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1 Switch Transcript and Rad51 Probes1
Departments of
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Molecular Biophysics and Biochemistry and
Genetics, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 switch transcript and Rad51
mRNA, we have visualized B cells that are newly activated for
switch recombination and characterized the spatial and temporal
patterns of I1 and Rad51 mRNA expression. Within 2 days after
immunization with (4-hydroxy-3-nitrophenyl)acetyl-chicken
gamma-globulin, expression of I1 switch transcripts and Rad51
mRNA was evident and was localized to B220+ B cells
clustered within the T cell-rich periarteriolar lymphoid sheath (PALS)
and surrounding follicles. By Ab staining, we have shown previously
that cells switching from IgM to IgG expression can be visualized at 3
to 5 days postimmunization and colocalize to clusters of
Rad51+ cells. Hybridization of adjacent sections with
probes for Cµ and C1 mRNA now shows that switching from µ to
expression occurs within Rad51+I1+ regions
of the PALS and peaks between days 3 and 5. Colocalized expression of
I1 and Rad51 transcripts was observed from days 2 through 12 of the
immune response. I1 and Rad51 transcripts were down-regulated but
still detectable at 12 days postimmunization, when they were evident in
peanut agglutinin-positive germinal center B cells. Taken
together, these observations show that B cells are first activated for
switch recombination in the T cell-rich PALS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Switch recombination in vivo occurs within the secondary lymphoid tissues, such as the spleen and lymph nodes. These tissues are comprised of red pulp, which contains large numbers of E, and white pulp, which contains mostly lymphocytes. In a naive animal, the histology of these tissues is relatively simple. The white pulp is sequestered into numerous follicles, which contain a network of follicular dendritic cells filled with surface IgM-expressing, recirculating B cells. Within each follicle is a central arteriole surrounded by a T cell-rich zone known as the periarteriolar lymphoid sheath (PALS)4 (12, 13). Nonrecirculating surface IgM+ B cells populate the marginal zones that surround the follicle, dividing white pulp from red. The histologic appearance of the spleen or lymph node changes dramatically when an animal is challenged by Ag. B blasts appear within the follicular dendritic cell network and proliferate rapidly, filling the follicle and displacing the recirculating B cells to the periphery, where they form the follicular mantle. Shortly afterward, the follicle becomes polarized, and the classical appearance of the germinal center (GC) becomes apparent. The rapidly dividing blast cells (centroblasts) cluster in the region adjacent to the PALS that is termed the dark zone. The nonproliferating progeny of these cells (centrocytes) fill the dendritic network and form the light zone of the GC.
T and B lymphocytes collaborate in the response to most protein Ags (reviewed in 14 ; following primary immunization, T cell-B cell interactions are first observed in the PALS, where B cells take up Ag, process it by fragmenting it into peptides, and present the resulting peptides to Th cells via class II molecules of the MHC (15). Recognition of the peptide-MHC complex by the TCR and its CD4 coreceptor then triggers the B cell response, which further depends upon functional interactions between CD40 molecules constitutively expressed by B cells and CD40 ligand expressed by activated Th cells (14, 16, 17, 18). B cell activation results in a burst of cell proliferation and the differentiation of many B lymphocytes into plasma cells secreting low-affinity Abs (19, 20, 21). It is selected progeny of these newly expanded T and B cell populations that are recruited from the PALS to the adjacent B cell-rich follicles, where they become founders of GCs, the specialized microenvironments in which somatic hypermutation occurs (Refs. 13, 20, 22, and 23; reviewed in Refs. 24 and 25).
Although the changes that occur in the spleen and lymph node during the primary response to Ag have been well characterized histologically, the site within these tissues at which B cells first become activated for switch recombination has not been defined. Previous reports using several different approaches have suggested the importance of the T cell-rich extrafollicular zones (19, 26, 27, 28). It has also been proposed that GCs provide the primary sites at which switch recombination occurs (22, 29, 30, 31).
Analysis of murine B cells that have been induced to switch in vitro by
culture with LPS has shown that switching to a given CH
region is preceded by production of a noncoding switch transcript from
both regions targeted for recombination (reviewed in 8 . The
switch transcript initiates at the IH (initiate) exon
promoter upstream of the targeted CH region and proceeds
through the IH exon, S region, and CH region
(see Figure 1
). Splicing of the primary
transcript removes the repetitive S region sequences and results in the
joining of the IH exon and CH exons. Analysis
of mice carrying targeted deletions has shown that recombination to a
given isotype depends upon switch transcription and splicing of the
switch transcript (32, 33, 34, 35, 36, 37, 38). Extracellular signals delivered by
cytokines and lymphokines target switch recombination to specific
isotypes by inducing transcription at the corresponding IH
exon promoter. The lymphokine IL-4, which is a potent activator of IgG1
and IgE expression, induces synthesis of I1 switch transcripts in
primary resting B cells cultured in vitro in the presence or absence of
LPS (39, 40, 41, 42, 43, 44). Induction of switch transcripts is rapid and begins
within 4 h of culture with IL-4 (41). As the production of a
specific I transcript is a prerequisite to recombination, I
expression should identify cells activated for switching in vivo.
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transcripts to identify switching B cells.
We have now defined the spatial and temporal patterns of I1 switch
transcript and Rad51 mRNA expression in newly activated splenic B
cells, using in situ hybridization to observe mRNA expression in
splenic sections of C57BL/6 mice immunized with
(4-hydroxy-3-nitrophenyl)acetyl (NP)-chicken gamma-globulin
(CGG). By analysis of serial sections with several different probes, we
have shown that cells expressing I1 and Rad51 transcripts colocalize
within the T cell-rich PALS. We have also correlated I1 and Rad51
expression with the switch from Cµ to C1, as assayed both by in
situ hybridization, as reported here, and by staining with IgM- and
IgG-specific Abs, as we reported previously (45). Therefore, our
results show that B cells are first activated for switch recombination
in the T cell-rich PALS.
We further report that cells expressing switch transcripts are still visible at late timepoints following primary immunization, when they are localized to GCs. Others have identified switch transcripts in GC B cells using different experimental approaches (28, 51, 52), and GC may consequently be a secondary site of switch recombination. A low level of Rad51 mRNA expression is evident in GC cells. We have reported previously that the peak of Rad51 staining of splenic sections occurs at about day 5, well before GC formation, and that GCs are not stained with Rad51 Abs (45). Taken together with our previous results, our current data suggest that expression of Rad51 protein is specifically down-regulated, or that its distribution is relocalized before GC development and somatic hypermutation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice (6 to 12 wk old) were immunized by i.p. injection of 100 µg alum-precipitated NP-CGG together with 2 x 109 heat-killed Bordetella pertussis cells (53). Mice were sacrificed on days 0, 2, 3, 5, 7, 9, and 12 following immunization; their spleens were removed, embedded in Tissue-Tek OCT (Baxter Scientific, Boston, MA), frozen on dry ice, and stored at -70°C. Cryostat sections (10 µm) were mounted onto ProbeOn Plus slides (Fisher Scientific, Pittsburgh, PA), fixed in 3% paraformaldehyde for 60 min, and dehydrated by sequential rinses (5 min each) in water and in 30%, 75%, and 95% ethanol. The air-dried sections were stored at -70°C until use.
Immunocytochemical staining of sections
Sections were rehydrated for 10 min in PBS, permeabilized by incubation in PBS containing 0.3% Triton X-100 for 30 min, blocked for 60 min in a blocking solution of PBS containing 10% goat serum (Life Technologies, Gaithersburg, MD), and incubated for 60 min with 100 µl of either the appropriate Abs or biotinylated peanut agglutinin (PNA) (Pierce, Rockford, IL) diluted 1/100 in blocking solution. All sections were rinsed for 10 min in fresh blocking solution.
PNA-staining, biotinylated anti-B220/CD45R Abs (PharMingen, San
Diego, CA) and biotinylated anti-Thy-1.2 Abs (PharMingen)
were detected using streptavidin-bound horseradish peroxidase.
B220/CD45R is a B cell-specific form of the T200 glycoprotein family;
the Thy-1 glycoprotein is expressed by Th cells located outside GCs.
Sections were incubated for 60 min with 100 µl of streptavidin-bound
peroxidase, prepared by incubating 20 µg/ml of streptavidin (Pierce)
and 40 µg/ml of biotinylated horseradish peroxidase (Pierce) for 30
min in blocking solution. Following a 10-min rinse in PBS, sections
were incubated with 1 ml of 3,3'-diaminobenzidine (DAB) substrate
solution (Sigma, St. Louis, MO; 0.8 mg/ml DAB, 0.009%
H2O2, and 100 mM Tris-HCl (pH 7.5)). Alkaline
phosphatase-conjugated goat anti-mouse Abs specific for IgM, IgG1,
and 
light chain (Southern Biotechnology Associates, Birmingham,
AL) were detected by incubation with 1 ml of 4-nitroblue
tetrazolium chloride (NBTC)/5-bromo-4-chloro-3-indolyl-phosphate
solution (BCIP) (Boehringer Mannheim, Philadelphia, PA).
Appropriate color development was visualized in <60 min, and the
reactions were stopped by rinsing the sections in distilled water (DAB)
or TE buffer (NBTC/BCIP) (TE is 10 mM Tris and 1 mM EDTA (pH
7.9)). Sections were air-dried and mounted in Aquamount (Fisher
Scientific). Rabbit polyclonal anti-human Rad51 Abs (a generous
gift of Dr. C. M. Radding, Yale University School of Medicine)
were detected by a 60-min incubation with 100 µl of Texas
red-conjugated goat anti-rabbit IgG Abs diluted 1/100 in blocking
solution. FITC-conjugated goat anti-mouse IgM or IgG Abs were
incubated with the relevant sections in the same way. Sections were
rinsed for 10 min in PBS and 5 min in distilled water and left to air
dry before mounting in antifade.
Riboprobes
A probe specific for the murine light chain was generated
using pMC linearized with either SpeI or NcoI
as template plasmid. pMC, cloned in the pGEM-T vector (Promega,
Madison, WI), carries a 432-bp region encompassing the 3'
portion of the leader sequence, V, J, and the 5' portion of
C, which is produced by RT-PCR amplification using total RNA from
hybridoma B1-8 as template (54) as well as primers
5'-TTCACTTATACTCTCTCTCCTGGCTCTC ( leader sequence, codons -16 to
-7) and 5'-GAGCTCTTCAGAGGAAGGTGGAAACA (C sequence, codons
128–121). A probe specific for the murine Cµ C region was generated
using plasmid pBPC3HP600 linearized with either HindIII or
XbaI as template. pBPC3HP600, cloned in the pBluescribe
vector (Stratagene, La Jolla, CA), contains a 600-bp
HindIII/PstI fragment including a Cµ genomic
sequence from exons 3 and 4. A probe specific for the murine C C
regions was generated using plasmid pSB650 linearized with
HindIII as template. pSB650, cloned in the pSP65 vector
(Promega), contains a 650-bp BamHI/SacI fragment
of C2b including sequences from the CH1 through CH3 domains that are
highly conserved within the IgG subfamily. Most Abs produced in the
C57BL/6 response to NP-CGG are IgG1 (53), and their transcripts are
efficiently visualized by hybridization with this probe. A probe
specific for the I1 region was generated using plasmid pMCI1
linearized with either SpeI or NcoI as template.
pMCI1, cloned in the pGEM-T vector, carries a 361-bp insert obtained
by PCR using as template a 442-bp PCR product comprising a portion of
the mouse I1 exon spliced to a portion of the C1 sequence (55)
(kindly provided by Dr. W. Dunnick, University of Michigan, Ann Arbor,
MI) and the I1 region primers 5'-GACGGCTGCTTTCACAGCTT
(nucleotides 35–54) and 5'-CTCTCAACCTGTAGTCCATGC (nucleotides
395–375). The entire nucleotide sequence of this I1 probe is
contained within the sequences of I1 probes A and B that were used
previously by Turaga et al. (56) and shown to be highly specific. A
probe specific for Rad51 was generated using the plasmid pEG986
linearized with NotI or SpeI as template. pEG986
contains the entire 1.1 kb-mouse Rad51 cDNA (47) in the pCRII vector
(Invitrogen, San Diego, CA) and was kindly provided by Drs. E.
Golub and C. M. Radding (Yale University). In vitro transcription
reactions to generate digoxigenin-labeled sense and antisense
riboprobes were conducted as described in the Dig RNA-labeling kit
using SP6, T7, or T3 RNA polymerases (Boehringer Mannheim).
In situ hybridization
In situ hybridization studies were performed using a modification of the method of Yang et al. (21); all solutions were prepared with water treated with diethylpyrocarbonate (Sigma). Frozen sections were thawed; rehydrated for 10 min in PBS; placed in 0.2 N HCl for 20 min; washed in water; and digested with 2 µg/ml of proteinase K (Boehringer Mannheim), 2 mM CaCl2, and 10 mM Tris-HCl (pH 7.4) at 37°C for 10 min. Sections were postfixed in 3% paraformaldehyde for 5 min; rinsed twice in 2x SSC; acetylated for 10 min in 0.1 M triethanolamine-HCl (pH 8.0) containing 0.25% acetic anhydride; rinsed in 2x SSC; incubated in 0.1 M Tris-HCl and 0.1 M glycine (pH 7.0) for 30 min; washed twice in 2x SSC; dehydrated via sequential washes in 70%, 80%, 90%, and 100% ethanol; and air-dried. Sections were prehybridized for 2 h with a solution containing 50% deionized formamide, 4x SSC, 50 mM sodium phosphate buffer (pH 6.5), 0.1% SDS, 1% glycine, 1x Denhardt’s solution, 200 µg/ml of yeast transfer RNA, and 500 µg/ml of heat-denatured herring sperm DNA (Sigma) at 56°C in a humid environment. Hybridization was performed at 56°C overnight with a solution containing 50% deionized formamide, 4x SSC, 20 mM sodium phosphate buffer (pH 6.5), 0.1% SDS, 1x Denhardt’s solution, 10% dextran sulfate, 500 µg/ml of heat-denatured herring sperm DNA, and 20 µg/ml of heat-denatured, digoxigenin-labeled probe. After hybridization, the slides were soaked for 15 min each in 1x SSC at 37°C and subsequently at 45°C, for 15 min in 0.5x SSC at 45°C, for 15 min in 0.5x SSC at 56°C, for 1 h in 50% deionized formamide and 0.2x SSC at 65°C, and subsequently for 5 min in 0.1x SSC at room temperature. Hybridized digoxigenin-labeled probe was visualized using the Dig wash and block buffer set, alkaline phosphatase-conjugated anti-digoxigenin Ab, and NBTC/BCIP substrate (Boehringer Mannheim). Slides were incubated for 5 min in washing buffer, for 1 h in blocking solution, for 1 h in blocking solution containing anti-digoxigenin Ab (diluted 1/5000), twice for 30 min each in washing buffer, for 5 min in detection buffer, and finally for 5 h (except where indicated) in detection buffer containing 0.45 mg/ml of NBTC and 0.175 mg/ml of BCIP. Color development was stopped by rinsing in TE buffer (10 mM Tris, pH 7.9, 1mM EDTA) and the slides were air-dried and mounted in Aquamount (Fisher Scientific). In situ hybridization experiments were routinely conducted in parallel using sense probes as a control. No hybridization was observed in any experiments using the sense probes.
Because a mature switch transcript contains sequences from the I and
C exons, a C probe can detect not only IgG mRNA but also switch
transcripts. However, we found that by decreasing incubation time for
color development from 5 to 2 h, we could detect intense signals
from cells expressing high levels of C mRNA and IgG protein but
essentially no signal from cells expressing an I1 switch transcript
and IgM protein. This observation was confirmed by the demonstration
that identical populations of cells in adjacent sections either
hybridized with the C probe after 2 h of development or were
stained by anti-IgG1 Abs (e.g., Fig. 2). Turaga et al. (56) have observed
previously the preferential detection of plasma cells in primary
cultures of LPS/IL-4-stimulated B cells using a 35S-labeled
C1 riboprobe. Under our conditions for C visualization, we
obtained clear signals after brief incubation, probably because high
levels of the IgG transcript are present in most newly activated cells.
It is pertinent to point out that our in situ hybridization studies do
not appear to give quantitative estimates of the absolute numbers of
cells synthesizing C-containing transcripts, because cells producing
low levels of the C transcript, such as memory cells, may not be
visualized. However, within an individual experiment it is appropriate
to compare the signal intensities achieved with each probe in different
areas of the same section and in sections taken at different times
postimmunization. We attempted to minimize variations in signal
intensity by routinely developing slides for a period of 5 h when
visualizing I1 hybridization.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 transcripts is evident before immunization
Splenic sections from C57BL/6 mice housed under clean conditions
were analyzed before immunization. In accordance with previous reports
(12, 13), we found that these sections contained characteristic zones
of Thy-1.2-expressing T cells surrounded by follicular regions of
B220+ B cells (Fig. 2
A, left).
Expression of IgM transcripts by follicular B cells was visualized by
Cµ hybridization (Fig. 2A, right). The field
shown in Figure 2 was taken at x40 magnification, and dark dots
correspond to individual cells that express high levels of Cµ mRNA.
At day 0, an I1-specific riboprobe did not hybridize to any cells
(Fig. 2A, right), nor were there clear signals
from hybridization with either a Rad51-specific (Fig. 2A,
right) or a -specific (data not shown) riboprobe. The
absence of IgG and light chain expression at day 0 was confirmed by
staining with anti- and anti-IgG1 Abs; both produced a
negligible staining pattern (data not shown).
Localized induction of I1 transcripts precedes switch
recombination
At 2 days after immunization with NP-CGG, both µ+
and + cells were readily identified by the hybridization
of splenic sections with specific riboprobes (Fig. 2B).
light chain expression is a specific marker for newly activated B cells
in C57BL/6 mice immunized with NP-CGG, as this Ag induces a
strain-specific response dominated by 1 light chains; most murine B
cells express 
light chains (19, 53, 57). In the consecutive serial
sections shown (Fig. 2B), adjacent PALS regions give the
appearance of hourglass-shaped clusters of cells. Each PALS
region surrounds a central blood vessel, which can be seen as a small
circular region from which stain has been excluded.
Each panel in Figure 2B was originally taken at x40
magnification and corresponds to an area that includes 
23,000
individual cells. We have chosen to present these panels at this level
of magnification to give a more accurate impression of the whole spleen
section and to show the consistency of observations across numerous
PALS and follicular regions. In situ hybridization identifies
individual positive cells as darkly stained dots against a background
of cells that are negative for staining. By counting the numbers of
darkly staining cells in the larger field, we can estimate the number
of activated B cells at each day. There are 23,000 cells in a
1.6 x 1.1 mm field; at day 2, we estimate that a field this size
contains 100 to 300 + cells and approximately twice
that number of µ+ cells. The number of the very darkly
staining µ+ and + cells appeared to
correlate well with the number of newly activated cells previously
reported by Jacob et al. (19).
It is clear from comparing the panels in Figure 2B that the
morphology of the PALS is preserved in consecutive sections. Moreover,
both Cµ+ cells and + cells were found
localized predominantly within the PALS, as predicted from results from
other laboratories (19, 28). Analogous results were obtained when
sections were analyzed by staining with anti- and anti-IgM
Abs instead of by hybridization (data not shown).
To determine whether expression of the I1 switch transcript could be
observed at day 2 postimmunization, a riboprobe specific for this
transcript (referred to as the I1 probe) was hybridized to splenic
sections taken at this timepoint. Specificity of hybridization was
assayed by hybridization of the complementary riboprobe (I1 sense).
Figure 2B shows the typical pattern of hybridization
observed with each probe, presenting consecutive sections from the
series shown hybridized with the µ and probes as examples. As
this example illustrates, there was clear hybridization by the I1
probe. Moreover, hybridization was localized to regions of B cells
that, in adjacent sections, displayed the characteristic phenotype of
PALS-associated clusters. As with µ and hybridization, there were
some very concentrated sites of I1 staining that appeared to
correspond to single cells. In contrast to the µ and signals,
there was a considerable level of I1 staining throughout the PALS.
The probe for the complementary strand produced only faint staining,
demonstrating the specificity of the I1 staining pattern.
Hybridization with a C probe (see Materials and
Methods) allowed us to determine whether any switch
recombination had occurred at day 2. A typical pattern of hybridization
is shown in the fifth consecutive section from the series in Figure 2B. A small number of cells hybridized to the C probe,
and a comparable number of IgG1+ cells were evident upon
staining with anti-IgG1 Abs (data not shown). Therefore, switching
has occurred in a small but significant fraction of newly activated
cells at day 2.
It is clear from this series of panels that I1 expression is
specific to regions containing B lymphocytes that have migrated to the
PALS following immunization and that express Cµ and mRNA. No
hybridization was obtained using a riboprobe specific for the
complementary strand of the switch transcript (Fig. 2B),
showing that the observed signal is specific for the transcript. These
results suggest that I switch transcription is initiated in newly
activated B cells of the PALS soon after primary immunization.
In addition to the large clusters of cells that hybridized intensely
with the Cµ, , and I1 probes, there were also regions in the
red pulp where hybridization was apparent. These regions may represent
cells in the periphery of PALS clusters located above and/or below the
plane of the sections shown, or they may derive from small numbers of
cells that have migrated to the red pulp. Cells labeled within the red
pulp also expressed mRNA and protein, identifying them as newly
activated B cells rather than memory cells.
As noted above, a certain level of I1 staining is visible throughout
the PALS at day 2. While this may represent background that is not
readily removed during the wash protocols, the fact that no comparable
background is seen in hybridization with the µ, , or probes
argues against this possibility. As shown below, we consider another
possibility (see Discussion), namely that this staining may
reflect a generalized activation of B cells and an initiation of I1
switch transcript synthesis throughout the PALS at early timepoints
after immunization.
Switch recombination occurs in PALS before formation of GCs
At day 5, hybridization with a C-specific probe showed
that considerable switch recombination had occurred. In the example
shown (Fig. 2C, left), C+ cells
can be seen tracking a blood vessel, which has been sliced along its
length during sample preparation. In an adjacent serial section, the
I1-specific probe hybridized to cells clustered along this same
arteriole. If the blood vessel had been sliced transversely, the
staining would be visible as clusters of cells around a central hole.
The staining of adjacent sections with PNA revealed no PNA+
GCs at this timepoint (Fig. 2C, left), nor were
there any diffuse regions of PNA staining that colocalized with regions
of C or I1 expression. In additional serial sections, the regions
of C and I1 hybridization were shown to contain both
B220+ and Thy-1.2+ lymphocytes (data not
shown), confirming that C and I1 expression was localized to the
PALS. The specificity of I1 and C hybridization was confirmed by
comparing C hybridization with the pattern of staining by
anti-IgG1 or anti-IgM Abs (Fig. 2C,
right). These results demonstrate that I1 and C
signals are colocalized in regions within the PALS where expression of
IgG protein is also evident.
Rad51 mRNA expression correlates spatially and temporally with
I1 switch transcript expression
The patterns of expression of Rad51, I1, and C transcripts
were compared in experiments that analyzed hybridization to adjacent
sections at several timepoints following immunization with NP-CGG. The
Rad51 transcript was not expressed at day 0 (Fig. 2A), but
Rad51 expression was clearly evident by day 2. At this timepoint and
through day 5, Rad51 specific hybridization colocalized with cells
expressing I1 switch transcripts (Fig. 3A). Probes for both
transcripts produced a similar hybridization pattern of intensely
labeled individual cells surrounded by a more diffusely stained
positive region. In contrast, no hybridization was obtained using a
riboprobe specific for the complementary strand of Rad51 (data not
shown). At day 3, some C expression is evident; this expression
increases dramatically by day 5, which is consistent with the
considerable switch recombination occurring during this interval. At
day 5, cells expressing Rad51 mRNA and I1 transcripts were clustered
in the PALS and follicles and colocalized to regions characterized by
intense hybridization with the C probe (Fig. 3A,
center). Previously, we have visualized the colocalization
of Rad51 and IgG-expressing cells at day 5 by staining with specific
Abs (45). At day 12, Rad51 expression is evident but appears to have
diminished, as have I1 and C expression (Fig. 3A,
bottom). Calculations of the splenic area occupied by cells
expressing I1 transcripts at various timepoints following
immunization indicated that I1-specific hybridization increased to a
peak at day 7 and then decreased, suggesting that I1 expression is
similarly down-regulated after switching is completed.
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1 switch transcripts are present in GC B cells
Somatic hypermutation occurs in GCs, which are readily visualized
by staining with PNA. Consistent with our previous findings (45),
essentially no GCs were evident before about day 9. By day 12, GCs were
very abundant and exhibited a characteristic pattern of staining.
Figure 3B shows an example of consecutive serial sections
stained with PNA, anti-B220, and anti-Thy-1.2 Abs, respectively
(Fig. 3B, left). Staining revealed T cell-rich
PALS zones located adjacent to the B cell-rich GCs. The black dots in
these panels correspond to nonspecific deposition of the DAB4 color
substrate solution. The PALS did not show significant levels of
anti-B220 staining at day 12, perhaps because the B cell population
in these regions was mostly comprised of plasma cells. A low level of
B220 staining by plasma cells in vitro has been observed
previously (58, 59).
Cells expressing I1 switch transcripts were still evident at day 12.
An example of a typical hybridization pattern is shown in Figure 3B (top right), where intensely
hybridizing individual I1+ cells are scattered against a
background of more diffuse staining localized to the GC and adjacent
PALS. Hybridization of an adjacent section with a C probe revealed
large numbers of C+ cells (Fig. 3B,
center right) that were predominantly located in the
Thy-1.2+ T cell-rich PALS (Fig. 3B, bottom
left). Essentially no hybridization to a Cµ probe was observed
(Fig. 3B, bottom right), indicating that
switching from µ had been completed by day 12.
Figure 3C shows analysis of consecutive sections through two
adjacent PNA+ GCs. Photographs were taken at x100
magnification and illustrate the colocalization of I1 and C
hybridization in consecutive serial sections. Intense signals are
visible in the PALS region that separates the two GCs, and more diffuse
staining is apparent within the two GCs. A clear C signal is evident
in the PALS between the two GCs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 switch
transcripts by in situ hybridization to splenic sections. Newly
activated B cells expressing I1 switch transcripts were first
evident at 2 days postimmunization. At this timepoint, there was
considerable expression of Cµ but not C mRNA. Therefore, induction
of I1 switch transcripts is evident in splenic B cells before the
occurrence of switch recombination, as expected from analysis of
switching in primary cultured cells (39, 40, 41, 42, 43, 44). The rapid induction of
switch transcripts that we have observed in vivo parallels the rapid
induction of switch transcripts that occurs in activated primary B
cells cultured in vitro (40, 41, 56).
The appearance of newly activated B cells corresponded both temporally
and spatially with the development of PALS-associated clusters of
Ag-specific B cells. As measured by the appearance of C+
cells, active switch recombination began as early as day 2 and peaked
at about day 5. During this interval, the number of C+
cells increased dramatically. This picture of the kinetics of switch
recombination in vivo during the NP-CGG response is consistent with
kinetics determined using plaque assays to quantitate IgM- and
IgG-secreting B cells (60). It is also consistent with the kinetic
picture of switching in the NP response as visualized in splenic
sections by Jacob et al. (19) and by our own laboratory (45). The
possibility that switching occurs in the PALS is further supported by
studies showing that mice which had been tolerized with soluble NP
preimmunization developed significantly lower numbers of NP-specific
GCs but showed no reduction in PALS-associated clusters of NP-specific
cells or levels of switching (27). Likewise, lymphotoxin-
-deficient
mice immunized with high doses of NP-OVA mounted a high affinity IgG1
response even though they failed to produce GCs (26).
The hypothesis that switch recombination occurs in GCs has been put
forward by several laboratories (22, 29, 30, 31, 52). In some instances,
this notion was based on experiments demonstrating that switch
transcripts were present in cells bearing surface markers of GC B
cells. Liu et al. (52) observed both switch transcripts (by RT-PCR) and
switch circles (by PCR) in analysis of a FACS-sorted population with
the surface phenotype of GC centrocytes. Islam et al. (51) used RT-PCR
to analyze I
and C mRNA levels in PBMCs from patients being
treated for schistosomiasis and found I transcripts present at day
12. Toellner et al. (28) used RT-PCR to analyze switch transcript
levels in splenic sections at various timepoints after immunization;
while they found switch transcripts present at late timepoints, they
observed no correlation between levels of switch transcripts and the
size of GCs in adjacent sections and concluded that GCs were not the
primary sites of switch recombination (28). Consistent with these other
studies (28, 51, 52), we did find some level of I1 expression in
GCs. We cannot distinguish whether these transcripts are evident
because they persist within B cells that have completed recombination
in the PALS and migrated to the GCs or because I transcription is
newly activated in GC B cells. Switch transcripts may persist in
activated B cells because they have a relatively long
t1/2 for degradation. Alternatively, just as the
presence of Rag1 and Rag2 transcripts may be evidence that V region
replacement occurs in the GCs (61), the presence of I1 switch
transcripts may indicate that switch recombination is ongoing in the GC
cells. As essentially no µ mRNA expression was evident at these late
timepoints, if recombination is occurring it almost certainly involves
other isotypes (for example, switching from one subclass to
another).
We have shown previously that intense Rad51 staining can be observed in splenic sections during a brief window following primary immunization, and that staining correlates temporally and spatially with cells switching from IgM to IgG expression (45). While the evidence linking Rad51 to switch recombination is thus far only correlative, expression of Rad51 protein is clearly induced at early timepoints (days 2–5) following immunization and then down-regulated before GC formation and somatic hypermutation. Rad51 appears to function in both recombination and repair pathways. If hypermutation in the GC involves a DNA lesion (62, 63, 64), high levels of Rad51 similar to those present in switching B cells might even interfere with the mutagenic mechanism.
The number of µ+ and + cells we observed
at day 2 correlated with estimates others have made of the number of
newly activated cells present at early timepoints after immunization
with NP-CGG (19). However, we observed a surprisingly intense I1
staining pattern at day 2, and significantly more cells appeared to be
expressing I1 transcripts than either or µ transcripts. This
signal could represent background hybridization, but several
observations argue against such an interpretation: the signal was
specific to the I1 probe; it was not evident in sections hybridized
with its complement (I1 sense); and there was little hybridization
of the C probe. Moreover, the I1 hybridization signal at day 2
represented a clear increase from day 0, when almost no hybridization
was evident. We suggest that there is another more interesting
interpretation of this observation. Primary resting B cells can be
stimulated to express I1 switch transcripts and switch to 1 by
culture with IL-4 in the absence of LPS (40, 41, 42, 43). IL-4 is a rapid and
potent inducer of I1 transcripts in such cultures, suggesting that a
significant fraction of resting B cells respond directly to this
lymphokine. We hypothesize that, at early timepoints after
immunization, a generalized induction of I1 transcription may occur
in vivo, as B cells respond to IL-4 secreted by activated Th cells.
This early burst of I1 transcription may not necessarily lead to
productive switch recombination but would prime the cells to respond to
additional signals necessary for commitment to switching. This
hypothesis is also favored by the observation that the frequency of
primary murine B cells expressing either germline 1 or
transcripts after 48 h in culture with LPS and IL-4 is higher than
the number of cells that ultimately switch to the respective isotypes
(65). In addition, evidence is accumulating for the requirement of
additional factors to promote switching: isotype commitment can be
influenced after IL-4-mediated germline transcription by signals from
cell surface molecules (e.g., Lyb2 (66)), and IgE secretion can be
suppressed by IFN- and IFN- without altering germline C
transcription (67). Additional signals required may depend upon
physical contact with activated T cells and ligation of the CD40
molecule (68, 69).
Having established that I1 is an early marker for imminent switch
recombination in splenic B cells, it should be possible to identify
additional genes that are specifically induced in B cells activated for
switching. It is invaluable to be able to carry out such analysis in
vivo, where critical cell-to-cell contacts and unique microenvironments
are preserved.
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Acknowledgments |
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1 exon DNA. |
Footnotes |
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2 Current address: Department of CNS Discovery, Pfizer Central Research, Eastern Point Road, Groton, CT 06340.
3 Address correspondence and reprint requests to Dr. Nancy Maizels, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 266 Whitney Avenue, New Haven, CT 06520-8114. E-mail address:
4 Abbreviations used in this paper: PALS, periarteriolar lymphoid sheath; DAB, 3,3'-diaminobenzidine; GC, germinal center; PNA, peanut agglutinin; NP, (4-hydroxy-3-nitrophenyl)acetyl; CGG, chicken gamma-globulin; NBTC, 4-nitroblue tetrazolium chloride; BCIP, 5-bromo-4-chloro-3-indolyl-phosphate.
Received for publication April 10, 1998. Accepted for publication June 17, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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