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* Department of Pathology and Committee on Immunology, University of Chicago, Chicago, IL 60637;
Sir William Dunn School of Pathology, Oxford, United Kingdom;
Serono Pharmaceutical Research Institute, Geneva, Switzerland; and
Department of Microbiology and Immunology, University of California, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R- and
LT
-deficient mice, suggesting that signaling by membrane LT is
required for the presence of CR-Fc+ DCs in the spleen.
Interestingly, analysis of mutant mice that lack T, B, or NK cells
demonstrates that B cell-derived membrane LT is essential for the
unique localization of CR-Fc+ DCs in the spleen. Using bone
marrow transfer and ligand-blocking approaches, we provide evidence
that B cell-derived LT acts indirectly on CR-Fc+ DCs
through LTR+ stromal cells. In analogous fashion to
certain Ag-activated T and B cells, CR-Fc+ DCs, expressing
CXCR5, localize to primary lymphoid follicles in response to CXC ligand
13 (B lymphocyte chemoattractant). Together, we propose that B
cells play a central role in establishing the chemotactic gradient that
attracts not only Ag-activated T and B cells but also Ag-carrying
CR-Fc+ DCs. In turn, CR-Fc+ DCs and T cells
home to B cell follicles to interact with B cells in the developing
germinal center. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Humoral immune responses require the physical contact of APCs and rare Ag-specific B and T lymphocytes in secondary lymphoid organs (4, 5). Recent data strongly implicate the intimate involvement of chemokines and their receptors in both immune cell trafficking and the compartmentalization of lymphoid organs (4). DCs are known to migrate to T cell zones to interact with T cells (12, 13). Extensive studies have focused on the mutual regulation of T cells and DCs and the mechanisms controlling the migration of maturing DCs to the T cell zone (5). The coordinated action of several chemokines expressed in the T cell zones of secondary lymphoid organs, including EBV-induced gene 1 ligand chemokine/CCL19 and secondary lymphoid tissue chemokine/CCL21, attracts mature DCs and naive T lymphocytes (14, 15, 16). They are likely to play a key role in facilitating the requisite encounter between Ag-bearing DCs and specific T cells. In contrast to the T/DC interaction, the interaction between B cells and DCs in vivo is significantly less well defined, as is the mechanism underlying the migration of DCs into B cell follicles.
Lymphotoxin (LT) is required for the establishment of a
microenvironment necessary for the localization of immune cells in the
spleen (4, 17, 18, 19). LT may exist in a secreted form as
a homotrimeric protein (LT3), binding to the two defined TNFRs,
TNFRI and TNFRII (20, 21). LT also exists in a
heteromeric form with membrane LT, as LT12 (22, 23). Membrane LT is expressed on activated T, B, and NK cells
and signals through a high-affinity interaction with LTR, found
predominantly on nonlymphoid cells (21, 22, 23, 24, 25). This signal
is critical for lymph node and Peyer’s patch development and formation
of microenvironments in the secondary lymphoid tissue (18, 26, 27, 28, 29, 30, 31, 32). The necessity of membrane LT and its receptor LTR on
normal lymphoid organ development and architecture can be attributed,
at least in part, to its ability to induce the expression of various
chemokines in lymphoid organs (19). The lack of chemokines
may contribute to disorganized T cells, B cells, and DCs in
LT-/- mice (17). Membrane LT
has been shown to be required for stromal cell expression of CXC ligand
(CXCL)13 (B lymphocyte chemoattractant (BLC)) in the B cell area of the
spleen (19). CXCL13 is strongly expressed in the follicles
of Peyer’s patches, the spleen, and lymph nodes (33). The
interaction between CXCL13 and its receptor CXCR5 on B cells is
required for B cell migration into lymphoid follicles
(33). A subset of B Th cells termed follicular B Th cells
also expresses CXCR5, which may help direct these cells to the B cell
follicles to support Ig production (34).
We now demonstrate that CR-Fc+ DCs, expressing CXCR5, localize to primary lymphoid follicles in response to CXCL13. We demonstrate that B cell-derived, but not T or NK cell-derived, membrane LT is essential for the regulation of CR-Fc+ DCs by controlling CXCL13 expression on the follicular stromal cells. This study reveals a novel mechanism by which a subset of DCs migrates toward the B cell follicle to meet lymphocytes in response to the same B cell-mediated chemokine. Therefore, B cells bring Ag-specific T cells, B cells, and Ag-carrying APCs together for effective IgG responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6), B6 µMT (BCR-/-), B6
TCR-/-
-/-
(TCR-/-), and B6 recombination-activating gene
(RAG)-1-/- mice as well as CD3
-transgenic
mice were purchased from The Jackson Laboratory (Bar Harbor,
ME). LT-/- mice (backcrossed to B6 mice for
seven generations) and their wild-type littermates on a B6 background
were bred under specific pathogen-free conditions.
CXCL13-/- mice were described previously
(35). LTR-/- mice were kindly
provided by Dr. K. Pfeffer (Technical University of Munich, Munich,
Germany) (28). Animal care and use were in accordance with
institutional guidelines.
Reagents and in vivo blockage of membrane LT or LIGHT activity
Production and preparation of anti-LT Ab
(25) LTR-Ig fusion protein (17) used in
this study have been previously described. Chinese hamster ovary cells
transfected with pMIgV expression vector containing DNA coding sequence
of herpesvirus entry mediator (HVEM) extracellular domain fused to that
of Fc portion of murine IgG2a were kind gifts of Dr. L. Chen (Mayo
Clinic, Rochester, MN). The HVEM-Ig chimeric protein was purified from
supernatants of transfected cells by protein A chromatography. To block
membrane LT or LIGHT (homologous to LT, inducible expression, compete
with herpesvirus glycoprotein D for HVEM on T cells or TNF superfamily
14) activity in mice, the LTR-Ig and anti-LT Ab or HEVM-Ig
(100 µg/injection) were given i.p., and the presence of
CR-Fc+ DCs was determined 7–14 days later by
immunohistology. The CR-Fc fusion protein was generated as described
before (10). In brief, the CR domain of the
mannose receptor was amplified by RT-PCR and cloned into the pIG
expression vector. The resulting plasmid was transfected into COS-7
cells. The chimeric protein consisting of CR fused to the Fc portion of
human IgG1 was purified from supernatants of transfected cells by
protein A column and stored at -20°C.
BM transplantation
Bone marrow (BM) was harvested and recipients were prepared as described previously (32, 36). Recipient mice were lethally irradiated with 1050 rad and reconstituted with 5 x 106 donor BM cells. Six weeks after transplantation, recipients were sacrificed and the presence of CR-Fc+ DCs in the spleen was determined by immunohistology.
Transfer of lymphocytes
Whole spleen cell suspensions were prepared from a single mouse donor by mincing with scissors and teasing between two frosted microscope slides. B cells were enriched using a nylon wool column as described (37) and further purified by using a column-based purification technique (StemCell Technologies, Paisley, U.K.) according to the manufacturer’s instructions. In brief, B cell-enriched spleen cell suspensions were incubated with a mixture containing anti-CD3, CD11b, TER119, and Gr-1 Abs. After the incubation at 4°C, magnetic colloids were added to cell-Ab complexes to deplete cells stained with the above Abs by using a column and a magnet. The purity of cells after column depletion was determined by flow cytometric analysis of purified cells. These preparations contained 95–97% B cells, defined as IgM+B220+ by flow cytometry. There were no detectable contaminating T cells. A total of 107 purified B cells was injected i.v. into each recipient. Three, 4, or 5 wk after transfer, spleens from these mice were analyzed by immunohistology.
Immunohistology
Spleens were harvested, embedded in OCT compound (Miles-Yeda, Rehovot, Israel) and frozen at -70°C. Frozen sections (6–10 µm thick) were fixed in cold acetone. For immunohistochemical staining, endogenous peroxidase was quenched with 0.06% H2O2 in methanol. The sections were preblocked with 2% normal mouse serum, rabbit serum, and goat serum in PBS for half an hour at room temperature in a humidified chamber. Staining for B cells and CR-Fc+ DCs was done by first incubating with FITC-conjugated anti-B220 (BD PharMingen, San Diego, CA) at a 1/100 dilution and CR-Fc at a 17.5 µg/ml concentration (1/200 dilution of the stock preparation) in blocking buffer. HRP-conjugated rabbit anti-FITC (DAKO, Carpinteria, CA) and alkaline phosphatase-conjugated rabbit anti-human IgG (H and L chains) (Southern Biotechnology Associates, Birmingham, AL) were added 1 h later. Staining for metallophilic macrophages was done with MOMA-1 (Serotec, Oxford, U.K.) at a 10 µg/ml concentration followed by a secondary incubation with HRP-conjugated goat anti-rat IgG (Southern Biotechnology Associates) (38). Staining of CD11c+ DCs was done with biotin-conjugated anti-CD11c (BD PharMingen) at a 1/100 dilution. Alkaline phosphatase-conjugated streptavidin (Vector Laboratories, Burlingame, CA) was used as second-step Ab. Color development for alkaline phosphatase and HRP was performed with an alkaline phosphatase reaction kit (Vector Laboratories) and with 3,3'-diaminobenzidine (Sigma-Aldrich, St. Louis, MO), respectively. For immunofluorescence staining, sections were blocked with 2% normal mouse serum, rabbit serum, and goat serum in PBS for half an hour at room temperature in a humidified chamber. Blocking solution was replaced with 50 µl of primary Abs, PE-conjugated anti-B220 (BD PharMingen), CR-Fc, or biotin-conjugated anti-CD11c (BD PharMingen), diluted 1/100 in blocking solution, and sections were incubated for 1 h at room temperature in a humid chamber. FITC-conjugated donkey anti-human Ig (Jackson ImmunoResearch Laboratories, West Grove, PA) and PE-conjugated streptavidin (ImmunoTechnologies, Cedex, France) were used as secondary reagents for CF-Fc and biotin-conjugated anti-CD11c, respectively. Specimens were mounted in Mowiol 4-88 (BD Biosciences, La Jolla, CA) containing 10% 1,4-diazobicyclo [2.2.2] octane. Samples were analyzed within 48 h using a Zeiss Axioplan microscope (Zeiss, Oberkochen, Germany) and a Photometrics PXL CCD camera (Photometrics, Tucson, AZ). No-neighbor deconvolution was performed using Openlab v2.0.6 (Improvision, Lexington, MA).
Enrichment of DCs
B6 mice were injected s.c. in each limb (50 µg per limb) and at the end of the tail (100 µg) with chicken egg OVA (Sigma-Aldrich) emulsified in IFA. Forty-eight hours later, the draining lymph nodes were collected and digested with 2 mg/ml collagenase and 100 µg/ml DNase for 30 min at 37°C and then gently pipetted in the presence of 0.01 M EDTA for 1 min. CD11c+ DCs were positively enriched by magnetic cell sorting (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. In brief, single-cell suspensions were sequentially incubated with biotinylated mouse anti-CD11c (BD PharMingen) at 10 µg/ml and MACS streptavidin MicroBeads (Miltenyi Biotec) before proceeding to magnetic separation. Positive selection column type MS+RS+ was used. CXCR5 expression on CR-Fc+ cells was analyzed on FACScan (BD Biosciences) by staining positively enriched CD11c+ DCs.
FACS analysis
The hybridomas 2.4G2 (anti-CD32) were obtained from American Type Culture Collection (Manassas, VA) and used to block FcR binding. A polyclonal rabbit antiserum prepared by immunization with CXCR5 peptides has been described previously (39). The enriched CD11c+ DCs were stained and analyzed by two-color flow cytometry on a FACScan (BD Biosciences). In brief, the enriched CD11c+ DCs were first blocked with anti-CD32 before being incubated with anti-CXCR5 Ab at a 1/50 dilution and CR-Fc at a 10 µg/ml concentration. The secondary reagents were goat anti-rabbit IgG (H and L chains) conjugated to PE (Southern Biotechnology Associates) and donkey anti-human IgG (H and L chains) conjugated to FITC (Jackson ImmunoResearch Laboratories).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the spleens of wild-type mice, CR-Fc+ DCs
are found predominantly in the marginal zones, with a small proportion
inside the B cell follicles (Fig. 1
A). Pathogenic products such
as LPS trigger the migration of the majority of
CD11high DCs into the T cell areas of lymphoid
organs (40). To study the migration pattern of
CR-Fc+ DCs in response to LPS, we analyzed serial
splenic sections of wild-type mice 24 h after LPS injection. We
found dramatically decreased numbers of CR-Fc+
DCs in the marginal zone and increased LPS-activated
CR-Fc+ DCs in the B cell area (Fig. 1A). Staining of spleen sections with anti-CD11c
verified the migration of the majority of CD11c+
DCs into the T cell zone, in the center of the white pulp (Fig. 1B). Double staining of these sections with CR-Fc and
anti-CD11c confirmed the unique response of this
CD11c+CR-Fc+ DC subset in
migrating to the B rather than T cell zones (Fig. 1B). These
results suggest that activated CR-Fc+ DCs in
marginal zone may respond to trafficking cues differently from the
majority of conventional DCs.
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We have previously reported that LT-/-
mice present with disorganized DC distribution and disrupted T/B cell
segregation, and fail to generate both primary and secondary IgG
response to foreign Ags (17, 32, 41). Therefore, LT is a
candidate cytokine for regulating the positioning of
CR-Fc+ DCs. To explore this possibility, spleens
from LT-/- mice were stained for
CR-Fc+ DCs. Interestingly, no
CR-Fc+ cells were detectable in the spleen (Fig. 2A), suggesting an essential
role for LT in regulating CR-Fc+ DC presence in
the spleen. As LT-/- mice lack both soluble
LT3 and membrane LT12, it was important to isolate the
contributions of each. To study whether LT3 is required for the
presence of CR-Fc+ DCs, spleens from
TNFRI-/- mice were analyzed. Staining with
CR-Fc showed a pattern of distribution of CR-Fc+
cells indistinguishable from wild-type (data not shown), pointing to
the dispensability of LT3 signaling through TNFRI. To isolate the
involvement of LTR, spleens from LTR-deficient mice were stained
for CR-Fc+ DCs. These mice showed
CR-Fc+ DC staining in neither the marginal zone
nor the B cell follicle (Fig. 2A). These experiments suggest
that both LT and LTR are essential for the presence of
CR-Fc+ DCs in the spleen.
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We have previously reported that LT participates in the
organization of lymph nodes and Peyer’s patches early in development
and actively maintains lymphoid microenvironments throughout life. To
determine whether membrane LT is required for the maintenance of
CR-Fc+ DCs in the spleen, we treated adult B6
mice with 100 µg LTR-Ig fusion protein. Within a few days,
CR-Fc+ DCs had completely disappeared from these
spleens (Fig. 2
B), suggesting that LTR ligands are
required. However, soluble LTR-Ig can bind both membrane LT and
LIGHT (TNF superfamily 14), a newly defined TNF superfamily member
(42, 43). To confirm the role of membrane LT12 in
the maintenance of CR-Fc+ DCs in the spleen, mice
were treated with anti-LT Ab, which specifically blocks membrane
LT but not LIGHT. CR-Fc+ DCs were undetectable in
both the marginal zone and B cell area (Fig. 2B). To exclude
a possible contribution by LIGHT, wild-type mice were treated with
soluble HVEM-Ig. HVEM-Ig blocks LIGHT function without any interference
with membrane LT12 (42, 43). Immunohistochemical
analysis of HVEM-Ig-treated mice showed unperturbed
CR-Fc+ DC distribution (Fig. 2B).
These results substantiate our finding that membrane LT, but not LIGHT,
is essential for the maintenance of CR-Fc+ DCs in
the spleen.
Control of CR-Fc+ DCs in the spleen by B cells but not T cells or NK cells
LT12 is expressed predominantly on T, B, and NK cells. To
test whether T and B cells are required for the regulation of
CR-Fc+ DCs in the spleen,
RAG-1-/- mice, which lack both T and B cells,
were used. RAG-1-/- mice showed barely
detectable CR-Fc+ cells in spleen (Fig. 3A), suggesting that either T
or B cells, or both, may regulate splenic CR-Fc+
DCs in the adult mice (Table I). To
investigate whether T cells were required for the homeostasis of
CR-Fc+ DCs, spleens from
TCR-/- mice were stained with CR-Fc. Spleens
from TCR-/- mice manifest a strong staining of
CR-Fc, similar to that of wild-type mice (Fig. 3A). Normal
staining of CR-Fc+ DCs in the CD3-transgenic
mice, which are defective in NK and T cells, further verifies that
neither NK nor T cells are involved in the regulation of
CR-Fc+ DCs (Table I). Interestingly,
BCR-/- mice show undetectable staining of
CR-Fc+ DCs in the spleens (Fig. 3A).
These data indicate that B cells, but not T or NK cells, are essential
for the regulation of CR-Fc+ DCs.
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12 and B cells are required
for the regulation of CR-Fc+ DCs. To further
substantiate the role of membrane LT12 on the B cells in the
control of CR-Fc+ DCs,
TCR-/- mice were administered with 100 µg of
LTR-Ig fusion protein and the spleens were collected 7 days later.
No CR-Fc+ cells could be detected in either the
splenic sinus or the follicular DC (FDC) area in
TCR-/- mice treated with LTR-Ig fusion
protein (Fig. 3A), confirming that constant signaling from
LT-expressing B cells is required for regulation of
CR-Fc+ DCs. To exclude the possibility that the
absence of CR-Fc+ DCs is due to the absence of
the marginal zone, we stained metallophilic macrophages normally
present in the marginal zones with MOMA-1 Ab.
MOMA-1+ cells were clearly present (Fig. 3A), suggesting that there are intact marginal zones in the
spleens of both TCR-/- and
BCR-/- mice. Therefore, the lack of
CR-Fc+ DCs in the BCR-/-
mice may not be attributed by the absence of marginal zone. It also
suggests that MOMA-1+ cells are not the same
cells as CR-Fc+ DCs. In fact, many
MOMA-1+ cells did not colocalize with
CR-Fc+ DCs when double staining was
performed (data not shown). Whether the two types of cells are derived
from the same lineage remains to be determined.
BM from TCR-/- mice may contain other type of
cells different from those of BCR-/- mice. To
investigate whether mature B cells are sufficient for the regulation of
CR-Fc+ DCs, purified B cells from the splenocytes
of TCR-/- mice were transferred into
RAG-1-/- mice. Three weeks after transfer,
lymphoid tissues from these mice were analyzed. CR-Fc expression was
restored in the reconstituted mice (Fig. 3B), suggesting
that mature B cells are sufficient to support the presence of
CR-Fc+ DCs in the absence of T and NK cells. To
further prove that it is LT derived from B cells that restored the
CR-Fc+ DCs in the
RAG-1-/- mice, purified B cells from the spleen
of LT-deficient mice were transferred into
RAG-1-/- mice. CR-Fc staining in the spleens of
the recipients was analyzed by histology 3, 4, or 5 wk after transfer.
We found that B cells were present in the spleens in numbers similar to
RAG-1-/- recipients of wild-type B cells.
However, CR-Fc+ DCs clearly did not recover in
mice that received LT-deficient B cells (Fig. 3B). These
experiments clearly demonstrate that LT derived from B cells is
sufficient to support the presence of CR-Fc+ DCs
in the spleen.
LTR on stromal cells is required for the presence of
CR-Fc+ DC in the spleen
Membrane LT signaling through LTR is necessary for the presence
of CR-Fc+ DCs in the spleen. To determine whether
BM-derived cells or radio-resistant stromal cells are the membrane
LT12 responding cells, reciprocal BM transplantation was
performed. In lethally irradiated wild-type mice reconstituted with BM
from wild-type mice, CR-Fc staining was maintained in the FDC area and
marginal sinus (Fig. 4). While no
CR-Fc+ cells were detected in the spleens of
LTR-/- mice reconstituted with BM from
wild-type mice, CR-Fc staining was normal in wild-type mice that
received BM from LTR-/- mice (Fig. 4). These
data indicate that LTR on BM-derived cells is not required for the
presence of CR-Fc+ DCs in the spleen. In
contrast, LTR expressed on stromal cells in the spleen is important
for the presence of CR-Fc+ DCs in the spleen. It
is likely that LT does not act directly on CR-Fc+
DCs, a BM-derived population, but rather on LTR-positive stromal
cells.
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It has been reported that membrane LT is required for the CXCL13
(BLC) production by follicular stromal cells (19). CXCL13
is thought to pair exclusively with CXCR5 to attract activated
Ag-specific T and B cells into B cell follicles (4). We
wondered whether CXCL13 is also required for the normal presence of
CR-Fc+ DCs in the spleen. To test that
hypothesis, spleens from CXCL13 (BLC)-deficient
(CXCL13-/-) mice were stained for
CR-Fc+ cells. Interestingly, few
CR-Fc+ were detected in the spleens of
CXCL13-/- mice (Fig. 5A). This result confirms the
requirement for CXCL13 for the presence of CR-Fc+
DCs in both the marginal zones and B cell follicles in the spleen. It
was interesting to test whether CR-Fc+ DCs express CXCR5
(Burkitt’s lymphoma receptor-1) so that they can directly respond to
CXCL13 gradient. To test the hypothesis, we enriched for
CR-Fc+ DCs from the lymph nodes after
immunization with OVA and stained the CR-Fc+ DCs
with anti-CXCR5 Ab. Most of the purified
CR-Fc+ DCs were positive for CXCR5 (Fig. 5B). This result supports our hypothesis about the possible
mechanism explaining the role of LT in regulating
CXCR5+CR-Fc+ DCs via a
CXCL13 gradient established in B cell follicles by B cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- nor
LTR-/- mice have
CR-Fc+ DCs in the spleen (Fig. 2A),
while TNFRI- or TNFRII-deficient mice have relative normal staining of
CR-Fc (P. Yu and Y.-X. Fu, unpublished data). 2) Transfer of
LT-expressing lymphocytes into RAG-1-deficient mice restores the
presence of CR-Fc+ DCs in the spleen while
LT-deficient lymphocytes fail to do so (Fig. 3B). 3)
Preexisting CR-Fc+ DCs in wild-type mice
disappeared completely within 7 days after blockade of LTR
signaling, by either LTR-Ig or anti-LT Ab (Fig. 2B). In contrast, total CD11c+ cells
were only slightly reduced after 7 days and reduced up to 60% in the
second week of treatment.
We have also determined that B cells, but not T or NK cells, are
essential for the regulation of CR-Fc+ DCs. 1)
BCR-/- but not TCR-/-
mice show undetectable CR-Fc staining (Fig. 3A). 2) CR-Fc
staining disappears completely after the blockade of LT signaling in
TCR-/- mice (Fig. 3A). 3) CR-Fc
staining in RAG-1-/- mice recovers completely
after LT-expressing B cells are transferred (Fig. 3B). It is
possible that T cells, mostly located in T cell zones, have little
opportunity to physically contact stromal cells in B cell zone via
membrane LT. This is consistent with the result that B cell-derived
membrane LT, rather than soluble LT, plays an important role in
establishing a microenvironment inside B cell follicle conducive for
the formation of GCs.
We have further demonstrated that B cell-derived LT does not act
directly on CR-Fc+ DCs to regulate its
localization, but rather acts indirectly through LTR-expressing
stromal cells. It has been shown that membrane LT is required for
stromal cell expression of CXCL13 in the B cell area of the spleen
(19). We show in this study that
CR-Fc+ DCs express CXCR5, and that the chemokine
CXCL13 is required for the normal presence of
CR-Fc+ DCs. These lines of evidence strongly
support the proposal that B cell-derived LT acts on follicular stromal
cells to form a CXCL13 gradient, which brings Ag-carrying
CR-Fc+ DCs and Ag-specific T and B cells
together. In addition to providing Ag, CR-Fc+ DCs
also produce high levels of lymphocyte chemoattractant, macrophage
inflammatory protein-1, and other factors to attract and activate T
cells (11). The dynamic direct contact is also required
for the interaction between membrane ligands and receptors, such as
CD40 and CD40 ligand, for effective GC formation and IgG response
(3, 5). A recent report has shown that B cells can contact
APC directly to form a B synapse and acquire Ags from APCs
(9). Therefore, B cells can play a central role in
bringing relevant DCs and rare Ag-specific T and B cells together via
the same chemokine to coordinate them for developing GC.
Accumulating evidence indicates that B cell function may not be limited to Ab production. B cells may also actively create lymphoid microenvironments that promote their interaction with other cells. A recent study demonstrated that early T cell responses to Listeria monocytogenes in µMT (BCR-/-) mice is impaired despite the transfer of serum from Listeria-infected wild-type mice, even though B cell Ag presentation is not involved in this model (44). Others have recently reported that BCR-/- mice are more susceptible to acute infection, and this cannot be completely restored by the transfer of additional sera from wild-type mice (45). We also found BCR-/- mice were much more susceptible to acute L. monocytogenes infection and harbor higher titers of Listeria in the spleen 2–4 days after infection (P. Yu and Y.-X. Fu, unpublished data). It is possible that B cells are also important for the establishment of appropriate microenvironments in the spleen to clear Listeria infections. For example, FDC development is also dependent on signals provided by B cell-derived membrane LT (18, 46), as is the maintenance of the expression of marginal zone mucosal addressin cell adhesion molecule-1 and other adhesion molecules. The marginal zone area may play a role in host response to infection and Ab response (38). The impaired marginal zone in BCR-/- mice may also increase their susceptibility to infection. B cells actively regulate and organize lymphoid structure and microenvironments to facilitate effective immune responses by providing membrane LT and possibly other signals. LT-deficient B cells have no intrinsic defects in generating Abs (32, 47). Rather, the lack of LT from B cells impairs the formation of a proper microenvironment for effective IgG responses.
LT-deficient mice fail to generate both primary and secondary IgG
response to foreign Ags (32, 41). Although FDCs play an
important role in presenting Ag-Ab complexes to activated B cells in
the GC, the lack of FDC in LT-/- mice cannot
be the major reason for the absence of early primary IgG response. In
contrast to LT-/- mice,
TNF-/- mice also lack FDC/GC, yet show
minor reductions in IgG responses. Initial Ag may be carried by a
subset of DCs that are also able to stimulate T and B cells (T and B
synapse). We suspect that a subset of Ag-carrying DCs may migrate into
B cell follicles to facilitate a positive feedback loop for developing
GCs. Chemokines, such as CXCR13, generated from B cell follicles may be
required for such migration. CR-Fc+ DCs that
traffic to primary lymphoid follicles are suitable candidates important
for the early activation of B cells. They were initially identified as
Ag-carrying cells. Most of them are strategically localized around the
marginal zone, an area critical to the transport of Ags into the B cell
follicles, and are capable of moving into B cell zones soon after
immunization to promote GC formation. Not coincidentally, they express
CXCR5 and only move into B cell follicles, even after the LPS
stimulation that drives most other DCs into the T cell zone. We have
found marginal changes in BLC production in the B cell follicle or
CXCR5 expression in CR-Fc+ DCs after LPS
stimulation. LPS may have multiple effects on the activation and
maturation of DCs. What LPS triggers to allow the migration of
CR-Fc+ DCs into B cell zone remains to be
determined.
In summary, B cell-derived LT controls the development and maintenance
of CR-Fc+ DCs, leading to the formation of
secondary B cell follicles. Therefore, this study presents an example
of B cells playing multiple roles in orchestrating efficient IgG
responses by constantly interacting with their surrounding cells. More
specifically, we propose that B cell-derived membrane LT controls
CR-Fc+ DCs by regulating CXCL13 expression on the
LTR-expressing follicular stromal cells. In turn,
CXCR5+CR-Fc+ DCs respond to
the B cell-mediated CXCL13 gradient and home to B cell follicles to
regulate and present Ag to B cells for an Ab response to occur. Better
understanding of such interactions may help us to design proper
approaches to alter unwanted immune responses and promote a more
desired effect.
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Acknowledgments |
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R-Ig and HVEM-Ig were produced in the National Cell Culture
Center (Minneapolis, MN). We thank James Lo and Sumit Subudhi for their
critical comments. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Yang-Xin Fu, Department of Pathology, University of Chicago, MC3083, 5841 South Maryland Avenue, J541, Chicago, IL 60637. E-mail address: yfu{at}midway.uchicago.edu
3 Abbreviations used in this paper: DC, dendritic cell; FDC, follicular DC; CR, cysteine rich; HVEM, herpesvirus entry mediator; LT, lymphotoxin; CXCL, CXC ligand; BM, bone marrow; RAG, recombination-activating gene; GC, germinal center; BLC, B lymphocyte chemoattractant; LIGHT, homologous to LT, inducible expression, compete with herpesvirus glycoprotein D for HVEM on T cells or TNF superfamily 14.
Received for publication August 15, 2001. Accepted for publication March 18, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are ligands for herpesvirus entry mediator. Immunity 8:21.[Medline]
-dependent fashion. J. Exp. Med. 187:1009. and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue. Eur. J. Immunol. 27:2600.[Medline]This article has been cited by other articles:
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