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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 701-712
Reactive Plasmacytoses Are Expansions of Plasmablasts Retaining the
Capacity to Differentiate Into Plasma Cells
By
Gaëtan Jego,
Nelly Robillard,
Denis Puthier,
Martine Amiot,
Françoise Accard,
Danielle Pineau,
Jean-Luc Harousseau,
Régis Bataille, and
Catherine Pellat-Deceunynck
From INSERM U463, Laboratoire Central d'Hématologie, Institut
de Biologie, Service d'Hématologie Clinique, Hôtel-Dieu,
Nantes, France.
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ABSTRACT |
Circulating plasma cells in 10 cases of reactive plasmacytosis had a
shared phenotype with early plasma cell (CD19+
CD38+ CD138+ CD40+
CD45+ CD11a+ CD49e
CD56). In most cases, a minor subpopulation of
CD28+ plasma cells was also detected. Reactive plasma
cells were highly proliferative, suggesting the presence of circulating
progenitors (plasmablasts). After CD138+ plasma cell
removal, highly proliferative CD138 plasmablasts
differentiated into CD138+ plasma cells within a few
days. This differentiation, which was associated with increased CD38
and decreased HLA-DR expression, was further confirmed by a large
increase in intracellular Ig content (associated with Ig secretion) and
was concomitant with extensive secretion of interleukin-6 (IL-6). The
addition of neutralizing anti-IL-6 and anti-CD126 (IL-6 receptor)
monoclonal antibodies totally prevented Ig secretion and cell
differentiation by inducing apoptosis of plasmablasts, which indicates
that IL-6 is an essential survival factor for plasmablasts. This report
provides the first characterization of normal plasmablasts and shows
that their phenotype is not exactly that of multiple myeloma cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
LONG-LIVED NONPROLIFERATING bone marrow
plasma cells (PCs) are generated during immune response, essentially
memory immune response.1,2 Memory B cells activated by the
antigen in secondary lymphoid organs in close contact with dendritic
cells differentiate into plasmablasts that migrate into the bone marrow and finally become PCs.1-3 Plasmablasts, unlike PCs, are
short-lived proliferating cells.2 In well-controlled immune
response, plasmablasts represent less than 0.1% of peripheral blood
mononuclear cells, and no PCs are detected in normal peripheral blood.
For these reasons, the study of terminal PC differentiation remains
difficult, although it is especially critical if progress is to be made
in the biology of myeloma cells, the malignant counterpart of normal PCs.4 Although several models of PC differentiation have
been developed in vitro to overcome these difficulties,5-10
most of them are limited by the low number of PCs obtained. Yet, recent studies have shown that CD27 and/or CD40 triggering of memory cells
allows more than 50% of PCs to be obtained.9,10
Unfortunately, the study of plasmablast differentiation into PCs is not
easy with these models, because the culture is a mix of B cells,
plasmablasts, and PCs. Interestingly, a large number of nonmalignant
PCs can be obtained in cases of reactive plasmacytosis (RP), thereby
allowing more detailed study of normal PCs. RP has been reported in a
variety of infections and neoplastic conditions,11-14 and
the PCs involved are polyclonal.11-13 However, data
concerning the biology of reactive PCs remain sparse. In vivo, reactive
plasmacytoses are known to be proliferative and to disappear
spontaneously and rapidly, but no data are available concerning the
mechanisms controlling their proliferation and cell survival or death.
More recently, reactive plasmacytoses have been observed in patients
bearing interleukin-6 (IL-6)-producing tumors in cases of cardiac
myxoma and gastric carcinoma.12,14 Furthermore, the
proliferation of reactive polyclonal PCs in cardiac myxoma has been
shown to be IL-6-dependent.12 Because IL-6 has been
extensively described as a B-cell differentiation factor15
and as a growth factor for myeloma cells,4 the study of the
mechanisms regulating the proliferation and death of PCs from RP could
be of major interest in improving our understanding of myeloma cells.
We report here the phenotype of PCs from 10 reactive plasmacytoses. In
all 4 cases evaluated, reactive plasmacytoses involved highly
proliferating cells, suggesting that available circulating plasmablasts
were present. In fact, plasmablasts in vitro differentiated into PCs,
which facilitated studies of their phenotypic and functional characterization and of the differentiation process. The implications of plasmablast characterization are considered with respect to multiple
myeloma (MM) as an expansion of malignant plasmablasts and plasma cells.
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MATERIALS AND METHODS |
Reactive Plasmacytoses
Ten cases of plasmacytosis were collected over a 3-year period. In all
cases, transient polyclonal circulating PCs were observed in peripheral
blood (from 8% to 79% PCs) in different contexts, mainly infections
(n = 5, including 2 Epstein-Barr virus [EBV] infections in
immunocompromised hosts, designated as RP5 and RP7) or tumors (n = 3;
Table 1). A diagnosis of reactive
plamacytosis was established initially by cytological identification of
PCs on spins, followed by flow cytometry PC identification
(intracellular  and  staining). For patient RP5, plasmacytosis
was quite extensive in terms of the absolute number of cells and the
percentage of plasma cells allowing the cryopreservation of peripheral
blood mononuclear cells (PBMNCs).
In vitro, cells were cultured in RPMI 1640 (GIBCO BRL,
Cergy-Pontoise, France) supplemented with 10%
heat-inactivated fetal calf serum (FCS; GIBCO BRL), 2 mmol/L glutamine,
100 µg/mL streptomycin, 100 U/mL penicillin, and 2 × 105 mol/L 2-mercaptoethanol.
Reagents and Antibodies
Recombinant human fibroblast growth factor basic (FGF
basic) was obtained from R&D (Minneapolis, MN).
Except for anti- (), which were rabbit polyclonal antibodies, all
others were mouse monoclonal antibodies (MoAbs). Anti-CD138 (B-B4), anti-CD40-phycoerythrin (PE),
anti-CD95 (B-G27), and anti-IL-6 (B-E8) MoAbs were obtained from
Diaclone Research (Besançon, France); APO2.7-PE, unconjugated or
PE- or fluorescein isothiocyanate (FITC)-conjugated IgG1
control, anti-CD11a-PE, anti-CD19-FITC/-PE, anti-CD20, anti-CD21,
anti-CD23-FITC, anti-CD45-FITC, anti-CD45RA-FITC, anti-HLADR-PE,
anti-CD95 (CH-11), anti-CD126-PE, and FITC-conjugated goat antimouse
(GAM) MoAbs from Immunotech (Marseille, France); anti--PE and
anti-CD45RO-FITC from Dako (Glostrup, Denmark); anti-CD20-FITC,
anti-CD22-PE, anti-CD25-PE, anti-CD28-PE, anti-CD38-PE, anti-CD56-PE,
and anti-CD80-PE MoAbs from Becton Dickinson (San Jose, CA);
anti-CD86-PE MoAb from Pharmingen (San Diego, CA); anti--PE ()
MoAb from Tebu (Le Perray en Yvelines, France); anti-CD49e-PE (SAM-1)
MoAb from Serotec Ltd (Oxford, UK); goat neutralizing antihuman FGF
basic Ab from R&D; and Steptavidin-Quantum Red Conjugate (PECY5) from
Sigma (St Louis, MO). Anti-CD138, anti-CD95 (B-G27), and IgG1 control
MoAbs were conjugated to biotin, as previously described.16
IL-6 and Ig Enzyme-Linked Immunosorbent Assay (ELISA)
IL-6 concentrations were determined in cell supernatants using an ELISA
from Diaclone Research. Ig (IgA + IgG + IgM) concentrations were also
determined by an ELISA. Briefly, a 96-well plate (MaxiSorp Plate; Nunc,
Glostrup, Denmark) was coated in phosphate-buffered saline
(PBS) overnight at 4°C with 5 µg/mL mouse antihuman chain MoAb (clone 6E1; Immunotech) or antihuman chain MoAb (clone C4;
Immunotech), washed 3 times in PBS 0.5% Tween 20, saturated for 1 hour
at 4°C with PBS containing 3% bovine serum albumin (BSA),
incubated with standards or samples for 2 hours at 20°C, washed 3 times in PBS 0.5% Tween 20, incubated with peroxidase-conjugated affinipure goat antihuman Ig (IgA + IgG + IgM; Jackson ImmunoResearch Laboratories, West Grove, PN) for 1 hour at 20°C, washed 3 times in
PBS 0.5% Tween 20, and incubated for a few minutes with 50 µL
peroxidase substrate (0.5 mg/mL o-phenylene diamine dihydrochloride; Sigma) in 0.1 mol/L sodium acetate (pH 4) containing 0.003%
H2O2. The reaction was stopped by adding 50 µL of 1 N H2SO4, and absorbance was
determined with a spectrophotometer at 490 nm. Standardization was
performed with human gammaglobulin (Jackson ImmunoResearch Laboratories).
Cell Staining and Flow Cytometry
For immunostaining, cells were incubated with the different MoAbs in
simple, double, or triple staining in PBS containing 1% BSA, 0.02%
NaN3, and 20% AB serum at 4°C for 30 minutes, washed, and fixed in PBS 1% formaldehyde. For unconjugated and biotinylated MoAbs, a second incubation was performed before fixation with FITC-GAM
and PECY5-conjugated streptavidin, respectively. and staining
was performed without AB serum. For intracellular and staining,
cells were fixed and permeabilized using IntraPrep Permeabilization
Reagent (Immunotech), stained for 30 minutes at 4°C in PBS
containing 1% BSA, washed, and fixed in 1% formaldehyde. Fluorescence-activated cell sorting (FACS) analysis was
performed on a FACScalibur apparatus (Becton Dickinson). Acquisition
and analysis were performed with Cell-Quest software (Becton
Dickinson). The fluorescence intensity ratio (r) was obtained by
dividing the fluorescence of each antigen by the fluorescence of the
appropriate control. The levels of this ratio were defined as follows:
 , r < 1.2; +/, 1.2 < r < 2; +, 2 < r < 20; ++,
20 < r < 50; +++, r > 50.
Determination of Cell Proliferation by BrdU Incorporation
Cells were incubated for 2 hours with 50 µmol/L BrdU
(5-bromo-2'-deoxyuridine; Sigma) at 37°C in culture medium,
washed in PBS, and permeabilized overnight in PBS containing 0.01%
Tween 20 and 1% paraformaldehyde. Double staining with FITC anti-BrdU- and PE-conjugated anti- or anti- Abs was performed as previously described.17
Immunomagnetic Bead Purification/Removal of Plasma Cells and B Cells
MACS LS+ separation columns (Miltenyi) were
used for immunomagnetic bead depletion of PCs, according to the
protocol of the supplier (Milteny: Biotec, Parish, France). Briefly,
cells were labeled with anti-CD138 B-B4 IgG1 MoAb, washed,
and then incubated with rat antimouse IgG1 MACS MicroBeads. Unlabeled
cells were not retained by the column and constituted the
CD138 fraction. Retained cells (CD138+
cells) were then flushed out, consituting the CD138+
fraction. These separation procedures allowed us to obtain pure populations (>95%): CD138+ purification was checked by
cytological analysis on cell cytospins, and CD138
purification was monitored by CD138 expression analysis. When indicated
in the text or legends to figures, B lymphocytes were removed by the
same method, using B9E9 anti-CD20 IgG2 MoAb (Immunotech) and rat
antimouse IgG2 MACS MicroBeads. The removal of B cells was performed
before CD138+ population purification or removal.
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RESULTS |
Peripheral Blood Plasma Cells From RP Have a Shared Phenotype With the
CD49e Plasma Cell Precursor
Ten reactive plasmacytoses were analyzed in the two different contexts
of infections and tumors (Table 1). The diagnosis of RP was established
by cytological detection of PCs in peripheral blood. In flow cytometry,
PCs were identified by CD138++, CD38bright, and
intracytoplasmic + or +
(Fig 1). The phenotype of PC was determined
in double staining with CD138 (syndecan-1). As shown in Fig 1 and
summarized in Table 2, circulating PCs were
polyclonal and presented a shared phenotype: CD19+
CD38+++ CD138+++ CD40+
CD45int CD49e CD56
CD11a+. Moreover, a very minor population of
CD28+ PC (range, 3% to 12%) was observed in 6 of 9 evaluated cases. The expression of CD45 was intermediate, ie, inferior
to lymphocyte expression but superior to malignant PCs from patients
with MM (not shown). The phenotype of reactive PCs proved to be very
similar to that of CD49 immature PCs, ie, PC
precursor.19,20
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| Fig 1.
Phenotype of plasma cells from RPs. The phenotype of
plasma cells was determined in triple staining with anti-CD138-PECY5,
anti-CD45-FITC, and the PE-conjugated MoAb indicated. Acquisition was
performed on all cells (dot-plots A and B) or on CD138+
gated cells (dot-plots C and D). Fluorescence analysis was performed on
gated cells (R1 or R2 or R3), as indicated in each dot-plot. Dot-plots:
A, RP4; B, RP1; C, RP4; D, RP7.
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Peripheral Blood Plasma Cells From RP Result From the Maturation of
Plasma Cell Progenitors (Plasmablasts) In Vivo and In Vitro
Maturation of polyclonal reactive plasma cells in vitro.
In most cases of RP, CD138 was not expressed by all PCs (Table 2). As
shown in Fig 2, not all CD38 bright cells
(PCs)21 expressed CD138. Furthermore, the absolute number
of cytoplasmic plus positive cells was always superior to that
of CD138+ cells, which confirms that CD138 was not
expressed by all PCs (Table 2). This failure of anti-CD138
B-B4 MoAb to stain all circulating PCs prompted us to
search for the presence of putative plasmablasts. When PBMC were
cultured for a few days in RPMI containing 10% FCS (Fig
2), most CD38 bright cells spontaneously acquired CD138 expression
within a few days (Fig 2A). The fraction of CD138+ cells
(13% at day 4 v 5% at day 0) and the fluorescence intensity ratio (560 v 133) were clearly enhanced. To exclude any
possibility that the increase in the CD138+ population
resulted from the death of CD138 cells,
CD138+ cells were removed at day 0 before in vitro culture.
As shown in Fig 2B, 15% of cells expressed CD138 (ratio [r] = 471)
after 4 days, compared with none at day 0. This upregulation of CD138 was accompanied by a significant enhancement of the CD38 expression level (Fig 2A).
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| Fig 2.
CD138 expression is upregulated during maturation of
plasma cells in vitro. (A) CD138 expression was determined in double
staining with CD38 in unseparated cells at day 0 and after a 4-day
culture. The percentage and fluorescence intensity ratio of CD138
expression were defined in the R1-gated cells and are indicated within
the dot-plots. (B) After immunomagnetic removal of CD138+
cells, CD138 cells were cultured for 4 days, and the
expression of CD138-PECY5 (FL3-H) (percentage and fluorescence
intensity ratio r) was determined in the R2-gated population.
CD138+ and CD138 cell separation was
performed as described in Materials and Methods (RP8).
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These phenotypic modifications were associated with morphogical
maturation into PCs, as indicated by dense chomatin clumping, well-developed cytoplasm, and an eccentrically placed nucleus (shown
for patient RP5 in Fig 3A and B). In vivo,
mature PCs (E) as well as occasional cells with prominent Russell
bodies (F), known as Mott cells,22 were only seen within
the bone marrow. In vitro, mature PCs, as well as occasional Mott
cells, were seen, but only after 6 days of culture (B). These data
suggest that PC precursors were present in peripheral blood and were
able to differentiate in vitro and in vivo.
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| Fig 3.
Plasma cell differentiation in vitro and in vivo.
May-Grünwald-Giemsa staining of cell cytospins at day 0 (A and C)
or 6 (B and D) from fresh (A and B) or thawed (C and D) PBMNCs or of
bone marrow smears (E and F) from patient RP5. From day 0, fresh or
thawed PBMNCs were cultured for 6 days in 10% FCS RPMI 1640. After
thawing, plasma cells (CD138+) and B cells
(CD20+) were removed, as described in Materials and
Methods. The CD19+ plasmablastic population (C) accounted
for 95% of cells; the remaining cells were T lymphocytes and
monocytes. (Original magnification × 1,000.)
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Maturation correlated with the extinction of proliferation.
The high proliferation of freshly explanted cells was apparent in the 4 cases evaluated (RP5, 7, 8, and 9). Figure
4 shows that 15% to 50% of c+ and 15% to 50% of
c+ incorporated BrdU in the case of RP7 and RP9.
However, cell maturation correlated with a dramatic extinction of
proliferation when cells were cultured in vitro. After only 48 hours of
culture, the percentage of BrdU+ cells was reduced sixfold
(dot-plot A), whereas no BrdU+ cells were detected after 3 days (dot-plot B). Taken together, the immature phenotype and the high
proliferation argue for the presence of a highly proliferative
plasmablastic compartment.
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| Fig 4.
High-rate proliferation of reactive plasma cells is
rapidly downregulated. Freshly explanted cells were incubated for 2 hours with 1 mmol/L BrdU at day 0 and at day 2 or 3, as indicated in
the figure. BrdU+ cells were determined in the
intracytoplasmic (c) or population by double staining with
anti--PE or anti--PE MoAb versus FITC-anti-BrdU MoAb, as
described in Materials and Methods. For each dot-plot, 10,000 events
were acquired. Dot-plots: A, RP9; B, RP7.
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Isolation and Characterization of Plasmablasts
Isolation and phenotypic characterization of plasmablasts.
To achieve their isolation, plasmablasts were negatively purified by
eliminating PCs using magnetic beads coupled to anti-CD138 (RP 5, 7, 8, and 9). RP5 and RP7 were observed in the context of EBV (re)activation.
To avoid any confusion between B lymphocytes (CD19+
CD20+) and plasmablasts (CD19+
CD20), the former were removed using magnetic beads
coupled to anti-CD20. Because plasmacytosis was quite extensive for RP5
(>89% of PBMC), the removal of plasma cells and B cells allowed us
to obtain a very pure population of plasmablasts (95%; Fig 3). The
remaining cells were T lymphocytes and monocytes. The plasmablasts were unrecognized cytologically either as B lymphocytes or as PCs (Fig 3C).
Moreover, these cells spontaneously differentiated into PCs, which was
the only cell population present within a few days (Fig 3D, day 6).
Cytological analysis showed that cells first became plasmacytoid (from
day 0 to day 2) and then PCs (after day 2), progressively reaching the
100% level at day 4 (data not shown). Few plasmablasts were also found
within bone marrow (not shown).
The phenotype of the remaining population obtained by negative
purification was defined in double staining with CD19
(Table 3). Table 2 clearly shows that these
cells were neither resting nor activated B lymphocytes, because they
did not express CD20, CD21, CD23, or CD25 and only weakly expressed
CD40 and CD80. They were not PCs, because CD138 was not expressed, CD38
was not very brightly expressed, and intracellular Ig was not highly
expressed.16,19-21,23 These data show that the plasmablast
phenotype was distinct from both resting and activated B-lymphocyte
phenotypes and from the plasma cell phenotype. Moreover, plasmablasts
expressed surface Ig.
Plasmablasts (CD138) are more proliferative than
early plasma cells (CD138+).
Because plasmablasts and plasma cells could be separated on the basis
of CD138 expression, the labeling index of CD138 and
CD138+ cells was determined. As shown in
Fig 5 for the + population,
BrdU+ cells were mainly located within the
CD138 fraction (25% for CD138
cells v 8% for CD138+ cells). The same result was
obtained for the + population (data not shown). Because
the survival of reactive plasma cells was dependent on paracrine IL-6,
3 ng/mL of IL-6 was added in each fraction at the time of initial
culture.
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| Fig 5.
Proliferative reactive plasma cells are mainly located in
the CD138 plasmablastic fraction. The labeling index was
determined by BrdU incorporation, as described in Fig 4. The labeling
index was determined in the unseparated population and the
CD138+ and CD138 populations.
CD138+ and CD138 cell isolation was
performed as described in Materials and Methods (RP10). Acquisition was
performed either in R1 gated cells (A and B) or R2 gated cells (C and
D). (A and C) -PE versus BrdU-FITC; (B and D) -PE versus control
IgG1-FITC.
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Characterization of Plasmablast Differentiation Into Plasma Cells
CD138 expression is upregulated during differentiation.
To determine critical phenotypic changes during the differentiation
process, we looked at the expression of different molecules, ie, CD38,
CD49e, CD138, HLA-DR, and cytoplasmic Ig. Plasma cells (CD138+) and B lymphocytes (CD20+) were removed
before culture.
Figure 6A shows that CD138 was not
expressed on plasmablasts (D0 dot-plots), but was quickly upregulated
from day 1 (20%, r = 25). Both the percentage and fluorescence
intensity ratio increased progressively (41%, r = 58 at day 2),
reaching 63% (r = 147) at day 7. The CD38 intensity ratio was highly
enhanced during the differentiation process, ie, from 332 at day 0 to
1,353 at day 4 (4-fold increase), as shown with RP5. The expression of
CD49e was negative in CD19+ plasmablastic cells and
remained negative in CD38+++ PCs (Fig 6). The intensity of
cytoplasmic Ig was enhanced, as shown in Fig 6B for light-chain
expression. The fluorescence intensity ratio increased from 16 to 75 within 2 days and then remained stable for 7 days. HLA-DR expression
was decreased from r = 6 to 3 within 4 days (RP8) and from r = 4.7 to
1.6 within 7 days (RP5; Fig 6C).
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| Fig 6.
Phenotypic changes during the differentiation process of
plasmablasts into plasma cells in vitro. (A) Analysis of CD138 and CD19
expression in double staining with anti-CD138-biotin-streptavidin-PECy5
and anti-CD19-PE MoAbs and analysis of CD38 expression in simple
staining during time-course differentiation (RP5). At day 0, CD138+ and CD20+ cells were removed as
described in Materials and Methods. The percentage of positive cells
and the fluorescence intensity ratio (r) are indicated. Intracellular
light-chain staining was performed, as described in Materials and
Methods. The histograms show the fluorescence profiles for or expression as indicated. (B) Analysis of CD49e expression at day 0 or 7 by double staining with anti-CD49e MoAb and anti-CD19-FITC MoAb or
anti-CD38-FITC MoAb, respectively (RP5). (C) Analysis of the expression
of HLA-DR by simple staining with anti-HLA-DR-PE MoAb. Overlay
histograms represent the immunofluorescence of HLA-DR and the control.
(Left) RP8; (right) RP5.
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Investigations in all 4 cases analyzed showed that CD138, CD38, cIg,
and HLA-DR were the only molecules whose expression was modulated
during the differentiation process. Our results indicate that CD138 was
the only antigen able to discriminate plasmablasts from PCs.
Because FGF basic has been reported to induce CD138 expression in
mesenchymal cells,24 we investigated whether FGF basic was
involved in the upregulation of CD138 expression in PCs. Neither the
addition of FGF basic (10 ng/mL) nor that of neutralizing anti-FGF Ab
(50 µg/mL) modified CD138 upregulation or Ig secretion, indicating
that FGF basic was not involved in this process
(Table 4).
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Table 4.
Neutralization of IL-6 (BE-8) and Its Receptor CD126
(PM-1) Prevented Differentiation by Inducing Apoptosis of
Plasmablasts
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Differentiation of plasmablasts into plasma cells is characterized by
enhanced Ig secretion and extensive apoptosis.
Cytologic and phenotypic differentiation was confirmed by studies of
cell capacity to secrete Ig. Figure 7A
shows that Ig accumulated rapidly in supernatant for 7 days. At day 0, 98% of cells were viable, but only 28% were viable at day 7. Thus,
the capacity of cells to secrete Ig was enhanced from day 0 to day 7.
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| Fig 7.
Kinetics of IgG secretion, apoptosis, and IL6-R
(CD126) expression. After thawing (RP5), CD20+
cells were removed and the population was separated into
CD138+ and CD138 cells. (A)
CD20 CD138 cells were cultured in RPMI
1640 10% FCS with 3 ng/mL IL-6 for 7 days. At days 0, 1, 2, and 7, supernatant was harvested and the amount of secreted Ig was determined
by ELISA. The number of viable cells was determined by trypan-blue
exclusion. (B and C) CD20 CD138+
cells (B) and CD20 CD138 (C) cells
were cultured in RPMI 1640 10% FCS with 3 ng/mL IL-6. Apoptotic cells
were determined by Apo2.7-PE staining. CD138-PECY5 and CD126-PE
expression was determined in double staining in the viable cell
population (R1 dot-plot FSC v SSC).
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IL-6 Is an Essential Survival Factor for Plasmablasts
This model of RP proved to be of particular interest, because
differentiation was spontaneous and required no exogeneous factor. In 3 evaluated cases, the determination of cytokines produced in vitro
showed that a large amount of IL-6 was secreted (range, 2 to 20 ng/0.2 × 106 cells × 24 hours). Purification of each
peripheral blood cell population (plasma cells, B lymphocytes, T
lymphocytes, and monocytes) indicated that IL-6 was produced
essentially by monocytes (data not shown). To determine its role in the
differentiation process, IL-6 was neutralized with an anti-IL-6 MoAb
(B-E8) or B-E8 and a blocking anti-CD126 MoAb (PM-1) at the time of
initial culture, which induced a dramatic inhibition of IgG secretion
(data not shown). CD138 expression was then evaluated to correlate
inhibition of IgG secretion with a lack of differentiation. It was
found that the proportion of CD138+ cells was highly
reduced among B-E8-treated cells (Table 4). Compared with untreated
cells, the level of CD138 staining was decreased, and
CD138++ cells were reduced from 25% to 9% at day 3 (RP5).
The lack of CD138 induction was associated with a large increase in the
number of apoptotic cells. APO2.7 MoAb, which recognizes 7A6
antigen,25 was used to characterize and count apoptotic
cells. Table 4 shows that the proportion of apoptotic cells was
enhanced threefold by B-E8 treatment (42% v 13% at day 3, RP5). When both B-E8 and PM-1 were added, a total inhibition of CD138
induction was observed (RP8 and 9). Once again, the absence of CD138
expression was clearly associated with an increase in apoptotic cells
(3-fold at day 3).
In view of the reduced upregulation of CD138 and the increase in
apoptosis, our data indicate that IL-6 is an essential survival factor
for plasmablasts.
Despite the presence or addition of IL-6, reactive plasma cells were
subject to apoptosis. Figure 7B and C show that both CD138 and CD138+ cells died by apoptosis
(as determined by Apo2.7 staining). In CD138+ cells,
positive CD126 expression was not modulated in culture. Although only
approximately 30% of CD138 cells expressed CD126 at
day 0, they were all CD126+ after 2 days. Despite this
overall expression of CD126, apoptotic cells kept accumulating,
reaching 72% at day 7. This suggests that loss of cell viability and
apoptosis were not related to CD126 expression.
| |
DISCUSSION |
In 10 cases of RP, circulating PC had a shared phenotype
(CD11a+ CD19+ CD281-12%+
CD38+++ CD40+ CD45int
CD49e CD56 CD138+++).
Circulating PCs can be regarded as PC precursors or early plasma cells,
as defined by Kawano et al,19 because they lack the CD49e expression characteristic of bone marrow PCs, the reference mature PCs.16,19,26 CD28 was expressed in a minor subpopulation of cells from 6 of 9 of the reactive plasmacytoses, whereas we previously showed that CD28 is not expressed in bone marrow PCs.16
CD138 expression, as compared with that of bone marrow PCs, was weaker and heterogeneous, ie, CD38+-reactive PCs were not all
CD138+.16 However, when cells were cultured a
few days in vitro, CD38+ CD138 PCs all
became CD138+, and the level of CD38 expression was
significantly enhanced (n = 4). Because CD138 expression is lost by
apoptotic malignant PCs (unpublished data and Jourdan et
al27), the disappearance of CD38+
CD138 cells could be due to apoptosis. In all 4 cases evaluated, CD138 cells were not stained by
Apo2.7 MoAb (data not shown).
In all investigated RPs (n = 4), circulating PCs proliferated highly ex
vivo. Proliferation was downregulated spontaneously and rapidly (within
4 to 7 days) after cytological maturation of cells in vitro, suggesting
that PC progenitors (plasmablasts) were present in peripheral blood and
able to differentiate into PCs in vivo and in vitro. To further
demonstrate this hypothesis, we removed CD138+ plasma cells
and CD20+ B cells and determined the phenotype of
plasmablasts in double staining with CD19. The CD19+
CD20 CD21 CD23
CD38+ CD40low CD45int
CD56 CD80+/ CD86+
CD138 HLA-DR+ sIg+
cIg+ phenotype clearly distinguished plasmablasts from B
lymphocytes and PCs. When the plasmablasts were cultured in vitro, they
spontaneously differentiated into PCs (Figs 2, 3, and 6). Quite
interestingly, CD138 was not expressed in plasmablasts but induced
early and progressively during the differentiation process. Our data
indicate that CD138 was the only differentiation antigen allowing PCs
to be distinguished from plasmablasts. The differentiation of
plasmablasts into plasma cells was also characterized by an increase in
CD38 and cytoplasmic Ig expression and a decrease in HLA-DR expression. The upregulation of CD38 during the differentiation of immature PCs
into mature PCs was in good agreement with a previous study, showing
that early blood PC CD49e CD38++
differentiated into mature PC CD49+ CD38+++
upon addition of IL-6 or stromal cells.19 Nevertheless, no upregulation of CD49e was observed during RP, even in the presence of
stromal cells and/or IL-6 (data not shown). The phenotype of reactive
short-lived bone marrow PCs remains undefined. In 2 cases (RP5 and
RP7), bone marrow smears were available and mature PCs were observed.
However, their phenotype, especially CD49e expression, could not be
determined. The expression of CD49e on plasma cells remains
controversial, requiring further investigation. Bone marrow and tonsil
plasma cells were found to be either CD49e+19,20,26 or
CD49e,3,28 even though the same MoAb was
used in all these studies (Sam-1, IgG2b isotype). In our hands, mature
reactive plasma cells (obtained in vitro after 6 days of culture) were
always found to be CD49e, regardless of the
anti-CD49e MoAb used (from the Fifth International Workshop on White
Cell Differentiation Antigens; data not shown). The decrease in HLA-DR
expression during differentiation is reminiscent of the previously
described downregulation of HLA-DR during IL-6-induced differentiation
of a few B cells into PCs (CD38++) in the CESS
EBV+ cell line.29 Nevertheless, the precise
role of IL-6 in this model is still uncertain, because IgG secretion
was found to increase when proliferation was inhibited by
hydroxyurea.6
Our data provide the first evidence that IL-6 (mainly produced by
monocytes) is the survival factor for human plasmablasts. Previous
studies demonstrated the critical role of IL-6 in PC differentiation
but did not indicate its precise role.15 More recently,
Kawano et al19 demonstrated that IL-6 was an essential survival factor for early circulating PC CD38+
CD49e. The ability of IL-6 to rescue plasmablasts
from apoptosis could explain why reactive plasmacytoses are observed in
vivo mainly in cases of excessive IL-6 production.12,14 We
confirmed that reactive plasmacytoses, as reported in 2 sparse
cases,11,14 are associated with detectable circulating IL-6
(4 of 4 cases). In fact, PCs were observed as long as IL-6 was
detectable in vivo, ie, with high C-reactive protein values. This
disappearance of circulating PCs was not simply the result of migration
(eg, to bone marrow), because the significant increase in total serum Ig (eg, IgG >40 g/L) disappeared within 2 to 4 weeks (data not shown). This rapid decrease in Ig serum is a further indication that
PCs are not long-lived, which is contrary to a recent report claiming
that bone marrow PCs in the mouse are very long-lived, ie,
t1/2 = 100 days.30
The majority of cells died in vitro within 1 week despite the addition
of IL-6 or stromal cells (data not shown). The inescapable nature of
this apoptosis prompted us to investigate the major molecules involved
in PC survival and apoptosis, ie, Bcl-2 family proteins and
CD95.31,32 We found that plasmablasts and PCs expressed
CD95 (Table 3), and observed in 2 of 3 cases that CD95 triggering
induced massive apoptosis of plasmablasts and PCs (data not shown).
Plasmablasts and PCs from reactive plasmacytoses expressed Bcl-2 very
weakly as compared with bone marrow PCs.33 Tonsillar PCs
have been reported to be Bcl-2+ CD95+ and
sensitive to CD95-induced apoptosis, whereas bone marrow PCs were
Bcl-2+ CD95.31,32 In this
context, Bcl-2+/ CD95+ reactive PCs were
distinct from both tonsillar and bone marrow PCs. In contrast to
tonsillar PCs, those from RP were not rescued from spontaneous
apoptosis by bone marrow fibroblasts, which suggests that the
mechanisms of cell death regulation were distinct (data not shown). The
very weak expression of Bcl-2 in RP could explain both the
proliferation and the apoptosis.33 Although reactive plasma
cells were CD95+, their phenotype was similar to that of
plasma cells generated in the EL4 system, ie, Bcl-2low and
CD49e.7
The characterization of nonmalignant plasmablast expansions has major
implications for the understanding of MM. MM is thought to be an
expansion of malignant plasmablasts and PCs, although the phenotype of
myeloma cells and cell lines, ie, CD19
CD28+ CD138+, is in total contrast with that of
the nonmalignant plasmablasts defined here, ie, CD19+
CD28 CD138.16,34 How
can this discrepancy be explained? It is now well known that the lack
of CD19 expression is directly related to that of Pax5.35
The expression of CD138 by myeloma cells and cell lines suggests that
the former could be expansions of early PCs rather than
plasmablasts.16,17,20,23,34 Concerning CD28, we have shown
that its expression is a hallmark of myeloma cells from patients with
advanced and terminal disease and of all MM cell lines. This would
suggest that CD28 expression is characteristic of the proliferative
malignant compartment.16,17,34 In reactive plasmacytoses,
we found that PCs were highly proliferative (Figs 4 and
5),11,12 although the population of CD28+ PCs
remained very low or even inexistent, which would indicate that no
correlation exists between proliferation and CD28 expression in
plasmablasts and PCs. The role of CD28 expression in malignant PCs is
still uncertain, although the constant expression of CD28 and its
ligand CD86 in myeloma cells could give them an advantage for survival
rather than growth.17
In conclusion, our study shows that reactive plasmocytoses are
expansions of PC progenitors (plasmablasts) and plasma cell precursors
or early plasma cells. Plasmablasts, in particular, are highly
proliferative in vivo, whereas progenitors and precursors are both
short-lived in vivo. However, plasmablasts in vitro stopped proliferating and differentiated into mature plasma cells. The survival
of plasmablasts and their differentiation were totally dependent on
endogenous paracrine IL-6. Maturation was characterized by a mature
plasma-cell morphology (including the appearance of Mott cells and
crystal-like structures within the cytoplasm), increased secretion of
Ig, and expression of plasma-cell antigens, mainly CD138. More
precisely, CD138 was the only antigen able to discriminate plasmablasts
(CD138) from early and mature plasma cells
(CD138+). It is noteworthy that this maturation process was
not observed in vivo in peripheral blood, although mature plasma cells
were seen within bone marrow. The phenotype of plasmablasts and early plasma cells and the demonstration of IL-6 as their survival factor are
important to an understanding of the process of plasma-cell generation
and the biology of MM resulting from the malignant transformation of
this lineage.
| |
ACKNOWLEDGMENT |
The authors thank Dr J. Wijdenes (Diaclone Research) for providing
B-B4, B-E8, and B-G27 MoAbs and Dr T. Kishimoto for
providing PM-1 MoAb.
| |
FOOTNOTES |
Submitted August 24, 1998; accepted March 21, 1999.
Supported by grants from the Ligue Nationale Contre le Cancer. G.J. is
supported by the Ligue Régionale des Deux-Sèvres de
Recherche Contre le Cancer and C.P.-D. is supported by the Association
pour le Développement de l'Etude des Leucémies et Maladies
du Sang (Adelmas).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Catherine Pellat-Deceunynck, PhD, INSERM
U463, Institut de Biologie, 9, quai Moncousu, 44093 Nantes Cedex 01, France; e-mail: cpellat{at}nantes.inserm.fr.
| |
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F. Zhan, E. Tian, K. Bumm, R. Smith, B. Barlogie, and J. Shaughnessy Jr
Gene expression profiling of human plasma cell differentiation and classification of multiple myeloma based on similarities to distinct stages of late-stage B-cell development
Blood,
February 1, 2003;
101(3):
1128 - 1140.
[Abstract]
[Full Text]
[PDF]
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K N Potter, C I Mockridge, A Rahman, S Buchan, T Hamblin, B Davidson, D A Isenberg, and F K Stevenson
Disturbances in peripheral blood B cell subpopulations in autoimmune patients
Lupus,
December 1, 2002;
11(12):
872 - 877.
[Abstract]
[PDF]
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A. Cerutti, H. Zan, E. C. Kim, S. Shah, E. J. Schattner, A. Schaffer, and P. Casali
Ongoing In Vivo Immunoglobulin Class Switch DNA Recombination in Chronic Lymphocytic Leukemia B Cells
J. Immunol.,
December 1, 2002;
169(11):
6594 - 6603.
[Abstract]
[Full Text]
[PDF]
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H. A. M. Wols, G. H. Underhill, G. S. Kansas, and P. L. Witte
The Role of Bone Marrow-Derived Stromal Cells in the Maintenance of Plasma Cell Longevity
J. Immunol.,
October 15, 2002;
169(8):
4213 - 4221.
[Abstract]
[Full Text]
[PDF]
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K. Tarte, J. De Vos, T. Thykjaer, F. Zhan, G. Fiol, V. Costes, T. Reme, E. Legouffe, J.-F. Rossi, J. Shaughnessy Jr, et al.
Generation of polyclonal plasmablasts from peripheral blood B cells: a normal counterpart of malignant plasmablasts
Blood,
July 30, 2002;
100(4):
1113 - 1122.
[Abstract]
[Full Text]
[PDF]
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B. P. O'Connor, M. Cascalho, and R. J. Noelle
Short-lived and Long-lived Bone Marrow Plasma Cells Are Derived from a Novel Precursor Population
J. Exp. Med.,
March 18, 2002;
195(6):
737 - 745.
[Abstract]
[Full Text]
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F. Medina, C. Segundo, A. Campos-Caro, I. Gonzalez-Garcia, and J. A. Brieva
The heterogeneity shown by human plasma cells from tonsil, blood, and bone marrow reveals graded stages of increasing maturity, but local profiles of adhesion molecule expression
Blood,
March 15, 2002;
99(6):
2154 - 2161.
[Abstract]
[Full Text]
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E. Arce, D. G. Jackson, M. A. Gill, L. B. Bennett, J. Banchereau, and V. Pascual
Increased Frequency of Pre-germinal Center B Cells and Plasma Cell Precursors in the Blood of Children with Systemic Lupus Erythematosus
J. Immunol.,
August 15, 2001;
167(4):
2361 - 2369.
[Abstract]
[Full Text]
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G. Jego, R. Bataille, and C. Pellat-Deceunynck
Interleukin-6 is a growth factor for nonmalignant human plasmablasts
Blood,
March 15, 2001;
97(6):
1817 - 1822.
[Abstract]
[Full Text]
[PDF]
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A. C. Rawstron, J. A. L. Fenton, J. Ashcroft, A. English, R. A. Jones, S. J. Richards, G. Pratt, R. Owen, F. E. Davies, J. A. Child, et al.
The interleukin-6 receptor alpha-chain (CD126) is expressed by neoplastic but not normal plasma cells
Blood,
December 1, 2000;
96(12):
3880 - 3886.
[Abstract]
[Full Text]
[PDF]
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A. Sampalo, G. Navas, F. Medina, C. Segundo, C. Camara, and J. A. Brieva
Chronic lymphocytic leukemia B cells inhibit spontaneous Ig production by autologous bone marrow cells: role of CD95-CD95L interaction
Blood,
November 1, 2000;
96(9):
3168 - 3174.
[Abstract]
[Full Text]
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TOP
ABSTRACT
INTRODUCTION