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Loyola University Chicago, Stritch School of Medicine, Department of Microbiology and Immunology, Maywood, IL 60153
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Abstract |
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4 to 8 wk of age, the IgH VDJ genes of essentially all rabbit
B lymphocytes have undergone somatic diversification. Some of this
diversification occurs in the appendix, which is a gut-associated
lymphoid tissue (GALT). To determine whether GALT is essential for
somatic diversification, we surgically removed the appendix, sacculus
rotundus, and Peyer’s patches from neonatal rabbits (designated
GALT-less) and examined the extent to which VDJ genes were somatically
diversified. We found that the IgM VDJ genes of peripheral B cells from
2- to 5-mo-old GALT-less rabbits had undergone considerably less
somatic diversification than those of control rabbits. Further, the
percentage of peripheral B cells in the GALT-less rabbits was generally
less than that of controls. Our data suggest that, in rabbits, the
primary Ab repertoire develops in GALT, and B cell expansion also
occurs there. Hence, GALT may function as a mammalian bursal homologue. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 to 3 mo of age (8, 9). Gut-associated lymphoid tissue (GALT)5 has a key role in somatic diversification of V(D)J genes in both chickens and sheep. In chicken, before hatching, the VDJ and DJ genes somatically diversify by gene conversion in the bursa (5, 10); in sheep, VJ genes somatically diversify in the ileal Peyer’s patch (6, 7). There is evidence to suggest that in rabbits, GALT may play a similar role. Cooper et al. (11) surgically removed the appendix, sacculus rotundus, and Peyer’s patches from neonatal rabbits and found that after immunization with several Ags, these rabbits responded to some of the immunogens with decreased Ab production. Further, Weinstein et al. (12) showed that VDJ genes undergo somatic diversification in the appendix of 6-wk-old rabbits, an age during which the primary Ab repertoire is forming in rabbits (9). Together, these studies led us to investigate further whether GALT is necessary in rabbits for the somatic diversification of Ig genes that results in the establishment of the primary Ab repertoire.
To determine whether GALT is necessary for generating the primary Ab repertoire, we performed experiments similar to those of Cooper et al. (11) and surgically removed the organized GALT (i.e., appendix, sacculus rotundus and Peyer’s patches) shortly after birth. Then we tested whether, in the absence of organized GALT, VDJ genes would undergo normal levels of somatic diversification within the first few weeks of life.
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Materials and Methods |
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The GALT of 10 rabbits was surgically altered in accordance with
institutional guidelines for animal welfare using the following
procedure: In nine 1-day-old rabbits, the appendix and the ileocecal
junction, in which the sacculus rotundus develops, were surgically
excised, and the ileum was religated to the cecum by an end to side
anastomosis. The rabbits were rested until 3 to 5 wk of age, at which
time the Peyer’s patches of the small intestine were surgically
removed using purse-string sutures. After surgery, these rabbits were
maintained under conventional conditions in our rabbit colony. We
designated these rabbits GALT-less, even though they probably harbor
macroscopically imperceptible lymphoid aggregates in the small and
large intestines. With the exception of two rabbits that died of
unknown causes at the ages of 3 and 9 mo, the GALT-less rabbits
appeared healthy, with no apparent signs of infection. The growth rate
of GALT-less rabbits was similar to that of control littermates (Fig. 1
).
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Nucleotide sequence analysis
PBLs were prepared from buffy coats of whole blood as previously described (9). Erythrocytes were lysed by hypotonic shock treatment, and RNA was prepared using TRIzol according to the manufacturer’s instructions (Life Technologies, Grand Island, NY). The cDNA was prepared from 3 µg of RNA and amplified by PCR (9). The PCR products were cloned into M13 mp19, and their nucleotide sequences were determined (13, 14). The sequences were compared with those of germline VH1, VHy and VHz as well as with the presumed germline sequence of VHx (15); the gene that was used in the VDJ gene rearrangement was identified by its similarity to the framework regions of these germline genes. Because VH1 is the only gene for which the nucleotide sequence is known for each of the three IgH allelic types, a1, a2, and a3, only the VH1-utilizing genes are reported in this study.
Flow cytometric analysis
PBLs, isolated from buffy coats as described above, and single cell suspensions from spleen and mesenteric lymph nodes (MLNs) were stained for light chain (L chain) expression with biotinylated goat anti-rabbit L chain and avidin phycoerythrin or stained for IgM expression with mouse anti-rabbit µ-chain mAb and FITC-goat anti-mouse Ig. The goat anti-rabbit L chain Ab was generated by immunizing a goat with purified secretory IgA and then affinity purifying the anti-L chain Ab on a rabbit IgG immunosorbant. The anti-rabbit µ mAb was generated by injecting a mouse with purified rabbit IgM (Accurate Chemical and Scientific Corp., Westbury, NY) and producing murine hybridomas by fusing spleen cells of the immunized mouse with the Ag8.653 fusion partner as described previously (16). Specificity of the anti-rabbit µ-chain mAb was established in an ELISA by showing that it did not react with purified IgG or IgA. Goat anti-mouse Ig was prepared by immunizing a goat with purified mouse IgG and then affinity purifying the specific Ab on a mouse IgG immunosorbant. Using immunofluorescence, we observed that this Ab did not react with rabbit B cells. According to ELISA, this Ab also did not react with purified rabbit IgG or IgA. The fluorescent-labeled cells were analyzed by flow cytometry (FACStar; Becton Dickinson, Mountain View, CA). The data were analyzed statistically using a Mann-Whitney U test (nonparametric).
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Results |
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Because Weinstein et al. (12) demonstrated that VDJ genes undergo
somatic diversification in germinal centers of the appendix of 6-wk-old
rabbits, we determined whether the appendix is the only immunologic
site in which somatic diversification occurs. We appendectomized a
rabbit on the day of birth and determined whether the VDJ genes
diversified within the same time span as in normal rabbits. The IgM
VDJ-Cµ genes from PBLs were amplified by RT-PCR, cloned, and the
nucleotide sequences were determined. We compared the nucleotide
sequences of the VDJ-Cµ genes with those of germline
VH1 to identify the extent to which
the VDJ genes were diversified. Because Crane et al. (9) reported that
the VH genes of PBLs of normal rabbits are extensively
diversified by 8 wk of age, we examined the level of somatic diversity
of IgM heavy chain VDJ genes in PBLs of the appendectomized rabbit at 9
wk of age. We found that the IgM VDJ genes were diversified (Fig. 2A) with an average of
9.1 nucleotide changes per VH region. This led us to
conclude that somatic diversification of VDJ genes during the first few
weeks of life can occur outside the appendix.
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4 wk of age. When these GALT-less rabbits
were 10 to 12 wk of age, we then analyzed the level of somatic
diversification of the VDJ genes. We amplified the IgM heavy chain VDJ
genes of PBLs by RT-PCR. By nucleotide sequence analysis, we found that
the VH regions of the VDJ genes of each of three GALT-less
rabbits had undergone little somatic diversification. The nucleotide
sequences differed from their corresponding germline sequences by an
average of 2.8 nucleotides per VH gene (Fig. 2
B). These data sharply contrasted with the data
obtained from the control littermate rabbits, in which peripheral B
cell VDJ genes underwent extensive somatic diversification with an
average of 24 nucleotide changes per VH region (Fig. 2C). We conclude that GALT is necessary for the
somatic diversification of VDJ genes that occurs shortly after
birth.
Each of the rabbits used in the study was examined surgically for the
visible presence of residual remnants of GALT. We found no remnants of
appendix in the appendectomized rabbit, and we found no visible
evidence of residual appendix, sacculus rotundus, or Peyer’s patches
in all but two of the GALT-less rabbits. One of these rabbits, 170L2,
had remnant appendix (approximately one-half normal size); the sacculus
rotundus and Peyer’s patches had been completely removed. We
PCR-amplified VDJ-Cµ genes from the PBLs of this rabbit at 10 wk of
age, and we found an average of 10 nucleotide changes per
VH region by nucleotide sequence analysis (Fig. 2D). We conclude that VDJ genes can diversify in the
appendix in the absence of both the sacculus rotundus and Peyer’s
patches.
The analyses described above focused on the VH region of
the VDJ genes. Because the D regions (complementarity-determining
region 3) also diversify extensively within a few weeks after birth
(17), we analyzed the D regions of the VDJ genes cloned from each of
the rabbits used in this study. The results from the D regions were
similar to those of the VH regions. That is, in the
GALT-less rabbits we found only a few mutations in the D regions (Fig. 3), whereas the D regions of VDJ genes
from 10-wk-old normal rabbits were so highly diversified that it was
difficult to identify the germline D gene segments used in the VDJ gene
rearrangements (data submitted to GenBank). We conclude that the
limited diversification of VH genes in GALT-less rabbits
extends through the entire VDJ gene.
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E). Moreover, the D
regions of most of these VDJ genes were relatively undiversified, and
we could readily identify the germline D genes used in the VDJ gene
rearrangements. By 28 wk of age, however, we found that the IgM VDJ
genes of peripheral B cells in GALT-less rabbits were extensively
diversified, with an average of 17.6 nucleotide changes per
VH region (Fig. 2F). We conclude that,
while removal of organized GALT in neonatal rabbits results in
decreased levels of somatic diversification of VDJ genes until early
adulthood, the VDJ genes can undergo somatic diversification later
in life. B cell expansion in GALT-less rabbits
The chicken bursa and the sheep ileal Peyer’s patch not only
function as sites for generating Ab diversity, but also as primary
lymphoid tissues for the expansion of B cells. Removal of the bursa and
the ileal Peyer’s patch at birth results in dramatically fewer
peripheral B cells in chicken and sheep, respectively (18, 19, 20, 21). To
investigate whether rabbit GALT functions as a primary lymphoid tissue
for B cell expansion, we used immunofluorescence to determine the level
of B lymphocytes in the periphery in a total of seven normal and seven
GALT-less rabbits. We found that at 12 wk of age the percentage of B
cells in PBLs, MLNs, and spleens of GALT-less rabbits was generally
<50% of that seen in littermate and age-matched normal rabbits (Fig. 4). The difference in the average
percentage of B cells in the two groups is statistically significant
for MLNs and spleen (p = 0.05). By analyzing
PBLs from two GALT-less rabbits at 28 wk of age and one GALT-less
rabbit at 16 mo of age, we again found a decreased percentage of B
cells in PBLs. Consistent with the decreased percentage of B
lymphocytes, we found nearly 50% fewer lymphocytes in GALT-less
rabbits as compared with control littermates when differential counts
of blood smears were used (Table I).
These data indicate that the total number of B lymphocytes in the
periphery of GALT-less rabbits is less than that of normal rabbits.
Hence, we conclude that early in life the rabbit GALT functions as a
primary lymphoid tissue for expansion of B cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 wk of age. If GALT
is necessary for development of the primary Ab repertoire, we expected
the VDJ genes in B cells of these GALT-less rabbits to undergo little
or no somatic diversification. We found that for the first 19 wk of
life, long after the time that VDJ genes undergo somatic
diversification in normal rabbits, the VDJ genes in the GALT-less
rabbits exhibited little or no somatic diversification. At 10 to 12 wk
of age, the number of mutations per VH gene averaged
2.8 for GALT-less rabbits compared with 24 mutations for 10-wk-old
littermate control rabbits that had undergone surgery to remove a small
piece of the intestine. We conclude that organized GALT is necessary
for development of the primary Ab repertoire early in life. Although we found dramatically reduced levels of Ab diversity in the GALT-less rabbits until they reached 19 wk of age, the level of Ab diversity by 28 wk of age was similar to that of normal rabbits. Where did this diversification occur? We suggest that some of the diversification occurred in B cells located in residual, macroscopically imperceptible, lymphoid aggregates in the gut that were not removed by surgery. Presumably, some of these cells expand, undergo somatic diversification of the VDJ genes, and exit to the periphery. However, the number of B cells in these aggregates is presumably small and would not account for the large pool of B cells in the periphery. It may be that the extensive somatic diversification of VDJ genes by 28 wk of age occurs in peripheral lymphoid tissues in response to antigenic stimulation as discussed below. However, because we found that the number of peripheral B cells in GALT-less rabbits was reduced, we cannot rule out the possibility that the limited diversity of VDJ genes until 19 wk of age is due simply to the delayed development, following surgery, of mature Ig-producing cells that diversified normally.
We propose that, in normal rabbits, B cells migrate from bone marrow to the gut, where they undergo expansion and somatic diversification (9). In the GALT-less rabbits, we suggest that B cells migrate instead to the secondary lymphoid tissues, including the spleen and regional lymph nodes; in these tissues, the B cells can be stimulated by Ag to form germinal centers and undergo Ag-driven somatic diversification. Several studies support this possibility. First, in the earlier experiments of Cooper et al. (11), who surgically removed the appendix, sacculus rotundus, and Peyer’s patches from young rabbits, germinal centers were found in the peripheral lymph nodes of immunized rabbits. They also found that these rabbits responded to several Ags, albeit at lower levels than normal rabbits. We immunized four of our GALT-less rabbits at 10 to 12 wk of age and found that each of them developed Ab to keyhole limpet hemocyanin (KLH) after both primary and secondary immunizations with KLH (our unpublished observations). Further, we found, in collaboration with William J. Simmons and Dr. Jeanette Thorbecke (New York University Medical Center, New York, NY), that the spleens of these immunized GALT-less rabbits had germinal centers that were equal in number and size to spleens from normal KLH-immunized rabbits (our unpublished observations). Because germinal centers are the sites at which VDJ genes undergo somatic diversification (22), we consider it likely that the VDJ genes in the germinal centers of GALT-less rabbits undergo somatic diversification. Hence, we suggest that much of the diversification of VDJ genes from the GALT-less rabbits at 28 wk of age occurred in the spleen and/or peripheral lymph nodes in response to antigenic stimulation. This idea needs to be tested directly.
The somatic diversity observed in 19- and 28-wk-old GALT-less rabbits offered the opportunity to determine whether IgH genes can undergo gene conversion outside GALT. We found many codon insertions and/or deletions in the diversified sequences that are characteristic of gene conversion events. We were unable, however, to identify potential VH gene donors for these codons, probably because the rabbits used in this experiment had the a1 haplotype and because only three potential donor VH genes 5' of the utilized gene, VH1, are cloned. Without identifying VH genes that could have served as donors for the diversified regions, we are reluctant to conclude that these genes diversified by gene conversion rather than by somatic hypermutation.
Separate observations indicate that the primary Ab repertoire can be generated if only a portion of GALT exists. The VDJ genes of the rabbit with both an appendix that was one half the normal size and no sacculus rotundus or visible Peyer’s patches were extensively diversified. Also, in the appendectomized rabbit with an intact sacculus rotundus and Peyer’s patches, the VDJ genes were extensively diversified. Although we did not directly test whether the sacculus rotundus and Peyer’s patches are able to individually promote somatic diversification, it seems likely that they can.
We found long-term deficits in the number of peripheral B cells in GALT-less rabbits. Cooper et al. (11) found lower than normal numbers of peripheral lymphocytes in rabbits with surgically removed GALT, although they did not show that the decrease was in the number of B lymphocytes. Similarly, there were fewer peripheral B cells in sheep with the ileal Peyer’s patch removed early in ontogeny (21). The mechanism for maintaining a normal level of B cells in mammals is not known. However, we suggest that GALT is required to develop and/or maintain a normal number of B cells in the periphery. If GALT is a site that serves as a reservoir of B cells that undergo extensive proliferation and migrate to peripheral tissues then, in the absence of GALT, we would expect fewer B cells in the periphery. We conclude that the long-term deficit in the number of peripheral B cells in the GALT-less rabbits results directly from the absence of GALT and, consequently, that GALT is required for the development of normal B cell numbers in the periphery.
The data from this study support the idea that in rabbits, GALT functions as a bursal homologue. Not only is GALT the site in which VDJ genes undergo somatic diversification resulting in the formation of the primary Ab repertoire, but it is also important for maintaining normal numbers of B cells. We suggest that these processes are stimulated by luminal Ags, such as viral or bacterial superantigens, that are taken up by M cells lining the follicle-associated epithelium in rabbit GALT. However, it is also possible that the expansion and somatic diversification may be a developmentally regulated process that occurs in the absence of exogenous luminal Ags. Experiments to address the potential role of luminal Ags in the expansion of B cells and diversification of Ig genes need to be performed.
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Acknowledgments |
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Footnotes |
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2 All sequences reported herein have been deposited in the GenBank database and assigned accession numbers AF029916-AF029969.
3 Current address: Children’s Hospital, Harvard Medical School, Boston, MA 02115.
4 Address correspondence and reprint requests to Dr. Katherine L. Knight, Loyola University Chicago, Stritch School of Medicine, Department of Microbiology and Immunology, 2160 S. First Ave., Maywood, IL 60153.
5 Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; MLN, mesenteric lymph node; L chain, light chain; KLH, keyhole limpet hemocyanin.
Received for publication July 18, 1997. Accepted for publication November 24, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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