From the Department of Biotechnology, Faculty of Engineering, Okayama University,
Tsushima-Naka, Okayama 700 Japan
V(D)J (V, variable; D, diversity; J, joining) combination of immunoglobulin (Ig) genes established in premature B cells has been thought to be conserved throughout differentiation at mature stages. However, germinal center (GC) B cells have been shown to reexpress recombination-activating gene (RAG)-1 and RAG-2 proteins in immunized mice. Here, we present
several lines of evidence indicating that RAG proteins thus induced are functional as the V(D)J
recombinase. DNA excision product reflecting V
1 to J1 rearrangement was generated in
parallel with the expression of RAG genes in mature mouse B cells that were activated in vitro with LPS and IL-4. Similar chain gene rearrangement was observed in the draining lymph
node of immunized mice. Further, B cells that underwent gene rearrangement were shown
by in situ PCR to be localized within GCs. Thus, secondary rearrangement of Ig genes (receptor editing) can occur in mature B cells.
 |
Introduction |
Variable regions of Ig genes are generated from three
classes of germline DNA segments, variable (V),1 diversity (D), and joining (J). These are assembled in a random fashion to generate a set of V(D)J in a given B cell
clone during its development in the primary lymphoid organ, thus giving rise to large varieties of rearranged V(D)J
genes in B cells (1). V(D)J rearrangement is mediated by
the products of recombination activating genes, recombination-activating gene (RAG)-1 and RAG-2 (2). A V(D)J
combination established in the primary lymphoid organ has
been thought to be conserved throughout B cell differentiation thereafter, although the nucleotide sequences are
modified by somatic hypermutations (3). One exception is
the secondary revision of Ig genes termed receptor editing
in which an original V(D)J rearrangement is replaced by a
secondary rearrangement in the bone marrow (4). This
phenomenon was first pointed out in mice transgenic for
genes encoding autoantibodies to self H-2K (5, 6) or double-stranded DNA (7). In these transgenic mice, B cell
clones that escaped deletion were found to lose the autoreactivity due to the replacement of the transgenic V(D)J by a
new endogenous rearrangement of L and/or H chain genes
(5). Further, bone marrow-derived IgM+ immature B
cells have been shown to retain RAG expression and to
undergo V-J recombination during culture (8) or when
the immature B cells were stimulated by surface Ig engagement in vitro (9).
However, mature B cells in which RAG-1 and RAG-2
expression is downregulated (8, 10), have been believed to
undergo no further Ig gene rearrangement. But we have
recently found that RAG-1 and RAG-2 proteins are reexpressed in IgD+ mature mouse B cells that are stimulated in
vitro with LPS or mAb to CD40 in the presence of IL-4 or
in germinal center (GC) B cells in the draining LN of mice
immunized in vivo (11, 12). Similar observations have
been made by Han et al. (13). However, it has remained
unclear whether the RAG proteins thus induced in mature
B cells are as functional as those expressed in premature B
cells. Recently, GC B cells were shown to have intermediate products of 
-L chain gene rearrangement (14, 15).
Further, mature B cells from mice with targeted VB1-8DJH2
and V3-83J2 replacement alleles were found to undergo rearrangement of endogenous H and -L chain genes in response to LPS plus IL-4 (15), which induce RAG gene expression in vitro (11). In this report, we describe that
mature mouse B cells undergo de novo rearrangement of
-L chain genes in vitro and in GCs of immunized wild-type mice in parallel with RAG gene expression.
| |
Materials and Methods |
Preparation and Culture of Mouse B Cells.
Spleen cells from
male C3H/HeN mice (7-9-wk of age; Japan Charles River,
Kanagawa, Japan) were treated with 1/1,000-diluted anti-Thy
1.2 mAb (SeroTec, Kidlington, UK) followed by incubation with
low toxic rabbit complement (Cederlane, Wesbury, NY) as described previously (11). The B cells thus obtained (3 × 106/ml)
were cultured for 2 d with 20 µg/ml LPS from Escherichia coli 055 B5 (Sigma Chemical Co., St. Louis, MO) and 10 ng/ml mouse recombinant IL-4 (PeproTech, Princeton, NJ) in 1 ml of RPMI-1640 medium containing 10% FCS, 10
5 M 2-ME, 100 U/ml
penicillin G, and 50 µg/ml streptomycin.
Analysis of RAG-2 Transcripts.
Total RNA was extracted from
106 cells and reverse transcribed as described previously (16). The
resultant cDNA was amplified by PCR using following sense and
antisense primers (11, 13): RAG-2, 5
-CACATCCACAAGCAGGAAGTACAC-3, 5-GGTTCAGGGACATCTCCTACTAAG-3; and GAPDH, 5-CCATCACCATCTTCCAGGAG-3, 5-CCTGCTTCACCACCTTCCTTG-3. Both primer
pairs span an intron to avoid the amplification of contaminating
genomic DNA. To heighten the specificity of the amplification,
time release PCR was performed using AmpliTaq Gold (Perkin-Elmer Corp., Foster City, CA) as the polymerase according to
the manufacturer's protocol. PCR was carried out as follows: for
RAG-2, at 94°C for 12 min followed by 40 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min; for GAPDH, 26 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The amplified products were electrophoresed on 7.5% polyacrylamide gel and
visualized by SYBER green I (FMC BioProducts, Rockland,
ME) staining. RAG-1 and RAG-2 expression in LPS/IL-4-stimulated mature mouse B cells has been confirmed by reverse transcriptase-dependent PCR (RT-PCR) followed by Southern
blotting as described previously (11).
Flow Cytometric Analysis.
Cultured B cells were incubated with
10 µg/ml biotinylated anti-mouse or chain mAb at 4°C for
30 min followed by staining with streptavidin-PerCP (Becton
Dickinson, San Jose, CA). The anti- mAb used is reactive with
1 and 2 chains. Additionally, the cells were stained with phycoerythrin-labeled anti-B220 mAb (PharMingen, San Diego, CA) and
FITC-labeled mAb to mouse IgG1 or IgM (PharMingen), respectively. Three-color analyses were carried out using FACSCalibur®.
Purification of + or + B Cells.
+ or + B cells were purified
from mouse spleen B cells using immunomagnetic beads. In brief,
108 spleen B cells were incubated with 5 µg/ml biotinylated mAb
to mouse or chain in 1 ml of phosphate-buffered saline at 4°C for 30 min. After washing, the cells were mixed with 1 mg/ml
streptavidin-coated magnetic beads (Dynal, Oslo, Norway) and
incubated at 4°C for 30 min. Rosetted cells were separated by
placing the tubes in magnetic particle concentrator (Dynal) for 2 min. This magnetic separation was repeated twice. Purity of each
cell preparation was >99% as assessed by flow cytometry.
Proliferative Response of B Cells.
Unseparated, + or + B cells
(each 105 cells) were cultured in 0.2 ml of the culture medium
with or without 20 µg/ml LPS and 10 ng/ml IL-4 for 2 d. Then
the cells were pulsed with 20 kBq [3H]thymidine (TdR; Daiichi
Chemical, Tokyo, Japan) for 12 h. The incorporated radioactivity
was measured by liquid scintillation counter (Aloka, Tokyo, Japan).
Detection of DNA Excision Product.
DNA excision product
derived from chain gene rearrangement was extracted from cultured B cells or LN cells, and amplified by PCR. 1,000,000 cells
were suspended in 200 µl of the lysis buffer, which consists of 10 mM Tris-HCl (pH 8.0), 10 mM KCl, 1.5 mM MgCl2, 0.5%
Tween 20, and 0.1 mg/ml proteinase K. The suspension was incubated at 56°C for 45 min and 95°C for 10 min. Then the supernatants were directly subjected to the analysis by PCR with 28, 30, and 32 cycles, which were in the exponential phase of the
amplification. AmpliTaq Gold was used as the polymerase. For amplification of V1-J1 excision product, the following primers described by Tiegs et al. (5) were used: 5-GCTGCATACATCACAGATGC-3 and 5-CAATGATTCTATGTTGTGCC-3. Each cycle consisted of 94°C for 30 s, 62°C for 30 s, and 72°C
for 90 s. Amplified products were electrophoresed and visualized
by Southern blotting using a 32P-labeled probe, which is the
HindIII-NspI 183-bp fragment derived from a DNA segment
spanning the V1-J1 signal joint of the excision product (provided by Dr. D. Nemazee, National Jewish Medical and Research Center, Denver, CO). Exon 4 of 
-actin gene was amplified as a control using a primer pair 5-ATGTTTGAAACCTTCAATAC-3 and 5-GGAAGGAAGGCTGGAAGAGT-3.
The product was visualized by staining with SYBER green I.
In Situ PCR Analysis.
For in situ PCR of LN sections,
8-µm-thick cryosections mounted on silane-coated slides were
allowed to air dry for 1 h and fixed in ice-cold 4% paraformaldehyde for 2 h. Then the slides were washed, dehydrated by ethanol
for 5 min, and air dried for 30 min. The thermal cycler designed
for in situ PCR (GeneAmp 1000; Perkin-Elmer Corp.) and core
PCR reagent kit (Perkin-Elmer Corp.) optimized for this apparatus were used according to the manufacturer's protocol. Digoxigenin (DIG)-labeled dUTP (Boehringer Mannheim GmbH,
Mannheim, Germany) was added to the reaction mixture at 2.5 µM.
DNA excision product derived from V1-J1 rearrangement was
amplified using the primer pair described above. PCR conditions were 20 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min 30 s at
72°C. The amplification was performed without primers or with primers for human IL-2 gene as a negative control. DIG-labeled amplified products were detected using DIG nucleic acid detection kit (Boehringer Mannheim GmbH). In brief, slides were
treated with Fab fragments of sheep anti-DIG Ab conjugated
with alkaline phosphatase, and visualized by the reaction with
5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt. Positive reaction gives blue/purple precipitates. The
slides were finally counterstained with methyl green.
| |
Results and Discussion |
Enhanced Expression of L Chains on B Cells Stimulated
with LPS Plus IL-4 In Vitro.
As reported previously (11),
stimulation of mature mouse B cells with LPS/IL-4, but
not with LPS alone resulted in the expression of RAG-2 in
vitro (Fig. 1 A). It was then investigated whether LPS/
IL-4-induced RAG gene products can mediate receptor
editing in mature mouse B cells. One of the criteria for the
occurrence of receptor editing is the induction of chains
in chain-bearing B cells since this process involves de
novo rearrangement of genes (5, 17). Thus, mouse spleen
B cells were stimulated in vitro with LPS/IL-4 and examined for the induction of chains after the culture. Before
the culture, >95% of B220+ B cells were IgM+, and
+/B220+ cells were <1% of total cells (Fig. 1 B). IgG1+
(
1+) cells were 2.3% before culture, but increased to
15.6% after culture with LPS/IL-4 due to IL-4-induced Ig
class switching (16). Interestingly, LPS/IL-4 stimulation resulted in an increase of +/B220+ cells from 0.94 to 4.67%,
a majority of which belonged to 1+ population. Enhanced
expression of chains was not observed in B cells stimulated with LPS alone that did not lead to RAG expression
(Fig. 1, A and B). B cells cultured without stimuli showed
similar flow cytometric patterns to those of the original cells (not shown). When purified + cells (>99% pure)
were stimulated with LPS/IL-4 for 4 d, 3-4% of the total
cells became positive for chains (our unpublished result).
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Fig. 1.
(A) Induction of RAG gene expression in activated mouse
mature B cells in vitro. Mouse spleen B cells were cultured with LPS or
LPS plus IL-4 for 2 d, and assessed for RAG-2 expression by RT-PCR.
(B) Enhanced expression of chains in mouse spleen B cells stimulated
with LPS/IL-4. After stimulation with LPS or LPS/IL-4 for 4 d, B220+
population of the cultured B cells was analyzed for the expression of , µ,
and 1 chains by flow cytometry.
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Detection of DNA Excision Product Derived from V1-J1
Rearrangement.
If the expressed chains are produced by
newly rearranged genes, circular DNA excision product
derived from V-J rearrangement might be detected (5).
Fig. 2 A shows that this is the case. PCR amplification of
the extracted DNA followed by Southern blotting with the
probe depicted in Fig. 2 B revealed that LPS/IL-4-stimulated B cells in which RAG-2 was expressed, were shown
to contain a definitely higher level of DNA excision product reflecting V1-J1 rearrangement compared with unstimulated or LPS-stimulated B cells. It has been reported
that the signal joint formation generates a restriction site
cleavable with ApaLI (18). The amplification product (302 bp) was cleaved by ApaLI into two fragments with expected lengths of 229 and 73 bp, respectively (Fig. 2 B),
thus confirming the identity of the amplified product. The
initial stage of V(D)J recombination is the generation of
broken signal ends and hairpin coding ends. It has been
reported that the joining of signal ends is slower than the
formation of coding joints, and may require the cell cycle
progression (19). V1-J1 excision product detected in
LPS/IL-4-stimulated B cells, however, may not be due to
the mere joining of the preexisting signal breaks since a
much lower level of the circular excision product was detected in B cells stimulated with LPS alone that progresses the cell cycle, but does not induce RAG expression (11).
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Fig. 2.
(A) Detection of circular DNA excision product derived
from V1-J1 rearrangement in mouse spleen B cells cultured with LPS/ IL-4. After the B cells were stimulated with LPS or LPS/IL-4 for 2 d, the
excision product derived from 106 cultured cells was amplified by PCR
with 28, 30, and 32 cycles. The amplified products were visualized by
Southern blotting using the probe depicted. The same cells were assessed
for RAG-2 expression by RT-PCR. Thymocytes or bone marrow cells
were used as a positive control. (B) Confirmation of an ApaLI restriction
site in the PCR-amplified segment of V1-J1 excision product. (C)
Comparison of proliferative response to LPS/IL-4 between purified + B
cells and + B cells. Purified + B cells, + B cells, or unseparated B cells
were cultured with or without LPS/IL-4 for 2 d followed by incubation
with [3H]TdR for an additional 12 h. Data were presented as the mean of
triplicate assays.
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As shown in Fig. 2 C, purified + B cells and + B cells
showed a comparable [3H]TdR uptake either in the presence
or absence of LPS/IL-4. Thus, LPS/IL-4-induced increase
of + B cells is not considered to be due to the preferential
expansion or survival of a small number of + cells present
in the original B cell preparation during the culture. Taken
together, chains expressed in response to LPS/IL-4 are thought to be generated from newly rearranged chain genes.
As shown in Fig. 1 B, chains were predominantly expressed on 1+ B cells. It remains unclear whether gene
rearrangement is undertaken more efficiently after isotype
switching, or whether these two events apparently proceed
in a synchronous manner due to their similar IL-4 dependence. It is interesting to note that Ku, a DNA-dependent
protein kinase activator protein complex that is involved
both in isotype switching (20) and in V(D)J recombination (21), was induced in mouse spleen B cells stimulated by
surface Ig engagement and IL-4 (22).
Chain Gene Rearrangement in GC B Cells In Vivo.
Do mature B cells also undergo chain gene rearrangement in vivo? We have reported that RAG-1 and RAG-2
are induced in the draining LN cells of immunized mice
(11, 12). It was confirmed that popliteal LN cells expressed
RAG-2 transcripts on days 6 and 8 after immunization
when mice were immunized in the footpads with TNP-KLH and alum (Fig. 3 A). Very interestingly, DNA excision product derived from V1-J1 rearrangement was detected by PCR in the same LN cells as those in Fig. 3 A on
days 6 and 8 after immunization, but not on day 0 (Fig. 3 B),
suggesting that the induced RAG proteins are functional
and able to mediate chain gene rearrangement in vivo.
Further, the localization in the LN of the cells that underwent V1-J1 rearrangement was examined in the LN
section by in situ PCR. On day 8 after immunization, well-developed GCs were observed in LN sections (Fig. 3 C,
a and b). A majority of the cells possessing DNA excision
product of V1-J1 rearrangement were found within
GCs in which RAG-expressing B cells have been reported
to be localized (11-13; Fig. 3 C, b and c). Intracellular deposition of the amplified DIG-labeled DNA fragments was
observed when in situ PCR was performed in the presence
of primers, but not in their absence. The primers for human IL-2 gene did not generate the DIG-labeled product
either (not shown), thus indicating the specificity of the
method. Thus, data presented here strongly suggest that
GC B cells undergo receptor editing in parallel with RAG
gene expression.
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Fig. 3.
Rearrangement of chain genes in the draining LN of immunized mice. (A) Mice were immunized in the hind footpads with 20 µg of TNP-KLH and 0.45 mg of alum. On days 0, 6, and 8 after immunization, popliteal LN cells were assessed for the expression of RAG-2 by RT-PCR. (B) The
same LN cells were examined for the occurrence of DNA excision product reflecting V1-J1 rearrangement by PCR followed by Southern blotting.
(C) Localization of B cells that underwent chain gene rearrangement in GCs of popliteal LNs. The DNA excision product derived from V1-J1 rearrangement was detected in cryosections prepared from the LNs on day 8 after immunization by in situ PCR, which was performed with (b and c) or
without (a) primers. GCs that stained dully with methyl green are observed in a and b. Dark blue/purple DIG-labeled deposits indicate the presence of
the excision product. Original magnification: a and b, 100; c, 400.
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|
These findings and recent observations made by other
investigators (14, 15) provide a new aspect in immunology
that V(D)J combination established in a given premature B
cell clone can be revised even at mature stages. What is a
biological role of RAG gene products expressed in GC B
cells? GC is a microenvironment in which somatic hypermutations, isotype switching, and clonal selection for affinity maturation of antibodies are undertaken (23, 24). Somatic hypermutations in GCs may produce not only high
affinity antibodies, but also generate autoreactive B cell clones. The high affinity clones will be selected positively
through interaction with follicular dendritic cells retaining
immune complexes on their surface, thus leading to affinity
maturation of antibodies (25). On the other hand, autoreactive clones must be either deleted or rendered anergic to
maintain self tolerance. Receptor editing in mature B cells
suggests another possible way to extinguish autoreactivity
in GCs. We have observed that a majority of RAG-expressing B cells in GCs are apoptotic and present in tingible bodies
(12). This may reflect, at least in part, a result of RAG-
dependent revision of antigen receptors and their subsequent selection in GCs. Further elucidation of the role of
RAG gene products in GCs will provide valuable clues to
understanding the onset of autoimmune diseases, lymphomas, and other immune disorders.
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Light Chain Genes in Mature B Cells
In Vitro and In Vivo. Function of Reexpressed
Recombination-activating Gene (RAG) Products
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