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*
Division of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, Osaka, Japan; and
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our group as well as Cooper’s colleagues independently cloned the cDNA
for murine BST-1 and BP-3, respectively, and found that both are
identical molecules (4, 5). In the hemopoietic system and lymphoid
tissues, the surface expression of murine BST-1/BP-3 is detected on T
or B cell progenitors, immature B cells, mature myeloid cells, and
reticular cells in the splenic white pulps, lymph nodes, and Peyer’s
patches (PP) (13, 14, 15, 16). The expression of murine BST-1 in fetus, is
maximally detected on thymocytes and liver B progenitors on days 15 to
16 and 17 of gestation, respectively, and gradually decreased until
birth. In adult mice, the surface expression of BST-1 on both B and T
cell progenitors begins around the stage when gene rearrangement of the
Ag receptors is initiated (15), suggesting its role in lymphopoiesis.
Actually, an anti-BST-1/BP-3 mAb, IF-7, has synergistic effects on
anti-CD3-induced proliferation of pre-T cells and promotes the
generation of TCR
ß+ cells in fetal thymic organ
culture (16). This may indicate that BST-1 functions as a receptor.
Along this line, we previously demonstrated that cross-linking of BST-1
induces tyrosine phosphorylation (17). These studies suggested that
BST-1 may affect B and T cell development.
Although the expression of BST-1 is down-regulated on mature B cells in the secondary lymphoid tissues, it should be noted that these B cells are surrounded by the reticular cells expressing BST-1. This suggests that BST-1 on the reticular cells may play an important role in supporting the growth, differentiation, and survival of mature B cells.
It is also interesting to note that BST-1 is highly expressed in gut (5, 14). However, the role of BST-1 in the intestinal immune system is unknown. The mucosal immune system has been shown to possess a unique immunoregulatory mechanism that distinctively differs from the systemic immune compartment for the induction of IgA Ab responses (18, 19). For example, immunity to Ags administered orally is achieved by the stimulation of Th cells and IgA committed sIgA+ B cells in the gut-associated lymphoreticular tissues. It has been shown that oral immunization of mice with tetanus toxoid and cholera toxin (CT) resulted in the generation of Ag-specific Th2-type CD4+ T cells for the induction of IgA-producing cells (20, 21). These Ag-triggered Th2 cells and sIgA+ B cells in gut-associated lymphoreticular tissues then migrate to distant mucosal effector sites, such as the lamina propria (LP) region of the intestine, where terminal differentiation of the sIgA+ B cell into IgA-producing plasma cells takes place under the influence of Th2 cytokines, including IL-5 and IL-6. Since BST-1 is expressed by the brush border of the intestinal epithelial cells and the reticular cells in PP (14), it is interesting to postulate that BST-1 may be involved in the Ag-specific mucosal immune response.
To evaluate the roles of BST-1 in vivo, our initial effort was aimed at the establishment of BST-1 knockout mice using homologous recombination. The lymphocyte development of BST-1-/- mice was almost normal, except for the delayed appearance of peritoneal B-1 B cells and the increase in CD38low/- B-lineage cells. Among humoral immune responses in the systemic compartment, the thymus-independent (TI-2) Ag-induced IgG3, but not the IgM, response was selectively impaired. Furthermore, oral immunization with CT resulted in low production of Ag-specific IgA and IgG Abs in fecal extracts due to the reduced number of Ag-specific Ab-producing cells in the intestinal LP. These findings demonstrated that BST-1 is an important molecule for the selected population of B cell responses associated with TI-2 Ag-induced IgG3 and thymus-dependent (TD) Ag-induced IgA in the systemic and mucosal compartments, respectively.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse genomic DNA clones were isolated by screening a genomic DNA library derived from the 129/SV mouse strain (Stratagene, San Diego, CA). The targeting vector was prepared by replacing the KpnI-SalI fragment containing exons with pMC1neo poly(A) in the opposite transcriptional orientation. The ClaI-linearized targeting vector was electroporated into the E14.1 embryonic stem (ES) cell line. G418- and ganciclovir-resistant clones were selected and screened by PCR with a primer corresponding to the 5' external sequence of the targeting vector (5'-TCCCTTTGACAAGGAAGCCCACTGAGTAGC-3') and a primer complementary to the neo gene sequence (5'-GAGGGGATCGGCAATAAAAAGACAGAATAAAAC-3'). Homologous recombination was also confirmed by Southern blotting. Genomic DNA from individually selected clones were digested with HindIII and hybridized with the 5'-flanking probe. A DNA restriction fragment of 6.4 kb corresponded to the wild allele, while a 4.8-kb fragment resulted from the targeted allele. Five clones containing both alleles were identified from 453 colonies resistant to G418 and ganciclovir. Targeted ES clones were then injected into C57BL/6 (B6) blastocysts. Chimeric offspring generated from one clone transmitted the mutation to their progeny and were mated to B6 females. Mice carrying the mutation in the heterozygous state (BST-1+/-) were intercrossed to produce homozygous mutants (BST-1-/-).
Flow cytometric analysis
Details of the method for flow cytometric analysis and most of
the mAbs used in this study were described previously (15). Briefly,
single-cell suspensions from spleen, thymus, bone marrow, and
peritoneal cavity were prepared and washed with ice-cold PBS without
Ca2+ and Mg2+ containing 2% FCS and 0.1%
sodium azide (washing buffer). Cells were blocked with a mixture of
culture supernatant of 2.4G2 (anti-Fc
receptor II/III) and 6%
rat serum for 10 min, and then stained with respective mAbs directly
labeled with FITC, phycoerythrin (PE), or biotin for 15 min on ice. Red
670-conjugated streptavidin (Life Technologies, Grand Island, NY) was
used to reveal biotin-coupled Abs. After the staining, samples were
analyzed with FACSort using the CellQuest program (Becton Dickinson,
Mountain View, CA). Abs used for the staining are as follows:
PE-conjugated RA3-6B2 (anti-CD45R); FITC-, PE- and
biotin-conjugated AM/3 (anti-µ); PE-11-26c-1
(anti-
); FITC-S7 (anti-CD43); PE-53-7.3 (anti-CD5);
FITC-90 (anti-CD38, PharMingen, San Diego, CA); PE-RM4-4
(anti-CD4, PharMingen); FITC-53-5.8 (anti-CD8ß, PharMingen);
and FITC- and biotin-M1/70 (anti-Mac-1, CD11b).
Immunizations
For the TD immune response, mice (2–3 mo old) were immunized i.p. with 100 µg of DNP-KLH (LSL, Tokyo, Japan) in CFA on day 0 and were boosted with 100 µg of DNP-KLH in IFA on day 21. For TI immune responses, mice were immunized i.p. with 50 µg of 2,4,6-trinitrophenyl-conjugated LPS (TNP-LPS) or 20 µg of TNP-Ficoll in saline. Mice were bled before and after immunization. TNP-Ficoll was donated by Dr. S. Ono (Osaka University, Osaka, Japan). For oral immunization, mice were deprived of food for 2 h and given 0.5 ml of a solution containing eight parts HBSS and two parts 7.5% sodium bicarbonate by gastric intubation to neutralize stomach acidity for 30 min. Then, the mice were immunized orally with 0.25 ml of PBS containing CT (25 µg/mouse; Sigma, St. Louis, MO) by gastric intubation. CT was administered to mice once per week for 3 consecutive wk.
ELISA assays
Fecal extracts or sera were collected for the analysis of Ag-specific and total Abs. The ELISA plates were coated with DNP-BSA, TNP-BSA, or CT for the Ag-specific Ab. To measure the isotypes and levels of Abs, plates were coated with isotype-specific rabbit or goat anti-mouse Abs. Diluted serum and secretion samples were incubated in the plates and revealed by alkaline phosphatase-labeled secondary Ab. The Abs were purchased from Zymed Laboratories (South San Francisco, CA), except for the unlabeled or alkaline phosphatase-labeled anti-IgG3 Abs, which were purchased from Southern Biotechnology Associates (Birmingham, AL).
ELISPOT assays
Mononuclear cells of spleen, PP, MLN, and LP of the small
intestine were isolated. Single cells were suspended in RPMI 1640
containing 10% FCS. An ELISPOT assay was used to detect cells
producing CT-specific IgM, IgG, and IgA Ab (22). Ninety-six-well
nitrocellulose-based plate (Millititer HA, Millipore, Bedfold, MA) were
coated with 2 µg/ml of CT (100 µl/well). Mononuclear cells (1
x 103 to 1 x 105/well) were added to
each well and then incubated at 37°C in air with 10% CO2
and 90% humidity for 4 h. After washing the plate, 1 µg/ml of
horseradish peroxidase-conjugated goat anti-mouse µ-, -, and
-specific Abs (Southern Biotechnology Associates) were added to
individual wells. Following overnight incubation at 4°C, the plate
was reacted with 3-amino-9-ethylcarbazole for the development of
isotype-specific spots.
B cell proliferation
Splenic B cells were enriched by eliminating T cells, macrophages, and NK cells using a Minimacs column (Miltenyi Biotec, Auburn, CA) following the manufacturer’s instruction. At first, T cells, macrophages, and NK cells were coated with the following rat mAbs: culture supernatant of RL172.4 (anti-CD4), GK1.5 (anti-CD4), 3.155 (anti-CD8), 53.6 (anti-CD8), and M1/70 (anti-CD11b) and purified 29B (anti-CD3, Life Technologies) and TMß1 (anti-IL-2R ß-chain, provided by Dr. T. Tanaka, Osaka University). The cells coated with rat mAbs were then incubated with colloidal superparamagnetic beads conjugated with goat anti-rat IgG and depleted by passing through a Minimacs column. The eluted cells were enriched with B cells (90% purity) and used in the proliferation assay. B cells were cultured in 96-well flat-bottom microtiter plates at 2 x 105/0.2 ml of RPMI 1640 medium supplemented with 10% FCS, 2-ME (50 µM), penicillin (50 µg/ml), and streptomycin (50 µg/ml). These cells were stimulated with LPS (Escherichia coli 0111:B4, Sigma), CS/2 (anti-CD38), 3/23 (anti-CD40, PharMingen), F(ab')2 goat anti-mouse IgM polyclonal Ab (Jackson ImmunoResearch Laboratory, West Grove, PA), and Con A (Pharmacia, Uppsala, Sweden). Cultured cells were pulsed for the last 6 h of a 48-h culture period with 0.5 µCi/well of 3H-labeled thymidine, followed by scintillation counting.
Immunohistochemical analysis
To make polyclonal anti-mouse BST-1 Ab, the chimeric molecule of mouse BST-1 and the human IgG1Fc portion (mBST-1Fc) was prepared as described previously (15). Rabbit antiserum was then obtained by immunization with mBST-1Fc. Abs to human IgG1Fc were depleted by passing through the column of human IgG Sepharose 4B (Pharmacia), and cross-reactive Abs to mouse CD38 (mCD38) were removed by the absorption with mCD38 transfectant BAFmCD38. The specificity of the antiserum was confirmed by Western blotting using human IgG and cell lysate of BAFmBST-1 and BAFmCD38 (23).
Frozen sections (6 µm) of spleens were air-dried and subsequently fixed in cold acetone. The sections were blocked with 20% goat serum, then incubated with the anti-mouse BST-1 serum diluted 1/200. After washing, the sections were treated with biotinylated goat anti-rabbit Ig, washed with 0.05% Triton X-100 in PBS, then visualized with FITC-avidin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To obtain genomic BST-1 DNA, we screened a genomic 129/SV
library using mouse BST-1 cDNA as a probe. Positive clones were mapped,
and a targeting vector was constructed as shown in Figure 1
A. In brief, the sequences
from the KpnI site in exon 1 up to the SalI site
in exon 3 were deleted and replaced by neomycin resistance gene. The
herpes simplex virus-thymidine kinase gene was placed at the end of the
targeting vector for negative selection against nonhomologous
integration. The targeting vector was linearized and introduced into
E14 ES cells by electroporation, and transfectants were selected with
G418 and ganciclovir. Homologous recombinants were identified by PCR
and Southern blot analysis. Five clones were selected and injected into
C57BL/6 blastocysts to generate chimeric mice. Chimeras from one
clone transmitted the mutated allele through germline. Homozygous mice
were generated by intercrosses of heterozygous mice (Fig. 1B).
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C). Moreover, production of BST-1 protein was examined by
immunohistochemical analyses of the spleen and PP using a polyclonal
antiserum reactive with BST-1 (Fig. 1D). In the wild-type
mice, BST-1 was expressed by the reticular cells of the spleen and PP,
as reported in the immunohistochemical analysis using BP-3-specific Ab
(14). However, in the BST-1-/- mice, BST-1 was
not detected, confirming that these mice are deficient in BST-1.
BST-1-deficient mice thrived and reproduced as well as their wild-type
littermates. Developmental defect in peritoneal B cells and abnormal expression of CD38 in B lymphocytes
Since the BST-1 expression is detected in the early ontogeny of lymphoid cells and is restricted at the critical stages of both B and T cell development, our experiment was aimed to elucidate whether BST-1 deficiency could affect lymphocyte development.
Interestingly, in 2- to 3-wk-old BST-1-/- mice,
the number of peritoneal µbright - B
cells, i.e., B-1 cells expressing CD5 or Mac1, was reduced by 30 to
70% compared with that in control mice (Fig. 2). However, the percentage of B-1 cells
was recovered to the normal level in adult
BST-1-/- mice older than 6 wk. Furthermore, the
surface expression of CD38 was compared using
BST-1-/- mice and control mice, since the
molecule has homology of amino acid sequences with BST-1 (1, 4, 5). In
mice, it is known that almost all B-lineage cells (CD45R+
cells) in the bone marrow and spleen express CD38 (24, 25, 26). An increase
in a minor B-lineage population expressing low to no CD38
(CD38low/-) was noted in the adult bone marrow and spleen
of BST-1-/- mice. In the wild-type littermates,
the CD38low/- populations in the bone marrow and spleen
were 1.5 and 0.6%, respectively, whereas in
BST-1-/- mice, the populations increased to 8.9
and 3.5%, respectively (Fig. 3a).
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b). However, the expression levels of CD38 by the
recirculating B cells (in the R3 fraction) of
BST-1-/- mice were normal, suggesting that the
delayed up-regulation of CD38 caught up during peripheral B cell
maturation.
Except for those defects, no difference was observed in the cell number
of bone marrow, thymus, and spleen between adult
BST-1-/- mice and wild-type littermates (data
not shown). We observed no differences in the populations of B lymphoid
progenitors defined by CD45R and IgM in the bone marrow and of T
lymphoid progenitors by CD4 and CD8 in the thymus (Fig. 4, a and b). The
mature B and T cell populations defined by IgM, IgD, CD4, and CD8 in
the spleen were also normal (Fig. 4c). Our analysis was
extended to other surface markers, including CD24, BP-1,
c-kit, CD19, Ig-ß, CD23, Ig
, Ig
, Thy1, IL-2R,
TCRß, Mac-1, and Gr-1 (data not shown). The differences in these
surface expressions were also indistinguishable between normal and
BST-1-/- mice, indicating that the major
pathway of lymphoid development was unaffected by depletion of the
BST-1 gene.
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The expression of murine BST-1/BP-3 on the reticular cells of
splenic white pulps, lymph nodes, and PP suggested that BST-1 can
support the functions of mature B cells. To evaluate the role of BST-1
in the induction of humoral immune responses, we first analyzed the
concentrations of serum Igs by IgM-, IgG subclass-, and IgA-specific
ELISA. The serum Ig levels of all isotypes examined in
BST-1-/- mice were the same as those in
wild-type littermates (Fig. 5a). In the next series of
experiments, the influence of BST-1 deficiency on the induction of
Ag-specific humoral immune responses was examined using a group of TD,
TI-1, and TI-2 Ags. When BST-1-/- mice were
systemically immunized with DNP-KLH as a TD Ag and with TNP-LPS as a
TI-1 Ag, BST-1-/- mice mounted normal levels of
Ag-specific primary and secondary responses of IgM as well as IgG
classes (Fig. 5, b and c). However, when
TNP-Ficoll, as a TI-2 Ag, was used, a reduction of TNP-specific IgG3,
but not IgM production, was observed (Fig. 5c).
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Anti-CD38 Ab induces potent proliferative and antiapoptotic
responses in splenic B cells, except for some B cell populations such
as xid and neonatal B cells in which these B cells were
unresponsive to anti-CD38 Ab and TI-2 Ag (24, 25, 27, 28). An
increase in CD38low/- splenic B cells and a defective TI-2
response in BST-1-/- mice prompted us to
examine whether B cells of BST-1-/- mice have
defects in responses to anti-CD38 Ab. However,
BST-1-/- B cells have no apparent defects in
response to anti-CD38 Ab (Fig. 6).
Also no differences were observed between the B cells of wild-type and
BST-1-/- mice in their responses to
anti-µ Ab, anti-CD40 Ab, or LPS (Fig. 6). Thus, the splenic
BST-1-/- B cells do not have inherent defects
in BCR, CD40, LPS, or CD38 signaling. Furthermore, immunohistochemical
analysis revealed that the structure of T and B cell areas in splenic
white pulps was intact in BST-1-/- mice (data
not shown). These data suggested that the defective IgG3 response to
TI-2 Ags in BST-1-/- mice was ascribed to the
lack of BST-1 normally expressed by splenic reticular cells.
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BST-1 is also expressed by the reticular cells in the PP and the
brush border of the intestinal epithelial cells (14). Such a
localization of BST-1 suggests the involvement of BST-1 in the mucosal
immune responses. To this end, we addressed the possible role of BST-1
in the induction and regulation of mucosal immunity. When the
concentration of total IgA in the sera and fecal extracts was examined
and compared between wild-type and BST-1-/-
mice, unaltered levels of total IgA were seen in both systemic and
mucosal compartments (Fig. 5a and data not shown). These
results indicated that normal numbers of IgA-producing plasma cells
were developed in BST-1-/- mice.
To examine possible influences of BST-1 deletion on the induction of
the Ag-specific mucosal immune response,
BST-1-/- and control mice were orally immunized
with CT, a strong immunogen as well as adjuvant for the induction of
mucosal IgA production. When the levels of CT in sera and fecal
extracts were examined, it was interesting to note that the levels of
anti-CT IgA and IgG Abs in the fecal extracts from
BST-1-/- mice were at least twofold lower than
those in wild-type littermates (Fig. 7a). However, in the sera no
difference was observed between BST-1-/- and
wild-type mice. To further examine the influence of BST-1 deficiency on
the CT-specific mucosal immune response, the frequency of CT specific
Ab-forming cells (AFCs) in different mucosa-associated tissues was
measured by ELISPOT assay. In BST-1-/- mice,
the number of CT-specific IgA AFC was low in the intestinal LP, which
is considered to be a major mucosal effector site for IgA production.
On the other hand, in the spleen and in MLN and PP, representing the
systemic compartments and the mucosal inductive tissues, respectively,
the frequencies of CT-specific IgA and IgG AFCs in
BST-1-/- mice were comparable to those seen in
wild-type mice (Fig. 7b). These results indicated that BST-1
could be an important molecule for the induction of the Ag-specific IgA
B cell response in the mucosa-associated effector tissue.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Role of BST-1 in lymphocyte development and its relationship to CD38
As described in the introduction, the accumulated evidence has indicated that BST-1 may play an important role in lymphoid cell development. The present study produced the interesting results that BST-1 gene deletion led to developmental defects in peritoneal B-1 cells and to an increase in the CD45R+CD38low/- population in bone marrow and spleen. Since B-1 cells are considered to be of fetal liver origin, and BST-1 is expressed on the B progenitor of fetal liver maximally on day 17 and is decreased on day 18 (15, 29), BST-1 may enhance a narrow window of the maturation or migration of B-1-lineage cells. It would be interesting to examine the expression of BST-1 in IL-5-/- or IL-5R-/- mice, since the deletion of IL-5 or IL-5R genes has been shown to influence B-1 cell development (30, 31).
BST-1 and CD38 molecules are members of a novel family of ectoenzymes (26, 32), but the functional relation or difference between BST-1 and CD38 is still largely unknown. To date, a different expression pattern on B cell development was observed between these molecules. The expression of CD38 is low on pro-B cells, up-regulated at the transition from pro-B to pre-B cells, and increased further during B cell maturation, while that of BST-1 is restricted on pro-B, pre-B, and immature B cells and then down-regulated at the mature B cell stage (15). However, in secondary lymphoid organs such as the spleen, PP, and lymph node, BST-1 is expressed on the reticular cells that may provide positive signals for the Ab production. Since both BST-1 and CD38 are expressed on B progenitor cells from pro-B to immature B cell stages, the functions of both molecules may complement each other at these stages. This may explain why BST-1-/- mice showed almost normal B cell development, except for CD38 expression. Unexpectedly, this study revealed that the loss of BST-1 was not compensated by the up-regulation of the expression of CD38. Instead, CD38 up-regulation at the pre-B stage was delayed in the bone marrow. Moreover, in the spleen of BST-1 knockout mice, the CD45R+CD38low/- population was increased. In normal immunized mice, the CD45R+CD38low/- population in peripheral lymphoid organs was considered to be germinal center B cells and mature plasma cells (33). These germinal center B cells have been shown to have down-regulated surface expression of IgM and IgD. However, the CD45R+CD38low/- population observed in BST-1 knockout mice expressed high levels of IgM and IgD (data not shown), suggesting that this population may not be germinal center B or mature plasma cells. Therefore, it is possible that some populations of splenic B cells could not express high levels of CD38 due to the lack of BST-1.
Involvement of BST-1 in systemic TI-2 Ag-induced IgG3 response
Analyses of the systemic humoral responses in BST-1 knockout mice revealed the decreased IgG3 response to a TI-2 Ag, TNP-Ficoll. The selective defects in the TI-2, but not the TD or TI-1, response, suggested that Ag-specific Ab responses that were dependent on BST-1 seemed to be limited. To interpret the relationship between the loss of BST-1 and the impaired TI-2 response, expression of BST-1 on the cells at the site of Ab production, the architecture of lymphoid tissues, and the responsibility of B cells to the stimuli important in differentiation to Ab-producing cells should be considered. BST-1 is not expressed on mature B cells, but is expressed on reticular cells in secondary lymphoid organs in normal mice. B cells of BST-1 knockout mice were essentially normal because the splenic B cell responses of BST-1 knockout mice to various proliferative stimuli were comparable to those of wild-type mice. Furthermore, the architectures of T and B cell areas in the secondary lymphoid organs were not altered in BST-1 knockout mice, except for the lack of BST-1 on reticular cells (data not shown). Taken together, these findings suggested that defective responses to TI-2 Ag in BST-1 knockout mice were probably due to the absence of BST-1 expression on the reticular cells of splenic white pulps, although this issue remains to be resolved.
Defective responses to TI-2 Ags were also observed in xid
mice, knockout mice of Btk, Ig-, IL-5R, protein kinase C, and CD22
(31, 34, 35, 36, 37, 38, 39). These molecules are expressed by mature B cells, and the
absence of these molecules affected signaling in B cells in response to
various proliferative stimulations. Among cytokines,
TNF-lymphotoxin- double-knockout mice lacked a TI-2 response (40).
The cell lineages affected by the deficient TNF-lymphotoxin- are not
clear, but the poorly organized architecture of lymphoid tissues seems
to be responsible for this defect. Compared with the molecules
affecting the TI-2 response in vivo, BST-1 is unique, in that BST-1 is
not a cytokine but, rather, a membrane protein expressed by the
reticular cells in microenvironment of Ab production and is required
only for the TI-2 response of the IgG3 class.
TI Ags are classified as the Ags that stimulate Ab production in the
absence of MHC class II-restricted T cell help. However, TI-2 Ag by
itself is not sufficient to stimulate B cells for subsequent Ig
synthesis, and it must therefore be accompanied by other
lymphoreticular cells that can interact with B cells either directly or
indirectly (41, 42). There is evidence implicating NK cells, T cells,
and follicular dendritic cells as playing important roles in response
to TI-2 Ag (41). Our results suggest that reticular cells expressing
BST-1 could be included in the auxiliary cells for the TI-2 response of
the IgG3 class. Although TI-2-specific IgG3 production was impaired in
BST-1 knockout mice, BST-1 is not an essential factor that induces the
class switch to IgG3 such as IFN-, because IgG3 production was
normal when TI-1 or TD Ags were immunized. IFN- is a switch factor
for IgG3 when B cells are activated with anti-Ig-dextran, a model
Ag for the TI-2 response in vitro (43, 44). Furthermore,
immnohistochemical analyses revealed that immunization with TNP-Ficoll
induced the appearance of IFN--producing T cells and NK cells in
the outer parts of periarteriolar lymphoid sheath, where TNP-AFC
localize (45). At present, we do not have any evidence that IFN-
production was impaired in BST-1-/- mice,
because they mounted the normal Th1 response in which IFN- plays a
major role (H. Tsutsui and K. Nakanishi, unpublished
observation). Moreover, IFN- has been shown to induce BST-1
expression on peritoneal macrophages in vitro (M. Itoh and K.
Ishihara, unpublished observation). These findings suggested
that BST-1 may be up-regulated in situ by IFN- and may function as
the effector molecule.
Although the exact mechanisms by which BST-1 supports the TI-2 response are currently unknown, it is possible that BST-1 transduces certain signals to activate the reticular cells to support B cells or that putative BST-1 ligands transduce signals to B cells. Actually, we showed that BST-1 facilitated the growth of a pre-B cell line (1) and recently found the expression of a BST-1 binding molecule on a B cell line (K. Ishihara, unpublished data). There is another possibility that the lowered expression of CD38, which is capable of augmenting BCR signaling (46), impaired the TI-2 response, since it was reported that marginal zone (MZ) B cells that respond to TI-2 Ag are CD38high (47). However, our findings do not support this possibility, since CD45R+CD38low/- cells were not typical MZ B cells, and the expression of CD38 by MZ B cells was not significantly altered in BST-1 knockout mice (data not shown). Since enzyme activity of CD38 has been suggested to affect the growth of B cells (7), it is likely that the enzyme activities of BST-1 on reticular cells are also involved in supporting the survival, growth of B cells, or class switching to IgG3. This issue remains to be resolved.
BST-1 is required for the full response of TD Ag-induced mucosal IgA responses
It was interesting to note that Ag (CT)-specific mucosal immune responses after oral immunization were impaired in BST-1 knockout mice. BST-1 is expressed on the reticular cells of PP, which is considered to be IgA-inductive tissue containing high numbers of IgA precursors cells (sIgA+ B cells) (14, 18). Following Ag stimulation, these sIgA+ B cells leave the tissue and migrate to IgA effector sites such as intestinal LP. When the frequency of CT-specific IgA AFC was compared in different tissues of BST-1-/- and control mice following oral immunization, the reduction of Ag-specific IgA-producing cells was most obvious in the intestinal LP of BST-1-/- mice. On the other hand, the numbers of CT-specific IgA AFC were comparable in the spleen, PP, and MLN in BST-1-/- and wild-type mice. In addition, it was important to indicate that the levels of CT-specific IgG serum responses induced by oral immunization were similar between these two mouse groups. These results indicated that Ag-specific mucosal, but not systemic, responses were impaired in BST-1-/- mice orally immunized with TD Ag such as CT.
Since PP-derived dendritic cells have the ability to promote commitment
to IgA (48), it is possible that BST-1 gene deletion may influence the
function of PP dendritic cells for their ability to induce
IgA-committed sIgA+ B cells. However, the committed
sIgA+ cells are comparable between
BST-1-/- and wild-type mice in PP, and serum
levels of IgA and systemic anti-CT Ab production in BST-1 knockout
mice were normal (Fig. 7), suggesting that the commitment of IgA B
cells was not impaired. Thus, BST-1 expressed in PP may support the
maturation or homing of B cell blasts that migrate to the LP.
Since the majority of B cells in the LP are B-1 cells, and there is traffic between the peritoneal cavity and the LP (49, 50), it is possible that the impaired development of peritoneal B-1 cells of BST-1-/- mice affects the function of peritoneal B-1 cells. This issue has yet to be examined.
Previous studies have demonstrated that cytokines such as IL-4, IL-5, IL-6, and TGF-ß regulate IgA B cell differentiation in vitro (51, 52, 53, 54, 55). For example, both mucosal and systemic Ab responses to orally administered TD (e.g., tetanus toxoid and KLH) Ags were impaired in IL-4 knockout mice (56, 57). Although conflicting results were generated in IL-6 gene-deleted mice (58, 59), a study demonstrated the reduction of IgA responses in both mucosal and systemic sites. In contrast to these studies, mucosal immunodeficiency in BST-1 knockout mice was unique, because only local immune responses to TD Ag were impaired.
Perspective
Although our current findings do not offer any explanation for the mechanisms of the partial immunodeficiency observed in BST-1 knockout mice, gene targeting clearly demonstrated that BST-1 is a unique surface molecule expressed by reticular cells and has a function to support the systemic B cell differentiation induced by TI-2 Ag. Furthermore, BST-1 knockout mice could be useful animal models that could be extended to demonstrate the importance of mucosal immunity, especially in the role of secretory IgA as a first line of defense against different infectious agents, since BST-1 knockout mice could be considered mucosal immunodeficient mice with intact systemic immune responses to orally administered Ags.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. T. Hirano, Division of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan. E-mail address:
3 Abbreviations used in this paper: BST-1, bone marrow stromal cell Ag-1; PP, Peyer’s patch; sIgA, surface IgA; CT, cholera toxin; LP, lamina propria; TD, thymus dependent; TI, thymus independent; ES, embryonic stem; PE, phycoerythrin; KLH, keyhole limpet hemocyanin; TNP, 2,4,6-trinitrophenyl; ELISPOT, enzyme-linked immunospot; MLN, mesenteric lymph node; mCD38, mouse CD38; AFC, Ab-forming cell; MZ, marginal zone.
Received for publication February 4, 1998. Accepted for publication June 16, 1998.
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