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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1199-1206
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Apoptosis or plasma cell differentiation of
CD38-positive B-chronic lymphocytic leukemia cells induced by
cross-linking of surface IgM or IgD
Simona Zupo,
Rosanna Massara,
Mariella Dono,
Edoardo Rossi,
Fabio Malavasi,
M. Elisabetta Cosulich, and
Manlio Ferrarini
From Servizio di Immunologia Clinica, Istituto Nazionale per la
Ricerca sul Cancro; Dipartimento di Oncologia, Biologia e Genetica,
Università degli Studi di Genova; Unità Anticorpi
Monoclonali, Advanced Biotechnology Center, Genoa, Italy; and Instituto
Biologia e Genetica, Università di Amcona, Italy.
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Abstract |
Previously, we demonstrated that B-chronic lymphocytic leukemia
(B-CLL) cells could be divided into 2 groups depending on the
expression of CD38 by the malignant cells. The 2 groups differed in
their signal-transducing capacities initiated by cross-linking of
surface IgM; only in CD38-positive cells was an efficient signal delivered, invariably resulting in cell apoptosis. In this study, we
investigated the effect of surface IgD cross-linking in 10 patients
with CD38-positive B-CLL. Exposure of the malignant cells to goat
antihuman  -chain antibodies (Ga-ab) caused
[Ca++]i mobilization and tyrosine kinase
phosphorylation in a manner not different from that observed after goat
antihuman µ-chain antibody (Gaµ-ab) treatment in vitro. However,
Ga-ab-treated cells failed to undergo apoptosis and instead
displayed prolonged survival in culture and differentiated into plasma
cells when rIL2 was concomitantly present. Cross-linking of surface IgD
failed to induce proliferation of the malignant cells in vitro.
Moreover, treatment with Ga-ab did not prevent apoptosis of B-CLL
cells induced by Gaµ-ab. Collectively, these experiments demonstrated that IgM and IgD expressed by the same cell may deliver opposite signals under particular circumstances and provide some clues for the
understanding of the pathophysiology of B-CLL.
(Blood. 2000;95:1199-1206)
© 2000 by The American Society of Hematology.
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Introduction |
IgD is expressed together with IgM on the surface of
most mature B-lymphocytes in humans and other animal
species.1 When expressed on the surface of the same cell,
the 2 isotypes share the same light chain type, idiotype, and antibody
specificity. Because of the description of IgD as a major B-cell
antigen-receptor (BCR), several questions have been raised concerning
its physiological function and its relationship with surface IgM. The
different constant region structures of the 2 immunoglobulins,2 the differential expression of IgM and
IgD during B cell development,1 and the identification of
proteins associated specifically with surface IgM or IgD3-5
all suggest that each membrane immunoglobulin isotype has a specialized
function. Indeed, a number of studies indicate that surface IgM, but
not IgD, can induce negative responses in B cells, such as growth
arrest, anergy, or apoptosis,6-8 and they suggest that the
negative selection of immature B cells may result from a relatively low
expression of surface IgD.9,10 A correlation between IgD
expression level and decreased sensitivity to tolerance was observed in
those studies.
There are, however, observations that contradict this notion. For
example, it has been observed that in irradiated mice
reconstituted with immature B cells11 or in certain
transgenic mice,12 both surface IgM and surface
IgD transduce signals that lead to the apoptosis of immature
murine B-lymphocytes. Furthermore, independent engagement
of either IgM or IgD of mature B-lymphocytes results in their
proliferation in vitro.13,14
In this study, performed on a particular subset of B-chronic
lymphocytic leukemia (B-CLL) cells characterized by the presence of
CD38 surface marker, we show that sIgM and sIgD can deliver different
signals. Specifically, though cross-linking of IgM was invariably followed by cell apoptosis, IgD cross-linking resulted in improved cell survival and the promotion of plasma cell
differentiation in vitro. The subset of CD38-positive B-CLL cells
studied was selected because we had previously demonstrated
that their malignant cells have a functional signal
transduction pathway initiated by BCR cross-linking, whereas the
malignant cells of CD38-negative B-CLL cells do not respond
to signals delivered by surface immunoglobulin.15 Collectively, the current data demonstrated that in particular circumstances, surface IgM and IgD can deliver signals that
activate different physiological functions of B cells.
Moreover, because B-CLL results from the expansion of malignant
cells frozen at a particular differentiation stage,16,17
these studies may also help trace the stages at which normal cells
display different responses to the stimulation of surface IgM
or IgD. This finding may contribute to the definition of the
role of isotypes in fine-tuning the humoral response.
Finally, CD38-positive B-CLL may constitute a suitable ex vivo
source of B cells for investigating the differences in the
biochemical events initiated by cross-linking of surface IgM or surface
IgD, respectively.
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Patients and methods |
Cell preparation
Ten patients (A-L) with B-CLL at Rai classification stages 0, 1, and
2 were studied at the time of diagnosis. These patients were selected
because of the high expression of CD38 on malignant B cells, as
determined by flow cytometry analysis. Purified B cells from the
peripheral blood of CD38-positive patients with CLL were obtained by
Ficoll-Hypaque density gradient centrifugation and by the depletion of
monocytes or T cells through adherence and erythrocyte rosette
formation methods, as described.15
Flow cytometry analysis performed on purified B-CLL cells showed that
these cells comprised more than 95% CD19+, CD5+ B cells and less than
3% CD3+ T cells and CD68+ monocytes. In each patient selected for this
study, comparable levels of membrane IgM and IgD were coexpressed, as
demonstrated by the similar relative fluorescent intensity values of
the IgM and IgD. In selected patients (A, I, L), B-CLL cells were
fractionated into CD38bright and CD38low by
magnetic cell separation. Briefly, cells were incubated with CD38
monoclonal antibody (mAb) for 30' at 4°C and then with goat antimouse immunoglobulin micro beads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10' at 4° C, and the cells were
separated on magnetic columns (Miltenyi Biotec).
Peripheral blood B cells from normal donors were purified as previously
described.15 Briefly, mononuclear cells were obtained by
F-H centrifugation and were depleted of monocytes and T cells by
leucine-methyl-ester treatment and erythrocyte rosette formation, respectively. The obtained cell suspensions were enriched in B cells
(80% CD19+) as demonstrated by flow cytometry analysis.
Immunofluorescence
The following mAbs were used: anti-IgM, anti-CD5, anti-CD23,
anti-CD38, anti-CD10, anti-CD95, anti-CD69, anti-CD71 (Becton Dickinson, San Jose, CA); anti-IgD, anti-Bcl-2, anti-Ki-67 (Dakopatts, Glostrup, Denmark); anti-CD79b, anti-CD86 (Ancell Europe,
Laüfelfingen, Switzerland); anti-CD44 (Janssen
Chimica, Beerse, Belgium); and anti-CD138 (Caltag Laboratories,
Burlingame, CA); and rabbit anti-CD77 (Immunotech, Marseilles, France).
Indirect immunofluorescence was performed with fluorescein
isothiocyanate (FITC) or phycoerythrin (PE)-conjugated
goat antimouse immunoglobulin isotype antibodies (Southern
Biotechnology, Birmingham, AL) as the second reagent. CD38 staining was
also performed using PE-conjugated CD38 mAb (Becton Dickinson).
Staining for intracellular molecules (Ki67, Bcl-2) was performed on
permeabilized cells as described previously.18 For control
staining, the cells were treated with murine immunoglobulin followed by
a second reagent conjugated with FITC or PE. Stained cells were
analyzed with a FACSort flow cytometer (Becton Dickinson). Data were
expressed as histograms of the fluorescence intensity versus cell
number or as relative fluorescence intensity calculated according to
the following formula: mean fluorescence intensity of cells stained
with the mAb/mean fluorescence intensity of control cells. The mean
fluorescence intensities were calculated by CELL QUEST software.
Cell cultures
Cells were cultured at the concentration of
1 × 106 cells/mL in RPMI 1640 medium supplemented
with 10% fetal calf serum (both from GIBCO, Paisley, UK) for various
time intervals. In the different experiments, the cells were exposed to
goat antihuman µ-chain antibodies (Gaµ-ab) (Southern
Biotechnology), goat antihuman -chain antibodies (Ga-ab) (Sigma,
Saint Louis, MO) at the indicated concentrations, rabbit (Fab')2
antihuman µ-chain antibodies (Sigma), rabbit (Fab')2
antihuman -chain antibodies (Caltag Laboratories), normal
goat immunoglobulin (NGI), phorbol myristate acetate (10 ng/mL) (Sigma)
in the presence of A23 187 (Sigma) at the concentration of 5 ng/mL,
SAC (1:10 000 vol/vol) (Calbiochem, Novabiochem, San Diego, CA). In
selected experiments, the cells were cultured with the stimuli listed
above in the presence of recombinant lymphokines in various
combinations: rIL2 (100 U/mL), rIL4 (100 U/mL), rIL6 (70 U/mL), and
rIL10 (100 U/mL) (Genzyme Diagnostics, Cambridge, MA). Morphology of
the cells was studied by either of 2 methods. Cytospin preparations
were stained with GIEMSA and analyzed by light microscopy or they were
stained with FITC-conjugated anti-IgM mAb (Becton Dickinson) and
analyzed by fluorescence microscopy. Immunoglobulin released by B-CLL
cells in the culture supernatants were measured by ELISA using
anti- or anti- chain-specific reagents (Southern).
Apoptosis
Apoptosis was measured by 2 different methods. Permeabilized cells
were stained with propidium iodide (PI, 50 mg/mL; Sigma), and the
amount of DNA fragmentation was calculated by flow cytometry analysis
as previously described.15 Results were expressed as the
percentage of fragmented DNA compared to total DNA. Alternatively, cultured cells were double stained with annexin V-FITC conjugate (ApoAlert Annexin V-FITC) (Clontech, Palo Alto, CA) and PI and then
analyzed by flow cytometry. Early apoptotic cells were positive for
annexin V-FITC conjugate but did not stain with PI because their
membranes were still intact. Late-stage apoptotic cells or dead cells
that had damaged permeable plasma membranes stained concurrently with
annexin V-FITC conjugate and PI.19 In selected experiments,
cell viability was determined by PI exclusion tests and flow cytometry analysis.
Cell cycle analysis and thymidine incorporation assay
Cell proliferation was measured by 2 methods. Cells were
permeabilized by exposure to hypotonic solutions, stained with PI, and
analyzed by flow cytofluorimetry.15 The percentage of cells present in each cell-cycle phase was calculated by the Modfit program
(Becton Dickinson). Alternatively, 3H-thymidine (Amersham
International, Buckinghamshire, UK) was added to the culture for 8 hours at various time intervals. Then the cells were harvested, and the
3H-thymidine incorporated by the cells was measured by a
 -counter.
Protein tyrosine phosphorylation assay
With the appropriate stimuli for the indicated times,
1 × 107 cells/mL were incubated, washed twice in
cold phosphate-buffered saline, pelleted by centrifugation, and lysed
in ice-cold lysis buffer (200 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 1% NP40, 2 mmol/L EDTA, 100 mmol/L phenylmethylsulfonyl
fluoride, 10 µmol/L leupeptin, 10 µmol/L apoprotein, and 1 mmol/L
sodium orthovanadate). Protein concentration of the cell lysates was
measured with high precision using the Micro BCA protein assay reagent
kit (Pierce, Milan, Italy). Equal amounts of protein (10 µg/lane) from each sample were separated by SDS-PAGE (10%
acrylamide) under reducing conditions and were transferred
electrophoretically to nitrocellulose (Hybond-C nitrocellulose
membrane; Amersham). Tyrosine-phosphorylated proteins were detected by
probing the filters with antiphosphotyrosine mAb 4G10 (UBI, Lake
Placid, NY) followed by horseradish peroxidase-conjugated goat
antimouse immunoglobulin, used as a developing reagent. The reaction
was detected by enhanced chemiluminescence detection reagents
(Amersham) and exposure to Hyperfilm-MP. In the immunoprecipitation experiments, the lysates were pre-cleared with protein A-Sepharose (Pharmacia, Uppsala, Sweden) and an irrelevant mAb and subsequently incubated with antiphosphotyrosine mAb followed by protein A-Sepharose. Specific immunoprecipitates were resolved by sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; 10%),
transferred to nitrocellulose, and probed with anti-syk or anti-PLC 1
mAb (UBI). The reaction was detected as above.
Calcium mobilization
Measurements of intracellular free Ca++ concentrations
([Ca++]i) were performed as described
previously.15 Briefly, 1 × 107 cells
were loaded with FURA2/AM (Calbiochem, Novabiochem) and exposed to the
indicated stimuli. [Ca++]i was detected by excitation of
the probe at 2 alternative wavelengths (340 and 380 nm) and by
measurement of the emitted fluorescence at 510 nm with a
spectrofluorometer (Perkin-Elmer Cetus, Norwalk, CT). A separate
calibration for each measurement was performed.
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Results |
Cell surface phenotype of CD38-positive B-CLL cells
Malignant cells of the 10 patients with B-CLL (A-L) selected for
this study were characterized by high levels of CD38 expression (relative fluorescent intensity, > 4) as assessed by flow cytometry. The cells expressed markers typical of B-CLL cells, including CD5,
CD23, CD44, and low levels of IgM and IgD (Figure
1). Bcl-2 was expressed at high levels, in
agreement with previous observations.15,20 Molecules
usually found on germinal center (GC) B cells were
consistently low or absent (CD95, CD77, and CD10), with the obvious
exception of CD38. Activation markers such as CD69, CD71, and CD86 were present, though variations in the staining intensity existed among the
patients (not shown). Cell-cycle analysis demonstrated that virtually
all the cells were constantly in G0 or G1 phase (see Figure 1). Because
most of the cells expressed Ki67, it is likely that they were activated
and probably arrested in the G1 phase of the cell cycle.
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| Fig 1.
Phenotypical analysis of CD38-positive B-CLL cells.
Solid lines: Freshly prepared B-CLL cells were stained with the
indicated mAb by indirect immunofluorescence. Dashes: Control stainings
with an irrelevant mAb. Cells were permeabilized before the staining
for Bcl-2, Ki-67, and cell-cycle analysis. Data are the results of 1 representative test (patient D) of the 10 performed (A-L) on patients
with CD38-positive B-CLL.
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Apoptosis of CD38-positive B-CLL cells induced by Gaµ-ab and not
by Ga-ab
Purified B-CLL cells were exposed to Gaµ-ab, Ga-ab, or NGI for
18 hours in vitro and tested for apoptosis by PI staining of
permeabilized cells. As shown in Figure 2A,
in the presence of Gaµ-ab, the percentages of apoptotic cells
increased compared to control cells treated with NGI (33% ± 15%
versus 8% ± 3%, mean ± SD of 10 patients;
P < .05). In contrast, exposure to Ga-ab did not induce
the apoptosis of B-CLL cells even when Ga-ab was used for a wide
range of concentrations (Figure 2B). Similar results were obtained when
Annexin-V FITC-conjugated staining was substituted for PI staining to
detect apoptosis (see below). Substituting Gaµ-ab and Ga-ab with
rabbit F(ab')2 antihuman µ-chain or -chain antibodies
yielded comparable results (not shown), ruling out the possibility that
signals potentially delivered through the Fc portion of the stimulating
antibodies could prevent or even induce apoptosis.21,22
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| Fig 2.
Apoptosis in CD38-positive B-CLL cells exposed to
Gaµ-ab or Ga-ab.
(A) B-CLL cells were cultured in the presence of Gaµ-ab, Ga-ab, or
NGI at the concentration of 10 µg/mL. After 18 hours, apoptotic cells
in the cultures were measured by PI staining of permeabilized cells.
Data represent the mean ± SD of the values obtained in the 10 patients with B-CLL (A-L). (B) B-CLL cells from patient G were
incubated with the indicated concentrations of Gaµ-ab ( ), Ga-ab
( ), or NGI ( ) for 18 hours, after which apoptosis was determined
as above. (C) B-CLL cells from patient D were cultured in the presence
of Gaµ-ab, Ga-ab, or NGI for the indicated time. At the end of the
cultures, cells were double stained in suspension with Annexin-V FITC
(to detect apoptotic cells) and PI (to detect late apoptotic or
nonviable cells) and then analyzed by flow cytometry. Data in B and C
represent 1 typical experiment of the 6 performed (patients B, C, G, D,
F, I).
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Time course experiments were carried out by exposing the cells to
Gaµ-ab or Ga-ab for different periods of time. The cells were
concomitantly stained with Annexin-V FITC-conjugate to measure apoptosis, and they were stained with PI to assess cell viability by
dye exclusion (Figure 2C). Gaµ-ab-induced apoptosis had already occurred in 25% or more of the cells after 24 hours and increased progressively. Later there was an accumulation of PI-adsorbing cells,
possibly cells that underwent lysis after apoptosis. As expected,
exposure to Ga-ab caused very low amounts of apoptosis at 48 hours
and a correspondingly small amount of cell lysis at 72 hours of
culture. Values observed with this reagent were similar to those
observed with NGI (Figure 2C).
Cells from 3 patients (A, I, L) were fractionated into
CD38high and CD38dim cells using a magnetic
cell separation method. Both fractions were exposed to Gaµ-ab or
Ga-ab and tested for apoptosis. Gaµ-ab induced apoptosis in both
cell fractions, though it was consistently more marked in the fraction
enriched for CD38high, perhaps suggesting a close
correlation between CD38 levels on the cell surface and a propensity to
apoptosis as described previously18 and in other cell
types.15,23 Exposure to Ga-ab had no effect on either
cell fraction (data not shown).
In another experiment, B-CLL cells from 4 different patients (C, G, D,
F) were exposed to Gaµ-ab in the presence or absence of increasing
concentrations of Ga-ab with the aim of investigating the potential
ability of Ga-ab to prevent Gaµ-ab-induced apoptosis. Ga-ab
treatment did not prevent Gaµ-ab-induced apoptosis (Figure 3). Some inhibition of apoptosis was
observed with high concentrations of Ga-ab (80 µg/mL) in the
cultures exposed to 50 µg/mL Gaµ-ab, but this was not significant.
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| Fig 3.
Failure of Ga-ab to prevent Gaµ-ab-induced
apoptosis.
B-CLL cells from patient C were exposed to Gaµ-ab at 3 different
concentrations in the presence of Ga-ab or NGI at the indicated
concentrations. Apoptotic cells were measured by Annexin-V FITC
staining after 18 hours in vitro. The experiment is representative of
the 4 carried out on patients with B-CLL (patients C, G, D, F).
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Activation of the early events in the signal transducing
pathway by Ga-ab and Gaµ-ab
In these experiments, we compared the ability of Ga-ab and
Gaµ-ab to activate tyrosine kinases and to induce changes in
intracellular free-calcium concentration ([Ca++]i). Cells
were stimulated with Ga-ab or Gaµ-ab for different times, lysed,
and analyzed for the induction of protein tyrosine phosphorylation. As
shown in Figure 4A, which reports 1 representative experiment (patient G) of the 3 performed on different
patients with B-CLL (G, H, F), Gaµ-ab and Ga-ab were able to
induce tyrosine phosphorylation of several proteins, the most prominent
of which were 2 proteins of 145 and 75 kd (see below).
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| Fig 4.
Ability of Gaµ-ab and Ga-ab to activate the signal
transducing pathway in CD38-positive B-CLL cells.
(A) B-CLL cells (patient G) were stimulated with NGI, Gaµ-ab, or
Ga-ab at the optimal concentration (10 µg/mL) for the indicated
time. Cell lysates (20 µg/lane) were separated on 10% reducing
SDS-PAGE gels and analyzed by immunoblotting with the
antiphosphotyrosine mAb, 4G10. Molecular weight in kilodalton is
indicated on the right. (B) B-CLL cells (patient H) were stimulated for
3 or 5 minutes with the indicated stimuli. Cell lysates were
immunoprecipitated with antiphosphotyrosine mAb (4 G10). The
immunoprecipitates were separated on 10% reducing SDS-PAGE gels and
probed with an anti-syk mAb (top) or anti-PLC1 mAb (bottom). (C)
Fura2/AM loaded B-CLL cells were stimulated with Gaµ-ab or Ga-ab
and studied for [Ca++]i mobilization. Means ± SD of
the experiments on the 10 different patients with B-CLL (patients A-L)
examined. Panels on the right show the typical profiles of
[Ca++]i response to the indicated stimuli (patient A).
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Although exposure of the cells to Ga-ab resulted in the rapid
(2-minute) phosphorylation of tyrosine followed by a sharp decline,
treatment with Gaµ-ab caused a progressive increase in phosphotyrosine that reached its maximum by 5 minutes and slowly decreased thereafter (Figure 4A).
Further studies were carried out to identify the 2 proteins of 145 and
75 kd. Because their molecular weights were similar to those of syk and
PLC1 proteins, respectively, B-CLL cells from 2 patients (G, H) were
stimulated as above, lysed, immunoprecipitated with antiphosphotyrosine
mAb, electrophoresed, and blotted. The blots were subsequently stained
with specific antibodies against syk and PLC1 proteins. As shown in
Figure 4B the 2 proteins, which were phosphorylated after the 2 stimuli, indeed were identified as syk and PLC1.
Next we investigated the ability of Ga-ab and Gaµ-ab to induce
[Ca++]i mobilization. Results of 10 experiments on
different B-CLL patients (A-L) are summarized in Figure 4C, which also
shows typical profiles observed in 1 experiment (patient A). Again,
though the observed increments in [Ca++]i induced by
surface IgM cross-linking were similar in amplitude to those obtained
with IgD cross-linking, differences were noted in the kinetics and in
the shape of the profiles of the [Ca++]i responses. Thus,
after treatment with Ga-ab, the observed peak in
[Ca++]i was followed by a sharp decrease. In contrast, a
slower decline in [Ca++]i was noticed in the cells
exposed to Gaµ-ab.
Ability of Ga-ab to prolong cell survival
The above experiments showing that surface IgD cross-linking induced
tyrosine phosphorylation of the cytoplasmic proteins and
[Ca++]i mobilization suggested that Ga-ab was perhaps
capable of delivering signals to the cells that resulted in
physiological functions other than apoptosis. We investigated whether
Ga-ab caused cell proliferation, increased cell survival, or both.
As shown in Table 1, no changes in the cell
cycle were observed in cells exposed to Gaµ-ab or to Ga-ab for 48 hours, even when different cytokines (rIL2, rIL4, rIL6, rIL10) were
added to the culture individually or in combination (data not shown).
However, the cells were recruited into the cell cycle on treatment with
phorbol 12-myristate-13-acetate (PMA) and A23187.
Similarly, no thymidine incorporation above control values was observed
by exposing the cells to Ga-ab for 24, 48, or 72 hours. Table 1
shows the results of 1 representative experiment out of the 3 performed
on different patients with B-CLL (A, E, L). In contrast, exposure of
normal peripheral blood B cells to Gaµ-ab and Ga-ab caused their
recruitment into the S-phase of the cell cycle with little or no
apoptosis (Table 2).
Next, the cells from 7 CD38-positive patients with B-CLL (patients B,
C, D, E, F, G, L) were cultured in the presence of Ga-ab, Gaµ-ab,
or NGI for different times, and their viability was measured by PI dye
exclusion and flow cytometry analysis. Exposure to Ga-ab resulted in
a substantial improvement in cell viability that became most apparent
after 4 to 5 days of culture (Figure 5).
After 10 days of culture, the mean of the cell viability
values in Ga-treated cells (30 ± 6) was more than 2-fold that
of cells treated with NGI (12 ± 6). As expected, incubation with
Gaµ-ab lowered cell viability, and no viable cells were detected
after 6 days of culture. The addition of increasing concentrations of
Ga-ab to the cultures exposed to Gaµ-ab did not modify their poor
viability (data not shown).
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| Fig 5.
Prolonged survival of CD38-positive B-CLL cells in vitro
on exposure to Ga-ab.
B-CLL cells were cultured with Gaµ-ab (), Ga-ab (), or NGI
() at 10 µg/mL for various time intervals. Viability was
determined by PI exclusion and flow cytometry analysis. Results are
shown as mean ± SD of the experiments carried out on 7 patients
with B-CLL (patients B, C, D, E, F, G, L).
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Ability of Ga-ab to induce plasma cell differentiation
We investigated whether exposure of CD38-positive B-CLL
cells to Ga-ab could induce their differentiation into
immunoglobulin-secreting plasma cells. Purified B-CLL cells from 3 different patients (B, F, E) were incubated with Ga-ab in the
absence or presence of rIL2 and harvested after 3, 5, and 7 days. They
were stained by 2-color immunofluorescence with anti-CD19 and anti-CD38
mAb to detect cells with the CD19-low/CD38-high surface phenotype
typical of plasma cells.24 Figure
6 illustrates the results of 1 representative experiment (patient B) in which the cells were stained
after 5 days of culture, the optimal incubation time for observing
plasma cell differentiation. As shown in Figure 6, cells incubated with Ga-ab and rIL2 displayed an increased percentage of CD19+/CD38+ (gate 1) and the appearance of CD19-dim/CD38+ bright cells (gate 2).
These cells, likely to represent cells undergoing plasma cell differentiation, were virtually absent from the cultures treated with
NGI, rIL2 (Figure 6), or Ga-ab alone (data not shown). In these
experiments, the analysis of FSC and SSC parameters (Figure 6) showed
that in the cultures exposed to Ga-ab and rIL2, there were large
cells with substantial granularity (circle in Figure 6). These cells
stained with the plasma cell-specific mAb (CD138)25 and
were brightly stained by the CD38 mAb. Moreover, they were consistently
negative for CD3 and CD11b, thus excluding that they represented
contaminant T cells or monocytes. Collectively, these data favor the
accumulation of newly formed plasma cells in culture (Figure 6). The
expression of CD138 was very low to absent in the cells outside the
indicated gate and in the cells cultured with NGI or rIL2 alone.
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| Fig 6.
Differentiation into plasma cells of CD38-positive B-CLL
cells after treatment with Ga-ab and rIL2.
CD38-positive B-CLL cells were cultured in the presence of NGI,
Ga-ab (10 µg/mL) plus rIL2 (100 U/mL), or rIL2 alone for 5 days.
Cells were recovered, stained, and analyzed by flow cytometry. (top)
Double staining for CD19 and CD38 of the cells cultured with the
indicated stimuli. Gates 1 and 2 show CD19-bright, CD38-bright, and
CD19-dim/CD38-bright cells, respectively, together with their
percentages. (middle) FSC and SSC parameters of the cultured cells.
Large cells with substantial cytoplasmic granularity were gated, and
their fluorescence relative to the CD138, CD38, CD3, and CD11b
stainings was recorded (bottom histograms). These cells were present
only in Ga-ab-stimulated cultures. Cytospin preparations from
Ga-ab rIL2-cultured cells (left) or rIL2 cultured cells (right)
were fixed and stained with FITC-conjugated anti-IgM mAb. Results are
from 1 representative experiment (patient B) of the 3 performed
(patients B, E, F).
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The presence of plasma cells was confirmed by morphologic analysis of
the 5-day cultures carried out by immunofluorescence with anti-IgM mAb
(Figure 6) and Giemsa staining (Figure 7).
In the cultures treated with Ga-ab plus rIL2, we observed many cells with the morphology of plasma cells or plasmablasts (Figure 7A) that
containing intracytoplasmic IgM (Figure 6). They were absent from the
cultures in medium (not shown) or with rIL2 alone, in which most small
lymphocytes and a few apoptotic cells were observed (Figure 7B). As
expected, apoptotic and necrotic cells were present in the culture
exposed to Gaµ-ab (Figure 7C).
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| Fig 7.
Morphological analysis of B-CLL cells exposed to various
stimuli for 5 days in vitro.
B-CLL cells (patient B) were exposed to Ga-ab + rIL2 (A), rIL2 alone
(B), or Gaµ-ab + rIL2 (C). Plasmablasts and plasma cells were
observed after culture with Ga-ab + rIL2. In contrast, the cells
treated with Gaµ-ab were primarily apoptotic or necrotic. Necrosis
occurred after apoptosis probably because of the prolonged time in
culture (see also Figure 2C). In the preparations exposed to rIL2 only,
there were a few surviving small lymphocytes.
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The ability to Ga-ab to induce the differentiation of CD38-positive
BLL cells to immunoglobulin-secreting plasma cells in vitro was
confirmed by measuring immunoglobulin molecules in the culture
supernatants. As shown in Table 3,
substantial amounts of immunoglobulin molecules were measured virtually
only after exposure of the cells to Ga-ab in the presence of rIL2.
Moreover, these immunoglobulins expressed the same - or -type
found at the surfaces of the malignant B cells.
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Table 3.
Immunoglobulin molecules of the or type in the
culture supernatants after exposure of B-CLL to Gaµ-ab or
Ga-ab
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Discussion |
This study demonstrated that cross-linking the surface IgM or IgD of
CD38-positive B-CLL cells has different physiological outcomes. Signals
delivered by IgM activate programmed cell death, whereas those released
by IgD promote cell survival and differentiation. Furthermore, signals
received through the IgM receptor appear to predominate over those
released by IgD because IgD cross-linking does not prevent
Gaµ-ab-induced apoptosis.
It is unlikely that these data can be explained on the basis of an
inferior ability of Ga-ab to trigger the appropriate signal transduction pathway. Ga-ab was able to induce [Ca++]i
mobilization and tyrosine kinase activation in malignant cells as
efficiently as Gaµ-ab. The different responses to Gaµ-ab and Ga-ab cannot be explained by any of several technicalities related to the anti-immunoglobulin reagents used. For example, differences in
the binding affinities do not seem to be involved because Gaµ-ab and
Ga-ab equally stimulated normal peripheral blood B cells to
proliferate in vitro (see Table 2). Moreover, Gaµ-ab or Ga-ab did
not have different capacities for modulating surface IgM or IgD
expression, at least as determined by the equal ability of the leukemic
cells to re-express both surface isotypes in vitro after their
modulation by capping with Gaµ-ab or Ga-ab (not shown). Finally,
neither IgD nor IgM expression was selectively lost in culture during
these experiments (not shown).
Previous studies using special cell lines in vitro demonstrated that
IgM, but not IgD, could deliver apoptotic signals.6,7,26,27 Our results were in line with those observations and demonstrated that
surface IgD was capable of delivering a signal that improved cell
survival and promoted differentiation. The latter phenomenon was not
seen in the cell lines used for the experiments cited above, probably
because of the elevated proliferative activity of the malignant cells
in vitro, which might have prevented maturation into terminally
differentiated cells. It should be stressed that in our experiments,
the capacity to differentiate immunoglobulin-secreting cells was a
feature of a relatively small proportion of the malignant B-CLL cells.
Whether this resulted from an ongoing process of intraclonal maturation
that rendered only a proportion of the cells susceptible to the signal
delivered by IgD remains to be established. Similarly, the requirement
for additional signals to improve cell differentiation or even to
promote cell proliferation, such as that delivered by CD40L, has to be
investigated.28
The choice of CD38-positive B-CLL for this study was indicated by the
observation that in CD38-positive B-CLL cells, the signal transduction
pathway initiated by cross-linking of IgM is generally preserved.15 It is of interest that CD38 per se does not
seem to be involved in the signal-transducing pathway of the B-CLL cells, at least based on the observation that its cross-linking is not
followed by [Ca++]i mobilization.15 Thus,
although studies in murine and human experimental systems have
demonstrated an involvement of CD38 in BCR signaling,29-33
in B-CLL cells CD38 should be considered a marker of a particular
physiological stage of the cell whose precise function has yet to be determined.
A crucial problem related to the current observations is to determine
why CD38-positive B-CLL present different IgM- and IgD-mediated responses. In principle, 2 hypotheses can be formulated. One implies that B-CLL cells are different from normal B cells in that neoplastic transformation causes a number of changes, including differential responses through IgM or IgD receptors. The other and, in our opinion,
more likely possibility is that, because of the malignant transformation, CD38-positive B-CLL cells remain frozen at a particular stage of differentiation in which normal B cells also have the same
physiological properties. A similar condition has been observed in
transgenic mice: B cells expressed BCR with anti-TNP
activity.9 These mice are characterized by a subpopulation
of B cells that remains frozen at a particular stage of
maturation in which surface IgM engagement causes clonal
elimination, whereas IgD engagement results in cell protection
and activation. Cells sharing the same features were not
observed, however, in transgenic autoimmune mice12 or in
irradiated mice reconstituted with normal splenic B
cells.11 This suggests that in anti-TNP transgenic mice,
conditions favored the accumulation of B cells at a stage that
might have been physiologically transient.
Studies are in progress to identify the normal counterpart of the
CD38-positive B-CLL cells in lymphoid tissues from patients without
neoplasia. The CD38-positive B-CLL cells described in this study did
not seem to correspond to either the normal germinal center B
cells34 or the germinal center founder cells.35
Both B-cell subsets share CD38 expression but differ in several aspects from CD38-positive B-CLL cells. For example, unlike B-CLL cells, both
of these cell types express CD10, CD77, and CD95, both are prone to
spontaneous apoptosis,34,35 and both have substantial somatic mutations in their VH and VL immunoglobulin
genes.35,36 In contrast, though many B-CLL cells display
somatic mutations in their VH and VL genes,37 CD38-positive
B-CLL cells seem to represent a remarkable exception in that they
express VH and VL genes that have not undergone mutation.38
The finding that opposing signals can be delivered by 2 receptors with
similar cytoplasmic tails and the same ability to associate with Ig-
and Ig- molecules raises a number of as yet unanswered questions
related to the mode of their signal transduction. Preliminary evidence
indicates an involvement of caspase 3 in the Gaµ-ab-induced programmed cell death of B-CLL cells. In contrast, caspase 8 does not
seem to be activated during this process, suggesting that apoptosis is
directly induced by signals delivered through the BCR and is not
mediated by Fas and FasL surfacing, as has been proposed to explain
apoptosis of several cell types.39 Consistent with this
observation is the failure to induce Fas expression of B-CLL cells by
Gaµ-ab. It is possible that IgM and IgD activate basically the same
signal-transducing pathway to the level of [Ca++]i
mobilization and PLC1 activation. From this point on, the 2 pathways
may diverge, as has been described for certain cell lines.40
Recent evidence indicates that surface IgM and IgD are associated with
different connecting molecules3,4,41 and that, though not
formally proven, the presence of these specific proteins could explain
the functional difference between the IgM and IgD BCR.
CD38-positive B-CLL cells may represent a useful model to clarify the
role of a number of apoptotic and antiapoptotic genes. For example, we
have preliminarily determined that c-myc, which predisposes
cells to apoptosis,23,42 is elevated in B-CLL. Moreover,
the antiapoptotic gene bcl-2 is up-regulated, and the induction of
apoptosis by Gaµ-ab is not followed by its prompt down-regulation
(preliminary data). The latter finding probably emphasizes the importance of other apoptotic genes such
as Bax, BcL-X, BAG1.43-46 Studies along this line are in progress.
Finally, a crucial problem is represented by the relative functions in
cell physiology of 2 surface isotypes that deliver opposite signals.
Although the current in vitro studies indicated that the signals
delivered by IgM always predominated and hence that a cell in the
physiological state, corresponding to a leukemic CD38-positive B cell,
would invariably be destined to apoptosis, this may not necessarily be
the case in vivo after antigen interactions. Under these conditions,
the relative ratios of IgM and IgD, the antigen-binding capacity of the
2 isotypes, and their different abilities to elicit help from T cells
may play a role in determining the outcome of the response to apoptosis
induction or antibody production. Therefore, different signaling by IgM
or IgD may contribute to the fine-tuning of responses under particular
circumstances.47,48
| |
Acknowledgments |
The authors thank Drs Nicholas Chiorazzi and John Monroe for helpful
discussions, Nicholas Chiorazzi for critical review of the manuscript,
Massimo Ulivi for technical advice, and Ms Teresa Tavilla for skillful
secretarial assistance.
| |
Footnotes |
Submitted June 1, 1999; accepted October 13, 1999.
Supported by grants from Associazione Italiana per la Ricerca sul
Cancro, Progetto di Ricerca di Ateneo, and Cofinanziamento Murst 1999.
Reprints: Simona Zupo, Servizio di Immunologia Clinica,
Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi N. 10, 16132 Genoa, Italy; e-mail: szupo{at}hp380.ist.unige.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction