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Blood, 15 August 2000, Vol. 96, No. 4, pp. 1612-1614
CORRESPONDENCE
To the Editor:
Monocytoid B cells
We read with interest the recent article by Stein et al studying
the monocytoid B cells in comparison to other B-cell subsets such as
mantle cells, germinal center B cells, and splenic marginal zone B
cells.1 In that study, 11 cases of Piringer Kuchinka lymphadenitis were selected for the study of monocytoid B cells. The
phenotype of the monocytoid B cells was investigated using immunohistochemistry and in situ hybridization on formalin-fixed paraffin-embedded tissues. Single-cell polymerase chain reaction (PCR)
was used to study the mutation pattern and immunoglobulin heavy chain
variable gene usage (VH) in monocytoid B cells from 5 cases. The study confirmed our findings that monocytoid B cells are
heterogeneous and comprise both naive and memory B cells, as well as
the fact that an important fraction of the monocytoid B cells but not
the splenic marginal zone B cells are proliferating.2 But
differences are reported by Stein et al with regard to the presence of
B-cell clones, as well as the level of somatic hypermutation in the
monocytoid B-cell and the splenic marginal zone. We previously reported
that the monocytoid B-cell zones and, to a lesser extent, the splenic
marginal zone may harbor B-cell clones.2 In contrast, no
B-cell clones could be identified by Stein et al.1 We
believe that the different results obtained are likely due to different methods that have been used to study the clonal relationship of the
monocytoid B cells. We performed a denaturing gradient gel electrophoresis (DGGE) analysis of the amplified immunoglobulin heavy
chain (IgH) complementarity determining region 3 (CDR3) products from
microdissected clusters of Ki-67 positive and negative monocytoid and
splenic marginal zone B cells.2 By using DGGE, small
clones of B cells were demonstrated in the monocytoid B-cell and
splenic marginal zones. In this study we analyzed about 500 cells in
each of the microdissected marginal zones. In contrast, Stein et al
investigated the rearranged immunoglobulin genes of single monocytoid B
cells by directly sequencing the amplified IgH products; 14 to 25 rearranged immunoglobulin heavy chain gene sequences were obtained per
case. The latter numbers of sequences may not be enough to demonstrate
the presence of B-cell clones. In addition, we observed that clones
were more frequent in zones with a higher number of proliferating
cells, a finding that we have recently confirmed by other methods
(manuscript in preparation). Indeed, the proliferating cell fraction in
monocytoid B-cell zones varies, a finding that is also reported by
Stein et al (between 10 to 35 percent of the monocytoid B cells, in
their study). Thus the selection of cells or zones for study can to a
great extent determine the outcome of the results on clonality and may
additionally explain the differences between Stein et al's and our findings. Stein et al also reports differences between their study and ours
regarding the percentage of cells with somatic mutations to be found
between monocytoid B cells and (splenic) marginal zone B cells. To
explain this, the authors claim that germinal center cells might have
been mistaken for monocytoid B cells in our study and therefore has
yielded a higher number of cells with somatic hypermutations. Even
without being experienced morphologists, this event would have been
unlikely in view of the combined staining for CD23 and IgD prior to
microdissection. This combined staining highlights both follicle
centers and their mantles. We argue that Stein et al's and our
findings are basically similar if one pools the cells with no or only
low-level somatic hypermutations and compares those with the number of
cells with a high level of somatic hypermutations. This seems a
relevant distinction in terms of the affinity maturation of the immune
response. As such, it is clear from both our studies, as well as that
of others, that the monocytoid B cells, as well as the splenic marginal
zone B cells, are heterogeneous with respect to the number of somatic
hypermutations.1-4 Stein et al further argues that the lower number of cells with
high-level somatic hypermutations among monocytoid B cells warrants
their distinction from (splenic) marginal-zone B cells. But analysis of
their data shows that the range of monocytoid B cells with somatic
hypermutations varies from 13.3 to 50 percent and the number of
accumulated mutations per rearranged immunoglobulin gene can reach up
to 14 depending on the case and the cells analyzed. This variability is
also seen in marginal-zone B cells in the spleen. Thus the relative
percentage of cells with low-level and high-level somatic
hypermutations differs between various monocytoid B cell zones, as well
as between monocytoid B cells and splenic marginal zone B cells. This
can be explained by different phases of the immune response at which
tissues are analyzed and by obvious differences in antigenic challenge
provoking the immune response. In that respect, it is relevant to note
that, both in Stein's study and ours, lymph nodes suspect for
toxoplasma infection are compared with normal spleen. The even higher
number of B cells with somatic mutations in the marginal zone of Peyer
patch, as well as the higher number of mutations in the rearranged
immunoglobulin genes of these cells as compared to the splenic marginal
zone B cells, can be explained similarly. Therefore, heterogeneity with
respect to the occurrence and number of somatic hypermutations in the
immunoglobulin genes of the B cells is a hallmark of the marginal zone,
including the monocytoid B-cell zone. Scoring this heterogeneity is not
appropriate to recognize different B-cell subsets. The authors further claim that weak signals for gamma transcripts are
another distinctive feature of monocytoid B-cells. But the authors have
not studied the presence of these transcripts in other marginal zone B
cells whereby the uniqueness of this feature for monocytoid B cells is
uncertain. In addition, in situ hybridization does not discriminate
beween germ-line gamma transcripts.5 Therefore, the
importance of this finding to define monocytoid B cells as
constituting a distinct B-cell subset is not established. In conclusion, we believe that Stein et al's data do not essentially
differ from our own and do not provide convincing evidence that
monocytoid B cells are different from marginal zone B cells at other sites.
Anne Tierens and Jan Delabie
Department of Pathology The Norwegian Radium Hospital and
Institute for Cancer Research Oslo, Norway
Chris De Wolf-Peeters
Department of Pathology University Hospitals of Leuven
Leuven, Netherlands
References
1.
Stein K, Hummel M, Korbjuhn P, et al.
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.
Blood.
1999;94:2800-2808[Abstract/Free Full Text].
2.
Tierens A, Delabie J, Michiels L, Vandenberghe P, De Wolf-Peeters C.
Marginal zone B cells in the human lymph node and spleen show somatic hypermutations and display clonal expansion.
Blood.
1999;93:226-231[Abstract/Free Full Text].
3.
Dunn-Walters DK, Isaacson PG, Spencer J.
Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells.
J Exp Med.
1995;182:559-566[Abstract/Free Full Text].
4.
Dunn-Walters DK, Isaacson PG, Spencer J.
Sequence analysis of rearranged Ig VH genes from microdissected human Peyer's patch marginal zone B cells.
Immunol.
1996;88:618-624[Medline]
[Order article via Infotrieve].
5.
Fujieda S, Lin YQ, Saxon A, Zhang K.
Multiple types of chimeric germ-line heavy chain transcripts in human B cells. Evidence for trans-splicing of human Ig RNA.
J Immunol.
1996;157:3450-3459[Abstract].
Response:
Monocytoid B cells are different from marginal zone B
cells
The assignment of monocytoid B cells to known B-cell subsets has
been a source of constant debate for many years now. More recent
molecular data provided both by Tierens et al1 and by our
group2 has further intensified this debate. In their letter and in their recent publication, Tierens et al stated that monocytoid B
cells are identical to nodal marginal zone B cells and belong to the
same B-cell compartment as marginal zone B cells of the spleen. As a
result, they have consistently used the term "nodal marginal B
cells" for monocytoid B cells. The authors have, however, overlooked
several important facts that would bring their concept into doubt. Tierens et al's statement that monocytoid B cells and marginal B cells
belong to the same B-cell compartment is based on the claim that the
mentioned cells have "similar" immunophenotypic features and a
"similar" immunoglobulin (Ig) rearrangement pattern. But our much
more detailed phenotypical analysis provides several pieces of evidence
to suggest that monocytoid B cells are distinct from marginal zone B
cells. The Ki-B3 epitope of CD45RA is consistently expressed by
monocytoid B cells, and DBA44 is detectable in approximately 20% of
these cells. In contrast, both molecules are completely absent from
splenic and nodal marginal zone B cells. Conversely, Bcl-2 and IgM,
which are consistently detectable in marginal zone B cells, are
completely (or predominantly) absent from monocytoid B cells. Further
striking differences between both types of B cells concern the
admixture of other cells. In contrast to marginal zones, areas of
monocytoid B cells are nearly completely devoid of T cells but contain
neutrophils in significant numbers. These clear-cut differences
prompted us to conclude that real monocytoid B cells represent a B-cell
subset unrelated to marginal zone B cells of the spleen and of the
lymph nodes. In harmony with this conclusion are our data concerning
rearranged Ig genes. Monocytoid B cells in most instances (75%) harbor unmutated Ig genes, indicating their derivation from pre-germinal center B cells. In contrast, marginal zone B cells originate, in the
vast majority, from mutated post-germinal center B cells as evidenced
by our and others' investigations.2,3 Tierens et al's
findings that the majority of nodal marginal zone B cells carry mutated
IgH genes and are thus derived from mutated memory B cells clearly
supports our conclusion that monocytoid B cells represent a different B
cell subset. Furthermore, Tierens et al found a relatively high number
of nonfunctional Ig rearrangements (37.5%) in their nodal marginal
zone B cells.1 Because there is no subset of B cells other
than germinal center B cells that carry nonfunctional IgH
rearrangements at that high frequency, it is reasonable to assume that
these cells most likely represent germinal center B cells. Because
Tierens et al also did not provide an alternative explanation in their
letter, a germinal center derivation of these nonfunctional B cells is
still a matter of high probability. Tierens et al's discussion concerning the "clonality" of
marginal zone B cells and monocytoid B cells is also unfortunately misleading. Generally speaking, proliferating cells always produce cell
clones. The extension of these cell clones depends on various factors
such as the number of cells in cycle, duration of proliferation, preferred (biased) mitotic division of certain cells, and so forth. We
estimated the growth fraction by determining the proliferation index
(Ki-67 staining) and found more monocytoid B cells in cell cycle
than marginal zone B cells. In line with this observation, by
single-cell analysis we found some identically rearranged
monocytoid B cells, whereas marginal zone B cells were unrelated in all
instances. In germinal centers many clonally related B cells have been
repeatedly demonstrated by many investigations, including our
own.2,4 Tierens et al performed denaturing gradient gel
electrophoresis (DGGE) analysis for the determination of clonality, but
we have several concerns about the reliability of their results: (1)
Germinal centers are known to consistently contain huge numbers of
proliferating B cells and large B-cell clones. Unexpectedly, in Tierens
et al's study they proved to be clearly polyclonal by DGGE analysis in many instances (see lanes 5, 7, 15, and 17 of their figure
5).1 (2) As stated in their paper, "clonal ... rearrangements could also be observed when analyzing marginal zones
containing only few or no cycling cells."1(p230) This
indicates that DGGE can create artificial clonal patterns. (3) As
stated in their letter, "small clones of B cells were demonstrated in
the monocytoid B-cell and splenic marginal zones." But upon comparison of the results provided in their paper (figures 4 and 5), the degree of clonality of the marginal zones clearly exceeded that of the germinal centers in several instances. To conclude, although the number of cells investigated is smaller in a single-cell approach, the results obtained with this technique are obviously more
representative and more reliable than those obtained by microdissection and DGGE. Finally, we are of course aware of the possibility that Ig
germ-line transcripts can occur in B cells. But these
germ-line transcripts are mainly found in germinal centers prior to
class-switch recombination and plasmacellular
differentiation.5 Because class switching and
plasmacellular differentiation have not been observed in monocytoid B
cells, the meaning of Ig transcripts in these cells requires further investigation. In summary, our data provide convincing arguments that monocytoid B
cells are distinct from marginal zone B cells and represent a unique
B-cell subset. It would appear most likely that differences in our
results and in Tierens et al's results are due to technical problems, analysis of different cells, and/or the selection of cases
unsuitable for the investigation of monocytoid B cells.
Michael Hummel, Karoline Stein, and Harald Stein
Institute of Pathology University Hospital Benjamin
Franklin Free University Berlin Berlin, Germany
References
1.
Tierens A, Delabie J, Michiels L, Vandenberghe P, Wolf-Peeters C.
Marginal-zone B cells in the human lymph node and spleen show somatic hypermutations and display clonal expansion.
Blood.
1999;93:226-234.
2.
Stein K, Hummel M, Korbjuhn P, et al.
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.
Blood.
1999;94:2800-2808.
3.
Dunn-Walters DK, Isaacson PG, Spencer J.
Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells.
J Exp Med.
1995;182:559-566.
4.
Kuppers R, Zhao M, Hansmann ML, Rajewsky K.
Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections.
EMBO J.
1993;12:4955-4967[Medline]
[Order article via Infotrieve].
5.
Xu MZ, Stavnezer J.
Regulation of transcription of immunoglobulin germ-line gamma 1 RNA: analysis of the promoter/enhancer.
EMBO J.
1992;11:145-155[Medline]
[Order article via Infotrieve].
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