction of cytokine production specifically related to CD40
engagement, as it was blocked with an anti-mCD154 mAb (Fig.
7). CD40-induced secretion of IL-10,
TNF-, LT-/TNF-ß, but not GM-CSF, involved TRAF3, as the DN
construct significantly inhibited production of the former cytokines,
but not that of the latter. Inhibition of cytokine secretion related to
the TRAF3 DN could not be ascribed to potential differences in the
number of cells, as the production of all cytokines was normalized to
the actual cell number at the end of the culture.
Finally, TRAF3 was not involved in CD40-mediated induction of nuclear
translocation of NF-B (Fig. 8A), as
previously shown (27, 28, 33), in that it was not blocked by expression
of the DN TRAF3 construct (Fig. 2). Moreover, TRAF3 appeared to play no
role in CD40-induced up-regulation of a variety of physiologically
relevant surface molecules that have been previously shown to be
regulated by NF-B (43, 44) including CD54, MHC class II, and CD95
(Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and LT-. These findings provide new
insight into the signaling pathway used by CD40 following
ligation-induced recruitment of TRAF3 to its cytoplasmic tail (26). The current results demonstrate that CD40 ligation on Ramos B cells induced TRAF3-mediated signaling uniquely linked to p38 activation and, in combination with other signaling pathways, coupled with JNK activation. Although these results are quite clear, and the cells are well characterized, it must be emphasized that the data were derived from a single cell line. Additional studies will be needed to determine whether other B cell lines or B cells at other stages of differentiation exhibit similar specific CD40 ligation response coupling.
Another important consideration is whether low level expression of
TRAF3 DN protein uniquely interfered with TRAF3 signaling. This is
likely to be the case for a number of reasons. First, TRAF2, -3, -5,
and -6 have closely approximated, but distinct, binding sites on the
cytoplasmic tail of CD40, with the binding sites for TRAF2, -3, and -5
clustered together (27, 28, 29, 30, 31) and the binding site for TRAF6 more
membrane proximal (32, 33). Secondly, CD40 engagement may initiate
several TRAF-mediated signaling pathways simultaneously and, therefore,
probably the recruitment of various TRAFs (33, 45, 46, 47, 48, 49). This is
confirmed by the observation that CD40 recruited TRAF2 and -3 (26) even
though they bind overlapping sites on its cytoplasmic tail (27, 28, 29, 30, 31, 32, 33).
Additionally, a transgenic mouse expressing a DN TRAF2 maintained a
degree of NF-B activation (48), implying that signaling through
TRAF5 and/or -6 was not inhibited and that cross-blocking of TRAFs did
not occur. Moreover, low level expression of TRAF3 DN protein did not
interfere with LMP1-mediated NF-B activation (50) that depends in
part upon signaling mediated by TRAF2, -5, or -6 (27, 28, 32, 33).
Similarly, in the current study, CD40-mediated activation of NF-B
and stimulation of NF-B-dependent functional events were not
inhibited by the TRAF3 DN construct (Figs. 7 and 8), indicating that
the linkage of TRAF2, -5, and/or -6 to CD40 was not blocked by low
level expression of TRAF3 DN protein. Of note, the low expression of
TRAF3 DN protein may have contributed to its specificity. Whereas
overexpression of DN or wt TRAF3 protein interfered with LMP1- or
CD40-induced NF-B activation (27, 28, 51, 52), a signaling event
previously shown to be TRAF3 independent (27, 28, 53), low level
expression of TRAF3 DN protein appeared to inhibit TRAF3-mediated
pathways specifically (
Figs. 3–8) (50). These findings are surprising
but may have to do with competing avidities of TRAFs for TNF receptor
family members as well as for cytoplasmic proteins such as
I-TRAF/TANK (54, 55) and other downstream effector molecules.
Thus, it is possible that the avidity of the TRAF3 DN protein for CD40
may be increased over that of the wt protein in the absence of the
N-terminal that interacts with downstream effector molecules. Together,
these observations suggest that low level expression of TRAF3 DN
protein inhibits TRAF3-mediated signaling pathways specifically.
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). In conjunction, these results demonstrate
that there is a difference in the degree of TRAF3 involvement linking
CD40 to these specific mitogen-activated protein kinases.
Regulation of p38 and JNK involves potentially overlapping as well as
selective upstream signaling cascades. MKK4 has been found to activate
both p38 and JNK in vitro (56), but only JNK in vivo (57). Moreover, in
vitro studies demonstrate that MKK3 (58, 59) and MKK6 (60, 61, 62, 63, 64, 65)
specifically activate p38, whereas MKK7/SKK4 specifically activates JNK
(66, 67, 68). These findings are consistent with the current findings that
TRAF3 is uniquely involved in p38 activation, but that JNK can be
activated by both TRAF3-dependent and -independent mechanisms. In this
regard, overexpression of TRAF2, -5, or -6 induces JNK activity
(45, 46, 47, 48, 49), and for TRAF2 this involves the MAPK/ERK kinase kinase
(MEKK-1)-MKK4 pathway (45). The involvement of TRAF2 in JNK
activation is further emphasized by the observation that its
stimulation following CD40 engagement on purified B cells from mice
expressing a DN TRAF2 protein is impaired compared with that in
controls (48). Besides a role for TRAF2, -5, and -6 in JNK activation,
TRAF3 is also involved, as indicated by the current studies (Fig. 3)
and by the finding that overexpression of TRAF3 was found to induce
activation of JNK, albeit to a lesser degree than that of TRAF2, -5, or
-6 (45, 49).
The current results suggest that CD40 engagement may initiate several
TRAF-mediated signaling pathways simultaneously and are consistent with
previous studies showing that immunoprecipitation of CD40 following
receptor engagement revealed recruitment of both TRAF2 and -3 (26) even
though they bind overlapping sites on the cytoplasmic tail of CD40
(27, 28, 29, 30, 31). Previous reports also support the conclusion that a single
TRAF may initiate several distinct independent signaling pathways. For
example, TRAF2 has been shown to induce both JNK and NF-B
activation. Since a DN JNK did not interfere with TRAF2-induced nuclear
translocation of NF-B, it is likely that independent signaling
pathways were activated (45, 46, 47, 48, 49). Moreover, TRAF6 has been shown to
induce both NF-B and ERK activation (33). The current data suggest
that, like TRAF2 and -6, TRAF3 may initiate distinct signaling pathways
leading to JNK and p38 activation. Finally, the low level of TRAF3 DN
protein stably expressed in the Ramos cells used in this study (Fig. 2)
would not be anticipated to interfere with docking of TRAF2, -5, or -6
to CD40 and subsequent initiation of downstream signaling events. In
this regard, TRAF3 DN did not interfere with CD40-induced NF-B
activation (Fig. 8), which depends in part on recruitment of these
adaptor molecules to CD40 (26).
JNK and p38 have overlapping and disparate substrate specificities, in that they both phosphorylate ATF-2, but only JNK phosphorylates Jun. Whereas ATF-2 homodimers and ATF-2/Jun heterodimers bind CRE promoter sites, Jun homodimers and Fos/Jun heterodimers associate with AP-1 binding sites (69, 70). Importantly, CD40 engagement has been shown to induce ERK (18, 19, 33), JNK (19, 20, 21), and p38 (22, 23) signaling pathways, leading to induction of Fos transcription as well as phosphorylation of Jun and ATF-2. In conjunction with the current data, these results suggest that CD40-induced, TRAF3-mediated activation of JNK and p38 may play a role in transcriptional regulation of genes controlled by AP-1 and/or CREB binding sites.
The current data also demonstrate that CD40-induced TRAF3-mediated
signaling is required for regulation of Ig secretion independent of the
control of proliferation and apoptosis (
Figs. 4–6) (4, 5, 12). The
data in the current study focused on inhibition of Ig production by
direct engagement of CD40 with recombinant CD154. However, Ramos cells
can be induced to secrete more Ig following stimulation with polyclonal
activators such as formalinized Staphylococcus aureus Cowan
I (data not shown) that have been shown previously to increase Ig
production by inducing low level expression of CD154 by B cells, with
subsequent homotypic CD40L-CD40 interactions enhancing Ig secretion
(39, 71). The increase in Ig secretion is dependent upon expression of
CD154 by Ramos cells, as it is blocked by a CD40-Ig construct or by mAb
to CD154. Of importance, S. aureus Cowan I does not increase
Ig production by Ramos cells expressing the TRAF3 DN, even though CD154
is up-regulated normally (data not shown). These findings imply that
CD40-mediated up- and down-regulation of Ig production are both
dependent on TRAF3-mediated signaling.
The physiologic importance of these findings is supported by the
observation that T cell-dependent Ab responses known to require
functional CD154-CD40 interactions (1) are absent in TRAF3-deficient
mice (53). Moreover, immunohistology reveals that TRAF3 is highly
expressed in Ig-secreting plasmablasts in the interfollicular regions
of secondary lymphoid tissues as in well as fully differentiated
Ig-secreting plasma cells in the bone marrow (72) supporting a
potential role for this effector molecule in regulation of Ig
secretion. Furthermore, mutation of a region of the cytoplasmic tail of
CD40 known to bind TRAF3 eliminates CD40-induced Ig secretion (73). Of
interest, mice deficient in MKK4, an upstream regulator of JNK and thus
a potential mediator between TRAF3 and JNK activities (Fig. 3), form
germinal centers and mount T cell-dependent Ab responses comparably to
those of their wt counterparts (74). Together with the findings that
either pharmacologic inhibitors of p38 activation (Fig. 6C)
or a DN version of TRAF3 abrogate CD40-mediated p38 activation (Fig. 3)
and control of Ig production (Fig. 6, A and B) by
Ramos B cells, these observations suggest that control of Ig secretion
following CD40 engagement is mediated by a TRAF3 signaling pathway that
is dependent upon p38, but is independent of JNK.
In vitro proliferation of B cells following CD40 ligation is unaffected
in mice deficient in either TRAF3 (53) or MKK4 (57), an upstream
regulator of JNK activation, compared with that in controls. By
contrast, CD40-induced proliferation, but not Ig secretion, is impaired
in mice deficient in the p52/p100/NF-B2 (75) and the
trans-activating c-Rel (76) and RelB (77) components of
NF-B, suggesting that regulation of proliferation resulting from
CD40 ligation is likely to involve TRAF3-independent activation of
NF-B. The current studies are consistent with this conclusion and
expand upon it by showing that CD40-induced inhibition of proliferation
and stimulation of apoptosis are independent of TRAF3 (Figs. 4 and 5)
and are increased by inhibiting NF-B with antioxidants such as
N-acetyl cysteine (data not shown). Together, these
observations confirm our previous findings that CD40-mediated Ig
secretion is regulated independently of proliferation (4, 5, 12) and
provide an explanation for this dichotomy by demonstrating that a TRAF3
signaling pathway exclusively controls Ig secretion with no influence
on control of apoptosis or cellular growth.
Recent studies have also suggested that TRAF2 may mediate
caspase-dependent apoptosis following engagement of TNF receptor family
members via association with TRADD-FADD complexes (78) and/or
FLIP/Casper (79, 80). The current data demonstrate that CD40
ligation also induces apoptosis that can be blocked by the caspase
inhibitor, zVAD-fluoro methyl ketone (data not shown), but is
independent of TRAF3 (Figs. 4 and 5). This finding is consistent with
the demonstration that engagement or overexpression of a variety of TNF
receptor family members, including CD120a/TNF receptor I, CD95, LT-ß
receptor, and LMP1 can induce apoptosis and may do so by a variety of
signaling pathways in different cells. Thus, apoptosis induced
following engagement of LT-ß receptor (81) or overexpression of LMP1
(82) in nonlymphoid cells involves a TRAF3-mediated signaling pathway,
whereas the current data show that CD40-induced apoptosis of Ramos B
cells is TRAF3 independent (Fig. 4). That the same signaling pathways
can mediate different functional outcomes in nonlymphoid vs lymphoid
cells is suggested by the finding that the TRAF2-associating protein
FLIP/Casper mediates apoptosis in nonlymphoid cells (79), whereas it
blocks apoptosis in cells of lymphoid origin (80). Together with the
current finding that TRAF3 plays no role in CD40-induced apoptosis of
Ramos cells, these observations suggest that apoptosis induced by
engaging TNF receptor family members in nonlymphoid cells may be
mediated by TRAF3, but the programmed cell death program may be induced
by the TRAF2-associating protein FLIP/Casper in lymphoid cells.
Whereas CD40-induced, TRAF3-mediated signals were uncharacterized
previous to this study, CD40-mediated nuclear translocation of NF-B
was shown to involve TRAF2, -5, or -6 (27, 28, 32, 33). The current
data extend the previous finding that TRAF3 is not involved in
CD40-mediated NF-B activation (Fig. 8A) (27, 28, 33) by
demonstrating that physiologic processes known to be regulated by
NF-B (Figs. 7 and 8B) (43, 44), such as GM-CSF secretion,
CD54, MHC class II, and CD95 expression, are not affected by
interference with TRAF3 signaling. These results are in agreement with
the finding that induction of genes regulated by NF-B, such as CD80
and CD23, are induced normally in B cells purified from TRAF3-deficient
mice compared with that in controls (53).
This report documents that CD40 engagement induces the production of a
variety of cytokines from Ramos cells (Fig. 7). The potential
physiologic relevance of CD40-induced cytokine secretion from Ramos
cells is emphasized by the detection of IL-10, TNF-, and LT- in B
cells isolated from secondary lymphoid tissues, a variety of
malignancies (83), and sites of inflammation, such as rheumatoid
synovium (84, 85). The current data extend earlier studies showing that
CD40 engagement increased the secretion of some cytokines (1) by
demonstrating that production of a large array of cytokines was
stimulated and that CD40-induced secretion of IL-10, TNF-, and
LT-, but not GM-CSF, was partially, but not completely, dependent
upon functional TRAF3. This finding suggested that TRAF3 as well as
other signaling molecules were likely to be involved in the regulation
of cytokine production by Ramos cells.
Transcriptional regulation of IL-10 and LT- has not been delineated,
so the role of TRAF3-mediated p38 and/or JNK pathways is unclear.
However, the presence of NF-B sites in these promoters suggests that
CD40-induced activation of these genes may be partially regulated by
nuclear translocation of NF-B, dependent on TRAF2, -5, or -6, along
with TRAF3-mediated signaling pathways. Although deletion of the
NF-B sites in the human TNF- promoter did not affect its
induction in B cells, optimal transcription has been shown to be
mediated by a high affinity NF-AT element and binding of ATF2/Jun to
CRE (86, 87). In addition, blocking nuclear translocation of
NF-ATc with cyclosporine inhibited CD40-mediated TNF-
secretion (88, 89). Of interest, analysis of the promoters for IL-10
(90, 91) and LT- (92) also revealed potential regulation by CREB.
Moreover, previous studies have demonstrated cooperativity between CRE
and either AP-1 or NF-B sites (43). These findings together with the
current results suggest that induction of these transcription factors
in B cells (24, 25) via a CD40-mediated, TRAF3-dependent signaling
pathway may regulate the secretion of TNF- as well as that of other
cytokines. Alternatively, or perhaps in parallel, the
membrane-proximal, proline-rich JAK3 binding BOX1 motif in the
cytoplasmic tail of CD40 (residues 202–209) may contribute to
CD40-induced production of LT- and other cytokines independently of
TRAF2 or TRAF3 (15) and may explain residual cytokine production when
TRAF3-mediated signaling is blocked by the DN construct.
Posttranscriptional regulation of cytokines may also be controlled by
TRAF3-mediated activation of JNK and/or p38. Of note, TNF- (93) mRNA
has been shown to be negatively regulated by the AU-rich motif found in
the 3'-untranslated region of this gene (94). Both JNK-mediated (95)
and p38-mediated (96) signaling events have been demonstrated to be
involved in posttranscriptional regulation of TNF-. Moreover,
TNF- production can be blocked at the posttranscriptional level by
specific pharmacologic inhibitors of p38 (96, 97). In conjunction,
these studies suggest that CD40-induced, TRAF3-mediated signaling
involving p38 and/or JNK may be involved in transcriptional and
posttranscriptional regulation of the production of cytokines such as
TNF-.
In summary, the current data link TRAF3-mediated activation of specific
kinases with physiologically relevant outcomes. Engagement of CD40 on
Ramos B cells regulates Ig, IL-10, TNF-, and LT- secretion by
TRAF3-mediated signaling pathways. Since CD40-mediated p38 activation
and control of Ig secretion are entirely dependent upon functional
TRAF3, it is possible that this member of the mitogen-activated protein
kinase family uniquely regulates CD40-dependent Ig production by Ramos
B cells. By contrast, activation of JNK and secretion of IL-10,
TNF-, and LT- following CD40 engagement are partially dependent
upon functional TRAF3, implying that signaling via other TRAF family
members may be involved.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter E. Lipsky, Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-8884. E-mail address:
3 Abbreviations used in this paper: MKK1, mitogen-activated protein kinase kinase; JNK, Jun N-terminal kinase; TRAF, TNF receptor-associated factor; AP-1, activating protein-1; DN, dominant negative; GaMIg, goat anti-mouse immunoglobulin; wt, wild type; CD40L, CD40 ligand; mCD154, murine CD154; GST, glutathione-S-transferase; GM-CSF, granulocyte-macrophage colony-stimulating factor; LT-, lymphotoxin-; EMSA, electrophoretic mobility-shift assay; LMP1, latent membrane protein-1; CRE, cyclic adenosine 3',5'-monophosphate response element; CREB, cyclic adenosine 3',5'-monophosphate response element binding protein; FLIP, FLICE-inhibitory protein.
Received for publication December 8, 1997. Accepted for publication April 1, 1998.
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