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Departments of
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Histology and Cell Biology and
Clinical Immunology, University of Groningen, Groningen, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MZ-B cells may be of mixed origin. Part of the MZ-B cells are presumably naïve (i.e., nonmemory) cells that develop in the absence of T cells and stimulation with exogenous Ags. This is suggested by the presence of MZ-B cells in spleen of Ag-free (10), germfree (11, 12), nude (13, 14), TCR-/- (7), or MHC class II-deficient animals (15). We and others showed that (naive) MZ-B cells in rat can develop directly from mature RF-B cells, a maturation step that does not require cell proliferation (2, 16). Kinetic studies imply that only a small proportion of RF-B cells eventually becomes MZ-B cell (17). B cells that become incorporated into the pool of MZ-B cells are most likely selected on the basis of their B cell receptor (BCR) specificity. First, as mentioned before, MZ-B cells are thought to be involved in Ab responses against TI-2 Ags and therefore carry BCRs specific for these types of Ags. Second, Chen et al. (18) demonstrated that, in VH81X Ig heavy (H) chain transgenic mice, the splenic MZ is selectively populated by a (polyreactive) B cell clone. Recently, Martin and Kearney (19) showed that the (positive) selection of this B cell clone into the MZ-B cell compartment is presumably due to its BCR specificity.
In addition to the presence of these so-called naïve MZ-B cells, also Ag-experienced memory cells may contribute to the MZ-B cell pool. Hapten-binding (memory) B cells reside in the splenic MZ of previously immunized rats (2, 20) and most MZ-B cells taken from human spleen carry somatically mutated Ig VH genes (21, 22). Moreover, virtually all MZ-B cells in man express CD27 (23), which is selectively expressed by somatically mutated memory B cells, but not by naive B cells (24). Memory MZ-B cells are most likely derived from germinal centers (25, 26).
In this study, we directly aimed at establishing the relative contribution of naive vs memory type of MZ-B cells and to explore whether (naive) MZ-B cells are indeed a selected population of cells. For this reason, we analyzed the relative representation and nucleotide sequences of VH genes belonging to the PC7183 VH gene family in several stages of B cell development in rats. Like in mice and man, Ig H chains and light (L) chains in rats are produced after sequential rearrangement of VH, D, and JH region genes, and VL and JL region genes, respectively (27). We recently showed that VH genes in rat can be subdivided into VH gene families similar to those of mice (17, 27, 28). At present, sequence analysis has revealed the existence of at least 10 VH gene families in rat (PC7183, X24, Vh11, S107, J606, Vh10, Q52, 3609, J558, and VGAM3.8) (17, 27, 28). Here, we analyzed the relative representation of VH genes belonging to the PC7183 VH gene family in four flow cytometry-defined stages of B cell development. In individual rats, the PC7183 VH gene repertoire expressed by MZ-B cells was compared with the repertoire expressed by mature splenic RF-B cells and that of the subsets of pro/pre-B cells and newly formed B cells (NF-B) from bone marrow. In addition, by analyzing the PC7183 VH genes for the presence of somatic mutations, we also determined to what extent memory B cells contribute to the MZ-B cell subset. Because representation of memory type of MZ-B cells may differ between flow cytometry-defined MZ-B cells and MZ-B cells defined on basis of their anatomical location in the spleen, we also analyzed productive PC7183 VH genes microdissected from follicular and MZ areas of rat spleen. Together, our data show that, in nonintentionally immunized rats, most MZ-B cells (defined either by flow cytometry or histology) express germline encoded PC7183 VH genes and possibly represent naive cells. Only a minor fraction of MZ-B cells appear to be memory B cells, as revealed by the presence of somatic mutations in their VH genes. The PC7183 VH gene repertoire of MZ-B cells differs from that of RF-B cells (and other B cells) and carry significantly shorter Ig H chain complementarity-determining region 3 (H-CDR3) regions, indicating that MZ-B cells are a selected population of cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone marrow cells and spleens were taken from two 6-mo-old male
PVG rats (R1 and R2, respectively), which were raised under clean
conventional conditions. The rats were obtained from and housed at the
Central Animal Facility of the Faculty of Medical Sciences (University
of Groningen, Groningen, The Netherlands). The preparation of cell
suspensions used for flow cytometry and cell sorting has been described
elsewhere (16). Bone marrow cells were collected from
either femur. Bone marrow and spleen cell suspensions were stained by a
combination of FITC-conjugated mouse anti-rat IgM (HIS40)
(29), PE-conjugated mouse anti-rat CD90 (HIS51;
anti-Thy-1) (30), and biotinylated mouse anti-rat
IgD (MaRD3; a generous gift from Dr. H. Bazin, University of Louvain,
Brussels, Belgium). Bone marrow cells were additionally incubated with
biotinylated mouse anti-rat TCR
(R73;
PharMingen, San Diego, CA) (31). Biotinylated mAbs were
revealed by streptavidin-allophycocyanin (PharMingen). Cell analysis
and sorting were performed on a dual laser Coulter Epics-Elite flow
cytometer with enhanced system performance upgrade (Coulter, Hialeah,
FL). Dead cell, plasma cell, monocyte/macrophage, and erythrocyte
contamination was excluded from the sorting by using forward and side
scatter profiles. Sorted cells were collected in sterile tubes
containing 
500 µl newborn calf serum (Life Technologies, Glasgow,
Scotland). FlowJo software (version 2.7; Tree Star, San Carlos, CA) was
used for flow cytometry data analysis.
RT-PCR and library construction of rearranged Ig H chain variable regions
Sorted cells were pelleted by 300 x g centrifugation for 10 min at 4°C. Equivalents of 2–4 x 105 cells were lysed in 0.5 ml TRIzol Reagent (Life Technologies) and stored at -80°C. Total RNA was isolated according to instructions of the manufacturer (Life Technologies). First-strand cDNA synthesis was performed on total RNA isolated from 2–4 x 105 sorted cells. Briefly, RNA was taken up in a 19-µl reaction volume containing 1,6 µg Oligo d(T)12–18 primer (Amersham-Pharmacia Biotech, Uppsala, Sweden) and 1.5 µl 10 mM each of dGTP, dATP, dTTP, and dCTP. This mixture was heated to 70°C for 10 min and quickly chilled on ice. First-strand cDNA reaction was performed in a final reaction volume of 30 µl, by adding 1 µl RNAquard (28 U/µl; Amersham-Pharmacia Biotech), 6 µl 5x first strand buffer (250 mM Tris-HCl (pH8.3), 375 mM KCl, 15 mM MgCl2), 3 µl 0.1 mM DTT, and 1 µl SuperScriptII reverse transcriptase (200 U/µl; Life Technologies). The cDNA reaction was conducted at 42°C for 50 min and finally inactivated by heating at 70°C for 15 min.
For construction of Ig
VHDJH-µ region cDNA
libraries, 2 µl of cDNA was amplified by PCR in a 50-µl reaction
mixture containing 0.6 µM VH PC7183
family-specific primer (VH PC7183; for primer
sequences see Table I
), 0.6 µM µ
constant-region primer (Cµ1.3), 20 mM Tris-HCl (pH 8.4), 50 mM KCl,
1.5 mM MgCl2, 2.5 U Taq DNA polymerase
(Life Technologies), and 0.2 mM each of dGTP, dATP, dTTP, and dCTP. The
PCR amplification program consisted of 35 cycles of 1 min at 94°C (2
min in first cycle), 1 min at 65°C, and 1 min at 72°C, followed by
an incubation at 72°C for 10 min. The size of the PCR products was
checked by agarose gel electrophoresis. PCR products were subsequently
cloned into pCR2.1-TOPO vector by using the TOPO TA cloning kit
(Invitrogen BV, Groningen, The Netherlands). Plasmid was obtained from
single white colonies, randomly picked, and grown in overnight cultures
(High Pure plasmid isolation kit; Boehringer Mannheim GmbH, Mannheim,
Germany). Aliquots of the isolated plasmids were digested by
EcoRI and subjected to agarose gel electrophoresis. Clones
containing a ±500-bp insert were subsequently sequenced at our local
sequence facility (Department of Pathology and Laboratory Medicine,
Division of Medical Biology, University of Groningen, Groningen, The
Netherlands). Sequence reactions were performed by cycle sequencing
using Thermo Sequenase primer cycle sequencing kit 7-deaza dGTP or
MegaBACE DYEnamic ET dye terminator kit (Amersham-Pharmacia Biotech).
Samples were run on the ALFexpress (Amersham-Pharmacia Biotech) or
MegaBASE-1000 system (Molecular Dynamics and Amersham-Pharmacia
Biotech, Sunnyvale, CA).
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Cryostat sections of 7 µm were made from snap frozen spleen tissue (liquid freon at -80°C) of rat number 1 (R1), air dried and fixed in acetone for 10 min at room temperature. Sections were stained for 1 h with mouse anti-rat IgM mAb (HIS40). After rinsing in PBS, the slides were incubated with polyclonal rabbit anti-mouse Ig conjugated to HRP (DAKO, Glostrup, Denmark). Peroxidase activity was revealed by 3,3'-diaminobenzidine tetra-HCl (Sigma, St. Louis, MO), containing 0.01% (v/v) H2O2. Sections were counterstained with hematoxylin according to Mayer and stored overnight in PBS at 4°C. In two separate experiments, three clusters of about 50 cells were microdissected from 6 different primary B cell follicles as well as from the MZ areas surrounding these follicles. Thus in total, 18 follicular and 18 MZ samples were taken. Microdissection was performed with a hydraulic micromanipulator (MMO-202; Narishige, Tokyo, Japan), as described by Küppers et al. (34). Microdissected cells were taken in 20 µl of PCR buffer (20 mM Tris-HCl (pH 8.4) and 50 mM KCl) and stored at -20°C. Microdissected cells were digested by 250 ng proteinase K (Boehringer Mannheim) by incubating for 60 min at 50°C, followed by a proteinase K inactivation step at 95°C for 10 min (reactions were covered with mineral oil). Primers used for PCR amplification were all HPLC-purified, and stock solutions were stored in aliquots at -20°C. In the first round of PCR amplification, the samples were taken in a 50-µl reaction volume containing 0.2 mM primer VH PC7183, 0.05 mM each of RJH-A,-B,-C,-D primer, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 2.5 U Taq DNA polymerase (Life Technologies), and 0.2 mM each of dGTP, dATP, dTTP, and dCTP. Samples were heated to 95°C before adding 5 µl Taq DNA polymerase (diluted to 0.5 U/µl in deionized water) to the reaction. The first round PCR amplification program consisted of 35 cycles of 1.5 min at 95°C (2 min in the first cycle), 1 min at 65°C (4 min in first cycle), and 1.5 min at 72°C, followed by an incubation at 72°C for 10 min. In the second round, PCR amplification reactions were conducted with each of the four nested 3' JH primers (RHJ-1, RHJ-2, RJH-3, and RJH-4) in combination with the VH PC7183 family-specific primer VH PC7183. Second round of PCR amplification was conducted in 50 µl containing 0.6 mM primer VH PC7183, 0.6 mM of either primer RJH-1, RJH-2, RJH-3, or RJH-4, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 2.5 U Taq DNA polymerase (Life Technologies), and 0.2 mM each of dGTP, dATP, dTTP, and dCTP. Hot start was performed by heating the reactions to 95°C before adding 5 µl Taq DNA polymerase (diluted to 0.5 U/µl in deionized water). Water overlaying the tissue section during the microdissection served as negative control for the PCR. Ten-microliter aliquots of the PCR samples were analyzed by agarose gel electrophoresis. Cloning, insert screening, and sequencing were conducted as described above.
Statistical analysis
Statistical analysis was performed using SPSS 9.0 for Windows
(SPSS, Chicago, IL). Frequency distributions of nominal variables such
as VH gene and JH gene
usage, and numbers of mutations per VH gene, were
compared using the likelihood ratio test for contingency tables. In
many cells, the frequencies were below 5%. Therefore, we calculated
the exact p values or, when this was not feasible, we
estimated them by a Monte Carlo method (±99% confidence interval
(CI)). Differences in PC7183 VH gene repertoire
were evaluated by taking the most frequently used members (sum of these
particular members over the B cell subsets compared (row count) >
5) and "all others" (row count 
5) as the categories. The
difference between mean H-CDR3 length and mean loss of
JH nucleotides between the B cell subsets was
statistically evaluated by a multiple comparison test (general linear
model) based on Bonferroni statistics. H-CDR3 length and loss of
JH nucleotides are normally distributed variables
(Kolmogorov-Smirnov test). For comparison of the frequency
distributions of H-CDR3 length and loss of
JH nucleotides, the Mann-Whitney U nonparametric
rank-order test was applied (p = Monte Carlo
significance ± 99% CI). Differences between groups were
considered significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Fig. 1, we show the
immunoperoxidase staining pattern of normal rat spleen for IgM, IgD,
and CD90 (Thy-1). B cells in MZs and follicles stain brightly for IgM,
whereas (bright) IgD staining is restricted to B cells located in the
follicles. Staining for CD90 (Thy-1), which is expressed in the B cell
lineage by immature B cells (35), clearly demonstrates
that virtually all cells in the MZ and most cells in the follicles are
CD90-negative. Only in follicles, low numbers of
CD90+ cells are present, thus most likely
representing immature B cells (NF-B cells and/or early recirculating
follicular B cells) (35).
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shows the flow
cytometry profiles of these B cell subsets in bone marrow and
spleen.
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. All sorted B cell fractions
were obtained with purities exceeding 98%.
VHDJH-µ transcripts
expressed in sorted B cell fractions were amplified by RT-PCR using a
PC7183 VH gene family-specific primer
(VH PC7183) and a µ constant-region primer
(Cµ1.3) (Table I). The VH PC7183 primer
sequence was deduced from the PC7183 VH genes
described previously (27). The VH
PC7183 primer is located in Ig H chain framework 1, at a position which
is highly conserved among members of the PC7183
VH gene family, but more diverged in
VH gene sequences from members of other families.
The position of the VH PC7183 primer was chosen
in such a way that no degenerated sites were necessary. This is of
advantage because competition between different
VH gene primers is now prevented, and therefore
no preferential amplification based on primer sequence constitution
will occur during the PCR. From either rat, about 20
VHDJH region sequences were
analyzed from each B cell subset, resulting in a total of 173 PC7183
VHDJH region sequences.
Establishment of germline PC7183 VH gene sequences of the rat
Previously, we obtained a number of germline PC7183 VH gene sequences from rat genomic liver DNA (27, 28). The large number of PC7183 VH gene sequences obtained in the present study offered the opportunity to identify more germline PC7183 VH genes of rat origin. New germline PC7183 VH gene sequences were established by comparing the VH gene sequences obtained from the present study with each other and with already established rat germline PC7183 VH gene sequences. Rearranged VH gene sequences were considered to be germline when two or more independently sampled, rearranged or genomic, VH gene sequence(s) were 100% identical. These rat VH PC7183 reference germline sequences are designated as "PC-n " (e.g., PC-9, PC-15). In a few cases, we established germline consensus sequences, which were derived from sequences containing Taq DNA polymerase errors and/or sequence ambiguities. These nontrivial VH gene sequences are followed by a "w" after their designation (e.g., PC-10w). Currently, we have established 28 individual germline VH gene sequences of the PC7183 VH gene family in rat (sequences are available at the IMGT database: http://imgt.cnusc.fr:8104 (32)). We assume that the PC7183 VH gene family in rat consists of more than 28 members, because a few VH gene sequences obtained from genomic liver DNA could not be confirmed at least twice.
Mutational analysis of productive Ig PC7183 VH genes expressed in the flow cytometry-defined B cell subsets
The VH gene of each sequenced VHDJH region was assigned to the most homologous germline PC7183 VH gene. Similarly, the JH genes of the rearrangements could be established after comparison to published genomic JH gene sequences of the rat (33). In contrast to VH and JH genes, very little sequence information is available on rat Ig H-chain diversity (D) genes; so far only one genomic D gene has been determined (DQ52) in this species (36). However, probably more D genes exist in rat, because many of the VH-D-JH junction sequences (H-CDR3) obtained in the present study contain identical stretches of nucleotides (data not shown). These identical stretches likely represent various D gene segments. In a previous study, we sequenced several D to JH rearrangements in which the 5'-terminal-end of the D gene was flanked by D gene specific recombination recognition sequences (nonamer, 12 bp spacer, heptamer) (28). Also these data revealed the existence of different D region-specific sequences. Knowledge of genomic D gene sequences is necessary to identify the number of N nucleotide additions between VH-D and D-JH boundaries. Because to date DQ52 is the only complete D segment known for rat, we could only establish N nucleotide additions for VH-D-JH junctions that carry the DQ52 gene.
In Fig. 3, we show the
VHDJH-µ transcripts
expressed in the flow cytometry-defined subsets of bone marrow
pro/pre-B cells and NF-B cells, and splenic RF-B cells, and MZ-B cells.
The vast majority (>90%) of the
VH-D-JH junction sequences
from R1 and all VH-D-JH
junction sequences from R2 represent unique rearrangements, as
indicated by their unique H-CDR3 region. In R1, four identical
VHDJH region sequences were
recovered from the MZ-B cell subset (MZ1801 series, clone 1, 16, 18,
and 20). These clonally related sequences were most probably sampled
from separate MZ-B cells, because two of these sequences carry more
mutations in the VH region than that can be
explained by Taq DNA polymerase (see further). Alignment of
these four clonally related sequences reveals that most mutations
occurred independently from each other (Fig. 4). Only clones MZ1801-1 and MZ1801-16
share one identical nucleotide exchange (G 
C mutation in codon 57;
Fig. 4A), which is indicative for their genealogical
relationship. Thus, these four
VHDJH region sequences are
most likely derived from distinct B cells that belong to a clonally
expanded B cell. We speculate that this clonal expansion took place
within a germinal center. Also, Tierens et al. (22)
observed clonally related B cells in the MZ of the human spleen. Two
pairs of identical VHDJH
region sequences were recovered from the R1 RF-B cell subset. However,
these sequences display no evidence for somatic mutations. Therefore,
we cannot determine whether these two pairs were sampled from one cell,
or represent independent, identical rearrangements of two different
cells. Furthermore, because we found no evidence for the existence of
dominant B cell clones within the RF-B cell subset (denaturing gradient
gel electrophoresis analysis of Ig H-CDR3 regions; data not shown), we
used only one of the two sequences for further (statistical)
analysis.
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summarizes the
characteristics of the productive
VH-DJH-µ transcripts
expressed in the various B cell subsets. The Taq DNA
polymerase mutation rate was about 0.1%, as established from the
±100-bp sequence of the µ constant-region (exon 1) adjacent to the
3' end of every VHDJH
region. Based on this Taq error rate,
VH genes were considered to be unmutated when no
more than two mutations were observed upon comparison with the germline
gene (chance of more than two mutations caused by Taq in a
239 bp VH region by an error rate of 0.1% is
<6% (=(239 bp x 0.001)2). By using this mutation
criterion, we found that the VH genes expressed
by all pro/pre-B cells and almost all NF-B cells and RF-B cells are
unmutated (Fig. 5). Only two
VH genes recovered from the NF-B cell fraction of
R1 carry three to four mutations per gene. We cannot, however,
completely exclude the possibility that this low number of mutations is
still the result of Taq errors, especially because the
number of sequences with this mutation rate is rather low. One
VH gene obtained from the RF-B cell subset of R1
reveals eight mutations when compared with the most homologous PC7183
germline gene.
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). In contrast, the number of mutated
VH genes expressed in MZ-B cells is significantly
higher compared to other B cell subsets (Table II). About 20% of the
VH genes recovered from MZ-B cells of either rat
carry mutations, ranging from 3 to 11 nucleotide exchanges per gene
(Fig. 5). The higher number of mutated VH genes
expressed in this subset results in a mutation frequency which is more
than three times higher than calculated for the other B cell subsets
(Table II). Two of eight mutated MZ-B cell VH
gene sequences have only three nucleotide exchanges with the germline,
and thus could have been possibly caused by Taq; the other 6
VH genes carry 5 to 11 mutations per gene, and
are therefore very likely the result of somatic mutations.
An Ag-driven response results in Abs with higher affinity. This is
generally reflected by a higher proportion of amino acid exchanges in
the CDR regions (Ag-binding sites) than in the FR regions of the
VH genes. For this reason, we calculated the
replacement over silent mutation (R/S) ratio of CDR and FR regions of
VH genes carrying more than four mutations. The
observed R/S mutation ratios were compared with the R/S mutation ratios
theoretically expected from a random distribution of mutations and the
chance (p) was calculated that the number (either an
excess or scarcity) of replacement mutations in the CDR and H-FR
regions was just the result of coincidence (39). Table III shows the observed and expected R/S
mutation ratios of seven VH genes (one from the
RF-B cell subset and six from the MZ-B cell subset) carrying more than
four mutations. In all these VH genes, mutations
in the H-CDR regions resulted in amino acid replacements. In five of
seven cases, the replacement mutations in H-CDR and H-FR regions are
randomly distributed (p > 0.05). However, in
VH genes MZ1801-17 and MZ1801-18, we observed an
excess of replacement mutations in the H-CDR regions
(p = 0.027 and p = 0.00015,
respectively) and a scarcity of replacement mutations in the H-FR
regions (p = 0.045 and p =
0.012, respectively) which cannot solely be explained by coincidence.
Therefore, the pattern of mutations observed in these two MZ-B cell
derived VH gene sequences resembles that of
VH genes encoding high affinity mAbs and provide
evidence that these MZ-B cells have undergone Ag-driven selection.
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In man, nearly all MZ-B cells are somatically mutated, as was
revealed by analysis of microdissected MZ B cells (21, 22). This is in marked contrast to our aforementioned findings
in flow cytometry-defined MZ-B cells in the rat. To exclude the
possibility that these differences can be explained by the technique
used to collect MZ-B cells (microdissection vs flow cytometry), we also
analyzed productively rearranged PC7183 VH genes
from cells microdissected from follicular and MZ B cell areas of the
spleen of R1. An example of a frozen section of the spleen after
microdissecting cells from the follicular and MZ B cell areas is shown
in Fig. 6. Cells were scraped from six
different follicles and from the MZs surrounding these follicles. From
these areas, we analyzed 13 and 10 productive PC7183
VHDJH sequences,
respectively. All VHDJH
regions carry unique
VH-D-JH junctions and are
thus derived from individual B cells (Fig. 7). In addition to the productive
VHDJH region sequences, two
sequences isolated from follicular areas and four sequences from the MZ
areas were nonproductive due to a frameshift and/or a stop codon in the
VH-D-JH junctional area
(Fig. 7). These nonproductive
VHDJH region sequences were
excluded from further analysis. The characteristics of the productive
sequences from these microdissected cells are shown in Table II. Except
for one sequence, all follicular PC7183 VH gene
sequences contain less than two mutations when compared with their most
homologous germline gene. One follicular VH gene
(F32-3) carries three mutations. Given the higher Taq error
rate (doubled number of PCR cycles), we assume that these three
mutations are most likely Taq errors (chance of three
mutations in 239-bp VH region with Taq
error rate of 0.2% = (239 x 0.002)3 = 0.11)) rather than
somatic mutations. Among the productive
VHDJH region sequences
recovered from the microdissected MZ B cell areas, only 1 of 10
VH gene sequences carries more mutations than
could be expected from Taq (M11-4, eight mutations upon
comparison to germline PC-5). Because of the excess of R mutations in
H-CDR regions (and their scarcity in the H-FR regions) in the
VH gene of M11-4 (Table III), we conclude that
this sequence is derived from an Ag-selected and somatically mutated
MZ-B cell. The frequency of mutated PC7183 VH
gene sequences from microdissected MZ B cells thus closely resembles
that of flow cytometry-sorted MZ-B cells, and confirms that the
difference in frequency of somatically mutated MZ-B cells observed
between man and rat, are not enforced by the technique used to isolate
the cells (microdissection vs flow cytometry).
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To establish whether the VH gene repertoire
expressed by MZ-B cells is the result of positive or negative selection
of certain Ig specificity’s, we determined the PC7183
VH gene usage in MZ-B cells and their precursors
(pro/pre-B cells, NF-B cells, and RF-B cells). To this end, we counted
the frequency of individual PC7183 VH family
members productively expressed in each B cell subset in either rat
(Fig. 8). In all four B cell subsets,
generally the same PC7183 VH members are used. In
addition, we observed no substantial difference in PC7183
VH gene usage between the two rats. However, the
PC7183 VH gene repertoire within the various B
cell subsets is not randomly expressed and certain members are more
frequently used than others. For example, PC7183 member PC-4 is
relatively frequently used by pro/pre-B cells and NF-B cells in bone
marrow and splenic RF-B cells (12.5%, 20.5%, and 9.3%,
respectively), whereas this gene is not observed among MZ-B cells.
PC-15 is also frequently expressed by pro/pre-B cells and NF-B cells
(20% and 11.4%, respectively) and less frequent by RF-B cells and
MZ-B cells (2.3% and 7.5%, respectively). Finally,
VHDJH-µ transcripts
encoded by PC7183 VH gene member PC-1 are
predominantly present in the RF-B cell subset (20.9%), whereas this
particular gene is used by 5.0% of both pro/pre-B cells as well as
MZ-B cells, and by 9.1% of the NF-B cells. Memory (MZ)-B cells are (by
definition) Ag-selected cells. To reveal whether the repertoire
expressed by naïve MZ-B cells is also selected, we
statistically evaluated the usage of unmutated PC7183
VH genes among the various B cells subsets. A
statistically significant difference (p =
0.040 ± 0.005) in PC7183 VH gene repertoire
is found between the B cell subsets when both rats are taken together.
When individual rats are analyzed, a difference in PC7183
VH gene usage is observed in R1
(p = 0.040 ± 0.005) and a nearly
significant difference in R2 (p = 0.102 ±
0.007). Given the linear developmental relationship between the four B
cell subsets (pro/pre-B cells NF-B cells RF-B cells MZ-B
cells), we further analyzed the repertoires between two subsequent B
cell stages. The difference in PC7183 VH gene
usage is most prominent between RF-B cells and MZ-B cells (R1 + R2,
pRF-B vs MZ-B = 0.020 ± 0.004). Our
data also indicate that the PC7183 VH gene
repertoire expressed by NF-B cells and RF-B cells might be different,
although confidence is no more than 85% (R1 + R2,
pNF-B vs RF-B = 0.146 ± 0.009). The
PC7183 VH gene repertoire expressed in pro/pre-B
cells does not differ from that of NF-B cells (R1 + R2,
ppro/pre-B vs NF-B = 0.655 ± 0.012).
Together, the data indicate that during B cell development in rats,
selection not only occurs between the stages of NF-B cells in the bone
marrow and RF-B cells in the periphery, but also between RF-B cells and
naïve MZ-B cells in spleen. In a previous report, studying the
kinetics of B cell subsets, we already proposed that significant
selection of cells might take place between NF-B cells and RF-B cells
(35, 40).
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The frequencies of the four JH genes used in
productively rearranged
VHDJH-µ transcripts of
bone marrow pro/pre-B cells and NF-B cells, and splenic RF-B cells and
MZ-B cells is shown in Fig. 9A. JH2
and JH3 are the most predominantly used
JH gene segments. In all B cell subsets (of the
two rats together), JH2 and
JH3 together encode for more than 70% of the
JH region in the
VHDJH-µ transcripts. In
pro/pre-B cells, this percentage is even 90%. Most likely, the
predominance of these two JH genes reflects the
preference of the recombination machinery for
JH-RSS (recombination signal sequence;
nonamer-spacer-heptamer) with a 23-bp spacer sequence over
JH-RSS carrying a 22-bp spacer (41).
In rats, both JH2 and JH3
have a RSS with a 23-bp spacer sequence, whereas
JH1 and JH4 carry a RSS
with a 22-bp spacer (28, 33). Interestingly, in both rats,
VHDJH-µ transcripts
expressed by MZ-B cells are most frequently encoded by the
JH2 gene (R1, 60% and R2: 80%), which is at
least 1.5 times higher compared with other B cell subsets (Fig. 9A). The p value for differences in
JH gene usage between the four B cell subsets is
0.068 ± 0.06.
|
The H-CDR3 region is the most diverse region of an Ig molecule,
both in length and amino acid constitution. Amino acids encoded by the
H-CDR3 play an important role in binding of the BCR with Ag. The length
of the H-CDR3 region becomes fixed during
VH-D-JH rearrangement and
is determined by the length of the D segment used, the number of N
nucleotide additions (by the enzyme TdT), and the number of nucleotides
removed at the site of recombination by exonuclease activity. As shown
in Table II, the mean H-CDR3 length and the distribution of the number
of H-CDR3 codons does not differ between pro/pre-B cells, NF-B cells,
and RF-B cells. However, the mean H-CDR3 length of MZ-B cells is
9.0 ± 2.8 codons and significantly shorter than that observed in
pro/pre-B cells (11.8 ± 2.6 codons), NF-B cells (11.2 ± 2.8
codons), and RF-B cells (10.6 ± 2.7 codons) (Bonferonni,
= 0.05). Also the distribution of the number of H-CDR3 codons expressed
by MZ-B cells differs from the other B cell subsets (Fig. 9B). Similar to flow cytometry-defined MZ-B cells,
also the VHDJH regions
of MZ-B cells microdissected from the MZ area of the spleen carry short
H-CDR3 regions (9.0 ± 3.4 codons; Table II). The mean H-CDR3
length of B cells microdissected from the follicular areas (9.7 ±
2.9 codons; Table II) is, however, only slightly higher compared with
MZ-B cells. This might be explained by the fact that follicles do not
consist exclusively of RF-B cells, but probably also contain MZ-B like
cells (sIgMhigh, HIS24low,
HIS57high) ( Ref. 16 , and P.M.D. and
F.G.M.K., unpublished observation).
The shorter H-CDR3 regions observed in MZ-B cells are not due to total
lack of N nucleotide additions or excess in exonuclease activity at
VH-D-JH junctions in these
cells. We show that MZ-B carrying
VH-DQ52-JH
rearrangements do contain N nucleotide additions (Fig. 3). Furthermore,
many homopolymer tracts of dG and dC residues are observed at the
VH-D and D-JH junctions,
which are typical for N nucleotide additions (42, 43). The
frequency of nucleotide deletions caused by exonuclease activity at the
VH-D-JH junctions was
measured by the number of nucleotides deleted at the 5'-terminal-end of
the JH segments. No difference is observed in the
distribution of 5'-JH nucleotide loss (Fig. 9B) and no significant difference is revealed between the
average number of deleted nucleotides at this site (Table II), between
all the investigated B cell subsets. Consequently, the use of shorter
H-CDR3 regions in VHDJH-µ
transcripts of MZ-B cells, compared with pro/pre-B cells, NF-B cells,
and RF-B cells, is most likely the result of a selection event rather
than a fundamental difference in recombination machinery.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) exhibit signs of
Ag-selection. Somatic mutations of VH genes in
conjunction with Ag-selection appear to be confined to germinal centers
(44). For this reason, we think that these somatically
mutated MZ-B cells are germinal center-derived memory B cells. The
presence of mutated MZ-B cells in our study confirms previous
observations that a proportion of B cells in the splenic MZ might be
germinal center-derived cells (2, 20, 25, 26). We like to
emphasize here that, in our experiments, the memory MZ-B cells, that
were collected by flow cytometry, are not (yet) isotype switched,
because 1) they were sorted on the basis of sIgM and sIgD expression
and 2) were amplified using a µ constant-region specific primer. As
shown recently, the frequency of memory MZ-B cells in mice also seems
to be rather low. Makowska et al. (15) reported that in
C57BL/6 mice nearly all J558 VH expressing
CD1high splenic B cells (mainly consisting of
MZ-B cells) were unmutated (zero to two mutations per
VH gene). These observations in rats and mice are
in striking contrast to the situation in human spleen where the
majority (more than 85%) of splenic MZ- B cells are somatically
mutated (21, 22). The difference in the proportion of
memory MZ-B cells between man and rodents is probably not explained by
differences in the technique used to isolate MZ-B cells. We did not
observe a difference in frequency of mutated VH
genes in MZ-B cells either collected by microdissection or sorted by
flow cytometry. In the studies performed by Dunn-Walters et al.
(21) and Tierens et al. (22), MZ-B cells were
exclusively collected from human spleen by microdissection. However,
one should realize that the microanatomical structure of the human
spleen differs from that of rats (and mice) with respect to
organization of the MZ and the presence of a so-called perifollicular
zone surrounding the MZ (45). The splenic MZ in man is
composed of an inner and an outer area and one cannot rule out the
possibility that these areas differ in composition of memory and naive
MZ-B cells. In the aforementioned studies, MZ-B cells could have been
scraped from only selected (inner or outer) areas of the MZ. Another,
probably more likely, explanation for the lower frequency of memory B
cells among MZ-B cells in rodents, is the lower antigenic exposure of
these animals, caused by the fact that they are housed in controlled
laboratory animal facilities, and the much shorter life history of
rodents in comparison to adult man. Although most MZ-B cells in adult rat spleen are naive B cells, our results support the hypothesis that MZ-B cells are selected on basis of the specificity of their BCR. Evidence for this follows from our observations that, in comparison to RF-B cells, NF-B cells and pro/pre-B cells 1) naive rat MZ-B cells express a different PC7183 VH gene family repertoire, 2) they tend to use more frequently JH2 genes, and 3) excitingly, MZ-B cells carry significantly shorter H-CDR3 regions. In rats, MZ-B cells most likely originate from the pool of RF-B cells (16). However, we cannot exclude the possibility that MZ-B cells can also develop directly from other B cell subsets. For example, Martin et al. (19) have shown in mice that MZ-B cells are recruited from (recirculating) IgMhighCD21int B cells rather than from the bulk of long-lived recirculating IgMlowCD21int B cells. Little is known about the nature of the ligands involved in the recruitment of naive MZ-B cells. MZ-B cells are thought to be involved in TI-2 Ab responses against (lipo-)polysaccharide Ags from encapsulated microorganisms (2, 3). Evidence for this is, however, largely circumstantial and remains to be established at the single cell level. On the other hand, recently, Kearney and coworkers showed that polyreactive B cells in Ig H chain transgenic mice, i.e., expressing a BCR that reacts to phosphorylcholine (PC) in addition to numerous of self-Ags, are positively selected into the MZ-B cell pool, whereas B cells highly specific for hen egg lysozyme (HEL) are not (18, 19). Possibly, MZ-B cells are primarily polyreactive B cells with Ig that bind with low affinities to a broad range of Ags, including bacterial-derived (TI-2) Ags as well as self-Ags. This notion suggests that self-Ags probably play an important role in establishing the B cell repertoire of the MZ-B cell subset, and thus also explains why in animals deprived of exogenous Ags (i.e., germfree and Ag free animals) MZ-B cells still develop (10, 11, 12, 15).
The hypothesis that MZ-B cells express primarily polyreactive Ig is
consistent with another intriguing finding of the present study, i.e.,
that MZ-B cells express PC7183
VHDJH-µ transcripts with
significantly shorter mean H-CDR3 length in comparison to other B cell
subsets, such as RF-B cells in spleen, and NF-B cells and pro/pre-B
cells in bone marrow. The mean number of amino acids in the H-CDR3
region of MZ-B cells (either defined by flow cytometry or
microdissection) is 9 amino acids, which is 2–3 amino acids shorter
than found among other B cell subsets. Several groups have reported the
importance of the H-CDR3 region in determining the polyreactive
behavior of Abs (46, 47, 48, 49, 50). By gene-shuffling experiments
using combinations of mono- and polyreactive mAbs that are encoded by
highly similar Ig H and L chain V genes, Ichiyoshi and Casali
(48) and Crouzier et al. (47) showed that
mAbs lose polyreactivity when their H-CDR3 region is replaced by the
corresponding region of their monoreactive counterpart, but not when
the polyreactive mAbs were grafted with either the H chain framework
1-framework 3 (VH) region or with the
V
L chain region of that monoreactive
counterpart. Although the H-CDR3 length of polyreactive Abs is
variable, there are several examples showing that polyreactive mAbs
carry relatively short H-CDR3 regions (46, 51).
Furthermore, from the data published by Tornberg and Holmberg
(52) we calculated that PC7183
VHDJH-µ transcripts
expressed by peritoneal B-1a and B-1b cells in mice also have
relatively short H-CDR3 lengths (mean ± SD: 8.6 ± 3.4 and
8.7 ± 2.9, respectively) in comparison to conventional B-2 cells
from spleen (11.3 ± 2.6). Similar to MZ-B cells, B-1 cells are
preferentially directed against bacterial coat Ags, and produce poly-
and self-reactive Abs (53, 54). Schroeder et al.
(55) reasoned that relatively short H-CDR3 regions would
probably generate "flat" Ab-binding sites, which would allow more
interactions possible between residues of the H-CDR3 and potential Ags,
resulting in polyreactivity. We propose that, at least for PC7183
VH gene encoded Ig H chains, polyreactive Ig is
frequently encoded by short H-CDR3 regions.
Studies reported over the past years have provided substantial evidence that BCR signaling is a prerequisite for proper development and maintenance of B cells. BCR signaling does not only play a role in early B cell differentiation (56) but does also determine the fate of B cells in later (more mature) stages of their development (19, 57, 58, 59, 60). The signals delivered by the BCR are subjected to positive and negative feedback regulation by accessory receptors like CD45, CD19, and CD22, and their secondary signal transducing molecules (61, 62). In this context, it is relevant to mention that Mason et al. (63) demonstrated in HEL/anti-HEL IgM/IgD double transgenic mice that anergic B cells cannot be recruited into the MZ-B cell pool, but persist in the lymphoid follicles. Possibly, B cells may become MZ-B cells when their BCR delivers a signal that in strength exceeds a certain threshold. By lowering the strength of BCR signaling (by diminishing the positive feedback of the BCR) this threshold is increased, resulting in reduced numbers or complete absence of MZ-B cells, as is the case in Xid (Bruton’s tyrosine kinase mutant) mice and in CD19- deficient mice, respectively (15, 19). We like to speculate here that selection of MZ-B cells is presumably based upon the degree of polyreactivity of their BCR, because B cells expressing a polyreactive BCR may reach the threshold levels required for MZ-B cell maturation more easily due to binding to multiple (self-)Ags.
In summary, data shown here clearly demonstrate that in rats the majority of cells populating the MZ area of the spleen are naïve B cells, which are most probably recruited from recirculating B cells by means of positive selection. Self-Ags may somehow be involved in the selection process of MZ-B cells. This process presumably favors the selection of polyreactive B cells, resulting in MZs populated with MZ-B cells expressing BCRs that bind with moderate affinity to various self-Ags, but in addition also exhibit reactivity toward (lipo-)polysaccharide (TI-2) Ags. In vivo, the polyreactive Abs produced by MZ-B cells may be of crucial importance in the first-line of defense against bloodborn infections with encapsulated bacteria. Polyreactive naturally occurring Abs have been shown to be essential in the primary immune response against pathogens, largely because of their neutralizing effect (64, 65, 66). Herewith naive MZ-B cells form an integral part of (innate) immunity by providing a strategically located buffer of polyreactive B cells in the spleen that can rapidly produce neutralizing Abs (mainly IgM) upon bloodborn and life threatening infections with encapsulated bacteria or other microorganisms.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: MZ-B, marginal zone B cell; BCR, B cell receptor for Ag; CDR, complementarity-determining region; NF-B, newly formed B cell; R, replacement mutation; RF-B, recirculating follicular B cell; S, silent mutation; TI, T cell independent; HEL, hen egg lysozyme.
Received for publication June 19, 2000. Accepted for publication August 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ and - B lymphocyte lineages in the rat. Ann. N. Y. Acad. Sci. 399:157.[Medline]
genes: implications for T cell lineages and for a novel intermediate of V-(D)-J joining. Cell 59:859.[Medline]
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=
unmutated VH gene from R1 (0–2 mutations/gene); 
=
mutated VH gene from R1). The PC7183 germline genes are
arranged in descending order of homology with PC-1 arbitrarily put on
top. The subgroups (I, II, and III) are formed by PC7183 members with
more than 90% sequence identity. A significant difference is revealed
between RF-B and MZ-B (p = 0.020 ± 0.004) and
a near significant difference between NF-B and RF-B
(p = 0.146 ± 0.009), using the most
frequently used members (sum of particular member over B cell subsets
(row count) >5) and "all others" (row count 