Abstract

Circulating plasma cells in 10 cases of reactive plasmacytosis had a shared phenotype with early plasma cell (CD19+CD38+ CD138+ CD40+CD45+ CD11a+ CD49eCD56). 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.

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,5-10most 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.9,10Unfortunately, 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 factor15and 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.

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 κ and λ 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).

Table 1.

Characteristics of Patients With RP

Diagnosis More Specific Context Age/Sex Duration% of PCs CRP (mg/L)*Ig (g/L)*
RP1  T-cell lymphoma —  69/F  ND  20  66  ND  
RP2  B-cell lymphoma  —  87/F  ND  11 ND  ND  
RP3 Acute myeloid leukemia  —  66/M  ND  11 ND ND  
RP4  Rheumatoid arthritis  At the time of cardiac surgery  78/F  ND  10 ND  ND  
RP5  Infectious mononucleosis  Treatment with cyclosporin A  8/M  10 d 50  21  36  
RP6  Acute respiratory infection B-cell lymphoma  42/M   7 d  20  20  43 
RP71-153 EBV reactivation  Aplasia1-155 27/M  NE  79 270  34  
RP8  Acute respiratory infection  —  54/M   6 d   8 ND  ND  
RP9  Acute respiratory infection  —  70/M  ND  12 108 28  
RP10  Primary amyloidosis  Aplasia1-155 49/M   5 d  12 ND  ND 
Diagnosis More Specific Context Age/Sex Duration% of PCs CRP (mg/L)*Ig (g/L)*
RP1  T-cell lymphoma —  69/F  ND  20  66  ND  
RP2  B-cell lymphoma  —  87/F  ND  11 ND  ND  
RP3 Acute myeloid leukemia  —  66/M  ND  11 ND ND  
RP4  Rheumatoid arthritis  At the time of cardiac surgery  78/F  ND  10 ND  ND  
RP5  Infectious mononucleosis  Treatment with cyclosporin A  8/M  10 d 50  21  36  
RP6  Acute respiratory infection B-cell lymphoma  42/M   7 d  20  20  43 
RP71-153 EBV reactivation  Aplasia1-155 27/M  NE  79 270  34  
RP8  Acute respiratory infection  —  54/M   6 d   8 ND  ND  
RP9  Acute respiratory infection  —  70/M  ND  12 108 28  
RP10  Primary amyloidosis  Aplasia1-155 49/M   5 d  12 ND  ND 

The percentage of plasma cells was either defined in the total population, ie, on blood smears (

) or in PBMNC, ie, on cytospins (

).

Abbreviations: ND, not done; NE, not evaluable (patient died during plasmacytosis).

*

Normal values: CRP, <3 mg/L; Ig, <15 g/L.

F1-153

This case has been extensively described elsewhere.18 

F1-155

Aplasia after stem cell transplantation (RP7, allogeneic; RP10, autologous).

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 × 10−5 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-κ (κ), 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-κ-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-λ-PE (λ) 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 κ chain MoAb (clone 6E1; Immunotech) or antihuman λ 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. κ and λ staining was performed without AB serum. For intracellular κ and λ 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-λ or anti-κ 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 CD138purification 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.

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 κ+ or λ+(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 CD56CD11a+. 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 

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.

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.

Table 2.

Phenotype of Plasma Cells From 10 RPs

RP1 RP2*RP3 RP4 RP5 RP6 RP7 RP8RP9 RP10 Total (n = 10)
% of cells expressing  
 CD38+++ 30%  15%  11% 20%  89%  37%  87%  27%  13%  8% +10/10  
 κ  ++ (16%) ++ (6%)   ND  ++ (16%)  ++ (63%)  ++ (20%) ++ (49%)  ++ (14%)  ++ (8%)  +++ (7%)  +9/9  
 λ  +++ (15%)  +++ (8%)  ND  +++ (4%)   +++ (28%)  +++ (17%) +++ (40%)  +++ (13%)  +++ (4%)  +++ (3%) +9/9  
 CD138  ++ (24%) +++ (17%)  +++ (6%)  +++ (14%)  +++ (50%) +++ (23%)  +++ (20%)  ++ (9%)   +++ (7%) +++ (6%)  +10/10  
Phenotype of CD138+cells  
 CD11a  + (90%)  +  ND  ND  ND  ND  ND  ND  ND  +3/3  
 CD19  +  ND  +  ++  +  +  +  +  +  +9/9  
 CD28 + (7%)   −  + (7%)  ++ (12%)  + (3%)   −  + (1%)   −  + (2%)  ND  +6/9 
 CD40  +  +  ND  ±  +  +  ND  +  ND  +7/7  
 CD45  +++  +++  +++ +++  +++  +++  +++  +++  +++  +++  +10/10 
 CD49e  ND  ND  ND  −  −  −  −  ND −  ND  −5/5  
 CD56  −  −  −  − −  −  −  −  −  ND  −9/9  
 CD80 ND  ND  ND  ND  ND  ±  ±  ±  ± ND  ±4/4  
 CD86  ND  ND  ND  ND  ND  +  ++  +  ND  +4/4  
 HLA-DR  ND  ND  ND ND  +  ND  ND  +  +  ND  +3/3  
 Apo2.7 ND  ND  ND  ND  ND  −  −  −  − ND  −4/4 
RP1 RP2*RP3 RP4 RP5 RP6 RP7 RP8RP9 RP10 Total (n = 10)
% of cells expressing  
 CD38+++ 30%  15%  11% 20%  89%  37%  87%  27%  13%  8% +10/10  
 κ  ++ (16%) ++ (6%)   ND  ++ (16%)  ++ (63%)  ++ (20%) ++ (49%)  ++ (14%)  ++ (8%)  +++ (7%)  +9/9  
 λ  +++ (15%)  +++ (8%)  ND  +++ (4%)   +++ (28%)  +++ (17%) +++ (40%)  +++ (13%)  +++ (4%)  +++ (3%) +9/9  
 CD138  ++ (24%) +++ (17%)  +++ (6%)  +++ (14%)  +++ (50%) +++ (23%)  +++ (20%)  ++ (9%)   +++ (7%) +++ (6%)  +10/10  
Phenotype of CD138+cells  
 CD11a  + (90%)  +  ND  ND  ND  ND  ND  ND  ND  +3/3  
 CD19  +  ND  +  ++  +  +  +  +  +  +9/9  
 CD28 + (7%)   −  + (7%)  ++ (12%)  + (3%)   −  + (1%)   −  + (2%)  ND  +6/9 
 CD40  +  +  ND  ±  +  +  ND  +  ND  +7/7  
 CD45  +++  +++  +++ +++  +++  +++  +++  +++  +++  +++  +10/10 
 CD49e  ND  ND  ND  −  −  −  −  ND −  ND  −5/5  
 CD56  −  −  −  − −  −  −  −  −  ND  −9/9  
 CD80 ND  ND  ND  ND  ND  ±  ±  ±  ± ND  ±4/4  
 CD86  ND  ND  ND  ND  ND  +  ++  +  ND  +4/4  
 HLA-DR  ND  ND  ND ND  +  ND  ND  +  +  ND  +3/3  
 Apo2.7 ND  ND  ND  ND  ND  −  −  −  − ND  −4/4 

The phenotype of plasma cells was defined in double staining with CD138. The intensity level was defined as indicated in Materials and Methods. CD38, intracytoplasmic κ and λ, and CD138+cells were determined in the total population. The phenotype of plasma cells was defined in the CD138+ cell population.

*

Immunological staining was performed after cells were cultured for 72 hours at 37°C in RPMI plus 10% FCS.

CD28 was either not expressed or in a minor subpopulation of plasma cells.

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 κ plus λ 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 (Fig2), 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).

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).

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).

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.

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.)

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.)

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). Figure4 shows that 15% to 50% of cκ+ and 15% to 50% of cλ+ 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.

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) λ or κ population by double staining with anti-λ-PE or anti-κ-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.

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) λ or κ population by double staining with anti-λ-PE or anti-κ-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.

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.

Table 3.

Phenotypic Analysis of the CD19+Plasmablastic Population

RP5 RP7 RP8 RP9
Antigens (Ag)  
 PC Ag  
  CD19  ++  +  +  ++  
  CD38  +++ ++  +++  ++  
  CD138  −  −  −  − 
  cIg  +  ++  +++  +++  
 B cells Ag 
  CD19  ++  +  +  ++  
  CD20  −  − ND  −  
  CD21  −  −  ND  −  
  CD22 +/−  −  ND  +/−  
  CD23  −  −  ND −  
  sIg  +  +  ND  +  
  CD40  +  ND  +  
  CD45  +++  +++  +++  +++ 
  CD45RA  +  ++  ND  +  
  CD45RO  −  − ND  −  
  CD80  +/−  +/−  −  − 
  CD86  +  +  +  +  
 Adhesion and activation Ag  
  CD25  −  −  ND  ND  
  CD28 + (3%)  −  −  ND  
  CD49e  −  − −  −  
  CD56  −  −  ND  − 
  CD95  +  +  +  +  
  HLA-DR  +  +++  ++ 
RP5 RP7 RP8 RP9
Antigens (Ag)  
 PC Ag  
  CD19  ++  +  +  ++  
  CD38  +++ ++  +++  ++  
  CD138  −  −  −  − 
  cIg  +  ++  +++  +++  
 B cells Ag 
  CD19  ++  +  +  ++  
  CD20  −  − ND  −  
  CD21  −  −  ND  −  
  CD22 +/−  −  ND  +/−  
  CD23  −  −  ND −  
  sIg  +  +  ND  +  
  CD40  +  ND  +  
  CD45  +++  +++  +++  +++ 
  CD45RA  +  ++  ND  +  
  CD45RO  −  − ND  −  
  CD80  +/−  +/−  −  − 
  CD86  +  +  +  +  
 Adhesion and activation Ag  
  CD25  −  −  ND  ND  
  CD28 + (3%)  −  −  ND  
  CD49e  −  − −  −  
  CD56  −  −  ND  − 
  CD95  +  +  +  +  
  HLA-DR  +  +++  ++ 

Fluorescence staining and analysis were performed as described in Materials and Methods. The phenotype of plasmablastic cells was defined in double staining with CD19 in the CD138 population (after removal of the CD138+ population).

Abbreviations: cIg, cytoplasmic Ig; sIg, surface Ig; ND, not done.

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 λ+ population, BrdU+ cells were mainly located within the CD138 fraction (25% for CD138cells v 8% for CD138+ cells). The same result was obtained for the κ+ 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.

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) λ-PE versus BrdU-FITC; (B and D) λ-PE versus control IgG1-FITC.

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) λ-PE versus BrdU-FITC; (B and D) λ-PE versus control IgG1-FITC.

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 λ 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).

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 λ or κ 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.

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 λ or κ 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.

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).

Table 4.

Neutralization of IL-6 (BE-8) and Its Receptor CD126 (PM-1) Prevented Differentiation by Inducing Apoptosis of Plasmablasts

RP5 RP8RP9
CD138+Apo2.7+CD138+Apo2.7+CD138+Apo2.7+
Treatment  
 Nil  25%  13% 14%     8%  6%     4%  
 Anti–IL-6 MoAb  9%  42%  ND  ND  ND  ND 
 Anti–IL-6 + anti-CD126 MoAbs  ND  ND  0.5%  25%  0.2%  10%  
 Anti-FGF basic Ab 24%  15%  ND  ND  7.5%  ND  
FGF basic  26% ND  ND  ND  6.8%  ND 
RP5 RP8RP9
CD138+Apo2.7+CD138+Apo2.7+CD138+Apo2.7+
Treatment  
 Nil  25%  13% 14%     8%  6%     4%  
 Anti–IL-6 MoAb  9%  42%  ND  ND  ND  ND 
 Anti–IL-6 + anti-CD126 MoAbs  ND  ND  0.5%  25%  0.2%  10%  
 Anti-FGF basic Ab 24%  15%  ND  ND  7.5%  ND  
FGF basic  26% ND  ND  ND  6.8%  ND 

Fluorescence analysis at day 3 of CD138 and APO2.7 expression in untreated or treated cells, as indicated (anti–IL-6, B-E8: 50 μg/mL; anti-CD126, PM-1: 10 μg/mL; anti-FGF: 50 μg/mL; rFGF basic: 10 ng/mL). The percentages of positive cells for CD138 or APO2.7 were determined in the total population.

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.

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).

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).

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+ CD45intCD49e 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.16CD138 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 CD23CD38+ CD40low CD45intCD56 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, CD19CD28+ 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 Pax5.35The 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 and5),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.

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.

REFERENCES

REFERENCES
1
MacLennan
ICM
Casamayor-Palleja
M
Toellner
K-M
Gulbranson-Judge
A
Gordon
J
Memory B-cell clones and the diversity of their members.
Semin Immunol
9
1997
229
2
MacLennan
ICM
Germinal centers.
Annu Rev Immunol
12
1994
112
3
Roldan
E
Garcia-Pardo
A
Brieva
JA
VLA-4-fibronectin interaction is required for the terminal differentiation of human bone marrow cells capable of spontaneous and high rate immunoglobulin secretion.
J Exp Med
175
1992
1739
4
Klein
B
Zhang
XG
Lu
ZY
Bataille
R
Interleukin-6 in multiple myeloma.
Blood.
85
1995
863
5
Jelinek
DF
Lipsky
PE
The role of B cell proliferation in the generation of immunoglobulin-secreting cells in man.
J Immunol
130
1983
2597
6
Vernino
L
McAnally
LM
Ramberg
J
Lipsky
PE
Generation of nondividing high rate Ig-secreting plasma cells in cultures of human B cells stimulated with anti-CD3-activated T cells.
J Immunol
148
1992
404
7
Grimaı̂tre
M
Werner-Favre
C
Kindler
V
Zubler
RH
Human naive B cells cultured with EL-4 T cells mimic a germinal center-related B cell stage before generating plasma cells. Concordant changes in Bcl-2 protein and messenger RNA levels.
Eur J Immunol
27
1997
199
8
Déchanet
J
Merville
P
Durand
I
Banchereau
J
Merville
P
The ability of synoviocytes to support terminal differentiation of activated B cells may explain plasma cell accumulation in rheumatoid synovium.
J Clin Invest
95
1995
510
9
Agematsu
K
Nagumo
H
Oguchi
Y
Nakazawa
T
Fukushima
K
Yasui
K
Ito
S
Kobata
T
Morimoto
C
Komiyama
A
Generation of plasma cells from peripheral blood memory B cells: synergistic effect of interleukin-10 and CD27/CD70 interaction.
Blood
91
1998
173
10
Arpin
C
Banchereau
J
Liu
Y-J
Memory B cells are biased toward terminal differentiation: A strategy that may prevent repertoire freezing.
J Exp Med
186
1997
931
11
Gavarotti
P
Boccadoro
M
Redoglia
V
Golzio
F
Pileri
A
Reactive plasmacytosis. Case report and review of the literature.
Acta Haemat
73
1985
108
12
Jourdan
M
Bataille
R
Seguin
J
Zhang
XG
Chaptal
PA
Klein
B
Constitutive production of interleukin-6 and immunologic features in cardiac myxomas.
Arthris Rheum
33
1990
398
13
Rosenthal
NS
Farhi
DC
Reactive plasmacytosis and lymphocytosis in acute myeloid leukemia.
Hematol Pathol
8
1994
43
14
Tatsuno
I
Nishikawa
T
Sasano
H
Shizawa
S
Iwase
H
Satoh
S
Interleukin-6 producing gastric carcinoma with fever, hypergammaglobulinemia, and plasmacytosis in bone marrow.
Gastroenterology
107
1994
543
15
Akira
S
Taga
T
Kishimoto
T
Interleukin-6 in biology and medicine.
Adv Immunol
54
1993
1
16
Pellat-Deceunynck
C
Bataille
R
Robillard
N
Harousseau
J-L
Rapp
M-J
Juge-Morineau
N
Wijdenes
J
Amiot
M
Expression of CD28 and CD40 in human myeloma cells: A comparative study with normal plasma cells.
Blood
84
1994
2597
17
Robillard
N
Jego
G
Pellat-Deceunynck
C
Pineau
D
Puthier
D
Mellerin
M-P
Barillé
S
Rapp
M-J
Harousseau
J-L
Amiot
M
Bataille
R
CD28, a marker associated with tumoral expansion in multiple myeloma.
Clin Cancer Res
4
1998
1521
18
Milpied N, Coste-Burel M, Accard F, Moreau A, Moreau P, Garand R, Harousseau J-L: Epstein Barr virus associated B-cell lymphoproliferative disease after non-myeloablative allogeneic stem cell transplantation. Bone Marrow Transplant (in press)
19
Kawano
MM
Mihara
MK
Huang
N
Tsujimoto
T
Kuramoto
A
Differentiation of early plasma cells on bone marrow stromal cells requires interleukin-6 for escaping from apoptosis.
Blood
85
1995
487
20
Pellat-Deceunynck
C
Barillé
S
Puthier
D
Rapp
M-J
Harousseau
J-L
Bataille
R
Amiot
M
Adhesion molecules on human myeloma cells: Significant changes in expression related to malignancy, tumor spreading and immortalization.
Cancer Res
55
1995
3647
21
Terstappen
WMM
Johnsen
S
Segers-Nolten
IMJ
Loken
MR
Identification and characterization of plasma cells in normal human bone marrow by high-resolution flow cytrometry.
Blood
76
1990
1739
22
Alanen
A
Pira
U
Lassila
O
Roth
J
Franklin
RM
Mott cells are plasma cells defective in immunoglobulin secretion.
Eur J Immunol
15
1985
235
23
Wijdenes
J
Vooijs
WC
Clement
C
Post
J
Morard
F
Vita
N
Laurent
P
Sun
RZ
Klein
B
Dore
JM
A plasmocyte selective monoclonal antibody (B-B4) recognizes syndecan-1.
Br J Haematol
94
1996
318
24
Jaakola
P
Vihinen
T
Maatta
A
Jalkanen
M
Activation of an enhancer on the syndecan-1 gene is restricted to fibroblast growth factor family members in mesenchymal cells.
Mol Cel Biol
17
1997
3210
25
Zhang
C
Ao
Z
Seth
A
Schlossman
SF
A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells.
J Immunol
157
1996
3980
26
Harada
Y
Kawano
MM
Huang
N
Mahmoud
MS
Lisuko
IA
Mihara
K
Tsujimoto
T
Kuramoto
A
Identification of early plasma cells in peripheral blood and their clinical significance.
Br J Haematol
92
1996
184
27
Jourdan
M
Ferlin
M
Legouffe
E
Horvathova
M
Liautard
J
Rossi
J-F
Wijdenes
J
Brochier
J
Klein
B
The myeloma cell antigen syndecan-1 is lost by apoptotic myeloma cells.
Br J Haematol
100
1998
637
28
Brieva
JA
Roldan
E
Rodriguez
C
Navas
G
Human tonsil, blood and bone marrow in vivo-induced B cells capable of spontaneous and high-rate immunoglobulin secretion in vitro: Differences in the requirements for factors and for adherent and bone marrow stromal cells, as well as distinctive adhesion molecule expression.
Eur J Immunol
24
1994
362
29
Altmeyer
A
Simmons
RC
Krajewski
S
Reed
JC
Bornkamm
GW
Chen-Kiang
S
Reversal of EBV immortalization precedes apoptosis in IL-6-induced human B cell terminal differentiation.
Immunity
7
1997
667
30
Slifka
MK
Antia
R
Whitmire
JK
Ahmed
R
Humoral immunity due to long-lived plasma cells.
Immunity.
8
1998
363
31
Medina
F
Segundo
C
Rodriguez
C
Brieva
JA
Regulation role of CD95 ligation on human B cells induced in vivo capable of spontaneous and high-rate Ig secretion.
Eur J Immunol
27
1997
700
32
Merville
P
Déchanet
J
Desmouilière
A
Durand
I
de Bouteiler
O
Garrone
P
Banchereau
J
Liu
Y-J
Bcl-2+ tonsillar plasma cells are rescued from apoptosis by bone marrow fibroblasts.
J Exp Med
183
1996
227
33
Puthier
D
Pellat-Deceunynck
C
Barillé
S
Robillard
N
Rapp
M-J
Juge-Morineau
N
Harousseau
J-L
Bataille
R
Amiot
M
Differential expression of Bcl-2 in human plasma cell according to proliferation status and malignancy.
Leukemia
13
1999
289
34
Zhang
XG
Gaillard
JP
Robillard
N
Lu
ZY
Boiron
JM
Jourdan
M
Bataille
R
Klein
B
Reproducible obtaining of human myeloma cell lines as a model for tumor stem cell study in human multiple myeloma.
Blood
83
1994
3654
35
Mahmoud
MS
Huang
N
Nobuyoshi
M
Lisukov
IA
Tanaka
H
Kawano
MM
Altered expression of Pax-5 gene in human myeloma cells.
Blood
87
1996
4311

Author notes

Address reprint requests to Catherine Pellat-Deceunynck, PhD, INSERM U463, Institut de Biologie, 9, quai Moncousu, 44093 Nantes Cedex 01, France; e-mail: cpellat@nantes.inserm.fr.