Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0603279 on March 12, 2004

Published online before print March 12, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0603279v1
75/6/1022    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arce, S.
Right arrow Articles by Manz, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arce, S.
Right arrow Articles by Manz, R. A.
(Journal of Leukocyte Biology. 2004;75:1022-1028.)
© 2004 by Society for Leukocyte Biology

CD38 low IgG-secreting cells are precursors of various CD38 high-expressing plasma cell populations

Sergio Arce*, Elke Luger*, Gwendolin Muehlinghaus*, Giuliana Cassese*, Anja Hauser*, Alexander Horst{dagger}, Katja Lehnert*, Marcus Odendahl*, Dirk Hönemann{ddagger}, Karl-Dieter Heller§, Harald Kleinschmidt§, Claudia Berek*, Thomas Dörner{ddagger}, Veit Krenn{ddagger}, Falk Hiepe{ddagger}, Ralf Bargou{ddagger}, Andreas Radbruch* and Rudolf A. Manz*,1

* Deutsches Rheuma-Forschungszentrum, Berlin, Germany;
{dagger} Miltenyi Biotec GmbH, Bergisch Gladbach, Germany;
{ddagger} Charite’ Humboldt University, Berlin, Germany; and
§ Orthopädische Klinik Braunschweig, Germany

1 Correspondence: Deutsches Rheuma-Forschungszentrum, Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany. E-mail: manz{at}drfz.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the important role immunoglobulin G (IgG)-secreting plasma cells play in memory immune responses, the differentiation and homeostasis of these cells are not completely understood. Here, we studied the differentiation of human IgG-secreting cells ex vivo and in vitro, identifying these cells by the cellular affinity matrix technology. Several subpopulations of IgG-secreting cells were identified among the cells isolated from tonsils and bone marrow, particularly differing in the expression levels of CD9, CD19, and CD38. CD38 low IgG-secreting cells were present exclusively in the tonsils. A major fraction of these cells appeared to be early plasma cell precursors, as upon activation of B cells in vitro, IgG secretion preceded up-regulation of CD38, and on tonsillar sections, IgG-containing, CD38 low cells with a plasmacytoid phenotype were found in follicles, where plasma cell differentiation starts. A unitary phenotype of migratory peripheral blood IgG-secreting cells suggests that all bone marrow plasma cell populations share a common precursor cell. These data are compatible with a multistep model for plasma cell differentiation and imply that a common CD38 low IgG-secreting precursor gives rise to a diverse plasma cell compartment.

Key Words: antibodies • B lymphocytes • cellular differentiation • memory


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eventually, activated B cells can differentiate into nonproliferating, mature plasma cells. Directed by chemokines [1 , 2 ], early plasma cells can leave the secondary lymphoid organs to migrate into the bone marrow [3 ] or into inflamed tissues [4 ]. Although most plasma cells found in secondary lymphoid tissue are short-lived, bone marrow plasma cells can live for extended periods of time [5 ] with an estimated half-life of ~6 months in mice [6 ].

Human plasma cell differentiation has been studied extensively in vitro [7 8 9 ] and on plasma cells isolated from blood [10 , 11 ]. The studies on normal plasma cells isolated from tonsils, blood, and bone marrow indicate that these tissues contain plasma cells expressing high levels of CD38 but heterogeneous levels of other surface molecules [12 , 13 ]. Although early plasma cells and their precursors are found in tonsils, more mature plasma cells are present in the bone marrow, possibly reflecting the different functions of these tissues in inducing and maintaining plasma cell responses, respectively. Immunoglobulin G (IgG) is the dominant Ig isotype secreted by these cells in T-dependent immune responses [14 ]. It has been indicated that most IgG-secreting cells present in the tonsils are proliferating cells (B cell/plasma blasts), and IgG-secreting cells in the bone marrow are nonproliferating cells (plasma cells) [12 ]. However, the exact relationship between phenotypically distinct plasma cell populations is not clear yet.

Here, we used the cellular affinity matrix technology to identify human IgG-secreting cells, i.e., plasma cells and their immediate precursors. This technology had been applied initially to identify murine antibody (Ab)-secreting plasma cells and cytokine-secreting T cells [15 ]. We identified subpopulations of IgG-secreting cells, including a CD38 low plasma cell precursor population present in tonsillar germinal centers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues and lymphocyte isolation
Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density-gradient centrifugation [16 ]. Tonsils and bone marrow were obtained from patients undergoing tonsillectomy and surgical removal of the head of the femur, respectively. After cutting the tonsils and bone into pieces, tissue cells were washed out by vigorous pipeting with phosphate-buffered saline (PBS)/0.5% bovine serum albumin (BSA) and were filtered through a cell strainer (Becton Dickinson, San Jose, CA). Erythrocytes were lysed with ammonium chloride. All samples were taken with the patient’s consent. To minimize background staining, PBMC were depleted of CD3-, CD14-, and CD16-positive cells before the staining for IgG. The depletion was achieved by using direct microbeads to these antigens and a type CS column, as indicated by the manufacturer (Miltenyi Biotech, Germany). To analyze IgG-secreting cells from tonsils and bone marrow, no depletion step was necessary.

Cellular affinity matrix technology
Single-cell suspensions were biotinylated with 0.5 mg sulfo–normal human serum–Langerhans cell–biotin (Pierce, Rockford, IL) in 1 ml PBS (37°C, for 10 min), and then the same volume of RPMI 10% fetal calf serum (FCS) was added, and cells were incubated for another 10 min. After three washing steps with PBS/0.5% BSA, cells were incubated with a Cy5-coupled anti-human IgG (PharMingen, BD, San Diego, CA, 10 µg/ml, 15 min on ice). Subsequently, the cells were washed, and unconjugated anti-human {lambda} [clone HP6054 (American Type Culture Collection, Manassas, VA), 50 µg/ml, 0.2 ml, 4°C] was added; 5 min later, 50 µg/ml HP6054 conjugated to avidin [catching Ab (catch-Ab)] was added. After 10 min incubation on ice, cells were resuspended in 2 ml RPMI/10% FCS at a final concentration of 3 million cells/ml and incubated for 30 min at 37°C. Under these conditions, Ig was secreted, and those bearing {lambda} light chains were captured on the surface of the Ig-secreting cells. Thereafter, the cells were cooled on ice for 10 min, and 5 µg/ml digoxigenized (DIG) anti-human IgG conjugate was added (PharMingen, BD). After washing, cells were stained with a titrated concentration of anti-DIG-phycoerythrine (PE). To allow discrimination among membrane bound and secreted-captured IgG, cells were stained for surface IgG before capturing the secreted IgG.

Monoclonal Ab (mAb) and fluorescein-activated cell sorter (FACS) analysis
The following fluorescein-labeled antibodies were used for phenotyping: CD3, CD5, CD14, CD16, CD19, CD20, CD22, CD25, CD27, CD28, CD35, CD40, CD44, CD45, CD49d, CD49e, CD70, CD71, CD72, CD86, CD95, CD100, CD154, and human leukocyte antigen (HLA)-DR (PharMingen, BD); CD9 and CD38 (Immunotech, Coulter, FL); and CD10 and CD138 (AMS Biotechnology GmbH, Germany). A FACScalibur (Becton Dickinson) flow cytometer was used for analysis. Propidium iodide (PI; 1 µg/ml) was used to exclude dead cells according to uptake of the dye. For intracellular staining, cells were fixed in 2% formaldehyde and incubated with anti-IgG-fluorescein [fluorescein isothiocyanate (FITC)] in PBS/BSA containing 0.5% saponin.

In vitro stimulation of PBMC
CD38/CD138-depleted PBMC were labeled with carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) as described [17 ] and were subsequently stimulated with 1 µg/ml tetanus toxoid [18 ]. To deplete CD38/CD138-positive cells, PBMC were labeled with CD38-PE (PharMingen, BD) and thereafter, with anti-PE and CD138 microbeads. Cells were depleted by magnetic cell sorter (MACS; Miltenyi Biotech). The depleted fraction did not contain any detectable IgG-secreting plasma cells, as tested by the IgG-secretion assay. CD38 high cells were also not detectable by FACS analysis.

Histology
Freshly ectomized human tonsils were cut in convenient pieces of ~300 mm3 and embedded in Tissue-Tek® Cryomold® intermediate. Cryosections (7 µm) were performed and acetone-fixed. Before specific Ab staining, unspecific binding sites were blocked with PBS/2% BSA for 20 min at room temperature. Sections were stained with FITC-labeled mouse anti-human CD38 mAb (Dako, Carpinteria, CA; clone AT13/5) and biotinylated mouse anti-human IgG mAb (PharMingen, BD, clone G18-145), respectively. After washing, sections were incubated with rhodamin-labeled streptavidin. Nuclei were counterstained with diamidinophenyl indole (DAPI).

Polymerase chain reaction (PCR)
FACS sorted cells directly into PCR tubes. PCR was performed for ß-actin and Blimp-1 (primers, Blimp-1: 5'-TCGGGTCGTTTACCCCATC-3' and 5'-CACAGCGCTCAGGCCATTA-3'). Reactions were annealed at 55°C and amplified for 35 cycles.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To detect all IgG-producing plasma cell and plasma cell precursor populations, including those possibly not expressing commonly used markers, we used the cellular affinity matrix technology [15 ]. This technique allowed us to identify cells secreting Ig of the IgG isotype. Single-cell suspensions from tonsils, blood, and bone marrow were analyzed for the presence of IgG-secreting cells. In all samples including the controls without catch-Ab, a population of "background"-stained cells appeared, which was excluded from the further analysis by using the gate as shown in Figure 1A . The population that was brightly stained for secreted IgG but was absent in control samples lacking catch-Ab was considered to be plasma cells, as all these cells exhibited intense staining for cytoplasmic IgG (Fig. 1B) . Following isolation by FACS, the capacity of these cells to secrete IgG was confirmed by enzyme-linked immunospot technique [19 ]. Spots were formed by more than 90% of cells stained for secreted IgG (data not shown). Thus, the cells identified by the cellular affinity matrix technology were IgG-secreting plasma cells or immediate plasma cell precursors. In healthy individuals, the average frequencies of these cells were ~0.47 ± 0.2%, 0.01 ± 0.1%, and 0.2 ± 0.1% (n≥8 for all tissues) among the nucleated cells in tonsils, peripheral blood, and in bone marrow, respectively. Although frequencies in tonsils and bone marrow showed little variation between different donors, frequencies of blood IgG-secreting cells could reach up to 0.1% in a few samples. The expression of various surface markers on IgG-secreting cells was analyzed (Fig. 2 ). A substantial fraction of the tonsillar IgG-secreting cells expressed HLA-DR and thus can be considered to be early plasma cells [20 , 21 ]. CD38 was expressed at high levels on all IgG-secreting cells derived from blood and bone marrow. However, an additional IgG-secreting cell population was detected in the tonsils, expressing only low levels of CD38. A third population of IgG-secreting cells, present in only very low numbers, expressed even less CD38.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 1. Detection of IgG-secreting cells. Bone marrow, blood, or tonsil-derived single-cell suspensions (here, cells from bone marrow are shown) were stained for surface and secreted IgG as described in the text. (A) Left dot plot: Cells were stained for surface and subsequently for secreted IgG as described in Materials and Methods. Right dot plot: Negative control treated with all staining reagents but lacking catch-Ab. The IgG-secreting cell population (left plot) is indicated by the square. (B) Cells stained for surface and secreted IgG as shown in A were fixed and stained for intracellular IgG. Saponin was used to allow the staining Ab to penetrate the cell membrane. Nonsecreting/surface IgG+ cells were excluded from the analysis by gating (gate shown in A, left plot). The cells stained for secreted IgG were specifically labeled for intracellular IgG (left) but neither for an isotype control (right) nor for IgG without permeabilization of the cell membrane (middle). Dead cells were excluded from the analysis by PI staining. About 2 x 105 cells are shown in each dot plot. Representative data of 12 independent experiments are shown.

 


View larger version (47K):
[in this window]
[in a new window]
  		Figure 2. Identification of IgG-secreting plasma cell subpopulations. Single-cell suspensions from tonsils (left column), blood (middle column), and bone marrow (right column) were stained for surface and secreted IgG and counterstained for the markers indicated. Dead cells were excluded from the analysis by PI staining. Nonsecreting/surface IgG+ cells were excluded from the analysis by gating (shown in Fig. 1A , left plot). About 2 x 105 cells are shown in each dot plot. Representative data of six independent experiments are shown.

 
Heterogeneity within the CD38 high plasma cell compartment had been described [13 ]. However, IgG-secreting cell populations expressing no or low levels of CD38 have been missed in earlier analyses of tissue samples, as these studies had been restricted to CD38 high cells. Recently and in accordance with our findings, it had been demonstrated that stimulation of memory B cells in culture could result in the formation of CD38 low IgG-secreting cells [22 ].

To further characterize IgG-secreting subpopulations, as a measurement for cell size, we compared the mean forward-scatter profile of these cells with that of B cells (Table 1 ). Tonsilar CD38-negative/very low IgG-secreting cells had about the same forward-scatter profile as B cells, i.e., ~245 linear units. The CD38 low and CD38 high population showed an increased scatter profile with 302 and 317 linear units, respectively. The differences in scatter among all three populations were statistically significant (P<0.05). These values were still below those measured for IgG-secreting cell populations isolated from blood or bone marrow. As an increase in cell size is probably associated with an increase in plasma cell maturation [23 ], these results already suggested that the relatively small, CD38 low Ab-secreting cells were early plasma cell precursors. To further test whether CD38 low IgG-secreting cells resemble a temporary stage of the early plasma cell development rather than a distinct plasma cell subpopulation, we analyzed the expression of this marker on stimulation of B cells in vitro. PBMC were depleted of plasma cells and activated B cells by MACS using CD38 and CD138 magnetic beads. The remaining cells, consisting mainly of resting B cells (memory and naive), were activated by tetanus toxoid as described [18 ]. When IgG-secreting cells first appeared at day 3 in culture, these cells expressed no or only low levels of CD38 (Fig. 3A ). Later, at day 6, all IgG-secreting cells had up-regulated CD38 to high levels. During the same period of time, the frequencies of these cells increased from 0.2 to 1.8%. The staining for secreted IgG on individual cells also increased between days 3 and 6 in culture, suggesting an increase in IgG secretion and level of maturation. Labeling the cells with CFSE allowed the analysis of their proliferation profile [17 ]. Already at day 3, the early-appearing IgG-secreting cells had lost some CFSE, indicating that proliferation precedes differentiation into IgG-secreting cells (Fig. 3B) . Until day 6, the IgG-secreting cells underwent further rounds of proliferation, indicated by a further decrease in CFSE labeling. These data support the idea that plasma cell differentiation starts already at a CD38-negative stage.


View this table:
[in this window]
[in a new window]
  		Table 1. Mean Forward Light-Scatter Analysisa

 


View larger version (49K):
[in this window]
[in a new window]
  		Figure 3. Plasma cell differentiation in vitro. (A) PBMC were depleted of plasma cells and stimulated with tetanus toxoid. IgG-secreting cells were identified by the cellular affinity matrix technology as described in the text and Figure 1 . At the time-points indicated, the IgG-secreting cells were stained for CD38. (B) PBMC were labeled with CFSE, and cultures were initiated as described in A. Cells were analyzed at the indicated time-points. Dead cells were excluded from the analysis by PI staining. Representative data of three independent experiments are shown.

 
CD9 is expressed at high levels on murine plasma cells in spleen [24 ]. Expression of this marker was also characteristic for the tonsilar IgG-secreting CD38 high and CD38 intermediate populations described here (data not shown). Based on this result, we aimed to identify plasma cells and their precursors by staining tonsilar cells for CD9 together with CD19. This marker combination stains for less than 3% tonsilar CD19-positive B cells, all of them expressing CD38 at high or intermediate levels (Fig. 4A ). All CD38-intermediate, CD9-positive tonsilar B cells express CD27 at intermediate levels, and CD38-high, CD9-positive plasma cells express CD27 at high levels. CD138, a marker for plasma cells, was expressed on a subfraction of 8% of CD38-intermediate, CD9-positive tonsilar B cells and on 59% CD38-high, CD9-positive plasma cells. As determined by PCR, the transcription factor blimp-1, which is specifically up-regulated during plasma cell differentiation and not expressed on mature B cells, was also expressed in both populations (Fig. 4B) . Thus, with respect to the expression of CD27, CD138, and blimp-1, the population of CD38-intermediate, CD9-positive B cells resembles intermediates between B cells and plasma cells. In accordance with the identification of CD38-intermediate, IgG-secreting cells, this result shows that tonsilar plasma cell differentiation starts before CD38 is expressed at high levels.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 4. Phenotype of CD38-intermediate CD9/CD19-positive tonsilar B cells. (A) Tonsillar cells were stained for CD9, CD19, and CD38 together with CD27 or CD138. CD19-negative cells were excluded from the analysis. (B) FACS sorted tonsilar cell populations, and PCR was performed on the indicated number of cells. Lanes 4/5 and 6/7 represent probes obtained from two different donors analyzed in one experiment. Data shown are representative of four independent experiments.

 
The enlarged cytoplasm of IgG-secreting cells containing high amounts of Ig allows the identification of these cells on tissue sections. Localization of CD38 high cells and IgG expression was compared on tonsilar sections by immunofluorescence staining (Fig. 5 ). In accord with previous studies [25 ], the vast majority of CD38 high plasma cells in the tonsils was found in extrafollicular regions. Only a few were detected within the follicles. The majority of IgG-containing cells was also found in the extrafollicular regions. Additionally, IgG-containing cells were found in the light zone of some but not all germinal centers. These cells were colocated with interstitial IgG.



View larger version (77K):
[in this window]
[in a new window]
  		Figure 5. CD38 and IgG staining on tonsillar sections. Cryosections were stained with mouse anti-human mAb for CD38 (FITC, green) and IgG (rhodamin, red); nuclei were stained with DAPI (blue). Cells expressing high levels of CD38 together with IgG appear in yellow.

 
As shown by double-staining for IgG (rhodamin, red) and CD38 (FITC, green), the majority of cells containing large quantities of IgG located inside the follicles was not brightly stained for CD38 (Fig. 5) . In contrast, the IgG-containing cells found in a ring adjacent to the follicular areas express high levels of CD38, as indicated by the yellow color, resulting in an overlay of the green staining for CD38 and the IgG staining in red. Although differing in their levels of CD38 expression, IgG-containing cells inside and outside the follicular areas exhibited a plasmacytoid phenotype, suggesting that both cell types resemble plasma blasts or plasma cells.

Colocalization of interstitial IgG and CD-38 low IgG-containing cells indicates that these cells already had started to secrete IgG and thus were committed to plasma cell differentiation. This result is in accord with the observation that Blimp-1, a master factor for plasma cell differentiation, is expressed in a fraction of 4–15% of germinal center B cells [26 ].

We further characterized the phenotype of blood IgG-secreting cells in donors not recently vaccinated. In contrast to the observed heterogeneity within the tonsilar and bone marrow IgG-secreting cell compartment, IgG-secreting cells in the blood were homogenous with respect to the expression of most markers analyzed (Table 2 ). The phenotype of steady-state peripheral blood IgG-secreting cells observed here does not differ from that described for blood plasma cells induced by immunization or that described for peripheral blood plasma cells in systemic lupus erythematosus patients [10 , 13 , 27 ].


View this table:
[in this window]
[in a new window]
  		Table 2. Phenotype of Peripheral Blood Ig-Secreting Cellsa

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, for the first time, we used the cellular affinity matrix technology, which allows the direct identification of Ab-secreting cells to study human plasma cells and their immediate precursors, i.e., plasma blasts, in tonsil, blood, and bone marrow samples. In secondary lymphoid tissues, e.g., tonsils, B cells are activated to form plasma cells. We show that the differentiation of IgG-secreting cells in the tonsils starts before CD38 is expressed. IgG-secreting cells expressing only little CD38 are present in human tonsils and early in B cell cultures, where these cells precede IgG-secreting cells with a CD38 high phenotype. As shown on tissue sections, cells containing high quantities of IgG in their enlarged cytoplasm and expressing only little CD38 are located in tonsillar follicles. In contrast, CD38 high IgG-containing plasma cells are mainly present outside the follicular areas. The early IgG-secreting plasma cell precursors seem to be present, particularly in the light zone of germinal centers. This idea is in accord with the finding that in mice, antigen-specific cells with a plasmacytoid phenotype are present in splenic follicles early following immunization, i.e., within 36–60 h [28 , 29 ]. During their course of differentiation, CD38 low plasma cell precursors possibly leave the follicles and increase the expression of CD38. A few CD38 high plasma cells were detected in the follicles, possibly indicating that up-regulation of CD38 precedes their translocation into the extrafollicular areas. The presence of IgG in the interstitium adjacent to the IgG-containing CD38 low plasma cell precursors in the tonsillar light zone led us to speculate that these cells are the source of the soluble IgG molecules involved in the selection of germinal center B cells. Tangye and colleagues [22 ] recently identified Ab-secreting cells with a CD38-negative phenotype in cultures of B memory cells. In these cultures of CD40 ligand-activated cells, IgA-, IgM-, and IgG-secreting cells had been detected in comparable numbers among the CD38-positive and CD38-negative populations. It is interesting that it had been shown in these experiments that the two populations differing in the expression of CD38 have different survival requirements, allowing the assumption that CD38-negative, Ab-secreting cells may resemble short-lived plasma cells. Tangye et al. [22 ] could not resolve the question whether the CD38-negative, Ab-secreting cells resemble precursors of the CD38 high cells or whether both cell types belong to different lineages. Our data suggest that CD38 high plasma cells are descendants of CD38 low Ab-secreting cells. However, we also detected some CD38 low plasmacytoid IgG-containing cells outside the follicles. It remains to be elucidated whether these cells belong to a distinct plasma cell linage, e.g., of short-lived plasma cells.

A fraction of the plasma cells formed in secondary lymphoid tissues can enter the bloodstream to migrate to the bone marrow, an organ, which is of particular importance for the survival of long-lived plasma cells. That bone marrow plasma cells are the progeny of B cells activated in secondary lymphoid tissues has been known [3 ]. Also, it has been shown that Ab-secreting cells present in secondary lymphoid tissues following immunization are equipped with chemokine receptors, allowing their emigration into the bone marrow, intestinal lamina propria, or into inflamed tissues [1 , 2 , 30 ], suggesting that the formation of Ab-secreting cells and their early differentiation occurs in secondary lymphoid tissue. It is interesting to note that peripheral IgG-secreting cells in the blood showed a homogeneous phenotype, and their progeny and their precursors, i.e., bone marrow plasma cells and tonsillar plasma cells, are heterogeneous with respect to CD9, CD19, CD38, and CD45. This suggests that plasma cell interaction with stromal cells, extracellular matrix components, and/or cytokines present in lymphoid tissues may regulate the expression of various surface molecules on plasma cells. That plasma cells are responsive to extrinsic signals had been shown earlier. Tonsillar plasma cells, e.g., can be rescued from apoptosis by stromal cells [31 ]. Also, the interaction with their environment allows the survival and proliferation of plasma blasts [32 ] and the entrance of a certain number of these cells into the long-lived plasma cell compartment in the splenic red pulp [33 ].

As a result of the absence of CD19 on myeloma cells, plasma cell differentiation has been considered to be accompanied by the loss of this molecule [34 , 35 ]. In accord with other reports [13 , 27 ], we show that CD19 is expressed on all nonmalignant, IgG-secreting cells including the later plasma cell stages in the bone marrow. However, the level of CD19 expression on IgG-secreting cells differs in the analyzed tissues. All tonsillar IgG-secreting cells, at least partially resembling early plasma cell stages, express CD19 at high levels, comparable or higher than other CD19-positive cells in this tissue. IgG-secreting cells in the blood, possibly on their way from secondary lymphoid tissues to the bone marrow and other peripheral tissues, show reduced levels of CD19. In the bone marrow, however, two populations of plasma cells are found expressing high or low levels of CD19. The later seem to express CD19 in levels comparable with those expressed on plasma cells isolated from blood. As plasma cells migrating through the blood are at least partly precursors of bone marrow plasma cells, this may indicate that CD19 is lost transiently during plasma cell translocation through the blood but is up-regulated on mature bone marrow plasma cells. This hypothesis is in accord with the observation that the level of CD19 expression on bone marrow plasma cells correlates with their cell size (Table 1) and thus with an increase in the level of maturation [23 ]. However, this issue remains to be further elucidated.

Our data show that plasma cell differentiation can be dissected in distinct, developmental stages of CD38 low and CD38 high cells. The presence of distinct, sequential steps during early plasma cell differentiation in the tonsils may possibly mark controlled checkpoints regulating this process in secondary lymphoid tissues. The presence of distinct subpopulations of bone marrow IgG-secreting cells representing later stages of the plasma cell development may indicate the presence of distinct types of plasma cells, e.g., short-lived and long-lived ones. However, we did not find subpopulations among the bone marrow plasma cell precursors, i.e., the IgG-secreting cells in the peripheral blood, arguing against different plasma cell lineages. An alternative explanation for the presence of multiple subpopulations of bone marrow plasma cells is the presence of a "checkpoint" controlling the entrance into the long-lived plasma cell compartment. Such a mechanism would explain the distinct bone marrow plasma cells by the presence of a population, which has entered the bone marrow just recently but is not allowed to enter the long-lived compartment yet, and a plasma cell population resembling the long-lived compartment. This idea is in accord with the hypothesis that plasma cell longevity is not guaranteed by entering the bone marrow but depends on specific niches providing survival signals [36 37 38 39 ].


   ACKNOWLEDGEMENTS
 
The Deutsche Forschungsgemeinschaft, Clinical Research Group, "Growth Control of Neoplastic B-Cells," Grant No. KFO 105/1, and by the European Community (Grants ERBFMBICT 983532 and QLK2-CT-2001-01205 Memovax) supported this work. We thank Anette Peddinghaus and Sailly Canal for excellent technical support and Dörte Huscher for helping in the statistical analysis of the data.

Received June 18, 2003; accepted January 21, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hargreaves, D. C., Hyman, P. L., Lu, T. T., Ngo, V. N., Bidgol, A., Suzuki, G., Zou, Y. R., Littman, D. R., Cyster, J. G. (2001) A coordinated change in chemokine responsiveness guides plasma cell movements J. Exp. Med. 194,45-56[Abstract/Free Full Text]
  2. Hauser, A. E., Debes, G. F., Arce, S., Cassese, G., Hamann, A., Radbruch, A., Manz, R. A. (2002) Chemotactic responsiveness toward ligands for CXCR3 and CXCR4 is regulated on plasma blasts during the time course of a memory immune response J. Immunol. 169,1277-1282[Abstract/Free Full Text]
  3. Benner, R., Hijmans, W., Haaijman, J. J. (1981) The bone marrow: the major source of serum immunoglobulins, but still a neglected site of antibody formation Clin. Exp. Immunol. 46,1-8[Medline]
  4. Cassese, G., Lindenau, S., de Boer, B., Arce, S., Hauser, A., Riemekasten, G., Berek, C., Hiepe, F., Krenn, V., Radbruch, A., Manz, R. A. (2001) Inflamed kidneys of NZB/W mice are a major site for the homeostasis of plasma cells Eur. J. Immunol. 31,2726-2732[CrossRef][Medline]
  5. Manz, R. A., Thiel, A., Radbruch, A. (1997) Lifetime of plasma cells in the bone marrow Nature 388,133-134[CrossRef][Medline]
  6. Slifka, M. K., Antia, R., Whitmire, J. K., Ahmed, R. (1998) Humoral immunity due to long-lived plasma cells Immunity 8,363-372[CrossRef][Medline]
  7. Arpin, C., Dechanet, J., Van Kooten, C., Merville, P., Grouard, G., Briere, F., Banchereau, J., Liu, Y. J. (1995) Generation of memory B cells and plasma cells in vitro Science 268,720-722[Abstract/Free Full Text]
  8. Agematsu, K., Hokibara, S., Nagumo, H., Shinozaki, K., Yamada, S., Komiyama, A. (1999) Plasma cell generation from B-lymphocytes via CD27/CD70 interaction Leuk. Lymphoma 35,219-225[Medline]
  9. Jelinek, D. F., Lipsky, P. E. (1983) The role of B cell proliferation in the generation of immunoglobulin-secreting cells in man J. Immunol. 130,2597-2604[Abstract]
  10. Odendahl, M., Jacobi, A., Hansen, A., Feist, E., Hiepe, F., Burmester, G. R., Lipsky, P. E., Radbruch, A., Dorner, T. (2000) Disturbed peripheral B lymphocyte homeostasis in systemic lupus erythematosus J. Immunol. 165,5970-5979[Abstract/Free Full Text]
  11. Horst, A., Hunzelmann, N., Arce, S., Herber, M., Manz, R. A., Radbruch, A., Nischt, R., Schmitz, J., Assenmacher, M. (2002) Detection and characterization of plasma cells in peripheral blood: correlation of IgE+ plasma cell frequency with IgE serum titre Clin. Exp. Immunol. 130,370-378[CrossRef][Medline]
  12. Brieva, J. A., Roldan, E., De la Sen, M. L., Rodriguez, C. (1991) Human in vivo-induced spontaneous IgG-secreting cells from tonsil, blood and bone marrow exhibit different phenotype and functional level of maturation Immunology 72,580-583[Medline]
  13. Medina, F., Segundo, C., Campos-Caro, A., Gonzalez-Garcia, I., Brieva, J. A. (2002) 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 99,2154-2161[Abstract/Free Full Text]
  14. Snapper, C. M., Mond, J. J. (1993) Towards a comprehensive view of immunoglobulin class switching Immunol. Today 14,15-17[CrossRef][Medline]
  15. Manz, R., Assenmacher, M., Pfluger, E., Miltenyi, S., Radbruch, A. (1995) Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix Proc. Natl. Acad. Sci. USA 92,1921-1925[Abstract/Free Full Text]
  16. Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g Scand. J. Clin. Lab. Invest. Suppl. 97,77-89[Medline]
  17. Lyons, A. B., Parish, C. R. (1994) Determination of lymphocyte division by flow cytometry J. Immunol. Methods 171,131-137[CrossRef][Medline]
  18. Volkman, D. J., Allyn, S. P., Fauci, A. S. (1982) Antigen-induced in vitro antibody production in humans: tetanus toxoid-specific antibody synthesis J. Immunol. 129,107-112[Medline]
  19. Czerkinsky, C. C., Nilsson, L. A., Nygren, H., Ouchterlony, O., Tarkowski, A. (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells J. Immunol. Methods 65,109-121[CrossRef][Medline]
  20. Halper, J., Fu, S. M., Wang, C. Y., Winchester, R., Kunkel, H. G. (1978) Patterns of expression of human "Ia-like" antigens during the terminal stages of B cell development J. Immunol. 120,1480-1484[Abstract/Free Full Text]
  21. Manz, R. A., Lohning, M., Cassese, G., Thiel, A., Radbruch, A. (1998) Survival of long-lived plasma cells is independent of antigen Int. Immunol. 10,1703-1711[Abstract/Free Full Text]
  22. Tangye, S. G., Avery, D. T., Hodgkin, P. D. (2003) A division-linked mechanism for the rapid generation of Ig-secreting cells from human memory B cells J. Immunol. 170,261-269[Abstract/Free Full Text]
  23. Terstappen, L. W., Johnsen, S., Segers-Nolten, I. M., Loken, M. R. (1990) Identification and characterization of plasma cells in normal human bone marrow by high-resolution flow cytometry Blood 76,1739-1747[Abstract/Free Full Text]
  24. Won, W. J., Kearney, J. F. (2002) CD9 is a unique marker for marginal zone B cells, B1 cells, and plasma cells in mice J. Immunol. 168,5605-5611[Abstract/Free Full Text]
  25. Bhan, A. K., Nadler, L. M., Stashenko, P., McCluskey, R. T., Schlossman, S. F. (1981) Stages of B cell differentiation in human lymphoid tissue J. Exp. Med. 154,737-749[Abstract/Free Full Text]
  26. Angelin-Duclos, C., Cattoretti, G., Lin, K. I., Calame, K. (2000) Commitment of B lymphocytes to a plasma cell fate is associated with Blimp-1 expression in vivo J. Immunol. 165,5462-5471[Abstract/Free Full Text]
  27. Harada, Y., Kawano, M. M., Huang, N., Mahmoud, M. S., Lisukov, I. A., Mihara, K., Tsujimoto, T., Kuramoto, A. (1996) Identification of early plasma cells in peripheral blood and their clinical significance Br. J. Haematol. 92,184-191[CrossRef][Medline]
  28. Jacob, J., Kassir, R., Kelsoe, G. (1991) In situ studies of the primary immune response to (4-hydroxy-3- nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations J. Exp. Med. 173,1165-1175[Abstract/Free Full Text]
  29. Liu, Y. J., Zhang, J., Lane, P. J., Chan, E. Y., MacLennan, I. C. (1991) Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens Eur. J. Immunol. 21,2951-2962[Medline]
  30. Bowman, E. P., Kuklin, N. A., Youngman, K. R., Lazarus, N. H., Kunkel, E. J., Pan, J., Greenberg, H. B., Butcher, E. C. (2002) The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells J. Exp. Med. 195,269-275[Abstract/Free Full Text]
  31. Merville, P., Dechanet, J., Desmouliere, A., Durand, I., de Bouteiller, O., Garrone, P., Banchereau, J., Liu, Y. J. (1996) Bcl-2+ tonsillar plasma cells are rescued from apoptosis by bone marrow fibroblasts J. Exp. Med. 183,227-236[Abstract/Free Full Text]
  32. Garcia De Vinuesa, C., Gulbranson-Judge, A., Khan, M., O’Leary, P., Cascalho, M., Wabl, M., Klaus, G. G., Owen, M. J., MacLennan, I. C. (1999) Dendritic cells associated with plasmablast survival Eur. J. Immunol. 29,3712-3721[CrossRef][Medline]
  33. Sze, D. M., Toellner, K. M., Garcia de Vinuesa, C., Taylor, D. R., MacLennan, I. C. (2000) Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival J. Exp. Med. 192,813-821[Abstract/Free Full Text]
  34. Harada, H., Kawano, M. M., Huang, N., Harada, Y., Iwato, K., Tanabe, O., Tanaka, H., Sakai, A., Asaoku, H., Kuramoto, A. (1993) Phenotypic difference of normal plasma cells from mature myeloma cells Blood 81,2658-2663[Abstract/Free Full Text]
  35. Mahmoud, M. S., Huang, N., Nobuyoshi, M., Lisukov, I. A., Tanaka, H., Kawano, M. M. (1996) Altered expression of Pax-5 gene in human myeloma cells Blood 87,4311-4315[Abstract/Free Full Text]
  36. Manz, R. A., Radbruch, A. (2002) Plasma cells for a lifetime? Eur. J. Immunol. 32,923-927[CrossRef][Medline]
  37. Manz, R. A., Arce, S., Cassese, G., Hauser, A. E., Hiepe, F., Radbruch, A. (2002) Humoral immunity and long-lived plasma cells Curr. Opin. Immunol. 14,517-521[CrossRef][Medline]
  38. Manz, R. A., Cassese, G., Thiel, A., Radbruch, A. (1999) Long-lived plasma cells survive independent of antigen Curr. Top. Microbiol. Immunol. 246,71-74[Medline]
  39. Minges Wols, H. A., Underhill, G. H., Kansas, G. S., Witte, P. L. (2002) The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity J. Immunol. 169,4213-4221[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
S. Sarantopoulos, K. E. Stevenson, H. T. Kim, N. S. Bhuiya, C. S. Cutler, R. J. Soiffer, J. H. Antin, and J. Ritz
High Levels of B-Cell Activating Factor in Patients with Active Chronic Graft-Versus-Host Disease
Clin. Cancer Res., October 15, 2007; 13(20): 6107 - 6114.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
G. Muehlinghaus, L. Cigliano, S. Huehn, A. Peddinghaus, H. Leyendeckers, A. E. Hauser, F. Hiepe, A. Radbruch, S. Arce, and R. A. Manz
Regulation of CXCR3 and CXCR4 expression during terminal differentiation of memory B cells into plasma cells
Blood, May 15, 2005; 105(10): 3965 - 3971.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. T. Avery, J. I. Ellyard, F. Mackay, L. M. Corcoran, P. D. Hodgkin, and S. G. Tangye
Increased Expression of CD27 on Activated Human Memory B Cells Correlates with Their Commitment to the Plasma Cell Lineage
J. Immunol., April 1, 2005; 174(7): 4034 - 4042.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
E. J. Blink, A. Light, A. Kallies, S. L. Nutt, P. D. Hodgkin, and D. M. Tarlinton
Early appearance of germinal center-derived memory B cells and plasma cells in blood after primary immunization
J. Exp. Med., February 22, 2005; 201(4): 545 - 554.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. Odendahl, H. Mei, B. F. Hoyer, A. M. Jacobi, A. Hansen, G. Muehlinghaus, C. Berek, F. Hiepe, R. Manz, A. Radbruch, et al.
Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response
Blood, February 15, 2005; 105(4): 1614 - 1621.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0603279v1
75/6/1022    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arce, S.
Right arrow Articles by Manz, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arce, S.
Right arrow Articles by Manz, R. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS