|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2830-2843
Prolonged Phenotypic, Functional, and Molecular Change in Group I
Burkitt Lymphoma Cells on Short-Term Exposure to CD40 Ligand
By
Matthew P. Baker,
Aristides G. Eliopoulos,
Lawrence S. Young,
Richard J. Armitage,
Christopher D. Gregory, and
John Gordon
From the Departments of Immunology and the Institute of Cancer
Studies, University of Birmingham, Birmingham, UK; and Immunex Corp,
Seattle, WA.
 |
ABSTRACT |
Group I Burkitt lymphoma (BL) cell lines (L3055, Elijah, Louckes,
BL2, and BL29) retaining the original biopsy phenotype were found to
undergo prolonged phenotypic, functional, and molecular change on
short-term exposure to soluble recombinant CD40L trimer. Sensitivity
to, extent of, and duration of change appeared to reflect passage
number in that the earliest passaged lines, L3055 and BL29, were
generally the most susceptible. Culture of group I BL lines with CD40L
resulted in significant growth arrest (without apoptosis) that, for
L3055 cells, was sustained for 7 to 9 days after 72 hours of exposure.
This was accompanied by the formation of large homotypic aggregates
together with gross changes in individual cell morphology and a
concomitant upregulation of CD54 (ICAM-1). Three of the five group I BL
lines exhibited rapid downregulation of the hallmark CD77 surface
antigen, which, for L3055 cells, was maintained for at least 12 days
after 72 hours of incubation with CD40L. With the exception of BL2, all
group I BL lines were induced to express CD40 homodimers on
CD40-stimulation, whereas only monomers were detected in unstimulated
cells. Experiments using CD40-transfected Rat-1 fibroblasts showed that
the ability to signal for dimer formation required Thr234
of CD40. For L3055 and BL29 cells, an initial 72 hours of exposure to
CD40L resulted in the maintenance of homodimers for up to 14 and 10 days, respectively. There was a close correlation between the induction
and duration of CD40 homodimers and the appearance of Bcl-2. For L3055
cells, which are sensitive to apoptosis-induction on BCR-engagement,
exposure to CD40L for 72 hours was found to provide considerable
protection from anti-IgM, which was still significant to 20 days. The
implications of such sustained effects on relatively short-term
exposure of tumor B cells to CD40L are discussed.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
BURKITT LYMPHOMA (BL) cell lines have
been established from biopsy material of patients from both endemic and
nonendemic regions. Epstein-Barr virus (EBV) is invariably associated
with the disease in endemic areas but is harbored by the cells in only
a minority of cases from nonendemic regions.1 Depending on
the expression of specific latent EBV genes, established BL lines
display a heterogeneous phenotype. Only EBNA1 protein can be detected
in recently established, biopsy-like BL lines, whereas EBNAs 1 and 2 and LMP1 and 2 can often be detected in long-term EBV+ve BL
lines that have undergone genotypic drift in culture.2,3 Retention of the original biopsy-like phenotype in
EBV ve lines or in EBV+ve lines that have
remained faithful to the starting genotype is identified from the
following expression of cell surface markers: CD10+,
CD77+, CD23, CD39,
CD44, LFA-1,
LFA-3, and ICAM-1. This profile
is remarkably similar to that associated with normal B cells found in
germinal centers (GC), and these lines are categorized as group
I.2 Where genotypic drift occurs, there is a corresponding change in cell surface markers with the acquisition of a
lymphoblastoid-like phenotype characterized as CD10,
CD77, CD23+, CD39+,
CD44+, LFA-1+, LFA-3+, and
ICAM-1+. BL lines with fully blown lymphoblastoid
characteristics are termed group III.2
In addition to expressing the full complement of EBV latent genes and
the surface marker changes, group III lines, unlike group I lines (or
normal GC B cells), express the antiapoptotic proto-oncogene
bcl-2.3 Either by comparing group I with group III
lines directly or by introducing bcl-2 into group I cells by
gene transfection, it has been shown that the Bcl-2 protein can afford
substantial protection of BL cells from a variety of otherwise
apoptotic stimuli. Particularly for early passaged group I lines, the
most physiological of these apoptotic triggers is provided by the
cross-linking of sIgM with anti-IgM antibodies.3-5 Stimulation through CD40 with either anti-CD40 monoclonal antibody (MoAb) or with CD40L protects, at least in the short-term, both group I
BL cells and GC B cells from apoptosis.5 For both cell types, CD40-mediated protection from apoptosis can be independent of
Bcl-2 induction as long as cells can be persuaded to remain in active
cell-cycle. It has been argued that, once out-of-cycle, Bcl-2
expression becomes a prerequisite to continued survival.5
CD40, a member of the tumor necrosis factor receptor
(TNF-R) superfamily, appears to play a central role in
B-cell responses to TD antigens impinging on B-cell activation, growth,
differentiation, isotype-switching, and survival at a number of key
stages.6 It shares extracellular domain homology with other
members of the TNF-R family, containing four homologous cysteine-rich
extracellular domains. Engagement of CD40 by CD40L expressed on the
surface of activated CD4+ T cells is believed to result in
receptor trimerization and is thought to be a prerequisite event
leading to signal transduction. Interestingly, preformed CD40
homodimers have been observed on a number of B-cell types, including BL
Raji cells and normal B cells, although they appear absent on carcinoma
lines or EBV-transformed cells.7 Although no function has
as yet been assigned to the CD40 dimers, it could be envisaged that,
given the presumed importance of receptor oligomerization, they may
serve to modify signaling requirements or outcomes on cells where they
are found.
Previous investigations into the consequences of engaging CD40 on BL
cells in vitro have tended to concentrate solely on the maintenance of
viability when the cells are confronted with apoptotic signals and,
moreover, have usually looked only at effects operative in the
short-term (<72 hours).8 Somewhat paradoxically, in contrast to the well-documented antiapoptotic actions observed in
vitro, studies on human B-cell lymphomas developing in SCID mice have
indicated that engagement of CD40 can provide protection from tumor
development.9 In light of these results, we felt that a
more detailed analysis of the response of group I BL cells to CD40L was
warranted, with an examination of both short-term and long-term
influences of CD40-ligation on morphology, phenotype, growth
characteristics, and protection from apoptosis-induction. Corresponding
analysis on the state of CD40 receptor dimerization together with the
dynamics of Bcl-2 expression was also performed. Given the reported
ability of the TH2-derived cytokine interleukin-4 (IL-4) to
augment CD40-dependent change at several stages of normal B-cell
development,10-13 comparisons were made in the responses of
group I BL cells when confronted with CD40L both in the presence and
absence of this cytokine.
| |
MATERIALS AND METHODS |
Cell lines, antibodies, and reagents.
BL cell lines L3055 (EBVve), Louckes
(EBVve), BL2 (EBVve), Elijah
(EBV+ve), and BL29 (EBV+ve) expressing a group
I phenotype, as previously described by Rowe et al,14-16
were used in this study. L3055 cells were from early passage, defined
as cells that had not exceeded an in vitro passage number of 60 from
initial establishment of the line from BL-biopsy tissue. Reference to
late passage cells, defined as lines that had exceeded an in vitro
passage number of 100, applies to the four other BL lines in this
study. Late passage group I BL cells had different passage numbers,
with the lowest being BL29 < BL2 < Elijah < Louckes. All BL cell
lines were assessed by fluorescence-activated cell sorting
(FACS) analysis before study with a panel of group I BL
cell markers to ensure that cells retained the original
phenotype.14-17 Cells were maintained in continuous culture
with RPMI 1640 supplemented with 10% prescreened fetal calf serum
(FCS; Bio-Whittacker, Wokingham, UK), 5,000 IU/mL penicillin, 5 mg/mL
streptomycin (GIBCO/BRL, Paisley, UK), and 200 mmol/L glutamine
(GIBCO/BRL). Rat-1 fibroblasts transfected with wild-type, full-length
human CD40 were as previously described.18 In addition,
Rat-I cells containing a Thr(234)-Ala mutation of CD40 were also
generated. Briefly, this substitution was introduced by site-directed
mutagenesis using the QuickChange site-directed mutagenesis kit of
Stratagene (Cambridge, UK) and the mutated oligonucleotide primer
5 -CAC TGC TGC TCC AGT GCA GGA GGC TTT ACA TGG ATG-3 and
its complimentary oligo. The presence of the above mutation was
verified by sequencing. Recombinant human IL-4 was purchased from R&D
Systems Ltd (Oxford, UK). Recombinant soluble trimeric CD40 ligand was
generated as described previously.8
The MoAbs used for phenotypic studies were obtained from the following
sources: phycoerythrin (PE)- and fluorescein (FITC)-conjugated negative
control isotype-matched Ab of irrelevant specificity, FITC-conjugated
anti-CD10, anti-CD23, anti-IgM, and PE-conjugated anti-CD20 were all
purchased from Dako Ltd (High Wycombe, UK). FITC-conjugated anti-CD40
EA5 MoAb and PE-conjugated anti-CD38 were purchased from Serotec and
Becton Dickinson (Cowley, UK), respectively. Ascitic fluid containing
rat IgM anti-CD77 (38.13) MoAb was a gift from Dr J. Weils (Institut
Gustav-Roussy, Villejuif, France). Mouse anti-CD44 (BU52), anti-CD11a
(BU17), anti-CD11b (OKM-1), anti-CD11c (BU15), and anti-CD54 (BU74)
MoAb were purified by diethyl aminoethyl (DEAE) anion
exchange from hybridomas grown in the Department of Immunology,
University of Birmingham (Birmingham, UK); these MoAbs were conjugated
to FITC by standard proceedures.
Immunodetection of CD40 and Bcl-2.
Cells (1 × 107) were lysed in 0.2 mL of 1% Triton
X-100 lysis buffer (25 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 1 mmol/L
EDTA, 50 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L
phenylmethyl sulfonyl fluoride [PMSF], 1 µg/mL
aprotinin, and 1 µg/mL leupeptin). Protein concentrations of each
sample were determined. After adding 40 µL of 5× gel sample
buffer (500 mmol/L Tris [pH 6.8], 12% sodium dodecyl sulfate
[SDS], 25% glycerol, 5 µmol/L EDTA, 0.01% bromophenol blue)
supplemented with 10% 2-mercaptoethanol for Bcl-2 lysates, the samples
were boiled for 5 minutes. Samples were fractionated after loading
equal protein concentrations onto 10% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) for CD40 blots and 12% SDS-PAGE for Bcl-2
blots. Proteins were transferred to Immobilon P membranes (Millipore,
Watford, UK) using standard techniques, and specific proteins were
detected using either mouse antihuman-Bcl-2 MoAb (100; Santa Cruz,
Santa Cruz, CA) or mouse antihuman-CD40 MoAb (G28.5).
Immunoblots were visualized using the ECL kit (Amersham International,
Amersham, UK) according to the manufacturer's instructions.
Measurement of apoptosis.
Cell samples (4 × 105 cells) were cytocentrifuged in
duplicate onto glass slides at 250 rpm for 3 minutes. After air-drying and methanol-fixing, the slides were stained using the Romanoski method.19 Briefly, slides were stained in 1/3 dilution of
Jenners (BDH/Merck, Lutterworth, UK) solution followed by 1/10 dilution of Giemsa (BDH/Merck) solution. Slides were air-dried and mounted using
DPX mountant (BDH/Merck). Slides were counted blind, and cells were
categorized according to apoptotic, viable, or lysed (late
apoptotic/necrotic) morphology.
DNA synthesis.
DNA synthesis in 105 cells/200 µL was determined by the
incorporation of [3H]thymidine ([3H]Thy;
Amersham) at 1 µCi/50 µL of culture medium/well for 5 hours after
24 hours of culture. Samples were cultured and assayed in triplicate
before harvesting using a Skatron cell harvester (Skatron, Newmarket,
UK).
Homotypic adhesion.
Cells were cultured in 24-well plates under the conditions indicated in
the text. Cell clumping was assessed under an inverted microscope
(Olympus LK2; Olympus, London, UK), and photographs were taken using a
mounted Nikon F302 camera (Nikon, Kingston-upon-Thames, UK) at the time
points indicated in the text.
Analysis of B-cell surface phenotype.
Cells were harvested after culture under the conditions indicated in
the text. Cells were washed once in phosphate-buffered saline
(PBS) with 0.1% bovine serum albumin (BSA) and 0.01% sodium azide
(FACS buffer) before direct or indirect immunofluorescence analysis was
performed. Briefly, cells were labeled by 30 minutes of incubation on
ice with either MoAb specific for cell surface markers conjugated
directly to fluorochromes or by an unconjugated MoAb that was detected
by an additional second-stage incubation with fluorochrome-conjugated
antimouse Ig Ab. Cells labeled either directly or indirectly were
washed once and resuspended in FACS buffer. Flow cytometry data were
obtained using a FACScan flow cytometer (Beckton Dickinson, Mountain
View, CA). Viable cells were gated according to forward (FSC) and
side (SSC) light scatter settings. Subsequent data were processed and
analyzed using the Cell Quest program (Becton Dickinson).
| |
RESULTS |
Establishment of CD40L dose-response kinetics for phenotypic and
functional change in group I BL cells.
First of all, the dose-response kinetics on the influence of soluble
CD40L trimer on three of the basic parameters analyzed in this study
was established for the EBVve L3055 group I BL cell
line: (1) the induction of homotypic adhesions; (2) the induction of
phenotypic change, assessed here by IL-4-dependent CD23 expression;
and (3) the capacity to reverse anti-IgM (BU1)-mediated inhibition of
[3H]-Thymidine incorporation, a measure of growth arrest
that preludes surface IgM-driven apoptosis in these cells.8
For the induction of all three parameters, a plateau effect was reached
with 1 µg/mL of CD40L (Table 1) and this
was the concentration chosen for all further experiments detailed in
this study. It is worthy to note that the induction of homotypic
adhesions was still being achieved, albeit suboptimally, with CD40L
when present at levels as low as 0.008 µg/mL. By
contrast, no significant change was observed in the other two
parameters at levels less than 0.2 µg/mL of CD40L.
CD40L-mediated induction of homotypic adhesions in group I BL cells.
With the exception of BL2 and Elijah, large homotypic aggregates were
observed in both early and late passage group I BL cells treated for 72 hours with CD40L (Fig 1A). CD40L-induced
cell clustering was enhanced by the presence of IL-4 (20 ng/mL) in all
three responding lines (data not detailed). Where, having washed out
the stimuli at 72 hours, cells were maintained in culture for a total
of 10 days, BL29 cells continued to display cell clusters comparable in
size and frequency to those observed after the initial 72 hous of
stimulation in the presence of CD40L, with or without IL-4 (Fig 1B).
Although for L3055 cells the size of cell clusters was considerably
reduced at 10 days, the cells were still forming large numbers of small
loose cell aggregates that were absent in cultures that had not been
precultured with CD40L (to effectively show small cell clusters,
CD40L-treated L3055 cells are displayed at a lower magnification; Fig
1B). By contrast, prestimulated Louckes cells at day 10 failed to show
levels of cell clumping above those observed in untreated controls
(data not detailed). It is worthy of note that, whereas the late
passage group I BL cells Elijah, BL29, and Louckes displayed some
background level of cell clustering, treatment with CD40L increased
this level of clustering in all except the Elijah line (Fig 1A).
Although Elijah cells failed to demonstrate cluster formation on
culture with CD40L, they did respond to CD40-stimulation by forming
large cells with pseudopodia-like extensions (Fig 1A). The formation of
large cells could also be seen in BL29 cells, although these cells
tended to extend fewer pseudopodia (Fig 1A). With the exception of
Louckes, in which a small enhancement of aggregate formation was
observed, culture with IL-4 alone failed to increase cell clustering in
the group I BL lines (data not detailed).
View larger version (97K):
[in this window]
[in a new window]
| Fig 1.
Phase contrast photomicrographs of BL cell lines cultured
at 37°C for 72 hours either alone (control) or with CD40L (1 µg/mL). Cell clumping was observed either after (A) the initial
72-hour treatment time or (B) after a total 10 days of culture, which
included the initial 72-hour treatment. For all experiments in which
cells were pretreated with reagents, cells at 5 × 105/mL
received an initial culture with CD40L (with or without IL-4) for time
stated and were then washed free of stimulants before replating at 5 × 105/mL in fresh culture medium in the absence of
stimulants. The scale bar represents 50 µm.
|
|
To identify surface molecules that may be involved in CD40-dependent
clustering of group I BL cells, the expression of a panel of adhesion
molecules was investigated on L3055 cells after the various treatments.
At 24 hours, there was a slight increase in CD11a (LFA-1) and a
substantial increase in CD54 (ICAM-1) expression on L3055 cells
cultured with CD40L alone. The addition of IL-4 did not further enhance
the CD40L-induced increase in expression of either of these molecules
(Fig 2A). After 72 hours of stimulation with CD40L alone, no further large increases in CD11a or CD54 expression were observed above those seen at 24 hours (Fig 2B). L3055
cells stimulated with CD40L alone for either 24 or 72 hours failed to
show increases in either CD11b (Mac-1) or CD11c (p150/95) expression
compared with untreated control cells (Fig 2). However, after 72 hours
of stimulation with CD40L in combination with IL-4, small but
reproducible increases in CD11b and CD11c surface expression as well as
large increases in CD11a and CD54 were observed (Fig 2B). Stimulation
with IL-4 alone for either 24 or 72 hours did not induce
any detectable changes in expression of all four adhesion molecules
studied (Fig 2).
View larger version (42K):
[in this window]
[in a new window]
| Fig 2.
L3055 cells were stained with MoAb against adhesion
molecules CD11a, b, c, and CD54 after (A) 24 hours or (B) 72 hours of
treatment with (i) CD40L alone, (ii) IL-4 alone, or (iii) CD40L with
IL-4. Untreated control cells are shown for comparison as a
superimposed thin line on each histogram.
|
|
Phenotypic changes in group I BL cells stimulated with CD40L.
The capacity of CD40 to trigger phenotypic differentiation in early and
late passage BL cell lines was investigated. Early and late passage
group I BL cells were cultured for 72 hours with the stimulants
indicated. Upon completion of the 72 hours of stimulation, cells were
washed free of the factors and further maintained in culture for the
periods indicated (which includes the initial 72-hour stimulation
period) before removing aliquots for the analysis of cell surface
phenotype. Preliminary experiments showed that, although CD40
stimulation induced a relatively early pattern of differentiation in
group I BL cells, the effects were more pronounced after day 5 (including the 72-hour stimulation time); therefore, in this study, day
5 was chosen to show the immediate effects of CD40 on BL cell
differentiation. Special attention is given to CD77, a hallmark group I
surface marker that is lost when BL cells progress to a group III
phenotype on long-term culture.2
Figure 3 shows FACScan analysis of early
passage L3055 cell surface phenotypes after 5 days (including the
72-hour preculture period). Every group I BL cell line tested in this
study showed distinct populations of cells when observed on forward
(FSC) and side (SSC) scatter dot plots (data not detailed). The large,
nongranular cell populations represented viable cells, which were gated
in every cell line before analysis of cell surface marker expression. Interestingly, group I BL cells stimulated with either CD40L alone or
CD40L with IL-4, but not with IL-4 alone, showed increases in cell size
and granularity that were detected by a shift in FSC and SSC dot plot
profiles (data not detailed). In control cultures, a bimodal
distribution of CD77 within the L3055 population with the presence of
both CD77high and CD77low expressing cells was
seen (Fig 3). Stimulation with CD40L induced a rapid
downregulation in CD77 expression, resulting in the formation of a
homogenous CD77low cell population (Fig 3). IL-4, although
appearing to have no affect by itself (data not detailed), augmented
the CD40-induced downregulation of CD77 expression (Fig 3). Two of the
late passage BL lines, Louckes and BL2, also downregulated CD77
expression in response to stimulation through CD40 (Fig 3), with this
effect again being enhanced by the presence of IL-4. Interestingly, for both Louckes and BL2 cells, culture with IL-4 alone could induce the
downregulation of CD77 expression (data not detailed).
View larger version (49K):
[in this window]
[in a new window]
| Fig 3.
BL cells were stained with MoAb against CD77 after 72 hours of stimulation with CD40L alone. Additional staining with MoAb
against CD77, CD23, and CD40 was performed after 72 hours of
stimulation with CD40L in combination with IL-4. FACS analysis was
performed after 5 days of culture (including the initial 72 hours of
stimulation). Untreated control cells are shown for comparison as a
superimposed thin line on each histogram.
|
|
L3055 cells expressed a CD77low phenotype for at least 12 days after stimulation with CD40L either with or without IL-4 (data not
detailed as day-12 FACScan histograms were essentially identical to
those at day 5 as shown in Fig 3); indeed, CD77 expression remained low
beyond the time scale of the experiment (>15 days). This finding is
in contrast to BL2 and Louckes cells, which maintained low level CD77
expression for 5 days on a 72-hour prestimulation, but by day 12, CD77
expression had returned to normal or elevated levels, respectively
(data not detailed). Changes in other cell surface markers were
detected on L3055 cells by day 5, with a transient increase observed in
the expression of CD40 itself after an initial 72 hours of culture with
CD40L in combination with IL-4 (Fig 3). Small but reproducible
increases in CD23 expression (a hallmark group III marker) were also
observed on culture with CD40L, with or without IL-4 (Fig 3).
Elijah cells failed to display any changes in cell surface phenotype
after culture with CD40L alone, at either 5 or 12 days with the initial
72 hours of stimulation (data not detailed). However, Elijah cells did
display a transient 5-day increase in CD23 and CD40 expression when
stimulated with CD40L in combination with IL-4 (Fig 3). These changes
could not be detected at 12 days (data not detailed).
Louckes cells displayed a pattern of change in cell surface phenotype
similar to that observed in L3055 cells (Fig 3), except that a more
marked increase in CD23 expression was observed in cells treated with
either CD40L alone (data not detailed) or CD40L in combination with
IL-4 (Fig 3). Decreases (that returned to normal levels within 12 days)
were also observed in the expression of CD40 when Louckes cells were
treated with CD40L alone (data not detailed) or CD40L in combination
with IL-4 (Fig 3). For BL2 cells, differences in surface CD40
expression were observed, depending on whether cells were previously
stimulated with CD40L alone or CD40L in combination with IL-4. Culture
with CD40L alone induced a large reduction in surface CD40 expression
(data not detailed), whereas the combination of IL-4 and CD40L resulted
in an increase in surface CD40 expression (Fig 3). Furthermore,
stimulation with CD40L alone failed to induce any changes in CD23
expression, whereas culture with IL-4 and CD40L resulted in the
increased surface expression of this molecule (Fig 3). The changes in
BL2 cell phenotype mediated by either CD40L alone or CD40L in
combination with IL-4 were also transient with levels returning to
normal by day 12 (data not detailed).
Stimulation through CD40 induces growth arrest in group I BL cells.
Next, we assessed both the short-term and long-term influences of
exposure to CD40L on DNA synthesis in group I BL lines. Culture with
CD40L induced growth arrest in L3055 cells within 48 hours (day 2) when
compared with untreated controls (Fig 4). The period for which L3055 cells then remained in growth arrest was
dependent on the length of time the cells had been prestimulated with
CD40L. Thus, L3055 cells stimulated through CD40 for 24 hours remained
in growth arrest for a maximum of 5 days (Fig 4A); by extending the
stimulation time to 72 hours, the duration of growth arrest was
increased to 7 to 9 days (Fig 4B). Furthermore, cells stimulated with
CD40L for 72 hours displayed a significant (P < .0001)
decrease in DNA synthesis for up to 7 days compared with cells treated
for just 24 hours (Fig 4). Reduction in cell proliferation was not
accompanied by an increase in the rate of apoptosis occurring in these
cultures as judged by examination of Romanowski-stained cytocentrifuge
preparations (data not detailed).
View larger version (18K):
[in this window]
[in a new window]
| Fig 4.
L3055 cells were incubated with CD40L alone, IL-4 alone,
or CD40L with IL-4 for either (A) 24 or (B) 72 hours. Aliquots were
removed at the times indicated and cell growth was evaluated by
incorporation of [3H]-Thy. Incorporation of
[3H]-Thy for each treatment is expressed as a percentage
of untreated control cultures. Data are presented as the mean with
standard deviation from three separate experiments. The range of
radioactivity incorporated in control (untreated) cultures for the
three experiments was 7,698 to 11,218 cpm. Treatment with either CD40L
alone or CD40L with IL-4 significantly (P < .0001)
decreases proliferation of L3055 cells where indicated (*).
|
|
For all four late passage BL lines, a significant (P < .0001)
reduction in DNA synthesis was observed in response to stimulation through CD40 via CD40L (Fig 5). Cell
proliferation was reduced within 24 hours (day 1) and with a 72-hour
exposure remained lower than in untreated cells to 4 days; however, by
day 7, proliferation had returned to normal levels (Fig 5). The
addition of IL-4 to CD40L-stimulated cultures induced further
significant (P < .0001) reductions in the DNA synthesis
occurring in Elijah and Louckes cells at 24 hours, although this effect
had disappeared in Louckes cells by day 4 (Fig 5). In contrast, for BL2
cells, the presence of IL-4 had a counteracting negative effect on
growth arrest where, at day 4, DNA synthesis was significantly
(P < .0001) higher in cells treated with both IL-4 and CD40L
compared with cells treated with CD40L alone (Fig 5). Interestingly,
BL2 cells also differed from both Elijah and Louckes in that
CD40L-induced growth arrest was not at its maximal until 4 days into
culture (Fig 5). The effects of CD40L on BL29 cells with regard to DNA
synthesis were less pronounced, where a modest (~30%) but still
significant (P < .0001) reduction was observed at 24 hours
that was maintained to day 4 (Fig 5). This reduction was not enhanced
by the presence of IL-4 (Fig 5).
View larger version (36K):
[in this window]
[in a new window]
| Fig 5.
Four late passage BL cell lines were incubated with CD40L
alone, IL-4 alone, or CD40L with IL-4 for 72 hours. Aliquots were
removed at the times indicated and cell growth was evaluated by
incorporation of [3H]-Thy, which is expressed as a
percentage of untreated control cultures. Data are presented as the
mean with standard deviation from three separate experiments. The range
of radioactivity incorporated in control (untreated) cultures in these
experiments for each cell line was: Elijah, 12,433 to 23,083 cpm;
BL2, 14,053 to 22,767 cpm; BL29, 19,353 to 24,662 cpm;
and Louckes, 13,471 to 23,622 cpm. Treatment with CD40L
significantly (P < .0001) reduces proliferation of all late
passage BL cell lines where indicated (*).
|
|
CD40 homodimer formation corresponds to an upregulation in the
expression of Bcl-2 in group I BL cells.
An upregulation in the expression of Bcl-2 was detected in all five BL
B-cell lines after stimulation with CD40L; with one exception (BL2),
this was accompanied by the formation of CD40 homodimers
(Figs 6 and 7).
Furthermore, in L3055 cells, CD40L induced low but detectable levels of
Bcl-2 protein within 24 hours (Fig 7). Both the levels of Bcl-2
expression and the formation of CD40 homodimers were maximal after 72 hours, and the presence of IL-4 enhanced both CD40 dimer formation and
Bcl-2 expression in L3055 cells (Fig 7). Stimulation with CD40L
resulted in an increase in Bcl-2 expression, which was sustained for up
to 14 days in L3055 cells (Fig 7) and up to 10 days in BL29 cells (Fig 6). Interestingly, IL-4 did not enhance Bcl-2 protein expression when
added with CD40L in all the late passage group I BL cells investigated
(Fig 6). Furthermore, culture with IL-4 alone failed to upregulate
Bcl-2 protein expression in both early and late passage BL cells (Figs
6 and 7). Late passage group I BL B cells showed a diminished ability
to maintain long-term increases in the levels of both Bcl-2 and CD40
homodimer expression. Indeed, with the exception of BL29, CD40
homodimers and Bcl-2 protein levels were either absent or had returned
to normal levels within 5 days in all late passage group I BL cells
(Fig 6).
View larger version (36K):
[in this window]
[in a new window]
| Fig 6.
BL cell lines were incubated either alone or with CD40L,
IL-4, or CD40L with IL-4 for 72 hours. Cell lysates were prepared at
either 5 or 10 days (including the initial 72-hour stimulation time),
and equal amounts of total cellular protein was resolved on 10%
SDS-PAGE. Western blotting was performed to determine the presence of
CD40 homodimers using anti-CD40 MoAb G28.5. In addition, the same
lysates were resolved on 12.5% SDS-PAGE, and Western blotting was
performed to detect the presence of Bcl-2 using anti-Bcl-2 MoAb 100. The numbers to the right of each panel set indicate the positions of
molecular weight markers.
|
|
View larger version (34K):
[in this window]
[in a new window]
| Fig 7.
L3055 cells were incubated either alone or with CD40L,
IL-4, or CD40L with IL-4 for 72 hours. During the 72-hour stimulation
time, the presence of CD40 homodimers and Bcl-2 was determined at 24, 48, and 72 hours. Immunoblots were also performed after cells were
incubated with the same additives for 72 hours, followed by an extended
14-day culture (including the initial 72-hour stimulation time) in
normal complete medium. Cell lysates were prepared and equal amounts of
total cellular protein resolved on either 10% SDS-PAGE for CD40
homodimers or 12.5% SDS-PAGE for Bcl-2. Western blots were performed
and CD40 homodimers were detected used anti-CD40 MoAb G28.5. Bcl-2 was
detected using anti-Bcl-2 MoAb 100. The numbers to the right of each
panel group indicate the positions of molecular weight markers.
|
|
In CD40-stimulated L3055 cells, the slow induction of Bcl-2 protein
expression mirrored the gradual increase in CD40 homodimer formation.
Both Bcl-2 protein and CD40 homodimers could be detected at low levels
for up to 14 days in these cells (Fig 7). Late passage group I BL cells
showed a similar pattern of Bcl-2 and CD40 homodimer expression (Fig
6), although CD40 homodimers and Bcl-2 were detected in the individual
cell lines for different lengths of time. Brief increases in CD40
homodimer expression were observed in Elijah and Louckes cells, each
lasting no more than 5 days; again, this was also seen with Bcl-2,
which also returned to normal levels after a transient 5-day increase
in expression (Fig 6). Louckes cells displayed an apparent increase in
Bcl-2 expression in untreated control cells after 10 days compared with
5 days, but this could be due to small variations in determining
protein concentrations of cell lysates. It should be noted that
observed differences in Bcl-2 expression are based on comparisons
between cells cultured with the additions indicated and untreated
control cells analyzed during the same time point. BL29 cells displayed
a similar pattern in the upregulation of CD40 homodimer expression, as
was seen in early passage L3055 cells. High levels of both homodimeric CD40 and Bcl-2 were detected in BL29 cells, and both CD40 homodimers and Bcl-2 expression levels remained elevated for a least 10 days (Fig
6). Despite the consistent production of CD40 homodimers in all other
CD40-stimulated group I BL cell lines, BL2 cells remained the
exception, where the failure to detect CD40 homodimers was reflected by
an upregulation in the expression of Bcl-2 protein, albeit a very
modest one (Fig 6).
The inability to detect CD40 homodimers within 24 hours suggests that
their formation is not simply a consequence of CD40L promoting direct
aggregation of CD40 into dimeric complexes on the cell surface (Fig 7).
This was further supported by the observation that Elijah cells
expressed large amounts of homodimeric CD40 only when treated with both
CD40L and IL-4 (Fig 6) and the fact that the BL2 cell line failed to
produce CD40 homodimers under identical conditions of culture with
CD40L, with or without IL-4 (Fig 6).
To gain some insights into the mechanism behind CD40-mediated dimer
formation, the Rat-1 fibroblast cell line transfected with wild-type or
mutated human CD40 was studied. Fibroblasts expressing wild-type CD40
could be induced to form homodimers after stimulation with CD40L, as
seen with the group I BL lines (Fig 8).
However, fibroblasts transfected with human CD40 mutated to substitute
Ala at residue Thr234 were unable to signal through CD40 to
produce high molecular weight CD40 immunoreactive material, including
that corresponding to homodimers (Fig 8). This finding suggests that
cells can only produce CD40 homodimers by transducing a signal through
CD40 and that the essential signaling residue Thr234 is an
important component of this pathway. These data are also consistent
with the notion that CD40 homodimers were not produced merely as a
result of nonspecific aggregation of the CD40 protein.
View larger version (26K):
[in this window]
[in a new window]
| Fig 8.
Rat-1 fibroblasts were transfected with either wild-type
human CD40 (Rat-1wCD40) or mutant human CD40 (Rat-1mCD40). Stable
transfectants were stimulated with CD40L for 72 hours. Cell lysates
were prepared and equal amounts of total cellular protein was resolved
on 10% SDS-PAGE. The presence of CD40 homodimers on immunoblots was
determined using anti-CD40 MoAb G28.5. The numbers to the right of each
panel set indicate the positions of molecular weight markers.
|
|
Exposure of L3055 cells to CD40L confers sustained protection from
anti-IgM-induced apoptosis.
Early passage L3055 cells are sensitive to apoptosis driven by soluble
anti-IgM, whereas later passage group I BL lines are relatively
resistant. L3055 was thus chosen for a detailed analysis of the
influence of short-term exposure to CD40L on the maintenance of
protection from this apoptotic signal. L3055 cells were incubated after
various times with anti-IgM MoAb (BU1) for 24 hours after an initial 72 hours of stimulation with either CD40L alone, IL-4 alone, or CD40L with
IL-4. Apoptosis was assessed by examining Romanowski-stained
cytocentrifuge preparations and enumerating the percentage of cells
possessing classical morphological features of apoptosis compared with
viable, intact cells. In addition, DNA synthesis was assessed by
[3H]Thy incorporation in response to cross-linking sIgM.
The validity of these techniques as measures of ant-IgM-induced
apoptosis in L3055 cells and their detailed assessment has been fully
documented elsewhere.8
Cells were again stimulated for 72 hours with the additives indicated,
followed by maintenance in continuous culture. [3H]Thy
incorporation was assessed at 14 and 20 days (which included the
initial 72-hour stimulation time) after 24 hours of incubation with
BU1. To determine the strength of survival signals delivered through
CD40 by CD40L, BU1 was used at a range of concentrations (0.01 to 10 µg/mL) for both assessment of apoptosis and [3H]Thy
incorporation in L3055 cells.
L3055 cells treated with CD40L displayed increased resistance to
anti-IgM-induced apoptosis within 72 hours compared with untreated
control cells (Fig 9A). Cells previously
stimulated with either CD40L alone or CD40L in combination with IL-4
retained a degree of resistance to apoptosis induced by anti-IgM for at least 14 days (Fig 9B). The most potent antiapoptotic effects induced
by CD40L were observed at 72 hours, where culture with CD40L conferred
protection against anti-IgM-induced apoptosis even when added at
concentrations of 10 µg/mL (Fig 9A). The addition of IL-4 in
combination with CD40L to L3055 cells appeared to have a synergistic
effect regarding protection, whereas L3055 cell viability in response
to anti-IgM increased approximately 40% above that when only CD40L was
providing protection (Fig 9A). IL-4 alone did not alter
L3055 cell viability compared with untreated control cells on the
addition of BU1 (Fig 9). At day 14, where cells had received an earlier
72 hours of exposure to either CD40L alone or CD40L in combination with
IL-4, they still displayed some degree of resistance to the effects of
BU1 when used at a concentration of 1 µg/mL (Fig 9B). Resistance to
apoptosis induced by treatment with 1 µg/mL of BU1 after 14 days was
enhanced in L3055 cells that had been stimulated previously with IL-4
in combination with CD40L compared with cells stimulated with CD40L
alone. No enhanced resistance to BU1 at 10 µg/mL was evident at 14 days with any of the treatments (Fig 9B).
View larger version (21K):
[in this window]
[in a new window]
| Fig 9.
L3055 cells were incubated either alone or with CD40L,
IL-4, or CD40L with IL-4 for 72 hours. Cells were then maintained in
complete medium for up to 14 days (including the initial 72 hours of
stimulation). During this time at (A) 3 and (B) 14 days of resistance
to apoptosis induced by anti-IgM MoAb, BU1 was assessed by counting
apoptotic cells from triplicate cultures on Romanoski-stained
cytocentrifuge preparations. Data are representative of two separate
experiments.
|
|
As mentioned above, it has been established that apoptosis promoted by
anti-IgM in L3055 cells is preceded by growth arrest as registered by a
decrease in the rate of DNA synthesis.8 In the present
study, it was seen that culture of L3055 cells with CD40L alone or
CD40L in combination with IL-4 engendered a high degree of resistance
to BU1 in this regard when compared with untreated control cells
(Fig 10). At day 14, where cells had been
cultured with CD40L for the first 72 hours and then had the stimulants
washed out, a significant (P < .0001) resistance to BU1-induced growth arrest was maintained even when they were confronted with high levels (10 µg/mL) of the antibody (Fig 10A). Whereas by day
20 the early 72-hour preculture with CD40L no longer appeared to be
protecting from 10 µg/mL of BU1 with regard to its ability to reduce
DNA synthesis, a significant degree (P < .0001) of protection was still being afforded against BU1 used at a lower dose of 1 µg/mL
(Fig 10B). The addition of IL-4 was not seen to confer any significant
additional increase in the resistance of L3055 cells to BU1 as measured
by reductions in DNA synthesis over and above that observed in cells
treated with CD40L alone at either 14 or 20 days (Fig 10).
View larger version (21K):
[in this window]
[in a new window]
| Fig 10.
L3055 cells were incubated either alone or with CD40L,
IL-4, or CD40L with IL-4 for 72 hours. Stimulants were washed out and
cells were maintained in complete medium for up to 20 days (including
the initial 72 hours of stimulation). [3H]-Thy
incorporation was assessed after a 24 hours of incubation
with anti-IgM MoAb BU1 at (A) day 14 and (B) day 20. The percentage of
incorporation [3H]-Thy for each treatment was determined
from untreated control cultures, which ranged from 4,674 to 8,522 cpm
in three experiments. Data are presented as the mean with standard
deviation from these three separate experiments. Treatment with either
CD40L alone or CD40L in combination with IL-4 significantly (P < .0001) increases L3055 cell proliferation after stimulation with
anti-IgM MoAb BU1 where indicated (*).
|
|
| |
DISCUSSION |
This study investigated the induction and maintenance of a variety of
phenotypic, functional, and molecular responses promoted in group I BL
cells by their stimulation through CD40. Of particular note was the
finding that exposure to CD40L not only protects group I BL cells from
surface Ig-promoted apoptosis, but also directly induces growth arrest;
furthermore, both outcomes could be relatively long-lived even after
the CD40 signal was withdrawn. Another novel finding was that
stimulation through CD40 induces the formation of CD40 homodimers in
group I BL cells and that this was concomitant with the induction or
increased expression of Bcl-2 protein, both of which could again be
sustained even after removal of the CD40 signal. Of the phenotypic
changes that were seen to accompany these events, which included the
induction of homotypic adhesions, the most profound was the rapid and
long-term downregulation of CD77, the hallmark group I BL marker.
Simultaneous addition of IL-4 to CD40-stimulated cultures augmented
many of these responses and, in some instances, IL-4 and CD40 signaling demonstrated marked synergy in determining both the intensity and
duration of the responses.
As yet, no functional significance has been attributed to the formation
of CD40 homodimers that has been detected on a number of B-cell types.
Previous studies have shown that Raji cell lysates treated with
alkylating agents such as N-ethylmaleimide or iodoacetimide fail to disrupt CD40 homodimers. This suggests that CD40 homodimers may
be formed in vivo and are not produced through nonspecific sulphydryl
interactions on CD40 monomers. This observation provides an important
insight into the way CD40 is expressed, particularly given the
requirement for CD40 to be cross-linked before receptor signaling can
occur. It is therefore tempting to speculate that CD40 homodimers can
enhance B-cell responses upon engagement with CD40L. This may be
achieved by reducing the requirement for large amounts of CD40L, either
in soluble or membrane-bound forms. It is possible that CD40 homodimers
are actively produced either on the cell surface or synthesized
intracellularly. From our present study, the slow induction of CD40
homodimers by CD40L leading to maximal homodimer expression at 72 hours
supports the notion that CD40 homodimers are produced by active protein
synthesis in vivo. Importantly, CD40L was unable to induce CD40
homodimer formation in Rat-1 fibroblasts transfected with CD40
expressing a point mutation at the essential signaling residue
Thr234, whereas the same cells transfected with wild-type
human CD40 could be induced to express homodimers in a manner analogous
to that observed with the group I BL lines. These findings strongly suggest that CD40 homodimers can only be formed in cells that have the
capacity to signal intracellularly through CD40. They also point to a
role for TRAF adaptor molecules in this process, because the binding of
some of them to the cytoplasmic domain of CD40 has been shown to
involve residue Thr234 of the cytoplasmic
domain.20 These findings potentially provide a mechanism
for the rewiring of CD40 during B-cell differentiation as has been
proposed previously.21 Further attempts to dissect the
processes involved in CD40 homodimer production using chemical inhibitors to either protein synthesis or transport have been hindered
by the toxic effects of these compounds on group I BL cells (our own
unpublished data).
The expression of CD40 homodimers was mirrored closely by an increase
in Bcl-2 protein levels in CD40L-stimulated group I BL cells. The
levels of both CD40 homodimers and Bcl-2 protein increased at
apparently similar rates over a 72-hour period. The ability of CD40
signals to induce a relatively late induction of Bcl-2 protein
expression have been detailed previously5 and were
confirmed in this study. It is difficult to speculate whether the
formation of CD40 homodimers is directly or indirectly linked to
induction of Bcl-2 expression or that the upregulation of these
molecules is simply coincidental. However, the importance of this
response may be reflected in the fact that (with the one exception of
BL2) both early and late passage group I BL cell lines that were
induced by CD40L, either alone or in combination with IL-4, to express
CD40 homodimers, showed a corresponding increase in Bcl-2 expression.
One of the most surprising features of this study was that a short-term
exposure to CD40L could lead to relatively long-term effects. In the
example of CD40 homodimer formation and Bcl-2 induction, a 3-day
culture with CD40L resulted in both these induced changes being
maintained to day 10 or day 14 for BL29 and L3055 cells, respectively.
Interestingly, these two lines showing the most durable changes were
also the earliest in terms of their passage number.
It is also of note that low levels of dimeric CD40 could be detected in
unstimulated Louckes cells, which had the highest in vitro passage
number and the highest constitutive level of Bcl-2 protein among the
cell lines studied. It is possible that the development of group I
lines toward a more stable phenotype in culture may be accompanied by
the constitutive expression of CD40 homodimers (and possibly also
Bcl-2) together with a relative refractoriness to further induced
long-term change. Indeed, as seen by us (unpublished
data) and in previous studies,7 many well-established B-lymphoma cell lines, such as Raji, express CD40 as
homodimers constitutively.
Given the duration of change in both CD40 homodimer formation and Bcl-2
protein expression, together with their previously reported sensitivity
to surface IgM-mediated apoptosis, L3055 cells were selected for a
study of possible long-term protection to apoptotic signals after
short-term exposure to CD40L. L3055 cells stimulated through CD40 were
resistant to apoptosis induced by high concentrations of anti-IgM from
72 hours and remained protected, albeit to a lesser extent, for up to
14 days even where CD40L had been removed from culture at day 3. Indeed, where stimulated via CD40 for 3 days, L3055 cells continued to
proliferate optimally when treated with low, but still normally
apoptosis-inducing, concentrations of anti-IgM at day 20. The
maintenance of low-level, induced expression of Bcl-2 at day 14 may
explain why L3055 cells were still afforded some degree of protection
from apoptosis when treated with high concentrations of anti-IgM at
this time. The lack of Bcl-2 expression at day 20 indicates that other
long-term protective mechanisms induced by CD40L may confer partial
protection from apoptosis. Earlier studies have shown that
interferon- protects Mutu BL cells from CaI-induced
apoptosis through a pathway that does not appear to involve the
upregulation of Bcl-2.22 It is possible that the long-term
protective action of CD40L may be provided by separate mechanisms, such
as those mediated by distinct members of the Bcl-2 family as has been
noted in other studies of B-lymphoma cell lines.23-26
Not only did stimulation through CD40 provide protection from induced
apoptosis, but it also promoted growth arrest directly in group I BL
cells. Treatment with CD40L induced growth arrest in both early and
late passage group I BL cell lines as measured by a reduction in
[3H]Thy uptake compared with untreated control cells.
Analysis of cell cycle profiles in previous studies on L3055 cells (and
confirmed here; our own unpublished data) has shown
relatively few cells arresting at any particular stage of the cell
cycle when confronted with CD40L.8,27 However, the
[3H]Thy incorporation experiments reported here show
clearly that L3055 cells are in growth arrest after exposure to CD40L,
due to their failure to synthesize DNA. Thus, it is likely that
CD40-mediated signals may inhibit L3055 cell progression through the
cell cycle by acting at multiple restriction points. Additional
evidence obtained in a similar way from another study confirms that
CD40L can induce growth arrest in B-lymphoma cell lines, including that of well-established BL lines such as Raji.9 Moreover, this type of growth inhibitory effect, without concommitant apoptosis, is
not restricted to B cells, because it has also been observed in
epithelial cells.18 Importantly, from our study, it is
clear that growth arrest in group I BL cells does not inevitably lead to apoptosis. This may relate to the ability of CD40L to turn on Bcl-2
expression in group I BL cells. It has been suggested that only once
cells have left the cell cycle does the potent protective action of
Bcl-2 becomes mandatory, and in its absence the eventual fate is one of
apoptotic death.5 The duration of the growth arrest
promoted by CD40L and the additional effects of adding IL-4 could be
quite variable among the five group I BL lines studied. Whereas IL-4
normally augmented CD40-dependent growth arrest, for BL2 cells it had a
counteracting effect. The basis for this heterogeneity is unclear.
Although for most of the cell lines growth arrest was relatively
short-lived once the CD40L stimulus had been removed, for the early
passage L3055 line, 24 hours of stimulation maintained growth arrest to
day 5; with 72 hours of exposure to CD40L, growth arrest was extended
to 7 to 9 days. This is considered remarkable for a cell line that is
normally growing exponentially with a usual doubling time of approximately 24 hours (data not detailed). Again, there was no morphological evidence of enhanced apoptosis in these long-term arrested cultures, which, as discussed above, is consistent with the
sustained expression of induced Bcl-2.
CD40L-promoted growth arrest, homodimer formation, induction of Bcl-2,
and conferment of resistance to apoptosis were seen to be accompanied
by changes in cell surface phenotype, cluster formation, and individual
cell morphology, which, again, could be relatively long-lived. Most
group I BL cell lines display little or no cell clustering, an
observation supported by the absence of detectable adhesion molecule
expression.28 However, it should be noted that late passage
BL cell lines can drift phenotypically and display modest amounts of
adhesion molecule expression, which can be accompanied by a small
degree of cell clustering.28 This was seen in the present
study for Elijah, BL29, and Louckes. As has been reported previously
for normal resting B cells,13 three of the five lines were
induced to form large homotypic aggregates when signaled through CD40,
and this was enhanced by the addition of IL-4. For the early passage
L3055 cells, this change in phenotype was once more relativley
long-lived in that, with 72 hours of exposure to CD40L, there was a
maintenance of small loose cell aggregates (which were absent in
control cultures) even to day 10. Of the adhesion molecules studied,
CD11a and CD54 were the ones that showed a corresponding upregulation
on CD40-signaling. Untreated group I BL cells, particularly those
recently established from biopsy tissue, display a resting cell
morphology, where cells have a small cytoplasmic to nuclear ratio and
the nuclear chromatin is relatively condense. Stimulation with CD40L
resulted in a series of dramatic morphological changes in both early
and late passage BL cell lines (full data not detailed). These changes
were characterized by the production of large blastoid-like cells that
often had pseudopodia-like extensions. These cells had a high
cytoplasmic to nuclear ratio, and detailed morphological observations
showed the presence of many cytoplasmic vesicles. The significance of these changes are unclear. However, the fact that nearly
every cell line tested responded to stimulation with CD40L by this
morphological criteria in an identical way highlights the importance of
CD40 signaling in inducing a unique differentiation pathway in BL cells even when they are at somewhat differing stages of developmental arrest.
Whether CD40 signaling in BL cell lines accurately reflects events
occurring in GC B cells, their presumed normal counterparts, is
unclear. The importance of CD40 signals in GC differentiation is
apparent in its requirement for the formation of either plasma or
memory B cells.29 Studies involving in vitro culture of
CD40-activated GC B cells have shown that withdrawal of CD40L
stimulation leads to the differentiation of activated B cells into
plasma cells, whereas prolonged CD40L stimulation results in the
generation of memory B cells.29 Little work has been
performed to investigate how CD40 signals influence the phenotype of
lymphomas, such as the group I BL, which carry GC markers. This study
has shown that stimulation through CD40 results in a heterogeneous
phenotypic response in early and late passage group I BL cells. The
addition of IL-4 to CD40L-treated cultures tended to enhance the
existing effects of CD40 signals on a number of surface markers
expressed by the BL cells. Interestingly, treatment of L3055 cells with both CD40L and IL-4, but not CD40L alone or IL-4 alone, resulted in a
marked decrease in CD20 expression (unpublished data). A particularly striking observation was that both early passage L3055
cells and late passage Elijah, Louckes, and BL2 cells increased their
expression of CD23 when treated with CD40L alone or CD40L in
combination with IL-4. This indicates that the cells were moving towards a more lymphoblastoid, or group III, phenotype (see below). The
fact that surface CD40 expression also increased on L3055, Elijah, and
BL2 cells when treated with the same stimuli indicates that BL cell
activation may involve an upregulation in the expression of various
markers, including CD40. Importantly, the increase in surface CD40
expression observed by FACS analysis did not correspond directly with
the ability of cells to form CD40 homodimers, in as much as BL29 cells
that expressed homodimeric CD40 failed to show increases in surface
CD40 expression, whereas BL2 cells that did not express CD40 homodimers
did show increased surface CD40 expression on culture with CD40L.
The three EBVve group I BL lines all showed a
reduction in surface CD77 expression in response to stimulation through
CD40 that, for the early passage L3055 cells, was dramatic and
prolonged. Unstimulated L3055 cells consisted of a mostly
CD77high population. However, upon treatment with CD40L,
the expression of CD77 skewed dramatically to produce an almost
exclusively CD77low population. Furthermore, CD40L-treated
L3055 cells failed to revert back to the normal phenotype by remaining
CD77low over the duration of the experiment (>15 days),
even though the stimulus had been removed at day 3. The importance of
this observed reduction in CD77 expression remains unclear, although it
should be noted that both the activation of CD40 and the downregulation of CD77 are effects observed during the in vitro progression of fresh
or early passage EBV+ve BL cells (group I cells) into the
differentiated and activated, lymphoblastoid-like cells, characterized
by group III BL cells.3,16,30,31 Unlike the early group I
counterparts, group III cells express the full repertoire of EBV latent
antigens of which EBNA-4 is a member. Indeed, recent reports suggest
that EBNA-4 is important in modulating CD40, vimentin, and CD77 on BL
cells.32 It was observed in the same study that
transfection of EBNA-4 into EBVve group I BL cells
(dG75) resulted in an increase in CD40 and a decrease in CD77
expression, respectively. It is of interest in our study that CD77
expression remained unchanged on CD40-stimulation of both the
EBV+ve BL lines. The expression of specific EBV-latent
genes in the late passage EBV+ve BL cells used in this
investigation has not been determined. However, it seems unlikely that
EBNA-4 was expressed in these cells, because phenotypic analysis showed
that all late passage BL cells expressed CD10 and CD77 but not CD23,
typically observed in BL cells categorized as a group I phenotype.
Taken together, these data suggest that stimulation through CD40
modulates CD77 expression in a manner similar to that observed in BL
cells expressing EBNA-4. It has been proposed that EBNA-4 may modulate
CD77 expression indirectly through intermediates of the glycolipid
pathway.32 This may be a common point at which the effects
of EBNA-4 in and EBV+ve BL cells and CD40 signals in
EBVve BL cells converge to regulate CD77 expression.
In common with CD40, CD77 may also be important in regulating apoptotic
mechanisms during GC B-cell development.32 The fact that
CD77 is expressed on GC B cells undergoing apoptosis could indicate a
possible counterstructure for this molecule that may be involved in the
induction of GC B-cell apoptosis. Therefore, CD40L-induced
downregulation of CD77, along with the coordinate upregulation of CD40
seen on a number of BL cell lines tested in this study, could have
important implications for cell survival. It should be noted that,
despite the absence of EBV latent genes, CD40-stimulated L3055 and
Louckes cells displayed a group III-like phenotype, in that CD77
expression was reduced, CD23 was upregulated, cells adopted a
lymphoblastoid morphology, and large homotypic cell aggregates were
produced. Some, or all, of these phenotypic responses were mirrored in
the EBV-positive BL, BL29, and Elijah cells, suggesting that CD40 may
have an important role in the progression of BL cells from a group I to
a group III phenotype.
In summary, stimulating CD40 on both early and late passage and
EBVve and EBV+ve BL cell lines results
in multiple phenotypic, functional, and molecular changes. It is
apparent that not all cell lines respond to CD40-meditated signals in
an identical manner, despite initial similarities in cell surface
phenotype and morphology. However, several of the cell lines follow
similar trends in many or all of the ways they respond to CD40: eg,
growth arrest, protection from apoptosis, coexpression of Bcl-2 and
CD40 homodimers, and morphological and phenotypic changes. Importantly,
the stimulation time during which cells are exposed to CD40L can
dictate the strength and the duration of the response to CD40 signals.
Most unexpectedly, and as highlighted by results obtained with the
early passage L3055 cell line, some of the induced changes were
maintained for 1 to 2 weeks after removal of the initial CD40L
stimulus. These are not only important factors to consider when
designing potential CD40L-based therapies for the treatment of B-cell
lymphomas,9,33,34 but also raise the possibility that a
transient exposure to CD40L, possibly via infiltrating T cells, may
influence the course and nature of B-cell lymphomagenesis.
| |
FOOTNOTES |
Submitted March 23, 1998;
accepted June 7, 1998.
Supported by the Medical Research Council (U.K.) and the Cancer
Research Campaign.
Address reprint requests to John Gordon, PhD,
Department of Immunology, Medical School, University of Birmingham,
Vincent Drive, Edgbaston, Birmingham, B15 2TT, UK; e-mail:
j.gordon{at}bham.ac.uk.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Michelle Holder for expert technical assistance.
| |
REFERENCES |
1.
Geser A,
Lenoir GM,
Anvret M,
Bornkamm G,
Klein G,
Williams EH,
Wright DH,
De The G:
Epstein-Barr virus markers in a series of Burkiit's lymphomas from the West Nile district of Uganda.
Eur J Cancer Clin Oncol
19:1393,
1983[Medline]
[Order article via Infotrieve]
2.
Gregory CD,
Dive C,
Henderson S,
Smith CA,
Williams GT,
Gordon J,
Rickinson AB:
Activation of Epstein-Barr virus latent genes protects human B cells from death by apoptosis.
Nature
349:612,
1991[Medline]
[Order article via Infotrieve]
3.
Gregory CD,
Rowe M,
Rickinson AB:
Different Epstein-Barr virus B cell interactions in phenotypically distinct clones of a Burkitt's lymphoma cell line.
J Gen Virol
71:1481,
1990[Abstract/Free Full Text]
4.
Gregory CD,
Milner AE:
Regulation of cell survival in Burkitt lymphoma: Implications from studies of apoptosis following cold-shock treatment.
Int J Cancer
57:419,
1994[Medline]
[Order article via Infotrieve]
5.
Holder MJ,
Wang H,
Milner AE,
Casamjor M,
Armitage R,
Spriggs MK,
Fanslow WC,
MacLennan ICM,
Gregory CD,
Gordon J:
Suppresssion of apoptosis in normal and neoplastic human B lymphocytes by CD40 ligand is independent of Bcl-2 induction.
Eur J Immunol
23:2368,
1993[Medline]
[Order article via Infotrieve]
6.
Van Kooten C,
Banchereau J:
CD40-CD40 ligand: A multifunctional receptor-ligand pair.
Adv Immunol
61:1,
1992
7.
Braesch-Andersen S,
Paulie S,
Koho H,
Nika H,
Aspenstrom P,
Perlmann P:
Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen Cdw40.
J Immunol
142:562,
1989[Abstract]
8.
MacDonald I,
Wang H,
Grand R,
Armitage RJ,
Fanslow WC,
Gregory CD,
Gordon J:
Transforming growth factor- 1 cooperates with anti-immunoglobulin for the induction of apoptosis in group I (biopsy-like) Burkitt lymphoma cell lines.
Blood
87:1147,
1996[Abstract/Free Full Text]
9.
Funakoshi S,
Longo DL,
Beckwith M,
Conley DK,
Tsarfaty G,
Tsarfat I,
Armitage RJ,
Fanslow WC,
Spriggs MK,
Murphy WJ:
Inhibition of human B cell lymphoma growth by CD40 stimulation.
Blood
83:2787,
1994[Abstract/Free Full Text]
10.
Holder MJ,
Liu Y-J,
Defrance T,
Flores-Romo L,
MacLennan ICM,
Gordon J:
Growth factor requirements for the stimulation of germinal center cells: Evidence of an IL-2-dependent pathway of development.
Int Immunol
3:1243,
1991[Abstract/Free Full Text]
11.
de Vries JE,
Gauchat J-F,
Aversa GG,
Punnonen J,
Gascan H,
Yssel H:
Regulation of IgE synthesis by cytokines.
Curr Opin Immunol
3:851,
1991[Medline]
[Order article via Infotrieve]
12.
Zhang K,
Clark EA,
Saxon A:
CD40 stimulation provides an IFN-gamma-independent and IL-4-dependent differentiation directly to human B cells for IgE production.
J Immunol
146:1836,
1991[Abstract]
13.
Gordon J,
Millsum MJ,
Guy GR,
Ledbetter JA:
Resting B lymphocytes can be triggered directly through the Cdw40 (Bp50) antigen. A comparison with IL4-mediated signaling.
J Immunol
140:1425,
1988[Abstract]
14.
Rowe M,
Rooney CM,
Rickinson AB,
Lenoir GM,
Rupant H,
Moss DJ,
Stein H,
Epstein MA:
Distinctions between endemic and sporadic forms of Epstein-Barr virus-positive Burkitt's lymphoma.
Int J Cancer
35:435,
1985[Medline]
[Order article via Infotrieve]
15.
Rowe M,
Rooney CM,
Rickinson AB,
Lenoir GM,
Rickinson AB:
Epstein-Barr virus status and tumour cell phenotype in sporadic Burkitt's lymphoma.
Int J Cancer
37:363,
1986
16.
Rowe M,
Rowe DT,
Gregory CD,
Young LS,
Farrel PJ,
Rupani H,
Rickinson AB:
Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells.
EMBO J
6:2743,
1987[Medline]
[Order article via Infotrieve]
17.
Rooney CM,
Gregory CD,
Rowe M,
Finerty S,
Edwards C,
Rupani H,
Rickinson AB:
Endemic Burkitt's lymphoma: Phenotypic analysis of tumour biopsy cells and of derived tumor cell lines.
J Natl Cancer Inst
77:681,
1986
18.
Eliopoulos AG,
Dawson CW,
Mosialos G,
Floettmann JE,
Rowe M,
Armitage RJ,
Dawson J,
Zapata JM,
Kerr DJ,
Wakelam MJO,
Reed J,
Kieff E,
Young LS:
CD40-induced growth inhibition is mimicked by Epstein-Barr virus-encoded LMP1-involvement of TRAF3 as a co-mediator.
Oncogene
13:2243,
1996[Medline]
[Order article via Infotrieve]
19.
Wyllie AH,
Kerr JFR,
Currie AR:
Cell death: The significance of apoptosis.
Int Rev Cytol
68:251,
1980[Medline]
[Order article via Infotrieve]
20.
Cheng G,
Baltimore D:
TANK, a co-inducer with TRAF2 of TNF-and CD40L-mediated NF- B activation.
Genes Dev
10:963,
1996[Abstract/Free Full Text]
21.
Gordon J:
CD40 and its ligand: Central players in B lymphocyte survival, growth, and differentiation.
Blood Rev
9:53,
1995[Medline]
[Order article via Infotrieve]
22.
Milner AE,
Johnson GD,
Gregory CD:
Prevention of programmed cell death in Burkitt lymphoma cell lines by bcl-2-dependent and -independent mechanisms.
Int J Cancer
52:636,
1992[Medline]
[Order article via Infotrieve]
23.
Choi MSK,
Boise LH,
Gottschalk AR,
Quintans J,
Thompson CB,
Klaus GGB:
The role of Bcl-xL in CD40-mediated rescue from anti-µ-induced apoptosis in WEHI-231 B lymphoma cells.
Eur J Immunol
25:1352,
1995[Medline]
[Order article via Infotrieve]
24.
Bargou RC,
Bommert K,
Weinmann P,
Daniel PT,
Wagener C,
Mapara MY,
Dorken B:
Induction of Bax- precedes apoptosis in a human B lymphoma cell line: Potential role of the bcl-2 gene family in surface IgM-mediated apoptosis.
Eur J Immunol
25:770,
1995[Medline]
[Order article via Infotrieve]
25.
Boise LH,
Gonzalez-Garcia M,
Postema CE,
Ding L,
Lindsten T,
Turka LA,
Mao X,
Nunez G,
Thompson CB:
bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death.
Cell
74:597,
1993[Medline]
[Order article via Infotrieve]
26.
Oltiva ZN,
Milliman CL,
Korsmeyer SJ:
Bcl-2 heterodimerises in vivo with a conserved homolog, Bax, that accelerates progarmmed cell death.
Cell
74:609,
1993[Medline]
[Order article via Infotrieve]
27.
Wang H,
Grand RJA,
Milner AE,
Armitage RJ,
Gordon J,
Gregory CD:
Repression of apoptosis in human B-lymphoma cells by CD40-ligand and Bcl-2: Relationship to the cell-cycle and role of the retinoblastoma protein.
Oncogene
13:373,
1996[Medline]
[Order article via Infotrieve]
28.
Gregory CD,
Edwards CD,
Milner A,
Weils J,
Lipinski M,
Rowe M,
Tursz T,
Rickinson AB:
Isolation of a normal B cell subset with a Burkitt-like phenotype and transformation in vitro with Epstein-Barr virus.
Int J Cancer
42:213,
1988[Medline]
[Order article via Infotrieve]
29.
Arpin C,
Dechanet J,
Van Kooten C,
Merville P,
Grouard G,
Briere F,
Banchereau J,
Liu Y-J:
Generation of memory B cells and plasma cells in vitro.
Science
268:720,
1995[Abstract/Free Full Text]
30.
Favrot MC,
Maritaz O,
Suzuki T,
Cooper M,
Philip I,
Philip T,
Lenoir G:
EBV-negative and positive Burkitt cell lines variably express receptors for B-cell activation and differentiation.
Int J Cancer
38:901,
1986[Medline]
[Order article via Infotrieve]
31.
Calender A,
Cordier M,
Billaud M,
Lenoir GM:
Modulation of cellular gene expression in B lymphoma cells following in vitro infection by Epstein-Barr virus (EBV).
Int J Cancer
46:658,
1990[Medline]
[Order article via Infotrieve]
32.
Silins SL,
Sculley TB:
Modulation of vimentin, the CD40 activation antigen and Burkitt's lymphoma antigen (CD77) by the Epstein-Barr virus nuclear antigen EBNA-4.
Virology
202:16,
1995
33.
Murphy WJ,
Funakoshi S,
Beckwith M,
Rushing SE,
Conley DK,
Armitage RJ,
Fanslow WC,
Roger HC,
Taub DD,
Ruscetti FW,
Longo DL:
Antibodies to CD40 prevent Epstein-Barr virus-mediated human B-cell lymphomagenesis in SCID mice given human peripheral blood lymphocytes.
Blood
86:1946,
1995[Abstract/Free Full Text]
34.
Dilloo D,
Brown M,
Roskrow M,
Zhong W,
Holladay M,
Holden W,
Brenner M:
CD40 ligand induces an anti-leukemia immune response in vivo.
Blood
90:1927,
1997[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Z. Stamataki, C. Shannon-Lowe, J. Shaw, D. Mutimer, A. B. Rickinson, J. Gordon, D. H. Adams, P. Balfe, and J. A. McKeating
Hepatitis C virus association with peripheral blood B lymphocytes potentiates viral infection of liver-derived hepatoma cells
Blood,
January 15, 2009;
113(3):
585 - 593.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Stewart, W. Wei, A. Challa, R. J. Armitage, J. R. Arrand, M. Rowe, L. S. Young, A. Eliopoulos, and J. Gordon
CD154 Tone Sets the Signaling Pathways and Transcriptome Generated in Model CD40-Pluricompetent L3055 Burkitt's Lymphoma Cells
J. Immunol.,
September 1, 2007;
179(5):
2705 - 2712.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. C. Hill, S. J. Youde, S. Man, G. R. Teale, A. J. Baxendale, A. Hislop, C. C. Davies, D. M. Luesley, A. M. Blom, A. B. Rickinson, et al.
Activation of CD40 in Cervical Carcinoma Cells Facilitates CTL Responses and Augments Chemotherapy-Induced Apoptosis
J. Immunol.,
January 1, 2005;
174(1):
41 - 50.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Reyes-Moreno, J. Girouard, R. Lapointe, A. Darveau, and W. Mourad
CD40/CD40 Homodimers Are Required for CD40-induced Phosphatidylinositol 3-Kinase-dependent Expression of B7.2 by Human B Lymphocytes
J. Biol. Chem.,
February 27, 2004;
279(9):
7799 - 7806.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. N. D'Souza, L. C. Edelstein, P. M. Pegman, S. M. Smith, S. T. Loughran, A. Clarke, A. Mehl, M. Rowe, C. Gelinas, and D. Walls
Nuclear Factor {kappa}B-Dependent Activation of the Antiapoptotic bfl-1 Gene by the Epstein-Barr Virus Latent Membrane Protein 1 and Activated CD40 Receptor
J. Virol.,
February 15, 2004;
78(4):
1800 - 1816.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. C. Davies, J. Mason, M. J. O. Wakelam, L. S. Young, and A. G. Eliopoulos
Inhibition of Phosphatidylinositol 3-Kinase- and ERK MAPK-regulated Protein Synthesis Reveals the Pro-apoptotic Properties of CD40 Ligation in Carcinoma Cells
J. Biol. Chem.,
January 9, 2004;
279(2):
1010 - 1019.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Serafeim, M. J. Holder, G. Grafton, A. Chamba, M. T. Drayson, Q. T. Luong, C. M. Bunce, C. D. Gregory, N. M. Barnes, and J. Gordon
Selective serotonin reuptake inhibitors directly signal for apoptosis in biopsy-like Burkitt lymphoma cells
Blood,
April 15, 2003;
101(8):
3212 - 3219.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Szocinski, A. R. Khaled, J. Hixon, D. Halverson, S. Funakoshi, W. C. Fanslow, A. Boyd, D. D. Taub, S. K. Durum, C. B. Siegall, et al.
Activation-induced cell death of aggressive histology lymphomas by CD40 stimulation: induction of bax
Blood,
June 17, 2002;
100(1):
217 - 223.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Challa, A. G. Eliopoulos, M. J. Holder, A. S. Burguete, J. D. Pound, A. Chamba, G. Grafton, R. J. Armitage, C. D. Gregory, H. Martinez-Valdez, et al.
Population depletion activates autonomous CD154-dependent survival in biopsylike Burkitt lymphoma cells
Blood,
May 1, 2002;
99(9):
3411 - 3418.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Batrla, M. Linnebacher, W. Rudy, S. Stumm, D. Wallwiener, and B. Guckel
CD40-expressing Carcinoma Cells Induce Down-Regulation of CD40 Ligand (CD154) and Impair T-Cell Functions
Cancer Res.,
April 1, 2002;
62(7):
2052 - 2057.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N J Gallagher, A G Eliopoulos, A Agathangelo, J Oates, J Crocker, and L S Young
CD40 activation in epithelial ovarian carcinoma cells modulates growth, apoptosis, and cytokine secretion
Mol. Pathol.,
April 1, 2002;
55(2):
110 - 120.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Serafeim, G. Grafton, A. Chamba, C. D. Gregory, R. D. Blakely, N. G. Bowery, N. M. Barnes, and J. Gordon
5-Hydroxytryptamine drives apoptosis in biopsylike Burkitt lymphoma cells: reversal by selective serotonin reuptake inhibitors
Blood,
April 1, 2002;
99(7):
2545 - 2553.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. S. Andersen, J. K. Larsen, J. Christiansen, L. B. Pedersen, N. S. Christophersen, C. H. Geisler, and J. Jurlander
Soluble CD40 ligand induces selective proliferation of lymphoma cells in primary mantle cell lymphoma cell cultures
Blood,
September 15, 2000;
96(6):
2219 - 2225.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. G. Eliopoulos, C. Davies, P. G. Knox, N. J. Gallagher, S. C. Afford, D. H. Adams, and L. S. Young
CD40 Induces Apoptosis in Carcinoma Cells through Activation of Cytotoxic Ligands of the Tumor Necrosis Factor Superfamily
Mol. Cell. Biol.,
August 1, 2000;
20(15):
5503 - 5515.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. Choe, L. Li, X. Zhang, C. D. Gregory, and Y. S. Choi
Distinct Role of Follicular Dendritic Cells and T Cells in the Proliferation, Differentiation, and Apoptosis of a Centroblast Cell Line, L3055
J. Immunol.,
January 1, 2000;
164(1):
56 - 63.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Segui, N. Andrieu-Abadie, S. Adam-Klages, O. Meilhac, D. Kreder, V. Garcia, A. P. Bruno, J.-P. Jaffrezou, R. Salvayre, M. Kronke, et al.
CD40 Signals Apoptosis through FAN-regulated Activation of the Sphingomyelin-Ceramide Pathway
J. Biol. Chem.,
December 24, 1999;
274(52):
37251 - 37258.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract