By
From the Department of Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700 Japan
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 V1 to J
1 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 V3-83J
2 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, 10Analysis 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, 5Flow Cytometric Analysis.
Cultured B cells were incubated with 10 µg/ml biotinylated anti-mousePurification of + or
+ B Cells.
Proliferative Response of B Cells.
Unseparated,Detection of DNA Excision Product.
DNA excision product derived fromIn 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 V ![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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 V
1-J
1 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). V
1-J
1 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).
|
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).
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 V
1-J
1 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 V
1-J
1 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 V
1-J
1 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.
|
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.
![]() |
Footnotes |
---|
Received for publication 15 October 1997 and in revised form 9 December 1997.
This work was supported by a grant-in-aid from The Ministry of Education, Science and Culture of Japan and the grant from Nagase Science and Technology Foundation. ![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Tonegawa, S.. 1983. Somatic generation of antibody diversity. Nature. 302: 575-581 [Medline]. |
2. | Schatz, D.G., M.A. Oettinger, and M.S. Schlissel. 1992. V(D)J recombination: molecular biology and regulation. Annu. Rev. Immunol. 10: 359-383 [Medline]. |
3. | Wagner, S.D., and M.S. Neuberger. 1996. Somatic hypermutation of immunoglobulin genes. Annu. Rev. Immunol. 14: 441-457 [Medline]. |
4. | Radic, M.Z., and M. Zouali. 1996. Receptor editing, immune diversification, and self-tolerance. Immunity. 5: 505-511 [Medline]. |
5. | Tiegs, S.L., D.M. Russell, and D. Nemazee. 1993. Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 177: 1009-1020 [Abstract]. |
6. | Lang, J., M. Jackson, L. Teyton, A. Brunmark, K. Kane, and D. Nemazee. 1996. B cells are exquisitely sensitive to central tolerance and receptor editing induced by ultralow affinity, membrane-bound antigen. J. Exp. Med. 184: 1685-1697 [Abstract]. |
7. | Chen, C., E.L. Prak, and M. Weigert. 1997. Editing disease-associated autoantibodies. Immunity. 6: 97-105 [Medline]. |
8. | Ghia, P., A. Gratwohl, E. Singer, T.H. Winkler, F. Melchers, and A.G. Rolink. 1995. Immature B cells from human and mouse bone marrow can change their surface light chain expression. Eur. J. Immunol. 25: 3108-3114 [Medline]. |
9. |
Hertz, M., and
D. Nemazee.
1997.
BCR ligation induces receptor editing in IgM+, IgD![]() |
10. | Grawunder, U., T.M.J. Leu, D.G. Shatz, A. Werner, A.G. Rolink, F. Melchers, and T.H. Winkler. 1995. Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement. Immunity. 3: 601-608 [Medline]. |
11. |
Hikida, M.,
M. Mori,
T. Takai,
K. Tomochika,
K. Hamatani, and
H. Ohmori.
1996.
Reexpression of RAG-1 and
RAG-2 genes in activated mature mouse B cells.
Science.
274:
2092-2094
|
12. | Hikida, M., M. Mori, T. Kawabata, T. Takai, and H. Ohmori. 1997. Characterization of B cells expressing recombination activating genes in germinal centers of immunized mouse lymph nodes. J. Immunol. 158: 2509-2512 [Abstract]. |
13. |
Han, S.,
B. Zheng,
D.G. Schatz,
E. Spanopolou, and
G. Kelsoe.
1996.
Neoteny in lymphocytes: RAG1 and RAG2 expression in germinal center B cells.
Science.
274:
2094-2097
|
14. |
Han, S.,
S.R. Dillon,
B. Zheng,
M. Shimoda,
M.S. Schlissel, and
G. Kelsoe.
1997.
V(D)J recombinase activity in a subset
of germinal center B lymphocytes.
Science.
278:
301-305
|
15. |
Papavasiliou, F.,
R. Casellas,
H. Suh,
X.-F. Qin,
E. Besmer,
R. Pelanda,
D. Nemazee,
K. Rajewski, and
M.C. Nussenzweig.
1997.
V(D)J recombination in mature B cells: a mechanism for altering antibody responses.
Science.
278:
298-301
|
16. | Hikida, M., T. Takai, and H. Ohmori. 1996. Requirements of a costimulus for IL-4-induced IgE class switching in murine B cells activated via antigen receptors. Effectiveness of 8-mercaptoguanosine. J. Immunol. 156: 2730-2736 [Abstract]. |
17. |
Chen, C.,
M.Z. Radic,
J. Erikson,
S.A. Camper,
S. Litwin,
R.R. Hardy, and
M. Weigert.
1994.
Deletion and editing of
B cells that express antibodies to DNA.
J. Immunol.
152:
1970-1982
|
18. | van Gent, D.C., J.F. McBlane, D.A. Ramsden, M.J. Sadofsky, J.E. Hesse, and M. Gellert. 1995. Initiation of V(D)J recombination in a cell-free system. Cell. 81: 925-934 [Medline]. |
19. | Ramsden, D.A., and M. Gellert. 1995. Formation and resolution of double-strand break intermediates in V(D)J rearrangement. Genes Dev. 9: 2409-2420 [Abstract]. |
20. |
Rolink, A.,
F. Melchers, and
J. Andersson.
1996.
The SCID
but not the RAG-2 gene product is required for Sµ-S![]() |
21. | Bogue, M.A., C. Wang, C. Zhu, and D.B. Roth. 1997. V(D)J recombination in Ku86-deficient mice: distinct effects on coding, signal, and hybrid joint formation. Immunity. 7: 37-47 [Medline]. |
22. | Zelazowski, P., E.E. Max, M.R. Kehry, and C.M. Snapper. 1997. Regulation of Ku expression in normal murine B cells by stimuli that promote switch recombination. J. Immunol. 159: 2559-2562 [Abstract]. |
23. | Pulendran, B., R. van Driel, and G.J.V. Nossal. 1997. Immunological tolerance in germinal centers. Immunol. Today. 18: 27-32 [Medline]. |
24. | Kelsoe, G.. 1996. Life and death in germinal centers (redux). Immunity. 4: 107-111 [Medline]. |
25. | Rajewsky, K.. 1996. Clonal selection and learning in the antibody system. Nature. 381: 751-758 [Medline]. |