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,
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Graduate Programs in
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Molecular Biology and
Immunology and Departments of
Microbiology and
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Internal Medicine, University of Iowa, Iowa City, IA 52242; and
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Veterans Affairs Medical Center, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B. Ligation of the endogenous B cell CD40 molecule also induces
these and other activated phenotypic changes. Here, we report that
expression of LMP1 also activates B cells to secrete Ig and IL-6 and
rescues them from B cell receptor-mediated growth arrest analogous to
CD40 signaling. Furthermore, an HLA-A2LMP1 chimeric construct
demonstrates that the oligomerization of the carboxyl-terminal 200
amino acids of LMP1 is sufficient for B cell signaling. Finally, we
demonstrate that LMP1 and CD40 signaling pathways interact
cooperatively in inducing B cell effector
functions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The virus has the capacity to produce at least 85 genes during the course of a normal infection, but during models of the latent stage of infection, the virus transcribes only 11 genes. Five of the genes encoding latent proteins have been shown to be essential for EBV to transform PBL into immortal lymphoblastoid cell lines in vitro. The required genes are Epstein-Barr nuclear Ags 1, 2, 3a, and 3c, as well as latent membrane protein 1 (LMP1)3 Refs. 4, 5, 6 ; for recent review, see 3 .
LMP1 is the only viral protein that has been shown to be oncogenic in
rodent fibroblast cell lines 7, 8 and is expressed in lymphoblastoid
cell lines and the majority of the EBV-associated malignancies. LMP1
consists of a short cytoplasmic N terminus, six transmembrane domains
connected by reverse turns, and a 200-amino acid (aa) carboxyl terminus
(CT), all of which lack any known enzymatic motifs. B cells expressing
LMP1 have an activated phenotype, as evidenced by the high surface
expression of the adhesion molecules and costimulatory molecules 9 ,
as well as activation of NF-B 10 and c-Jun kinase 11 . While the
molecular mechanisms of LMP1 function are still incompletely
understood, they are beginning to be elucidated. It has recently been
demonstrated that the CT cytoplasmic tail interacts with several
TNFR-associated factors (TRAFs), namely TRAF1, -2, -3, and -5 12, 13, 14 .
The N terminus and transmembrane domains form aggregates in the
cytoplasmic membrane, allowing LMP1 to act like a constitutively
activated receptor 15, 16 . It is also now apparent that the
oligomerization of the CT is all that is required for LMP1-induced
NF-B activation 15, 17 .
In normal B cells, an important mechanism of activation is CD40 ligation. CD40, a member of the TNFR family of proteins, is found on all B cells (except plasma cells) and is necessary for the proper formation of germinal centers and memory B cell production (reviewed in 18 . The CD40 cytoplasmic tail also lacks any known enzymatic motifs but does contain a PXQXT motif, shown to be important for interaction with TRAF proteins and has been shown to interact with TRAF1, 2, 3, 5, and 6 19, 20, 21, 22 . Recent work has demonstrated that different structural features of the CD40 cytoplasmic tail are required for different effector functions 23, 24 . However, the detailed mechanism by which CD40 activates B cells remains to be elucidated.
Because LMP1 and CD40 have been shown to interact with several, but not
all, of the same intracellular molecules and have many of the same
effector functions, we believe that LMP1 is able to exploit the CD40
signaling pathway in the B cell. To determine how far this mimicry
extends and to what extent LMP1 can influence CD40 signaling, we stably
transfected B cells with inducible LMP1 to study LMP1 in isolation from
other EBV latent genes in the major cell type of EBV latent infection.
Since CD40 is endogenously expressed on B cells, we are able to use
CD40 signaling as an internal control. As mentioned above, LMP1
initiates signaling by self-aggregation as it is expressed on the
plasma membrane. To control initiation of signaling by receptor
engagement, we created a chimeric molecule consisting of the
extracellular and transmembrane regions of HLA-A2 and the LMP1 CT,
which allows us to examine early events in the signaling cascade. With
these model systems, we demonstrate in both human and mouse B cells
that LMP1 induces not only NF-B activation and surface molecule
up-regulation, but also causes B cells to secrete Ig and IL-6 and can
rescue them from B cell receptor (BCR)-mediated growth arrest.
Aggregation of the 200-aa LMP1 CT is necessary and sufficient to induce
these functions. Furthermore, when LMP1 and CD40 signals are given
concurrently, the B cell responds more robustly than it does to either
signal alone.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M12.4.1, CH12.LX, and CHB3 are mouse B lymphoma cell lines,
which have been described previously 23, 25, 26 . Ramos, an
EBV-negative Burkitt’s lymphoma cell line, was obtained from the
American Type Culture Collection (ATCC; Manassas, VA). All B cells were
cultured in RPMI 1640 supplemented with 10% FCS, 10 µM 2-ME, and
antibiotics (BCM-10). CH12.LX transfected with the Lac repressor
(CH12.LAC) was selected and maintained in BCM-10 plus 200 µg/ml of
hygromicin, while M12.LAC required 400 µg/ml of hygromicin
(Calbiochem, La Jolla, CA). Cell lines transfected with A2LMP1, human
(h)CD40, or inducible LMP1 were maintained in 400 µg/ml of geneticin
(Life Technologies, Grand Island, NY) in BCM-10 (CH12 and M12
transfectants), 800 µg/ml of geneticin (CHB3 transfectants), and 1
mg/ml of geneticin (Ramos transfectants). Supertransfected cell lines
were maintained in BCM-10 plus geneticin. All transfectants were
generated by electroporation, as previously described 27 . Chinese
hamster ovarian cells (CHO-KI) obtained from the ATCC were cultured in
DMEM (high glucose) and supplemented with 10% FCS, 1x MEM
nonessential amino acids (Sigma, St. Louis MO), 10 µM 2-ME, and
antibiotics. CHO transfected with a plasmid encoding mouse CD40 ligand
(mCD40L) behind the EF-1
promoter plus the neoR gene
(produced in our laboratory, see below) were cultured in the
aforementioned DMEM medium additionally supplemented with 1 mg/ml
of geneticin. Sheep erythrocytes, used as a source of Ag for CH12.LX
cells, were purchased from Elmira Biologicals (Iowa City, IA). The
insect cell line Sf9, infected with wild-type baculovirus or a
baculovirus encoding mCD40L 28 , was used to stimulate the mouse B
cells in some experiments.
Abs and reagents
Isopropyl-B-D-thiogalactopyranoside (IPTG) was purchased from Amresco (Solon, OH) and propidium iodide (PI) was purchased from Molecular Probes (Eugene, OR). Recombinant mIL-6, and the monoclonal Abs 16/10A1 (FITC-labeled anti-B7-1-FITC, Armenian hamster IgG), G235-2356 (FITC-labeled isotype control, Armenian hamster IgG), HM40-3 (anti-mouse CD40, Armenian hamster IgM), G235-1 (isotype control, Armenian hamster IgM), Jo2 (anti-mouse Fas, Armenian hamster IgG), and the anti-human B7-1 (mouse IgM) were purchased from PharMingen (San Diego, CA). CH11 (anti-human Fas, mouse IgM) was purchased from Upstate Biotechnology (Lake Placid, NY). Goat-anti-Armenian hamster IgG-FITC was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). MOPC-21 (isotype control, mouse IgG1), TEPC 183 (isotype control, mouse IgM), and streptavidin-horseradish peroxidase (HRP), as well as the o-phenylenediamide dihydrochloride tablets were purchased from Sigma. Goat-anti-rabbit HRP was purchased from Bio-Rad (Hercules, CA), and goat-anti-mouse IgG1 and goat-anti-mouse IgM-FITC were purchased from Southern Biotechnology Associates (Birmingham, AL). The polyclonal rabbit Ab against LMP1 was a kind gift from Dr. Bill Sugden (University of Wisconsin, Madison, WI).
The following hybridomas were grown in our laboratory and were purchased from the ATCC or were the generous gifts from the indicated individuals: anti-mouse Kd (SF1-1.1.1, mouse IgG2a) from Dr. John Harty (University of Iowa, Iowa City, IA); anti-mouse IgM (Bet-2, rat IgG1) from Dr. John Kemp (University of Iowa); anti-mouse CD40 (1C10, rat IgG2a) from Dr. Frances Lund (Trudeau Institute, Saranac Lake, NY); anti-mouse IgE (EM95.3, isotype control, rat IgG2a) from Dr. Thomas Waldschmidt (University of Iowa); anti-HLA-A2 (CR11-351, mouse IgG1) from Dr. Charles Lutz (University of Iowa); anti-human CD40 (G28-5, mouse IgG1, ATCC); anti-mouse LFA-1 (M17/4.4.11.9, rat IgG2a, ATCC) and anti-mouse IL-6 hybridomas 20F3.11 (rat IgG, ATCC) and 32C11.4 (rat IgG1, ATCC).
DNA constructs
The Lac repressor plasmid p3'SS was purchased from Stratagene (La Jolla, CA). pOPRSVI.mcs1 was made by removing the chloramphenicol acetyltransferase gene from pOPRSVICAT (Stratagene) using NotI and replacing it with a synthetic double-stranded oligonucleotide encoding a multiple cloning site. LMP1 was subcloned from the p1281 plasmid generously provided by Dr. Bill Sugden (University of Wisconsin) into the pOPRSVI.mcs1 vector. To generate the A2LMP1 construct, primers containing BamHI and XbaI were used to amplify by the PCR the sequence corresponding to the 200 CT amino acids of LMP1 (5'-AAGGATCCCATGGACAACGACACAGTGA and 3'-AATCTAGAAAGCCTATGACATGGTAATGCC). The PCR product was cloned into the BglII and XbaI sites of the pA2CD45 plasmid, a kind gift from Dr. Gary Koretzky (University of Iowa), after removal of the CD8/CD45 insert. The A2LMP1 chimera was subcloned into the pRSV.neo plasmid 29 for stable expression in B cells. The plasmids used to generate the CHB3.hCD40 transfectant have been previously described 23 . The pEF-CD40L plasmid was constructed by substituting the pEF promoter from pEF-BOS 30 for the Rous sarcoma virus (RSV) promoter of pRSV.neo containing a mCD40L cDNA insert (M. Baccam and G.A.B., manuscript in preparation).
Western blot analysis
Cells (2 x 106) were grown in BCM-10 alone or in BCM-10 + 100 µM IPTG for the indicated time points. Harvested cells were lysed in 1% Nonidet P-40 lysis buffer (50 mM Tris (pH 7.5), 1% Nonidet P-40, 150 mM NaCl, 0.02% NaN3, 50 µg/ml PMSF, 50 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A) and incubated for 30 min on ice. After centrifuging at 14,000 x g for 10 min, the protein content of the supernatant was determined using the Bio-Rad protein assay. The lysates (100 µg/lane) were then separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. LMP1 was detected by blotting with a 1:1000 dilution of the polyclonal rabbit anti-LMP1, followed by the goat-anti-rabbit HRP, and visualizing with a chemiluminescent detection system (Pierce, Rockford, IL).
Surface molecule up-regulation
M12.LMP1 was stimulated, as previously described 23 , for 72 h with 0.5 µg/ml of isotype control or anti-mCD40 Ab (EM-95 or 1C10) and/or 100 µM IPTG. M12.A2LMP1 received combinations of 1 µg/ml of the following Abs, anti-A2 (CR11-351), anti-mCD40 (1C10), or isotype control (MOPC-21 and EM-95), for a total of 2 µg/ml Ab per stimulation. Ramos.A2LMP1 were stimulated with 1 µg/ml anti-A2 (CR11-351), 0.5 µg/ml anti-hCD40 (G28-5), and enough isotype control (MOPC-21) to yield a total Ab concentration of 1.5 µg/ml. Cells were stained with FITC-labeled mAb against surface markers or FITC-labeled isotype control mAb. Staining was detected by immunofluorescence flow cytometry, using a FACScan benchtop flow cytometer (Becton Dickinson, Mountain View, CA).
Nuclear extraction and electrophoretic mobility shift assay (EMSA)
Both nuclear extraction and EMSA were performed as previously
described 24 . Briefly, 5 x 106 viable cells were
stimulated for 1.5 h with 1 µg/ml of Ab or for 24 h with
100 µM IPTG at a concentration of 1 x 106 cells/ml.
Cells were lysed in sucrose buffer, centrifuged at 500 x
g at 4°C, and washed once with sucrose buffer minus the
Nonidet P-40. Nuclei were extracted in 30 µl of low-salt buffer, 30
µl of high-salt buffer added slowly in two equal aliquots, and
rotated for 30 min at 4°C. Extracts were clarified by centrifuging
for 15 min at 14,000 x g at 4°C, and the supernatant
was stored at -70°C in the presence of 25 µg/ml pepstatin A, 25
µg/ml leupeptin, and 80 µg/ml aprotinin. The double-stranded
NF-B probe, previously described 24 , was end-labeled with
-[32P]ATP using T4 polynucleotide kinase. Five
micrograms of nuclear extract were incubated with 0.25–0.5 ng of probe
for 30 min at room temperature before being separated on a 5% native
polyacrylamide gel at a constant current of 20 mA. The gel was dried
and exposed to x-ray film overnight at -70°C.
IL-6 secretion
CH12.LX cell lines were cultured at 1 x 105 cells/200 µl BCM-10/well in a flat-bottom 96-well microtitration plate for 48 h. Cells were cocultured with 2.5 x 104 untransfected CH0 or CH0-mCD40L cells and/or 100 µM IPTG as indicated. IL-6 present in the supernatant after 48 h was quantitated by ELISA. Immulon II flat-bottom plates (Dynex Technologies, Chantilly, VA) were coated overnight at 4°C with 10 µg/ml mAb 20F3.11 in a 15 mM Na2CO3, 40 mM NaHCO3, 3 mM NaN3 solution (pH 9.6). The plate was washed with PBS-T (PBS + 0.05% Tween 20), blocked for 3 h at room temperature with PBS + 10% FCS, and washed once more with PBS-T. Culture supernatant (undiluted, and diluted 1:2–1:16 in BCM-10) and recombinant mIL-6 standards (1:2 dilutions 10–0.15 ng/ml, and medium alone) were then allowed to bind overnight at 4°C. After washing, bound IL-6 was detected by incubating plates with biotinylated mAb 32C11.4 diluted in PBS-T + 0.5% BSA, washing with PBS-T, incubating with streptavidin-HRP diluted in PBS-T + 0.5% BSA, followed by further washing. Next, o-phenylenediamide dihydrochloride dissolved in 50 mM Na2HPO4, 25 mM citric acid, and 0.012% hydrogen peroxide solution (pH 5.0) was incubated at room temperature, in the dark, with the samples. The reaction was stopped with 0.67 M sulfuric acid and then read on a microplate reader at 490 and 600 nm. The reading at 600 nm was subtracted from the 490 nm reading to adjust for small volume differences in the wells. Values given represent the mean and SE of triplicate wells.
Cell cycle analysis
CHB3 cells (1 x 105 cells/2 ml) were cultured in the presence of the indicated amounts of Abs for 48 h. Cells were then pelleted and resuspended in 0.5 ml of 70% ethanol, on ice, and stored at -20°C until staining. Upon removal from -20°C, 4 ml of PBS was added to samples, and samples were pelleted and incubated in 300 µl of DNA extraction buffer (4 mM citric acid, 192 mM NaHPO4) for 5 min at room temperature. After centrifugation, cells were resuspended in 200 µl of staining solution (0.04 mg/ml PI, 0.2 mg/ml RNase A in PBS) and incubated for 30 min at room temperature before analysis by immunofluorescence flow cytometry.
[3H]thymidine uptake
CHB3 cells were cultured at 5 x 103 cells/200 µl/well in a flat-bottom 96-well microtitration plate for 48 h. Five hours before harvesting, cells were pulsed with 0.5 µCi of [3H]thymidine per well. Cells were harvested to glass fiber filters with a semiautomatic cell harvestor (Skatron Instruments, Sterling, VA). Liquid scintillation counting was then performed, and the values presented represent the mean and SE of triplicate samples.
Ab secretion assay
CH12.LX and its transfected subclones express surface IgM specific for phosphatidylcholine, an Ag found on the surface of SRBC. Enumeration of SRBC-specific IgM-secreting cells was by direct plaque assay, as described previously 31, 32 .
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the constitutive over-expression of LMP1 has been reported
to be toxic to cells 33 , LMP1 was expressed via an inducible system.
First, M12.4.1 and CH12.LX B cells were stably transfected with a
plasmid encoding the Lac repressor protein. Next, Lac
repressor-expressing subclones were supertransfected with an LMP1
construct containing two Lac repressor binding sites, one in the RSV
promoter region and one in the 5' intronic region, where the Lac
repressor binds and shuts off production of LMP1 at the level of
transcription. Expression of this construct leads to a low basal
expression of LMP1 that can be augmented greatly by addition of IPTG to
the medium (Fig. 1
A). Maximum
expression of LMP1 is achieved 24–48 h after the addition of IPTG, at
which time degradation products of LMP1 are detectable in the cell
lysate. Levels of LMP1 are elevated for at least 72 h with no
noticeable toxicity to the cells (data not shown).
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B shows constitutive surface expression
of this molecule in stable transfectants of both mouse and human B cell
lines. By using the anti-A2 mAb CR11-351 34 , we can aggregate the
chimera at will, study early signaling events, and determine the role
of the carboxyl cytoplasmic tail in LMP1 effector functions.
Total amounts of cellular induced LMP1 vs A2LMP1 hybrid molecules were
found to be similar when tested by Western blot analysis (data not
shown). However, levels of induced LMP1 specifically expressed on the
cell surface could not be directly tested, as the LMP1-specific Abs do
not bind to portions of the molecule that are surface-exposed. Up-regulation of activation markers
Both CD40 and LMP1 are reported to induce the up-regulation of
adhesion molecules LFA-1 and ICAM-1 as well as coactivation molecules
such as CD23 9, 23, 35 . CD40 signaling has also been shown to
up-regulate Fas and B7-1 in M12.4.1 cells 23 . M12.LAC cells inducibly
expressing LMP1 (M12.LMP1) were incubated for 72 h with IPTG in
the presence or absence of anti-mCD40 mAb. The cells were stained
for expression of B7-1, LFA-1, ICAM-1, CD23, Fas, and CD40 and analyzed
by flow cytometry. Fig. 2A
presents flow cytometry profiles of M12.LMP1, M12.A2LMP1, and
Ramos.A2LMP1 cells stained for B7-1 expression following
expression of inducible wild-type LMP1 or ligation of A2LMP1 molecules.
Fig. 2 summarizes up-regulation data for additional surface
molecules following induced expression of wild-type LMP1 (Fig. 2B) or ligation of A2LMP1 (Fig 2C). While IPTG
had no affect on surface molecule expression in M12.LAC cells (not
shown), IPTG treatment led to an up-regulation of surface molecules in
M12.LMP1, as did anti-CD40 stimulation. When LMP1 and CD40 signals
were given together, enhanced B7-1 up-regulation was seen in both mouse
and human B cells (Fig. 2, A-C). That is, provision of both
signals caused an up-regulation in B7-1 that was reproducibly (two
separate subclones, each tested 2–3 times) approximately double that
seen when a CD40 signal alone was given, and 5- to 10-fold greater than
that seen following induced expression of LMP1.
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, A and C). Stimulation
of the endogenous MHC class I molecule Kd had no effect on
surface molecule expression (Fig. 2C). When both LMP1 and
CD40 signals were provided concurrently to M12.A2LMP1 cells, an
enhancing effect was again seen in B7-1 up-regulation. The magnitude of
this effect was similar to that seen with induction of LMP1 expression
plus a CD40 signal (Fig. 2, A and C). A small but
reproducible enhancement of ICAM-1 and CD23 up-regulation was also
seen, although not as dramatic as that of B7-1 (Fig. 2C).
Enhancement was not seen in CD40, LFA-1, and Fas up-regulation. Because
no enhancement is seen in CD40 up-regulation, the cooperative effect of
LMP1 and CD40 signals in the up-regulation of the other surface
molecules cannot be due to the ability of LMP1 to up-regulate
CD40, resulting in a stronger CD40 signal. These studies demonstrate
that the CT 200 aa of LMP1 are sufficient for inducing surface molecule
up-regulation, and that LMP1 and CD40 signals can act cooperatively.
A2LMP1 was stably expressed in the EBV negative human B cell line Ramos
(Fig. 1B) to show that aggregation of the LMP1 CT was
sufficient for surface molecule up-regulation in human as well as mouse
B cells. Ramos.A2LMP1 cells were stimulated with the anti-A2,
anti-CD40, or both mAbs for 72 h. As shown in Fig. 2A, A2LMP1 or CD40 stimulation induced up-regulation of
B7-1, and the signals acted cooperatively. Similar up-regulation was
seen for Fas (data not shown). Thus, although mouse B cells lack the
receptor to which EBV binds and cannot be infected with EBV, the LMP1
molecule appears to signal similarly in mouse and human B cells.
Nuclear translocation of NF-B
Both CD40 and LMP1 have been reported to stimulate NF-B
activity 24, 36, 37, 38 . However, most of the work dealing with
LMP1-stimulated NF-B activity has been performed in 293 epithelial
cells, a cell type that is not normally infected by EBV. Fig. 3A shows a striking increase
in nuclear NF-B in B cells induced to express LMP1
(lane 13), while addition of IPTG has no affect on
nuclear NF-B in the parent cell line (lane 11).
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B reporter gene in 293 cells 15 and Jurkat T cells
17 . Here we show, in B cells, that the signal received via the A2LMP1
chimera is similar to that received by the endogenous CD40 molecule
(Fig. 3A). Similar results were seen in the CH12.A2LMP1 and
Ramos.A2LMP1 cell lines (data not shown). The time course of NF-B
nuclear translocation following A2LMP1 cross-linking is similar to that
seen with CD40 cross-linking 24 ; an increase is detectable by 20 min
(Fig. 3B), reaches a plateau by 1.5 h, and remains
elevated up to 24 h with continuous stimulation (data not shown).
Fig. 3B, lane 5 vs lanes 3 and
4, shows that enhancement is seen in the amount of nuclear
NF-B translocated at the early time point when both CD40 and A2LMP1
are engaged (73-fold increase in band intensity compared with the
isotype control lane), compared with anti-CD40 alone (50-fold
increase) or anti-A2 alone (23-fold increase). Stimulation of the
endogenous MHC class I molecule Kd had no effect on nuclear
NF-B levels (Fig. 3B, lane 1). Effects on Ab secretion
The CH12.LX cell line is induced to differentiate and secrete IgM
when stimulated via CD40 28 . CH12.LX does not respond to Ag-receptor
engagement by Ab secretion, however BCR and CD40 do synergize,
resulting in an augmented number of plaque forming colonies 23, 28 .
CH12.LAC cells do not respond to IPTG, either in the presence or
absence of Ag, by secreting Ab, but CH12.LAC inducibly expressing LMP1
(CH12.LMP1) respond similarly to CD40L and IPTG stimulation (Fig. 4A). This is the first
demonstration to our knowledge that LMP1 expression and signaling is
sufficient to drive a B cell to differentiate into an Ab-secreting
cell. Further, LMP1 is able to synergize with BCR signaling as does
CD40 (Fig. 4A).
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B). Furthermore, both the inducible LMP1 and the chimeric
LMP1 can act cooperatively with CD40 signals (Fig. 4C). Even
when a saturating CD40 signal was administered, there was an increase
in the number of plaque-forming colonies upon addition of LMP1
signaling. This suggests that LMP1 and CD40 may be using partially
distinct intracellular signaling pathways, or that intracellular
components of the pathway are not limiting. Induction of IL-6 secretion
CD40 signaling has been shown to induce IL-6 secretion in B cells
39 and epithelial and fibroblast cell lines 40, 41, 42 , while thus far,
LMP1 has only been shown to induce IL-6 secretion in epithelial cells
40 . Here, we show that LMP1 can also stimulate IL-6 secretion in B
cells (Fig. 5). CH12.LAC cells secrete
IL-6 only when stimulated with mCD40L. In contrast, all CH12.LMP1 cell
lines tested show a basal level of IL-6 secretion, presumably due to
low basal LMP1 expression, that is augmented 4- to 5-fold by IPTG
induction of increased LMP1 expression. When both mCD40L and LMP1
stimulation is given at the same time, slightly more than additive
levels of IL-6 are produced (Fig. 5).
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Rescue from BCR-mediated growth arrest
It has been previously shown that the mouse B cell line CHB3
receives a negative signal via the BCR 26 , just as do freshly
isolated immature B cells. Signaling through a transfected human CD40
molecule is sufficient to protect CHB3 from the anti-IgM-mediated
growth arrest (Fig. 6A).
Signaling through the A2LMP1 chimera (compared with transfected hCD40
because the two transfected molecules are expressed at comparable
levels on CHB3 cells) is also sufficient to block BCR-mediated growth
inhibition (Fig. 6A). While [3H]thymidine
assays revealed that CHB3 cells stop synthesizing DNA in response to
BCR signaling, the PI assay allowed us to determine whether the cells
degraded their DNA or are simply halted in some phase of the cell
cycle. There was a small increase in the number of apoptotic cells (not
shown), but this increase was barely detectable in comparison to a
substantial buildup of cells in the G1 stage of the cell cycle. Fig. 6B depicts the percent increase of cells in G1 following
sIgM ligation, and rescue from this arrest by either LMP1 or CD40
signaling.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B nuclear translocation, Ig secretion, and rescue from
BCR-mediated growth arrest. Both LMP1 and CD40 have been shown to bind
several of the same TRAF molecules, namely TRAF2 13, 43 , -3 12, 19 ,
and -5 14, 21 , although there has been one report that TRAF5 does not
bind CD40 44 . There are some reported differences in TRAF binding.
TRAF6 binds to CD40, but it does not bind to LMP1 22 , and TRAF1 and
TNFR1-associated death domain protein (TRADD) have been reported to
bind to LMP1 13, 45 but do not bind CD40 directly 43 . However, it
remains possible that TRAF1 and TRADD could be recruited to the CD40
signaling complex via interactions with TRAF2 since both molecules have
been shown to bind to TRAF2 46, 47 . TRAF6 has been reported to only
form dimers with other TRAF6 molecules 48 , reducing the possibility
that it would be brought into the LMP1 signaling complex through
associations with other TRAF molecules.
The exact role of TRAF molecules in LMP1 and CD40 signaling remains
unclear. TRAF molecules contain no known enzymatic motifs but do
contain amino-terminal zinc (Zn) finger and Zn RING finger
domains and a characteristic carboxyl TRAF domain through which they
homo- and hetero-dimerize as well as bind to receptors. Over-expression
of TRAF2, -5, and -6 leads to NF-B reporter activity in an
epithelial cell line (293), and expression of dominant negative
versions of these molecules that lack all or most of their RING and Zn
finger domains block NF-B activation in these cells 21, 22, 43, 44, 48 . However, B cells from transgenic mice lacking TRAF2 or expressing
a dominant negative version of TRAF2 are able to activate NF-B in
response to CD40 signaling. These B cells are unable to activate c-Jun
kinase in response to CD40 signaling 49, 50 , implicating TRAF2 as
critical in the stress-activated mitogen-activated protein
kinase stimulation but not NF-B activation. We have previously shown
that CD40 lacking 22 carboxyl amino acids or containing a point
mutation that changes the threonine at position 234 to alanine still
retain the ability to activate NF-B, up-regulate LFA-1 and ICAM-1,
and stimulate Ig secretion 23, 24 , although these mutations abrogate
the ability of CD40 to bind TRAF2, TRAF3 and presumably TRAF5 (Ref. 19,
and B. Hostager and G.A.B., manuscript in preparation).
Our findings also indicate that LMP1 can cooperate with CD40 signaling. This could mean either that intracellular molecules, such as the TRAFs, are not limiting or that CD40 and LMP1 use distinct, but converging, signaling pathways. LMP1 has long been associated with cell transformation, and there are at least two possible explanations of how LMP1 and CD40 cooperation in B cell signaling contribute to this association. In vivo, latently infected B cells expressing LMP1 can be stimulated through both CD40 and LMP1, perhaps resulting in a superactivated state predisposing the B cell to a second event causing cell transformation. The expression of LMP1 has been shown to be insufficient to maintain continuous proliferation of B cells 51 , so a second signal coming from another EBV latent protein or a mutation within the B cell would be required for transformation. Alternatively, the presence of LMP1 signals could lower the amount of CD40 signal needed to drive activation by a T-dependent Ag and cognate interactions with T cells, thus lowering the threshold for activation of B cells and perhaps predisposing them to transformation. It has been demonstrated that BCR and class II signals enhance CD40-induced differentiation of CH12.LX and proliferation of mouse splenic B cells, with the enhancement being most pronounced at limiting concentration of mCD40L 28 , thus establishing a precedent for such cooperation between CD40 and other signaling pathways.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gail A. Bishop, Department of Microbiology, University of Iowa, 3-570 Bowen Science Building, Iowa City, IA 52242. E-mail address:
3 Abbreviations used in this paper: LMP1, latent membrane protein 1; CT, carboxyl terminus (-terminal); BCR, B cell receptor; TRAF, TNF receptor-associated factor; IPTG, isopropyl-B-D-thiogalactopyranoside; EMSA, electrophoretic mobility shift assay; PI, propidium iodide; L, ligand; h, human; m, mouse; HRP, horseradish peroxidase; CHO, Chinese hamster ovary; RSV, Rous sarcoma virus.
Received for publication September 23, 1998. Accepted for publication November 12, 1998.
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