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B Required for Proper Secondary Lymphoid Organ Microarchitecture: Functions Enhanced by Bcl-3
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Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Serono Pharmaceutical Research Institute, Plan-les-Ouates, Geneva, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, or in Bcl-3,
an IB-related regulator that associates with p52 homodimers, share
defects in the microarchitecture of secondary lymphoid organs. The
mutant mice are impaired in formation of B cell follicles and are
unable to form proper follicular dendritic cell (FDC) networks upon
antigenic challenge. The defects in formation of B cell follicles may
be attributed, at least in part, to impaired production of the B
lymphocyte chemoattractant (BLC) chemokine, possibly a result of
defective FDCs. The p52- and Bcl-3-deficient mice exhibit additional
defects within the splenic marginal zone, including reduced numbers of
metallophilic macrophages, reduced deposition of the laminin-ß2 chain
and impaired expression of a mucosal addressin marker on sinus-lining
cells. Whereas p52-deficient mice are severely defective in all of
these aspects, Bcl-3-deficient mice are only partially defective. We
determined that FDCs or other non-hemopoietic cells that underlie FDCs
are intrinsically impaired in p52-deficient mice. Adoptive transfers of
wild-type bone marrow into p52-deficient mice failed to restore FDC
networks or follicles. The transfers did restore metallophilic
macrophages to the marginal zone, however. Together, the results
suggest that p52 carries out functions essential for a proper splenic
microarchitecture in both hemopoietic and non-hemopoietic cells and
that Bcl-3 is important in enhancing these essential activities of
p52. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
transcription factor family is intimately involved in numerous cellular
responses, especially those associated with stress, injury, and
pathogens (1, 2, 3, 4). Upon pathogenic challenge, NF-B
factors are activated and can induce the expression of many genes that
encode proteins critical to the defense of the organism. Mice rendered
deficient for various NF-B subunits exhibit diverse immunologic
defects (5, 6, 7, 8, 9, 10, 11, 12). The intimate relationship between this
transcription factor and immune responses is quite ancient in
evolutionary terms, since the Drosophila host defense responses to
pathogens have been shown to depend on NF-B protein homologues
(13). NF-B factors are present in many cell types,
where they may be activated by signals derived from pathogenic
challenge, either directly or indirectly.
NF-B is a family of dimeric complexes composed of closely related
polypeptides that share a Rel homology domain (1). This
domain of about 300 amino acids encodes DNA-binding, dimerization, and
nuclear localization functions. In mammalian systems, the family of
complexes is composed of the subunits p50, p52, RelA (p65), c-Rel, and
RelB. The prototypical complex is a p50-RelA heterodimer, but most of
the other possible dimer combinations have been observed as well,
depending on cell type and activation status. One exception is RelB,
which is known to dimerize with p50 or p52 only. RelA, c-Rel, and RelB
have definite trans-activation domains, while p50 and p52 do
not. Therefore, all dimers that include the former subunits can
directly trans-activate B site- dependent reporters.
NF-B activation is regulated primarily through inhibitory IB
proteins, which normally complex with and retain the transcription
factor in the cytoplasm. These inhibitors can be rapidly degraded in
response to a wide variety of signals, thus freeing the complexes to
translocate into the nucleus and carry out their functions
(14, 15, 16, 17).
Although the various NF-B factors have redundant functions, they
also harbor unique functions, as evidenced by defects in mice lacking
individual factors. We as well as others have previously generated mice
deficient in the p52 subunit (9, 10). These mice have
impaired Ab responses to T-dependent Ags. The reason for this may
relate to a partially impaired microarchitecture in spleen and lymph
nodes that no longer supports proper germinal center formation.
Germinal centers are the principal sites where Ag-activated B cells
undergo affinity maturation and isotype switching, and where both
plasma and memory B cells are generated (18). Other
subunits therefore are unable to substitute for some activities
conducted by p52, this despite the fact that the p50 subunit is
structurally and functionally very similar to p52. In addition, p50 is
abundantly and apparently ubiquitously expressed (1, 19).
Although p50 knockout mice have certain defects associated with B cells
(5), these mice do not appear to have the problems with
lymphoid microarchitecture noted in the p52 knockout mice (9, 10). One important way in which these two subunits may differ
relates to their interaction with the Bcl-3 protein. Based on its
structure, Bcl-3 is a member of the IB family, but functionally it
appears to be quite distinct. Rather than inhibit, Bcl-3 may be able
act as a transcriptional coactivator, as shown in transient
transfection experiments (20, 21). Although Bcl-3 can
complex with both p50 and p52, its interaction with p52 homodimers in
particular appears to lead to potent trans-activation of a
B-dependent reporter construct in some cell types. However, no
specific physiologic target of this unusual association of p52 and
Bcl-3 has been defined to date. Nevertheless, consistent with a shared
function, the defects observed in mutant mouse strains lacking p52 or
Bcl-3 are similar (9, 22, 23). In contrast, mutant mice
lacking p50 did not share any obvious defects with those lacking Bcl-3.
Thus, p52 and Bcl-3 may together have a unique function, one not shared
with p50.
In the present study, we explore the phenotypes of mice lacking p52 or Bcl-3 to identify the cells directly affected by the lack of these proteins. Ultimately this will set the stage to elucidate molecular mechanism(s). Adoptive transfer of wild-type bone marrow into p52 knockout animals revealed that at least two cell types are responsible for the observed defects. A non-hemopoietic cell, most likely the follicular dendritic cell (FDC)2 itself, is critically dependent on p52 in formation of B cell follicles and of FDC networks. In addition, certain marginal zone (MZ) defects appear to be the result of an impaired non-hemopoietic cell. On the other hand, a hemopoietic cell is critically dependent on p52 for the appearance of metallophilic macrophages in the MZs, a subset of macrophages that is lacking in p52 knockout mice (9, 10, 24). We also demonstrate that Bcl-3 significantly enhances the critical activities of p52 in these cells, but that it is not absolutely required.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice deficient in p52 or Bcl-3 protein were generated by targeted disruption in embryonic stem (ES) cells as described previously (9, 22).
Ags used for immunization
2,4,6-Trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH), SRBC, and HRP (Sigma, St. Louis, MO) were used to elicit T-dependent immune responses in mice. SRBC were washed three times in PBS, and then a 10% solution in PBS was made. Mice were injected with 250 µl i.p. of this solution and sacrificed 7 days later. TNP-KLH was prepared as described previously (9). Briefly, 20 mg of lyophilized KLH (Pierce, Rockford, IL) was dissolved in 4 ml of potassium borate buffer (0.25 M at pH 9.2), and 3 mg of TNBS (2,4,6-trinitro-benzyl sulfonic acid; Sigma) was added along with 16 µl of sodium carbonate (1 M). The reaction was allowed to take place overnight, after which the protein derivatives were dialyzed against PBS (pH 7.4). TNP-KLH conjugates were frozen at -20°C until use.
Abs used for immunohistochemical analyses
The following Abs/markers were used at a final concentration of 10 µg/ml: anti-B220 (clone RA3-6B2), anti-CD35 (clone 8C12), anti-mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (clone MECA-367) (all from PharMingen, San Diego CA), anti-mouse laminin ß-2 chain (Upstate Biotechnology, Lake Placid, NY), anti-p52, and anti-Bcl-3 (Santa Cruz, Santa Cruz, CA). MOMA-1 (BACHEM Bioscience, Torrence, CA) and HRP-conjugated peanut agglutinin (PNA) were used at a 1:20 dilution (PNA; Dako, Carpinteria, CA). Anti-FDC Abs FDC-M1 and FDC-M2 were diluted 1:150 and 1:200, respectively (produced in house). Biotinylated goat anti-rat and goat anti-rabbit Abs (Vector Laboratories, Burlingame, CA) were used as secondary Abs at a 1:1000 dilution.
Adoptive bone marrow transfers
Bone marrow cells isolated from femora of 6-wk-old wild-type mice were injected (3 x 106 cells per mouse) into either p52 knockout recipients or control littermates (6–18 wk old); recipient mice had been irradiated with 900 rad or 600 rad 24 h previously. Seven to eight weeks after the adoptive transfers, recipients were injected i.p. with TNP-KLH (100 µg) adsorbed to alum. For cryosections, spleen and lymph nodes were collected 9 days after immunization.
Preparation of FDCs
Peripheral lymph nodes were collected from mice immunized with SRBC and then digested with an enzyme mixture. Briefly, the lymphatic tissue was incubated for 45 min at 37°C with RPMI 1640 (Life Technologies) containing collagenase class IV (450 U/ml, Worthington, Freehold, NJ) and DNase I while being stirred constantly. At 15-min intervals, the released cells were collected, and a new stock of enzyme solution was added to the remaining nondissolved tissue fragments. The final cell suspension was washed with RPMI 1640 to remove any nondigested tissue debris, overlayed onto a continuous Percoll gradient, and centrifuged at 600 x g for 30 min. Low density cells were collected, twice washed in RPMI 1640, and incubated with FDC-M2 Ab for 15 min at 4°C. Subsequently, the cell preparation was washed in RPMI 1640 and incubated with goat anti-rat IgG Microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) for 15 min at 4°C and applied to a MiniMACS separation column. Unlabeled cells were eliminated with washes of RPMI 1640. Cells attached to the column by conjugation with the Microbeads were eluted after removal of the magnetic field by rinsing with RPMI 1640.
Immunohistochemical (IHC) analyses
Single color IHC. Spleens were extracted, placed in OCT freezing medium (Miles Laboratories, Elkhart, IN) and flash frozen. Ten-micrometer thick acetone-fixed sections were processed as described previously. Briefly, tissue sections were rehydrated in PBS and then blocked for 30 min with Dako protein block. After blocking, sections were incubated for 30 min in the presence of relevant Ab (60 min in the case of FDC-M2), diluted in Dako Ab diluent, washed in PBS, and incubated an additional 30 min with biotinylated goat anti-rat secondary Ab in Dako Ab diluent. After quenching endogenous peroxide activity in 0.3% H2O2 for 15 min, tissue sections were incubated for a further 30 min with HRP-conjugated streptavidin (Vector Laboratories). Slides were then washed in PBS, and the avidin/biotin complexes were revealed with diaminobenzidine (DAB) tetrachloride chromogen (Vector Laboratories) according to the manufacturer’s instructions. Finally, slides were rinsed, counterstained with Methyl green (Vector Laboratories), and mounted with Permount (Fisher Scientific, Pittsburgh, PA).
Two color IHC. Double IHC was performed on acetone-fixed cryosections as described previously (9). Briefly, tissue sections were dehydrated in PBS for 10 min, blocked for 20 min in Dako protein block, and subsequently incubated for 30 min in the presence of the first biotinylated primary Ab, followed by a 30 min incubation with alkaline phosphatase (AP)-conjugated streptavidin diluted 1:50 (Vector Laboratories). The sections were then incubated for a further 30 min in the presence of the next relevant Ab, conjugated with HRP (except for PNA, which was for 1 h). As a final step, HRP-conjugated streptavidin was applied for 30 min. Endogenous peroxidase activity was quenched with 0.3% H2O2 in PBS for 15 min. All washings were done as described above, and all incubations were at room temperature. Alkaline phosphatase and HRP enzymatic activities were finally revealed with the Fast Red (Dako Corporation) and DAB chromogens, respectively, and specimens were mounted in aqueous mounting medium (Dako Corporation).
Localization of laminin
Unfixed, frozen tissue sections were washed in PBS two times for 20 min and blocked in 8% BSA in PBS for 1 h at room temperature. Sections were then washed with PBS four times, 15 min each at room temperature. Anti-mouse laminin ß-2 chain Ab (10 µg/ml) diluted in 1% albumin in PBS was applied overnight at 4°C. Subsequently, sections were washed twice for 30 min, quenched with 0.3% H2O2, and further incubated in the presence of goat anti-rat secondary Ab diluted at room temperature for the next 1 h. After three washings, 15 min each, the HRP reaction was revealed with DAB.
Intracellular labeling of p52 and Bcl-3 proteins
Acetone-fixed tissue sections were washed in PBS two times for 10 min and then blocked in the same way as described for laminin. After extensive washings in PBS, sections were permeabilized with 0.5% Triton X-100 for 5 min at room temperature, followed by two washes in PBS, 15 min each. Rabbit anti-mouse p52 or rabbit anti-mouse Bcl-3 Abs diluted in PBS/1% BSA/0.01% Triton X-100 were then applied for 2 h at room temperature. Sections were quenched with 0.3% H2O2 in PBS for 15 min. Biotinylated goat anti-rabbit Ab diluted in PBS/1% BSA/0.01% Triton X-100 was applied for 30 min followed by HRP-conjugated streptavidin. All incubations were done at room temperature. After the final washes, the HRP reaction was revealed with DAB.
RT-PCR analyses
RNA from splenocytes was isolated using Trizol reagent (Life Technologies, Grand Island, NY). cDNAs were synthesized using the Clontech (Palo Alto, CA) PCR kit starting with the same quantity of RNA (1 µg). The following primers were used: for B lymphocyte chemoattractant (BLC), 5' primer, TCA CCT AGG ATG AGG CTC AGC ACA GCA, and 3' primer, CAC TCA TTC TCT TCT CGA CGG GAA TTC CAC (the amplified PCR fragment size is 364 bp); for B220, 5' primer, GTG TAC AGC TGA TCT GGG ACG TGA AC, and 3' primer, GAA GAT AAT AGT TGA AAG TTT ATT ATG G (the amplified PCR fragment size is 520 bp); and for GAPDH, 5' primer, GGT GAA GGT CGG TGT GAA CGG A, and 3' primer, TGT TAG TGG GGT CTC GCT CCT G. PCRs were optimized for each set of primers and were performed using different numbers of cycles to ensure that amplification occurred in a linear range. After amplification, the products were electrophoresed in a 1% agarose gel and detected by ethidium bromide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice deficient in the p52 subunit of NF-B were found to be
impaired in their Ab response to T-dependent Ags (9, 10).
In particular, the mutant mice lacked Abs with switched isotypes,
whereas the IgM response was higher than controls. Similarly, mice
lacking Bcl-3 were defective in the generation of Abs with a switched
isotype (IgG2a), in response to infection with flu (22).
The ability to switch, however, was not seriously impaired, since
challenge with high dose Ags in the presence of adjuvants allowed for
the generation of switched isotypes in both types of mutant
mice.
Further analysis of these mice by immunohistochemistry revealed defects
that underlie the impaired Ab responses. Spleens were isolated 7 days
after challenge with 100 µg of TNP-KLH Ag adsorbed to alum and
cryosections prepared. p52 knockout mice were unable to generate FDC
networks, as judged by the lack of cells expressing high levels of
CD35, confirming our previous data (9) (Fig. 1
A, wild-type control; Fig. 1B, p52 KO; CD35 red). Typical germinal centers were not
observed either in the p52 knockouts as judged by PNA labeling
(wild-type, Fig. 1A; p52 knockout, Fig. 1B; PNA
blue). Instead, PNA+ clusters were detected in
spleens of challenged p52 knockouts but were located exclusively in the
T cell zone and were profoundly reduced in numbers.
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D; p52
knockouts, Fig. 1E). FDC-M2 labels FDC networks in
challenged wild-type animals and, in addition, weakly stains some
reticular structures throughout the white pulp (25). We
have previously shown lack of labeling with FDC-M1 in these mice
(9), an Ab that recognizes a distinct Ag from FDC-M2
(M. H. Kosco-Vilbois, unpublished observation). Combined, these
data using the two anti-FDC mAbs indicate an apparent complete lack
of mature FDC networks in p52-deficient mice. Impaired splenic MZ structures in p52-deficient mice, including a defect in basement membrane
We further processed these sections for MAdCAM-1, an adhesion
receptor present on sinus-lining cells in MZs and present on some FDCs
(26, 27). MAdCAM-1 expression could not be detected in p52
knockout mice (Fig. 1H), whereas it was readily observed in
the MZ and in follicular areas of wild-type littermate controls (see
Fig. 1G). Therefore, together with the data presented above,
no mature FDCs could be detected in p52 knockout mice. Beyond this, p52
knockout mice appear to have a defect associated with sinus-lining
cells. Possibly related to the absence of MAdCAM-1 expression on the
sinus-lining cells, a lack of deposition of the laminin ß-2 chain was
observed (Fig. 2A, wild-type
control; Fig. 2C, p52 KO). This basement membrane component
is usually detected near sinus-lining cells in wild-type mice (Fig. 2D, MAdCAM-1; Fig. 2E, MAdCAM-1 and laminin ß-2
chain). In addition to possible defects in or of sinus-lining cells,
there were other defects associated with splenic MZs. As demonstrated
previously (9), p52 knockouts lack metallophilic
macrophages that reside in the MZs of wild-type animals.
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Bcl-3-deficient mice exhibit defects similar to those of
p52-deficient mice, except that these deficiencies are generally less
severe. Challenged Bcl-3 knockout mice did form
PNA+ centers in response to high-dose Ags plus
adjuvants, even though the numbers of these centers appeared to be well
below those seen in wild-type mice (see Ref. 22 and Fig. 1C; PNA brown). Furthermore, the PNA+
B cell clusters of Bcl-3-deficient animals were located in B
cell-enriched zones, although these zones did not have a normal
follicular appearance (22). However, Bcl-3-deficient mice
did not develop detectable FDC networks in response to a single
antigenic stimulus, as judged by the lack of expression of FDC-M1
(22) or of high levels of CD35 (Fig. 1C, CD35
red; compare with control in Fig. 1A). Spleens from Bcl-3
knockouts did contain some FDC-M2-expressing cells, but there was no
evidence for a network of such cells in the follicles (Fig. 1F). Instead, FDC-M2 expression was dispersed throughout the
white pulp and was highest around the periphery.
Bcl-3 knockouts also expressed some MAdCAM-1 in their MZs (Fig. 1I), but only at very low levels compared with wild-type
(Fig. 1G). Similarly, laminin ß-2 chain deposits near the
sinus-lining cells of the MZ were significantly reduced in these mutant
mice as well, although the reduction was not as severe as that seen in
p52-deficient mice (Fig. 2B).
Impaired trapping of immune complexes in p52- and Bcl-3-deficient mice
These data, together with previously presented results,
demonstrate that both p52 and Bcl-3 knockout animals do not form proper
FDC networks under the conditions used here for stimulation. But
whereas p52 knockout mice lack known markers for FDCs (CD35, FDC-M1,
MAdCAM-1, and FDC-M2), Bcl-3 knockouts appear to contain some
FDC-M2+ and MAdCAM-1+
cells, especially in the MZ areas. Therefore, it is conceivable that
the Bcl-3 knockouts contain FDC precursor-like cells and that full
differentiation of these cells into networks does not occur in response
to a single dose of Ag. To investigate this more rigorously, the
ability to retain Ag, a functional feature of FDCs, was tested. Mice
were injected s.c. with 100 µg of the Ag HRP, on three separate
occasions, and then sacrificed 24 h after the last injection. At
this time, Ag is known to be bound to FDCs in wild-type mice, largely
in the form of Ag-Ab complexes both on cell processes and iccosomes
(28, 29). The presence of the HRP Ag was then detected
with peroxidase anti-peroxidase (PAP), and sections were also
labeled for CD35. Fig. 3 shows the
presence of CD35 (A, red; C, brown) and HRP Ag
(A and B, brown) in wild-type mice, whereas p52
knockout mice not only lack CD35 clusters (G and
I) but also do not contain any deposits of Ag (G
and H). Thus, p52 knockout mice lack cells capable of
capturing Ag, and they also lack markers classically associated with
FDCs. In contrast, Bcl-3 knockout mice have some deposits of Ag
(D and E; brown) and, surprisingly, also contain
some CD35+ cell clusters (D, red;
F, brown), although much less so than the wild-type. The
presence of CD35 clusters was unexpected in light of the total absence
of such clusters in the previous experiments. However, in contrast to
these earlier experiments, Ag has been administered three times,
conditions that apparently allowed a weak but detectable development of
an FDC network. Therefore, Bcl-3 knockouts are not absolutely blocked
for the generation of an FDC network, whereas p52 knockouts appear to
be totally impaired.
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To better define the identity of the cell responsible for the
observed defects, p52 knockout mice were reconstituted with wild-type
bone marrow. Six to eight weeks after adoptive transfer into either
lethally or sublethally irradiated p52 knockout mice, the resulting
chimeric mice were challenged with 100 µg TNP-KLH adsorbed to alum
and were sacrificed 9 days later. IHC analyses of the recipient
knockout mice still presented a lack of proper B cell follicles (Fig. 4A; B cells red), whereas such
follicles readily formed in recipient wild-type mice (Fig. 4B). PNA+ B cell clusters still
localized in the T cell zone in these recipient mutant mice, rather
than in the B cell follicles like wild-type recipients (Fig. 4, A and B, respectively; PNA brown). The p52
knockout recipients also still lacked cell clusters highly positive for
CD35 (Fig. 4C; control in D), and cells positive
for MAdCAM-1 (Fig. 4E; control in F), and FDC-M1
(Fig. 4G; control in H). Although
FDC-M2+ cell clusters were not present either,
some weak and widely scattered staining for FDC-M2 was seen throughout
the white pulp (Fig. 4I; control in J). The level
of CD35 labeling in the MZs of p52 knockout recipients was quite strong
(Fig. 4C) (although still well below that of mature FDC
networks), which may reflect an increased number of B cells that now
accumulate in this location (Fig. 4A). Together, these data
clearly indicate that p52 knockout mice receiving wild-type bone marrow
were still unable to form B cell follicles, FDC networks, or proper
germinal centers.
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K; control in
L). Therefore, the wild-type bone marrow cells corrected a
specific defect of the original knockout mice. This unexpected result
suggests that the absence of metallophilic macrophages in p52 KO mice
is independent of the lack of FDCs or B cell follicles. In contrast to
the restored metallophilic macrophages in the MZs, MAdCAM-1 expression
was not restored to sinus-lining cells (neither was it restored to
FDCs; see above) (Fig. 4E; control in F),
indicating that not all defects in the MZ can be corrected. Impaired expression of the transcript for the BLC chemokine
The inability of naive B cells to form follicles into which
Ag-activated PNA+ B cells can migrate could be
due to a defect intrinsic to B cells in receiving appropriate signals,
or to a defect in the generation of such signals by other cells. Since
mature p52-deficient B cells migrate properly into B cell follicles
when placed into recombination-activating gene (RAG)-deficient animals,
it is more likely that p52 knockout mice lack the ability to send a
proper signal to B cells. BLC is a recently described chemokine that
may be important in directing B cells to migrate into the follicles of
the spleen (30, 31). PCR analysis of reverse transcribed
RNA extracted from spleens 48 h after administration of SRBC
demonstrated an apparent lack of BLC transcripts in p52 knockout mice,
whereas such transcripts were readily detected in the wild-type
controls (Fig. 5A). However,
when analyzed by PCR on day 7 after Ag stimulation, some BLC
transcripts could be detected in the p52 knockout animals (Fig. 5B). Therefore, the expression of BLC in p52 knockout mice
appears to be partially disrupted, which may contribute to the observed
lack of B cell follicles and FDC networks. Impaired expression of BLC
in p52 knockout mice is also consistent with an impaired generation of
FDC networks, given that FDCs/stromal cells have been suggested to
express BLC (30, 31, 32). In support of this hypothesis, we
detected BLC by PCR in cell preparations from wild-type mice enriched
for FDCs (Fig. 5C; left). Although the FDC
preparations contain significant numbers of B cells (see B220
transcripts in Fig. 5C; left), B cells were not
the source of BLC, since purified B cells lacked detectable levels of
transcripts for this chemokine (Fig. 5C; right).
Therefore, FDCs are the most likely source of BLC transcripts in
wild-type mice, but this remains to be demonstrated directly.
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Given the critical importance of p52 and, to a lesser degree, of
Bcl-3 for formation of FDC networks, we investigated the expression of
these proteins in splenic sections (Fig. 6). Bcl-3 (Fig. 6A) and p52
(Fig. 6C) have a high level of expression in the
follicles/FDC networks subsequent to antigenic challenge. To control
for the specificity of the Ab, the anti-p52 peptide Ab was competed
for with the reactive peptide, and in this case no specific staining
was observed (Fig. 6D). In the case of the polyclonal
anti-Bcl-3 Ab, the specificity was demonstrated by its lack of
labeling in Bcl-3 knockout animals (Fig. 6B). To confirm the
localization, we have stained sections for both CD35 and p52 as well
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the inability to form any FDC networks or B cell follicles, p52 knockout mice nevertheless generate switched Abs to T dependent Ags, but only in the presence of adjuvants, as noted previously (9). Most likely, this is due to small PNA+ B cell clusters that develop in the T cell zones under these conditions. The activated B cells within these clusters may receive sufficient local stimulation to partially overcome the lack of FDC networks; as a result they appear able to undergo some affinity maturation and switching. High dose Ag plus adjuvant may provide sufficient levels of Ag and locally produced cytokines to bypass the need for a proper germinal center. Less potent and presumably more physiologic antigenic stimulation, however, may require the formation of proper germinal centers in association with an FDC network, a condition met only in wild-type mice, but not in p52 knockouts. Therefore, one function of FDCs may be to lower the threshold of stimulation required for effective Ab responses.
The PNA+ B cells that form clusters in the T cell zones of p52 knockout mice in response to Ag plus adjuvant may not be able to migrate properly for the same reason that B cells do not form follicles in these mice. The BLC chemokine is thought to provide an important signal to localize B cells into follicles, and transcripts for this chemokine cannot be detected in spleens of p52 knockout mice early after antigenic stimulation, although they are detectable at later times, although at apparently lower levels. In contrast, wild-type animals contain easily detectable levels at early and later times after antigenic stimulation, whereas Bcl-3 knockout mice exhibit partially reduced levels at early times (our unpublished observations). It has been suggested that BLC is produced by FDCs or underlying stromal cells within the B cell areas (32), and our enriched populations of FDCs do express this chemokine. Therefore, the impaired expression of BLC in p52 knockouts is consistent with the absence of FDC networks in these cells.
An important question concerns the identity of the cells responsible
for the lack of an FDC network. Previously we had ruled out mutant
mature B or T cells as sources for this defect. Adoptive transfers of
bone marrow from mice lacking p52 or Bcl-3 resulted in near normal Ab
responses in recombination-activating gene-deficient recipients, as
well as normal germinal centers (9) and fully developed
FDC networks (L. Poljak and U. Siebenlist, unpublished observations).
It was important to rule out intrinsic defects in B cells, since FDC
networks have previously been shown to depend on the production of
lymphotoxin-
by B cells (33). We demonstrate now that
transfer of wild-type bone marrow into p52-deficient animals does not
correct the FDC defects. The recipient mice are still unable to form
FDC networks. Therefore, the source of the defects leading to lack of
FDCs cannot be readily transferable hemopoietic cells but instead
appear to be non-hemopoietic cells. It remains to be demonstrated
whether FDCs are intrinsically impaired or whether the defects reside
in stromal cells distinct from FDCs that are necessary for formation of
FDC networks. Consistent with intrinsic defects in FDCs/stromal cells,
IHC analyses of sections (Fig. 6) and of partially purified FDC
preparations (L. Poljak and U. Siebenlist, unpublished results)
revealed high levels of expression of both p52 and Bcl-3 proteins in
FDC-like cells. Indeed, p52 levels had previously been reported to be
high in FDCs (34).
In contrast to the continued inability to form FDC networks in transfers of wild-type bone marrow into p52-deficient mice, this procedure did result in the appearance of metallophilic macrophages in the MZ. Therefore, lack of metallophilic macrophages in the original mutant strain must be due to a defect in a transferable, presumably hemopoietic cell type, possibly the metallophilic macrophages themselves. Interestingly, Bcl-3 knockouts had a significant, but only partial reduction of metallophilic macrophages, suggesting that Bcl-3 may again play a modulating role in this instance as well (22). The data imply that p52 has critical functions in at least two distinct cell types, possibly in FDCs and metallophilic macrophages, and that these functions may be enhanced by Bcl-3.
Unlike metallophilic markers, the expression of the MECA-367 epitope of
the addressin MAdCAM-1 on sinus-lining cells in the MZ was not restored
by transfer of wild-type bone marrow into p52 knockouts. MAdCAM-1 is
reportedly expressed on FDCs and on sinus-lining cells in wild-type
mice (26, 27), and its expression in Peyer’s patches has
been reported to be necessary for extravasation of blood lymphocytes
into these lymphoid organs (35). Adoptive transfers do not
restore expression of the MECA-367 epitope to either FDCs or
sinus-lining cells. The defect associated with sinus-lining cells may
or may not be intrinsic to these cells. In this regard, it could be
relevant that laminin ß-2 chain deposits near sinus-lining cells are
significantly reduced in the p52 knockouts. Therefore, the basement
membrane may be disrupted, and this in turn may affect other cells,
including FDCs, that receive critical signals from this matrix via
their integrin receptors (36, 37, 38). In fact, signaling via
2-ß1 integrin
receptors has been reported to induce protein translation of preformed
Bcl-3 mRNAs in platelets, although it is not known whether such a
control mechanism also operates in other cells (39). It
remains to be investigated whether lack of expression of MAdCAM-1 on
sinus-lining cells is related to the defect in laminin ß-2 chain
deposition and/or the defect in FDC network formation.
Disruptions of the microarchitecture of secondary lymphoid organs have
been observed in several other mutant mice. In particular, mice
deficient in TNF-, lymphotoxin (LT), LTß, LTßR, or TNFRI all
lack FDC networks (40, 41, 42, 43). In addition, some of these
mice share other phenotypes with the p52 knockout mice, although all of
the mutants present with unique disruptions as well. Nevertheless, an
interdependent network of these TNF/LT ligands, their receptors, and
NF-B is suggested (p50/p52 double knockout mice have very severe
disruptions in lymphoid architecture). Although it is possible that
loss of p52 could impair the expression of these particular mediators
and thus cause the observed phenotypes, PCR analyses have not revealed
major changes in the mRNAs for these proteins in total splenocytes from
our knockouts (L. Poljak and U. Siebenlist, unpublished observations).
Furthermore, adoptive transfers of wild-type hemopoietic precursors
into the knockout mice did not cure the majority of the defects,
arguing against intrinsic defects in hematopoietic cells. Thus
activation of NF-B complexes downstream of the activities of the TNF
and lymphotoxin ligands may be critical to lymphoid architecture, with
the p52 protein in particular playing an essential role for B cell
follicle formation, FDC network development, and a proper MZ.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: FDC, follicular dendritic cell; BLC, B lymphocyte chemoattractant; TNP-KLH, 2,4,6-trinitrophenyl-keyhole limpet hemocyanin; PNA, peanut agglutinin; MZ, marginal zone; IHC, immunohistochemistry; PAP, peroxidase anti-peroxidase; LT, lymphotoxin.
Received for publication July 13, 1999. Accepted for publication September 30, 1999.
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