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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2800-2808
Monocytoid B Cells Are Distinct From Splenic Marginal Zone Cells
and Commonly Derive From Unmutated Naive B Cells and Less
Frequently From Postgerminal Center B Cells by Polyclonal
Transformation
By
Karoline Stein,
Michael Hummel,
Petra Korbjuhn,
Hans-Dieter Foss,
Ioannis Anagnostopoulos,
Theresa Marafioti, and
Harald Stein
From the Institute of Pathology, Consultation and Reference Centre
for Lymph Node Pathology and Haematopathology, University Hospital
Benjamin Franklin, Free University Berlin, Berlin, Germany.
 |
ABSTRACT |
Monocytoid B cells represent a morphologically conspicuous B-cell
population that constantly occurs in Toxoplasma gondii-induced Piringer's lymphadenopathy. Although widely believed to be closely related to splenic marginal zone B cells, neither this relationship, nor the B-cell differentiation stage of monocytoid B cells, nor their
cellular precursors have been established. We have therefore examined
monocytoid B cells for their expression of B-cell differentiation markers and the Ig isotypes at the RNA and protein level as well as for
rearranged Ig heavy chain (H) genes and somatic mutations within the
variable (V) region. The results obtained were compared with the
corresponding features of other B-cell populations. The monocytoid B
cells displayed immunophenotypical differences to all other B-cell
populations. IgM and IgD expression was absent from most monocytoid B
cells at the RNA and protein levels. Unrelated (polyclonal) Ig
rearrangements were found in 85 of the 95 cells studied. Seventy-four
percent of the rearranged VH genes were devoid of somatic mutations,
whereas the remaining 26% carried a low number of somatic mutations.
The majority of these showed no significant signs of antigen selection.
This finding in conjunction with the predominantly unrelated Ig gene
rearrangements indicates that most monocytoid B cells arise not by
clonal proliferation but by transformation of polyclonal B cells. The B
cells undergoing a monocytoid B-cell transformation are in the majority
(74%) naive B cells, and only a minority are (26%)
non-antigen-selected postgerminal center B cells. Thus, our data show
that monocytoid B cells represent a distinct B-cell subpopulation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MONOCYTOID B CELLS can be encountered in
various reactive lymphadenopathies and some lymphomas, including
Hodgkin's disease,1-12 but occur most consistently in the
form of cohesive sheets in Piringer's lymphadenopathy usually caused
by Toxoplasma gondii.13-17
Monocytoid B cells are characterized by an abundant pale cytoplasm,
indented nuclei with inconspicuous nucleoli, and open chromatin.13,14 They are located in the marginal zones
adjacent to the subcapsular and intermediary sinuses.14
Because these features were regarded as characteristic of immature
sinus histiocytes or blood monocytes, these cells were initially termed
immature sinus histiocytes14 or monocytoid
cells.15 When, thanks to the hybridoma technology,
macrophage-specific and B-cell-specific marker molecules became
available and background-free immunohistochemical staining methods were
developed, it was found that monocytoid cells did not carry markers of
the monocyte-macrophage system, but express B-cell antigens, suggesting
their relationship to the B-cell lineage.18-21 Hence, 2 new
designations were proposed: sinus B cells19 and monocytoid
B cells.20 The latter term has prevailed.
The assignment of the monocytoid B cells to the B-cell system raised
the question as to which B-cell subpopulation they are most closely
related to. Although in some studies18,19 the monocytoid B
cells could not be allocated to any of the established B-cell
populations, the study by Burke and Sheibani22 suggested a
close relationship to hairy cell leukemia cells and the study by Van
den Oord et al23 and others21 suggested a close
relationship to splenic marginal zone B cells. Whereas a relationship
to hairy cell leukemia cells was not supported by other studies, the
view that monocytoid B cells might represent the nodal counterpart of
splenic marginal zone cells became widely accepted.23 This view had significant impact on the classification of primary nodal lymphomas growing in the marginal zone, in that all of them were lumped
together under the term nodal marginal zone lymphoma, irrespective of
whether they resembled immunophenotypically monocytoid B cells or
splenic and Peyer's patch marginal zone cells.24-29 The
conclusion that monocytoid B cells and marginal zone cells represent
the same B-cell subset was mainly reasoned by the claim that both cell
populations resemble each other by a similar expression of IgM in the
absence of IgD, a similar homing to marginal zones, and a similar VH
gene mutation pattern. However, the close relationship was challenged
by other studies19,21,30,31 demonstrating that most
monocytoid B cells are, in contrast to splenic marginal zone cells,
vastly or completely devoid of IgM and BCL-2. Further differences
included the absence of intermingled T cells and the admixture of
neutrophils in monocytoid B-cell areas,13,14,19 whereas the
opposite finding was observed in marginal B-cell zones.
To clarify the B-cell differentiation stage of monocytoid B cells in
relation to other B-cell subsets, to identify the B-cell population
from which monocytoid B cells originate, and to determine whether
monocytoid B cells arise polyclonally from many cells, oligoclonally
from a few cells, or monoclonally from a single cell, we investigated
the immunophenotype, the Ig isotype expression, and the rearrangement
and VH gene mutation pattern of monocytoid B cells at the single-cell
level. Our results show that monocytoid B cells are distinct from
marginal zone cells of the spleen and mesenteric lymph nodes. They are
heterogeneous in their differentiation stage in that the majority
originate from unmutated naive B cells and a minority originate from
mutated postgerminal center memory type B cells. Finally, monocytoid B
cells are generated by the transformation of many cells, rather than by
expansion of a few cells or a single cell.
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MATERIALS AND METHODS |
Tissue samples.
Eleven cases of Piringer's lymphadenopathy from which paraffin blocks
and snap-frozen material were available were taken from the files of
the Institute of Pathology at the University Hospital Benjamin Franklin
of the Free University of Berlin. All 11 cases were used for
immunophenotypical analysis, 6 of them for in situ hybridization and 5 cases with the most prominent areas of monocytoid B cells were selected
for the isolation of single cells. For the comparative analysis of
mantle cells, germinal center cells, and marginal zone cells, 2 normal
spleens and 2 to 4 mesenterial lymph nodes from 2 patients were
included in our study.
Tissue processing and immunohistochemistry.
Immunohistochemical analysis of the monocytoid B cells was performed on
formalin-fixed paraffin-embedded tissues. Paraffin sections were
pretreated by heating in 0.01 mol/L citrate buffer, pH 6.0, for 2 minutes in a pressure cooker followed by cooling to room temperature
and washing with Tris-buffered saline. Frozen sections, immunostained
for single-cell isolation, were air-dried overnight and fixed in
acetone for 10 minutes. For identification of the monocytoid B cells as
well as B-cell subsets and T cells, antibodies directed
against the following antigens were applied: CD20 (L26), DBA44, CD25
(Ber-Act 1), CD103 (Ber-Act 8), CD11c (KB90), BCL-2 (124), BCL-6 (594),
IgM, IgD, IgA, IgE, CD3 (DAKO, Glostrup, Denmark), CD23 (1B12), CD10
(56C6), CD5 (4C7), CD4 (1F6; Novocastra, Newcastle-on-Tyne, UK), CD27
(1A4CD27; Beckman Coulter Inc, Fullerton, CA), Ki-67
(Mib-1; kindly provided by Prof J. Gerdes, Borstel Institute, Borstel,
Germany), and CD45RA (Ki-B332; kindly donated
by Prof M.R. Parwaresch, Kiel, Germany). The binding of antibodies was
made visible either by the immuno-alkaline phosphatase-antiphosphatase (APAAP) technique or by the biotin-avidin-method, as previously described.33,34
In situ hybridization.
In situ hybridization for the detection of mRNA specific for IgM, IgD,
and IgG was performed as previously described.35 In brief,
after linearization of plasmids containing the IgD, M, and G heavy
chain constant regions with appropriate restriction enzymes,
35S-labeled run-off antisense and sense (control)
transcripts were generated using T7 or SP6 RNA polymerases. Paraffin
sections were pretreated by exposing them to 0.2 N HCl and 0.125 mg/mL
pronase (Boehringer Mannheim, Mannheim, Germany), followed by
acetylation with 0.1 triethanolamine, pH 8.0/0.25% (vol/vol) acetic
anhydride and dehydration through graded ethanols. Slides were
hybridized to 2 to 4 × 105 cpm of labeled probes
overnight at 50°C. Washing and autoradiography was performed as
described.36 Cells with a 4-fold signal intensity, as
compared with background signal, were scored as positive. Hybridization with sense probes only showed homogeneously distributed low background signal. The probes for the Ig heavy chain constant regions were prepared from cDNA of peripheral blood lymphocytes or B-cell lines and
cloned into the appropriate plasmids. The nucleotide sequence of the
probes was confirmed by DNA sequencing.
Isolation of single cells.
Single cells, identified by their localization and their
immunophenotype (ie, monocytoid B cells by their negativity for BCL-2 and CD21; mantle cells by their expression of IgD; germinal center cells by their positivity for BCL-6/CD10 and/or Ki-67; and splenic marginal zone cells by their IgM+/IgD
isotype profile), were isolated from frozen sections, as previously described.37 In brief, immunostained frozen tissue sections were covered with a buffer containing salmon-sperm DNA (1 mg/mL in
phosphate-buffered saline [PBS]). Single cells were isolated from the
surrounding tissue and transferred into a polymerase chain reaction
(PCR) test tube by means of a manipulation capillary and reception
capillary, which were fixed to a hydraulic micromanipulator. The whole
isolation process was monitored by microscope. Isolated T cells and
aliquots of the overlaying buffer drawn after each cell isolation
process served as the negative controls.
Single-cell PCR.
PCR for the detection of IgH genes was performed as previously
described.38-40 In brief, after proteinase K digestion of
the isolated cells for 1 hour (1.0 mg/mL), a fully nested PCR using family-specific frame-work (FW) 1 primers for the first amplification and family-specific FW2 primers for reamplifications was used in
conjunction with 2 nested primers for the joining region (JH). The
first round of amplification was performed with 40 cycles consisting of
5 cycles of 15 seconds at 96°C, 30 seconds at 63°C, and 30 seconds
at 72°C, followed by 35 cycles of 15 seconds at 96°C, 30 seconds at
57°C, and 30 seconds at 72°C in 2.5 mmol/L MgCl2, 200 µmol/L each dNTP, 300 ng of the FW primers (50 ng each), 100 ng of
the JH primers, and 2 U Taq DNA polymerase (AmpliTaq; Perkin
Elmer, Weiterstadt, Germany). The second round of
amplification consisted of 35 cycles of 15 seconds at 96°C, 30 seconds at 63°C, and 30 seconds at 72°C. Six microliters of the
reaction mixture was analyzed on an ethidiumbromide-stained
polyacrylamide gel (PAGE, 6%). The visualized bands of the PCR
products were isolated and directly sequenced in both directions by
separate application of the PCR primers. The sequencing was performed
with an automated fluorescence DNA sequencer (377A; Applied Biosystems,
Weiterstadt, Germany) by using the Dye Deoxy Terminator method.
Sequence analysis.
The amplified VH rearrangements were analyzed for VH usage and for
somatic mutations by comparison with germline VH-segments in the VBASE
databank, and coding capacity was determined by translation of the
sequences into amino acids. To exclude contaminations, all amplified
sequences were also compared with our own and published data bank
sequences.40,41
Antigen selection.
To determine the pattern of somatic mutations compatible with antigen
selection, 2 methods were applied. First, the ratio of replacement to
silent mutations (R/s) in the CDR2 and FW3 region was
studied.42 A sequence was considered to be antigen selected when the R/s ratio in the CDR2 was higher than 2.9 and R/s ratio in the
FW3 region was lower than 1.5. Second, only the R/s ratio of the
somatic mutations in the FW3 region was considered. A sequence was
regarded as being antigen selected when the R/s ratio was less than
1.6.43
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RESULTS |
Immunophenotype and admixed cells.
To update the phenotype of the monocytoid B cells and to determine
their relationship to other B-cell populations, an immunophenotypical analysis was performed on 11 cases of Piringer's lymphadenopathy, including new marker molecules (Tables 1 and 2 and Figs 1 and 2).
This investigation confirmed that monocytoid B cells differ from mantle
B cells by a complete absence of BCL-2 (Fig 2b) and CD23 and a vast
absence of sIgM and sIgD (Fig 1d and e). They can be distinguished from
germinal center B cells by the absence of CD10, BCL-6, cytoplasmic Ig,
and partial presence of DBA44, a hairy cell leukemia-associated
molecule. From splenic and nodal marginal zone B cells, monocytoid B
cells can be discriminated by a partial presence of DBA44, a complete
presence of the Ki-B3 epitope of the CD45RA molecule, and additionally
by a complete absence of BCL-2 and a nearly complete absence of sIgM.
Different from all other B-cell subpopulations, the monocytoid B-cell
zone was devoid of T cells and follicular dendritic cells (FDC) (Fig 2a
and d), but contained a significant admixture of neutrophils. The
proliferation rate of monocytoid B cells, varying from 10% to 25%,
was significantly higher than that of mantle B cells (1% to 5%) as
well as of splenic and nodal marginal zone B cells (5% to 10%), but
considerably lower than that of germinal center B cells (50% to
100%), as demonstrated by the Ki-67 index (Fig 2c).
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Table 1.
Immunophenotype and Cellular Admixtures of Monocytoid B
Cells From 11 Cases of Piringer's Lymphadenopathy in Comparison With
Other B-Cell Populations
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Table 2.
Ig Isotypes at the Protein and RNA Level of Monocytoid B
Cells From 11 Cases of Piringer's Lymphadenopathy and Other B-Cell
Populations
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| Fig 1.
Piringer's lymphadenopathy. Morphological and
immunophenotypical features of monocytoid B cells in comparison with
other B-cell populations. (a and b) Giemsa staining. (c) CD20
immunostaining: mantle cells and germinal center B cells are less
intensively stained than the monocytoid B cells and display a slightly
different red tone. (d) IgM immunostaining: monocytoid B cells are
largely negative for IgM, which is different from the strongly
IgM-positive follicle mantle cells. (e) IgD immunostaining. Only some
monocytoid B cells are positive for IgD. Arrows show small clusters of
epithelioid cells characteristic of Piringer's lymphadenopathy. MCB,
monocytoid B cells; FM, follicle mantle; GC, germinal center.
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| Fig 2.
Piringer's lymphadenopathy. (a) CD3 immunostaining: the
monocytoid B-cell zone is nearly devoid of T cells, in contrast to the
follicle mantle and germinal center. (b) BCL-2 immunostaining:
monocytoid B cells are negative for BCL-2. (c) Ki-67 immunostaining:
approximately 10% to 20% of the monocytoid B cells are in cell cycle.
(d) CD21 immunostaining. Note the labeling of follicular dendritic
cells within the germinal center and their absence from the monocytoid
B-cell zone. MCB, monocytoid B cells; FM, follicle mantle; GC, germinal
center.
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Ig isotype expression.
To check the validity of the Ig labeling results and whether the
absence of Ig protein expression on most monocytoid B cells is due to a
downregulation at the transcriptional or translational level, the
expression of Ig mRNA (Igµ,  , and  ) was studied and compared
with the presence of the corresponding Ig protein (Table 2). Igµ and
Ig mRNA were detectable by in situ hybridization in approximately
20% of the monocytoid B cells, a finding in harmony with that obtained
by immunohistochemistry for protein detection (Fig
3a). An unexpected result was the finding
of an expression of Ig transcripts on the majority of monocytoid B
cells in the absence of IgG protein molecules. The level of this RNA
expression was low and corresponded with that of other B cells carrying
surface membrane bound Ig (Table 2 and Fig 3b and c).
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| Fig 3.
Piringer's lymphadeno- pathy. Radioactive in
situ hybridization for the detection of Ig heavy chain gene
transcripts. (a) Igµ in situ hybridization, antisense: the vast
majority of the monocytoid B cells are negative, whereas resting mantle
B cells are clearly labeled. Some plasma B cells in the germinal center
display extremely intensive labels. (b) Ig in situ hybridization,
antisense: most monocytoid B cells are weakly labeled, whereas mantle B
cells are completely negative. Some germinal center B cells display a
moderate labeling, whereas others are very intensely labeled (plasma
cells). (c) Ig in-situ hybridization, sense: negative control. No
cells are labeled. MCB, monocytoid B cells; FM, follicle mantle; GC,
germinal center.
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Ig gene rearrangements and VH mutation pattern.
Three hundred twenty-seven monocytoid B cells were isolated from the
lymph node sections of 5 cases of Piringer's lymphadenopathy. For
comparison, single cells were isolated from mantle zones and germinal
centers of the same and other cases, as well as from the marginal zone
of 2 spleens, and were subjected to a single-copy PCR and sequence
analysis for the demonstration of Ig heavy chain gene rearrangements
and their mutation pattern. Ninety-five sequences (29%) were obtained
from the 327 isolated monocytoid B cells, and 85 of these sequences
(89%) were unique. In addition, 5 unrelated sequences occurred twice
in 3 of the 5 cases of Piringer's lymphadenopathy. The sequence
comparison of the VH rearrangements with databank germline VH segments
showed that 67 (74.4%) of the monocytoid B cells displayed unmutated
VH genes ranging from 50% to 86.6% among the 5 cases investigated
(Table 3). Twenty-three sequences (25.6%)
harbored somatic VH mutations, with an average mutation frequency of
3.8% and ranging from 1 to 14 base substitutions (Table 3).
To check the validity of the results of our VH mutation analysis, 42 mantle zone B cells, 81 germinal center B cells, and 66 splenic
marginal zone B cells were isolated and their VH rearrangements were
amplified by PCR, resulting in 12, 25, and 21 amplificates, respectively. The sequence analysis of the PCR products showed that
none of the VH gene rearrangements amplified from mantle zone B cells
contained somatic mutations or a clonal relation. Germinal center B
cells displayed mutated VH rearrangements in most instances that were
partly clonally related with signs of ongoing mutations as evidenced by
their intraclonal diversity. All VH gene rearrangements amplified from
the isolated splenic marginal zone B cells were unique. The majority of
them harbored somatic VH mutations (Table
4).
Our single-cell isolation technique was controlled by the analysis of
36 single T cells and 552 buffer samples that were drawn after each
cell isolation. None of these controls gave rise to VH-specific PCR
products, indicating that the isolated monocytoid B cells and control B
cells were not contaminated by other cells or by their DNA.
Antigen-selection and VH-gene family usage.
Two methods were applied for the estimation of antigen selection in the
mutated B cells. Both methods agreed in that the majority of the
monocytoid B cells with VH mutations (88.8%; 16/18) lacked signs of
antigen selection when the CDR2 and FW3 region were analyzed and 55.5%
(10/18) lacked signs when only the FW3 proportion was drawn into
consideration. Five sequences with somatic Ig mutations could not be
used for the determination of antigen-selection due to their very low
number of mutations. In contrast to the monocytoid B cells, most of the
mutated splenic marginal zone cells showed signs of antigen selection.
Coding capacity.
Eighty-three of the 90 individual monocytoid B cells (93%) showed a
functional VH rearrangement without stop codons or frame shift
mutations. Four nonfunctional VH rearrangements occurred in the CDR3
proportion of the unmutated monocytoid B cells and consisted of 3 stop
codons and 2 frame shifts, with 1 cell simultaneously carrying a stop
codon in the FW3 region and a frame shift. The remaining nonfunctional
VH rearrangement occurred in a mutated monocytoid B-cell and consisted
of a deletion and 2 insertions in the FW3 region resulting in a frame shift.
| |
DISCUSSION |
In the present study, we have investigated monocytoid B cells to
identify the precursor B cells of monocytoid B cells and to elucidate
their relationship to other B-cell populations, including nodal and
splenic marginal zone cells. For this purpose, we selected cases of
Toxoplasma gondii-induced Piringer's lymphadenopathy with prominent,
and thus reliably identifiable, sheets of monocytoid B cells and
analyzed their antigen profile, their Ig isotype expression at the RNA
and protein levels, and their Ig gene rearrangement and VH gene
mutation pattern. The results obtained were compared with the features
of other B-cell populations with special reference to splenic marginal
zone B cells and, when technically possible, with the features of nodal
cells occurring in the marginal zone of mesenteric lymph nodes. Our
immunohistochemical investigations confirmed the vast absence of IgM
and the total absence of BCL-2 from monocytoid B cells and the presence
of both molecules on marginal zone cells of the spleen and mesenteric
lymph nodes. We could further show that the Ki-B3 epitope of the CD45RA
molecule and the DBA44 molecule (characteristically associated with
hairy cell leukemia cells) were totally absent from splenic and nodal marginal zone cells but were expressed either by all or by a proportion of monocytoid B cells, respectively. The proliferation rate was higher
in monocytoid B cells than in splenic and nodal marginal zone cells.
Our studies underscore the immunophenotypical differences between
monocytoid B cells and all other B-cell subsets (Table 1).
Because the immunohistological demonstration of Ig is prone to
artifacts due to high levels of soluble Ig in the serum,44 we extended our studies to the expression of Ig mRNA. The in situ hybridization experiments showed, for Igµ and Ig transcripts, a
distribution similar to that of the corresponding proteins, indicating
that the vast absence of IgM and IgD in monocytoid B cells is a true
finding and that their expression is regulated at the transcriptional
level. These findings confirm previous studies showing that monocytoid
B cells are distinct from all known B-cell populations, including
marginal zone cells of the spleen and mesenteric nodes, and dismiss the
concept that monocytoid B cells represent the nodal equivalent of
splenic marginal zone cells. An unexpected finding of our in situ
hybridization studies was weak, but distinct signals for Ig over the
monocytoid B cells in the absence of IgG protein labeling. However, a
similarly weak but distinct labeling for Ig transcripts was found
over most germinal center B cells, with the difference that, in the
germinal center light zone, there were some very strong additional
Ig transcript signals. These strong signals could be allocated to germinal center cells being in the process of plasma cellular differentiation. This finding demonstrates the absence of plasma cellular differentiation from monocytoid B cells and thus adds to the
differences between monocytoid B cells and germinal center cells. In
conclusion, the present and previous phenotypical
studies18,19,21 disclosed differences between monocytoid B
cells and established B-cell populations and thus proved to be
incapable of identifying the B-cell population that gives rise to
monocytoid B cells.
We therefore turned our studies to the genetic level. This included the
analysis of the rearrangement and mutation pattern of the VH genes in
single monocytoid B cells isolated from 5 different cases. Ninety-five
amplificates from 327 isolated monocytoid B cells could be obtained.
The detection of VH rearrangements in the monocytoid B cells formally
proved their B-cell nature. The finding that nearly all VH
rearrangements (85/95 [89%]) of the monocytoid B cells were unique
indicates that most, if not all, monocytoid B cells originate from many
(polyclonal) B cells and not from a few cells or one B cell by
oligoclonal or monoclonal expansion. This polyclonal origin of the
monocytoid B cells is further substantiated by a VH family usage
compatible with normal B cells excluding a derivation from B cells with
a particular type of VH rearrangement.
The sequence analysis of the IgH rearrangements showed that most
monocytoid B cells (95%) contained functional Ig genes. Of the
monocytoid B cells, 74.4% were devoid of VH gene mutation and 25.6%
carried somatic mutations at a frequency (3.8%) comparable to those of
IgG- and IgA-positive peripheral blood memory B cells.43,45 These findings indicate that the monocytoid B cells, despite their uniform morphological appearance, uniform antigen profile, and uniform
homing, are not homogeneous but consist of a mixture of B cells, with
the majority of them representing B cells at a pregerminal center stage
of differentiation, like mantle cells, and a minority representing late
B cells that have acquired somatic mutations during their passage
through a germinal center, like memory B cells. However, most of the
mutated monocytoid B cells lacked signs of antigen selection, drawing
their derivation from normally selected memory B cells into doubt.
These findings are in partial conflict with a recent
study29 reporting that splenic as well as nodal marginal
zone B cells (designated as monocytoid B cells in the mentioned study)
show somatic mutations and display clonal expansion. We therefore
extended our genetic single-cell studies to splenic marginal zone B
cells, mantle cells, and germinal center B cells. Unfortunately,
marginal zone cells of mesenteric lymph nodes could not be included
because of a lack of frozen tissue. In agreement with previous
investigations,43,46,47 mantle cells did not show any
clonal relationship or VH gene mutations, but clonally related VH
rearrangements with ongoing somatic mutations were detectable in the
germinal center cell population. Furthermore, our analysis of the
single splenic marginal zone cells showed only unrelated (polyclonal)
rearrangements with mostly antigen selected mutations, as shown in a
previous study.48 The different results concerning the
clonality and mutation pattern of the monocytoid B cells described by
Tierens et al29 might have been caused by an erroneous
comicrodissection of parts of the germinal centers leading to the
appearance of dominant PCR products. This assumption is supported by
the reported high frequency of crippling mutations (6 of 16 sequences
[37.5%]), because disrupted VH genes to that extent are so far only
reported for cells isolated from the inside but not from the outside of
germinal centers.43,46
In conclusion, the absence of related rearrangements from most
monocytoid B cells suggests that these cells arise by transformation of
many polyclonal B cells, eg, unmutated mantle cells, and to a lesser
extent of mutated immature (not fully antigen selected) memory B cells.
It is tempting to speculate that this transformation might be induced
by a B-cell superantigen expressed by Toxoplasma gondii. It has already
been shown that Toxoplasma gondii possess superantigen or
superantigen-like activity,49,50 which is effective on T
cells and leads to a selective expansion of a certain T-cell subset. It
could be that Toxoplasma gondii-expressed superantigens are also active
on a subset of pregerminal center and postgerminal center B cells. The
postulated superantigen(s) would induce a transformation of B cells of
various differentiation stages (ie, unmutated and/or mutated B cells),
including a change of their antigen profile and an isotype switch
similar to the one seen in germinal centers that is associated with a
downregulation of BCL-2, Ig, and CD23 and an upregulation of the
proliferation. Furthermore, this superantigen could be responsible for
the attraction of neutrophils into the monocytoid B-cell zone.
Taken together, our findings support the view that monocytoid B cells
represent a separate B-cell subset that is distinct from all
established B-cell subpopulations, including marginal zone cells of the
spleen and mesenteric lymph nodes. They are not homogeneous, because
74.4% of monocytoid B cells appear to derive from unmutated naive
pregerminal center B cells and 25.6% disclose VH mutations compatible
with a derivation from postgerminal center B cells. The predominant
expression of Ig transcripts points towards an uncommon dissociation
between isotype switch and somatic VH gene mutation, at least in the
unmutated proportion of monocytoid B-cell, further stressing the
distinctiveness of these cells.
| |
ACKNOWLEDGMENT |
The authors thank H. Lammert, E. Berg, H. Protz, D. Jahnke, and H.-H.
Müller for their excellent technical assistance and L. Udvarhelyi
for his help with the preparation of the manuscript.
| |
FOOTNOTES |
Submitted May 6, 1999; accepted June 14, 1999.
Supported by a grant of the Deutsche Forschungsgemeinschaft (STE
318/5-2, STE 318/9-1).
Our nucleotide sequences are available as GenBank accession nos.
AF167582 through AF167676.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Harald Stein, MD, Institute of Pathology,
Benjamin Franklin University Hospital, Free University Berlin,
Hindenburgdamm 30, 12200 Berlin, Germany.
| |
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