We show in this report a new regulatory role for IL-15 and IL-15R in the development of B-1 cells and their differentiation into IgA-producing cells. Mucosal IgA levels were found to be inhibited by anti-IL-15 mAb treatment in vivo, but enhanced by administration of rIL-15, while serum IgA levels remained unaffected. Mucosal B-1 cells preferentially proliferated in response to IL-15 in vitro. When mucosal B-1 and B-2 cells were separated into surface (s)IgM+sIgA and sIgMsIgA+ fractions, IL-15R-specific mRNA was found to be predominant in both sIgM+sIgA and sIgMsIgA+ B-1 cells at a much higher level than B-2 cells. Further, incubation of these different subsets of B-1 and B-2 cells with IL-15 resulted in greater enhancement of the corresponding receptor expression by B-1 subset when compared with B-2 fraction. Interestingly, de novo isolated sIgM+sIgA B-1, but not sIgM+sIgA B-2, cells were already class-switched cells because the germline Cα transcript was detected and was then further enhanced by IL-15. IL-15 also supported differentiation of both sIgM+sIgA and sIgMsIgA+ B-1 cells into IgA-producing cells. Taken together, these findings suggest that IL-15 is a critically important cytokine for the differentiation of both sIgM+,IgA and sIgMsIgA+ B-1 cells expressing IL-15R into IgA-producing cells in mucosal tissues.

Interleukin 15 is a novel cytokine that uses β- and γ-chains of IL-2 receptor for signal transduction, and though it shows no sequence homology with IL-2, shares many of its biological properties (1, 2). IL-2 is produced mainly by T cells, while IL-15 is secreted by placenta, skeletal muscle, kidney, and activated monocytes/macrophages (1). IL-2 and IL-15 have been shown to possess several important biological activities for T and B cells (1, 3, 4). However, these two cytokines differ in their controls of expression and secretion, their range of target cells, and their functional activities (5, 6, 7, 8). For example, IL-2 induces or inhibits T cell apoptosis in vitro, respective of the stage of T cell activation, whereas IL-15 inhibits cytokine deprivation-induced apoptosis in activated T cells (5). IL-15 supports human B cell proliferation and immunoglobulin synthesis in vitro, in combination either with CD40 ligand or immobilized anti-IgM (4, 6). Further, IL-15 appears more physiologically and immunologically relevant to extrathymic T lymphocyte development than IL-2 (7). IL-15 is also a potent growth and differentiation factor for NK cells (8).

Since the discovery that intestinal epithelial cells (i-EC)3 can produce IL-15 in mice, rats, and humans (9, 10), a focus of research in mucosal immunity has been to elucidate the possible role of IL-15 in the mucosal interaction between i-EC and intestinal intraepithelial lymphocytes (i-IEL). Upon infection with Listeria monocytogenes, i-EC in rats begin to produce IL-15, which in turn stimulates i-IEL to produce IFN-γ (11). IL-15 has also been shown to control the development of CD4 CD8αα+ i-IEL, a fraction of T cells considered to be extra-thymically developed mucosal T cells (12, 13). These experimental results suggest that IL-15 is an important cross-talk molecule for integrated i-EC and i-IEL functions in the mucosal immune system. Thus far, investigations into the role of IL-15 in the mucosal immune system have been focused on i-IEL T cells and NK T cells (11, 12, 13). However, it remains unclear whether i-EC-derived IL-15 plays a role in the growth and differentiation of B cells in the mucosal immune system.

Mucosal effector sites such as the lamina propria of the gut and salivary gland contain high numbers of plasma cells committed to the secretion of IgA Ab. The dimeric or polymeric forms of these IgA are transported across the epithelium into the gut lumen via secretory component to provide a first line of defense as secretory IgA against pathological microorganisms. Mucosal B cells can be classified into B-1 cells and conventional B (B-2) cells based on the expression of B220, IgM, IgD, CD5, and Mac-1 (14, 15, 16, 17). Based on surface CD5 expression, the B-1 cell population can be further divided into a CD5+ B-1a cell and a CD5 B-1b “sister” cell population. Our previous study demonstrated that B-1 cells constituted a major fraction of B cells in mucosal effector tissues including intestinal lamina propria (i-LP). Among enriched B-1 cells, the B-1b cell fraction showed a particularly strong expression of surface (s)IgA (18). In addition, it has been suggested that B-1 cells are a major supplier for IgA plasma cells in mucosal effector tissues (19, 20, 21). Furthermore, a selected cytokine produced mainly by Th2-type cells such as IL-5 has been shown to tightly regulate the differentiation of mucosal B-1 cells into IgA Ab-producing cells (22). A support for this regulatory role of certain Th2-type cytokines is provided by our separate study, which directly demonstrated that sIgA+ B-1 cells expressed higher levels of IL-5R than of IL-6R (18). Further, lack of the IL-5R gene resulted in the reduction of B-1 cell-originated sIgA+ B cells and IgA plasma cells in mucosal effector tissues (18).

In the present study, we hypothesize that epithelial cell-derived IL-15 could be an essential mucosal cytokine in addition to Th2-type cytokines for the development of IgA-committed B cells in mucosal effector tissues. Our study explores the role of IL-15 and the corresponding receptor (IL-15R) for B-1 cells in the development and differentiation of common mucosal immune system (CMIS)-independent IgA-producing cells in mucosa-associated tissues.

C57BL/6 mice were obtained from Charles River Japan (Atsugi, Japan). Mice were maintained in conventional animal facilities in the experimental animal facility of the Research Institute for Microbial Diseases, Osaka University (Osaka, Japan). All experiments were conducted with sex-matched, 6- to 10-wk-old mice.

Mice were treated i.p. with 250 μg of monoclonal rat IgG1, anti-mouse IL-2 or IL-15, or control mAb (Rat IgG1) once a week for three consecutive weeks (Fig. 1). Anti-IL-2 (S4B6), anti-IL-15 (G277-3588), and rat IgG1 Ab (R3-34) were purchased from PharMingen (San Diego, CA). The other groups of mice were injected i.p. with 1 × 105 U of recombinant mouse IL-2 or human IL-15 every 3 days for three consecutive weeks (Fig. 1). Recombinant mouse-IL-2 and human-IL-15 were also purchased from PharMingen. Serum, saliva, and fecal extracts were obtained 4 wk after the first Ab treatment or cytokine administration (Fig. 1). Following these in vivo treatments, isotype-specific Abs were measured in mucosal secretions and serum using a standard ELISA. Further, mice were sacrificed and levels of IgM-, IgG- and IgA-producing cells in spleen (SP), submandibular gland (SMG), and i-LP were determined by enzyme-linked immunospot (ELISPOT) assay (see below).

FIGURE 1.

A protocol for the elucidation of IL-15 and IL-15R as a new member of mucosal cytokine network for the induction of IgA response. The black arrow indicates the administration of cytokine or mAb. The white arrow demonstrates sampling of serum, mucosal secretions, and tissues (e.g., SMG and i-LP).

FIGURE 1.

A protocol for the elucidation of IL-15 and IL-15R as a new member of mucosal cytokine network for the induction of IgA response. The black arrow indicates the administration of cytokine or mAb. The white arrow demonstrates sampling of serum, mucosal secretions, and tissues (e.g., SMG and i-LP).

Close modal

Mononuclear cells of SP, SMG, or i-LP were prepared as described previously (23). Briefly, mononuclear cells from SP were isolated by the mechanical method using gentle teasing through stainless steel screens. i-LP and SMG mononuclear cells were isolated by the enzymatic dissociation procedure with collagenase type IV (Sigma, St. Louis, MO).

Levels of isotype-specific Ab in fecal extract, saliva, and serum were determined by ELISA, as described previously (23, 24, 25, 26). Briefly, 96-well plates (Nunc, Roskilde, Denmark) were coated with 100 μl of an optimal concentration (2 μg/ml) of goat anti-mouse Ig (Southern Biotechnology Associates, Birmingham, AL) in PBS. Wells were blocked with 200 μl PBS containing 10% normal goat serum (Life Technologies, Gaithersburg, MD) for 2 h at 37°C. After extensive washing, serial dilution of samples were added and incubated for 2 h at 37°C. After incubation and washing, the wells were treated first with 100 μl of a 1:1000 diluted biotinylated goat anti-mouse μ, γ, or α heavy chain-specific mAb (Southern Biotechnology Associates) and then with the detection solution containing a 1:2000 dilution of HRP-conjugated streptavidin (Life Technologies). After washing, the color reaction was developed at room temperature with 50 μl of tetramethylbenzidine reagent (Moss, Pasadena, MD). For the quantitation of Igs, purified IgM, IgG, and IgA (Chemicon International, Temecula, CA) were used as standards. Reactions were terminated by the addition of 50 μl 0.5 M HCl after a 15-min incubation. The color reaction was measured by an OD at 450 nm (OD450).

To determine the numbers of IgA-, IgG-, and IgM-producing cells in mucosal effector tissues (i-LP and SMG) and SP, the ELISPOT assay was used as previously described (23, 26, 27). Briefly, 96-well filtration plates with a nitrocellulose base (Millititer HA; Millipore, Bedford, MA) were coated with 5 μg/ml affinity-purified goat anti-Ig (Southern Biotechnology Associates). The plates were blocked with complete medium containing RPMI 1640 in the presence of 10% FBS, 50 μg/ml gentamicin, 50 μg/ml penicillin G, and 50 U/ml streptomycin. The mononuclear cells in complete medium were added at varying concentrations and were cultured at 37°C for 4 h in air with 5% CO2. After the incubation, the plates were thoroughly washed with PBS and then with PBS containing 0.05% Tween 20 solution. For the capture of Ab-producing cells, 1 μg/ml HRP-conjugated affinity-purified goat anti-mouse μ-, γ-, or α-specific Abs (Southern Biotechnology Associates) was added. After overnight incubation at 4°C, the spots were developed with 2-amino-9-ethylcarbazole (Polysciences, Warrington, PA) containing hydrogen peroxide. Spots were counted as Ab-forming cells (AFC) with the aid of a dissecting microscope. The data are expressed as the mean number of AFC ± SE per 105 cells, after triplicate determinations.

To separate B cell subsets to B-1 and B-2 cells (17, 19), lymphocytes from different tissues were incubated with FITC-conjugated anti-IgD (PharMingen, 11-26c.2a) and PE-conjugated anti-IgM (IgH-6b; PharMingen, AF6-78) for the IL-15-induced B cell-proliferation assay. FITC-conjugated anti-IgA (PharMingen, R5-140), PE-conjugated anti-CD45R/B220 (Phar-Mingen, RA3-6B2), and biotinylated anti-IgM (PharMingen, AF6-78) followed by streptavidin-conjugated PerCP (Becton Dickinson, Sunnyvale, CA) were used for the separation of sIgM+sIgA or sIgA+ B-1 and B-2 cells for the analysis of IL-15R and Cα expressions and IgA production (14, 16, 17, 28, 29, 30). In some cases, appropriate fluorescence conjugated anti-Mac-1 mAb (PharMingen, M1/70) was also used for multicolor FACS separation of B-1 and B-2 cells (16, 17, 19, 20). These samples were then subjected to flow cytometry analysis by using a FACScaliber (Becton Dickinson). Control cells were incubated with individual isotype control Ab, and these cells were used to set the lymphocyte gates. Each analysis was performed at least three to five times to verify the results obtained, and the results were expressed as the mean. For the purification of different subsets of B cells, samples underwent a similar staining procedure at 4°C and were then subjected to flow cytometry sorting separation using FACSvantage (Becton Dickinson). This procedure yielded cells that were >99% pure.

A B cell-proliferation assay was conducted in complete RPMI 1640 medium containing 10% heat-inactivated FBS at 37°C in a humidified atmosphere of 5% CO2 (4). A total of 1 × 105 cells/well were cultured in triplicate in U-bottom, 96-well microtiter plates (Corning, Corning, NY) for 72 h in the presence of different doses (0–1000 ng/ml) of IL-2, IL-5, or IL-15 (all from PharMingen). Cells were pulsed with 1 μCi/well [3H]TdR (Amersham, Arlington Heights, IL; 25 Ci/mmol) for the final 18 h of culture before being harvested. Levels of incorporated cpm were then determined by liquid scintillation counting. In another experiment, different subsets of B cells (e.g., sIgA+ B-1 and B-2 cells) were cocultured containing 10 μg/ml Escherichia coli LPS (Sigma, 0127:B8) with an optimal concentration of IL-15 (PharMingen; 100 ng/ml) and/or TGF-β1 (R&D Systems, Minneapolis, MN; 1 ng/ml) for 1 and 3 days to characterize isotype switching and IL-15R expression, respectively.

For quantitation of IL-15Rα-specific mRNA in freshly isolated and cultured B cells, quantitative RT-PCR was adapted using LightCycler (Roche Diagnostics, Mannheim, Germany) technology (31, 32). B cells were harvested and total RNA was purified by Trizol reagent (Life Technologies). To apply the same amount of synthesized cDNA from B cells, the amounts of synthesized cDNA labeled with digoxigenin were measured with a chemiluminescent image analyzer (Molecular Imager System; Bio-Rad, Hercules, CA). A detailed protocol for the synthesis of cDNA was previously reported by our laboratory (33). For the amplification of cDNA, 20 μl PCR mix was added to each tube to give a final concentration of 0.05 μM 5′ primer, 0.05 μM 3′ primer, 0.2 μM FITC labeled-probe, 0.2 μM LightCycler Red 640 labeled-probe, 2 mM MgCl2, and 1× LightCycler-DNA master hybridization probes mix (Roche Diagnostics). The oligonucleotide primers specific for the IL-15Rα (sense, 5′-ATGGCCTCGCCGCAGCTCCG-3′; antisense, 5′-CCTGAGGGAGGTGGAGGCTG-3′; Ref. 10), IL-15Rα detection FITC-labeled hybrid probe (5′-GTGACACCAAAGGTGACCTCACAGC-3′), and LightCycler Red 640-labeled hybrid probe (5′-AGAGAGCCCCTCCCCCTCTGCAAAA-3′) were prepared (34). After heating at 94°C for 2 min, cDNA were amplified for 40 cycles, each cycle consisting of 95°C for 0 s, 55°C for 30 s, and 72°C for 30 s. Once the cycle during which the log-linear signal can be distinguished from the background is identified, it is possible to compare the target concentrations (external standard) in samples (31, 32). The outer standard was constructed by cloning IL-15Rα-specific PCR products with a T-A cloning vector (pGEM-T Vector, Promega, Madison, WI) as described previously (24). After PCR has been completed, the LightCycler software (Roche Diagnostics) converts the raw data into copies of target molecules (31, 32).

For detection of Cα transcript-specific mRNA in different subsets of freshly isolated or cultured B cells (see below), a standard RT-PCR amplification protocol was used (25, 34, 35, 36). Total RNA was purified and then cDNA were synthesized as described above (31). For the amplification of cDNA, 25 μl PCR mix was added to each tube to give a final concentration of 1 U/25 μl AmpliTaq Gold DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT), 0.2 μM 5′ primer, 0.2 μM 3′ primer, 2 mM MgCl2, and 1× PCR buffer II (Perkin-Elmer Cetus). The oligonucleotide primers specific for germline Cα transcript (Iα-leader, 5′-GACATGATCACAGGCACAAGGC-3′; Cα, 5′-TTCCCCAGGTCACATTCATCGT-3′) were prepared (36). The sequence of β-actin used for this study has already been described in our previous paper (24, 25). After heating at 95°C for 9 min, cDNA were amplified for 40 cycles in Cα transcript or 35 cycles in β-actin, each cycle consisting of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and one cycle at 72°C for 10 min. PCR products were separated by electrophoresis in 1.8% agarose gels and visualized by UV light illumination following ethidium bromide (0.5 μg/ml) staining.

Highly purified murine sIgM+sIgA or sIgMsIgA+ B-1 (B220low) and B-2 (B220high) cells were isolated from i-LP lymphocytes by flow cytometry sorting as described above. In addition, fluorescence conjugated anti-Mac-1 mAb was also used as an additional maker to further ensure separation of those B-1 and B-2 subsets. Purified B cells (1 × 104 cells) were cultured in 100 μl complete RPMI 1640 medium containing 2 μg/ml LPS (Sigma) either in the presence or absence of an optimal concentration of IL-2 (100 ng/ml), IL-5 (100 ng/ml), IL-6 (500 pg/ml), or IL-15 (100 ng/ml; all from PharMingen) in U-bottom, 96-well plates (Falcon, Lincoln Park, NJ; Ref. 18). After 3 days of incubation, culture supernatants were harvested for the assessment of IgA production by isotype-specific ELISA as described above. In some experiments, a similar culture condition was established using 1 ng/ml TGF-β1 or 100 ng/ml IL-15 to analyze germline Cα transcript as described above.

The results were statistically analyzed by Student’s t test.

In the initial experiment, a group of mice was treated with anti-IL-15 mAb to determine the role of IL-15 in the induction of IgA synthesis. The two control groups of mice were injected with anti-IL-2 and rat IgG1 mAbs. The levels of IgA in mucosal secretions, but not serum, were more significantly reduced in anti-IL-15 mAb-treated mice than in anti-IL-2 mAb-treated and control mice (Fig. 2,A). As regards other isotypes, the levels of IgM and IgG were significantly reduced in anti-IL-2 mAb-treated mice but not in anti-IL-15 mAb-treated and control mice (Fig. 2,A). In addition, mononuclear cells were isolated from i-LP to directly determine whether the reduction of mucosal IgA levels was caused by the decrease of IgA-producing cells in mucosal effector tissues of mAb anti-IL-15 mAb-treated mice. When the isotype-specific ELISPOT assay was used to determine the frequency of IgA-producing cells in anti-IL-2- and anti-IL-15 mAb-treated mice and in control mice, the numbers of mucosal IgA-producing cells were significantly reduced in the anti-IL-15 mAb-treated mice (Fig. 2,B). In contrast, the numbers of IgM- and IgG-producing cells in SP were significantly decreased in the anti-IL-2 mAb-treated mice (Fig. 2 B). These results suggest that removal of IL-15 leads to the selective impairment of IgA production in mucosal compartments but not at systemic sites in vivo.

FIGURE 2.

The reduction of secretory IgA in anti-IL-15 mAb-treated mice. A, Levels of serum and secretory Abs in anti-IL-15- (□) and anti-IL-2 (▨) mAb-treated mice and control mice (rat IgG1 mAb treatment (▪)) were analyzed by ELISA. B, AFC of IgM, IgG, and IgA isotypes were determined in mucosa-associated tissues including the i-LP and SMG, and the SP by ELISPOT. The results represent the values (mean ± SE) from three experiments (three mice per group). ∗, p < 0.05, ∗∗; p < 0.01.

FIGURE 2.

The reduction of secretory IgA in anti-IL-15 mAb-treated mice. A, Levels of serum and secretory Abs in anti-IL-15- (□) and anti-IL-2 (▨) mAb-treated mice and control mice (rat IgG1 mAb treatment (▪)) were analyzed by ELISA. B, AFC of IgM, IgG, and IgA isotypes were determined in mucosa-associated tissues including the i-LP and SMG, and the SP by ELISPOT. The results represent the values (mean ± SE) from three experiments (three mice per group). ∗, p < 0.05, ∗∗; p < 0.01.

Close modal

To further examine the role of IL-15 in the induction and regulation of mucosal IgA synthesis, two groups of mice were treated with IL-2 and IL-15. Given the result obtained by anti-IL-15 mAb treatment (Fig. 2), one might expect that administration of IL-15 would lead to the enhancement of IgA synthesis in mucosal secretions. Our experiments showed that systemic injection of IL-15 resulted in increased levels of IgA Ab in saliva and fecal extracts but not in serum (Fig. 3,A). Further, mononuclear cells isolated from i-LP of IL-15-treated mice contained higher numbers of IgA AFC than did those from IL-2-treated and control mice (Fig. 3,B). This enhancing effect of IL-15 was selectively seen for mucosal IgA. In the case of IgM and IgG production, IL-2 treatment enhanced systemic IgM and IgG production but not IgA (Fig. 3, A and B). Taken together, our findings provide further evidence that IL-15 could be a key mucosal cytokine for the induction of mucosal but not serum IgA.

FIGURE 3.

The increase of secretory IgA in IL-15-treated mice. A, Levels of serum and secretory Abs in IL-15- (□) and IL-2- (▨) treated mice and control mice (▪) were analyzed by ELISA. B, AFC of IgM, IgG, and IgA isotypes were determined by ELISPOT in mucosa-associated tissues including i-LP and SMG, and the SP. The results represent the values (mean ± SE) from three experiments (three mice per group). ∗, p < 0.05; ∗∗, p < 0.01.

FIGURE 3.

The increase of secretory IgA in IL-15-treated mice. A, Levels of serum and secretory Abs in IL-15- (□) and IL-2- (▨) treated mice and control mice (▪) were analyzed by ELISA. B, AFC of IgM, IgG, and IgA isotypes were determined by ELISPOT in mucosa-associated tissues including i-LP and SMG, and the SP. The results represent the values (mean ± SE) from three experiments (three mice per group). ∗, p < 0.05; ∗∗, p < 0.01.

Close modal

To further specify IL-15-dependent IgA B cell development, the frequency of sIgA+ B cells in B-1 (B220low) and B-2 cell (B220high) subsets was measured in mononuclear cells isolated from the i-LP of IL-2-, IL-5-, IL-15-, and PBS-treated mice by FACS analysis. Because expressions of selected B-1 cell specific surface molecule on the B cells (e.g. B220) have been shown to change after isotype class switching (37), it was important to use an additional marker for the distinction of B-1 and B-2 cells. Thus, we also examined sIgA+ B220low and B220high fractions using Mac-1 specific mAb. As results, the sIgA+ B220low fraction also expressed Mac-1, but not sIgA+ B220high fraction (data not shown). Because Mac-1 expression has been shown to associate with B-1, but not B-2, subset (16, 17, 19, 20), these results further indicate that sIgA+ B220low and B220high fractions belong to B-1 and B-2 cells, respectively. In vivo treatment with IL-15 resulted in the preferential increase of sIgA+ B-1 cells when compared with B-2 cells (Fig. 4). Further, IL-5 treatment also resulted in the preferential enhancement of sIgA+ B-1 cells (Fig. 4). However, it should be noted that the slight increase of sIgA+ B-2 cell was also noted in IL-5-treated mice. To this end, a previous study (18) showed that the corresponding receptor is expressed on B-2 cells. In contrast, in vivo treatment with IL-2 showed no such enhancing effect for sIgA+ B-1 cells (Fig. 4). Instead, IL-2-treated mice showed a slight increase in the frequency of sIgA+ B-2 cells. These findings raise the interesting possibility that IL-15, like the well-known mucosal cytokine IL-5, could be an essential cytokine for the differentiation of mucosal sIgA+ B-1 cells.

FIGURE 4.

Two-color immunofluorescence analysis of sIgA+ B-1 and B-2 cells in mucosal effector tissues. i-LP lymphocytes were isolated from several types of cytokine-treated and untreated (control) mice. The cells were then stained with FITC-conjugated anti-mouse IgA and PE-conjugated anti-mouse CD45R/B220. The two subsets of sIgA+ B cells were classified into B-1 (B220low) and B-2 (B220high). The results represent the mean of three separate experiments (three mice per group).

FIGURE 4.

Two-color immunofluorescence analysis of sIgA+ B-1 and B-2 cells in mucosal effector tissues. i-LP lymphocytes were isolated from several types of cytokine-treated and untreated (control) mice. The cells were then stained with FITC-conjugated anti-mouse IgA and PE-conjugated anti-mouse CD45R/B220. The two subsets of sIgA+ B cells were classified into B-1 (B220low) and B-2 (B220high). The results represent the mean of three separate experiments (three mice per group).

Close modal

We next examined the effect of IL-15 on the proliferative responses of mucosal B cells in vitro. IL-15 caused an increase in the [3H]thymidine incorporation of intestinal B-1 cells (IgMhigh, IgDlow) in a dose-dependent manner. Approximately a 20-fold higher [3H]thymidine incorporation was noted in IL-15-treated intestinal B-1 cells (IgMhigh, IgDlow) than in B-2 cells (IgMlow, IgDhigh) at a dosage of 100 ng/ml (Fig. 5). Incubation with IL-5 resulted in a similar effect in which higher proliferation was observed in B-1 cells in comparison to B-2 cells. In contrast to IL-15 and IL-5, IL-2 did not induce high levels of cell proliferation in intestinal B-1 cells. According to the expression and intensity of sIgM and sIgD, B-1-like cells and B-1 cells were also found in marginal zone of SP and peritoneal cavity, respectively (38). Thus, marginal zone B-1-like cells (IgMhigh, IgDlow, CD5, Mac-1, CD23+, and B220high) and peritoneal B-1 cells (IgMhigh, IgDlow, CD5+, Mac-1+, CD23, and B220low) were isolated and cultured with IL-15. High levels of proliferation were noted in peritoneal B-1 cells (8650 ± 1560 cpm), but not marginal zone B-1 like cells (1050 ± 650 cpm). These findings suggest that, like IL-5, IL-15 is an effective growth and activation cytokine for intestinal B-1 cells. Further evidence in support of this view includes the increased proportion of large and blast cells in cultures containing IL-15 or IL-5 when compared with IL-2, as assessed by FACS analysis of forward scatter, cell size and side scatter, granularity (Fig. 6). In contrast, the frequency of those activated cells was low in intestinal B-2 cells cocultured with IL-15 or IL-5 (Fig. 6). These findings provide further evidence that IL-15 is a key cytokine for the growth and activation of intestinal B-1 cells.

FIGURE 5.

IL-15 preferentially induces proliferative responses in intestinal B-1 cells. B-1 (IgMhigh, IgDlow) or B-2 (IgMlow, IgDhigh) cells were isolated from i-LP by FACSorting. Purified B cells (1 × 105 cells/well) were cultured for 3 days with various concentrations of recombinant cytokines (IL-2 (○), IL-5 (□), and IL-15 (•)) at 37°C, and then assayed for [3H]thymidine incorporation during the final 18 h of incubation. The data represent the mean ± SE of three separate experiments.

FIGURE 5.

IL-15 preferentially induces proliferative responses in intestinal B-1 cells. B-1 (IgMhigh, IgDlow) or B-2 (IgMlow, IgDhigh) cells were isolated from i-LP by FACSorting. Purified B cells (1 × 105 cells/well) were cultured for 3 days with various concentrations of recombinant cytokines (IL-2 (○), IL-5 (□), and IL-15 (•)) at 37°C, and then assayed for [3H]thymidine incorporation during the final 18 h of incubation. The data represent the mean ± SE of three separate experiments.

Close modal
FIGURE 6.

FACS analysis of intestinal B-1 and B-2 cells induced by IL-15. B-1 or B-2 cells (1 × 105 cells) purified by flow cytometer sorting were cultured with IL-2, IL-5, or IL-15 for 3 days at 37°C. After culturing, blasted cells were gated in forward scatter, cell size and side scatter, granularity profile. The data represent the mean of three separate experiments.

FIGURE 6.

FACS analysis of intestinal B-1 and B-2 cells induced by IL-15. B-1 or B-2 cells (1 × 105 cells) purified by flow cytometer sorting were cultured with IL-2, IL-5, or IL-15 for 3 days at 37°C. After culturing, blasted cells were gated in forward scatter, cell size and side scatter, granularity profile. The data represent the mean of three separate experiments.

Close modal

When IL-15Rα expression was examined at the mRNA level in the different subsets (e.g., B-1 and B-2 cells) of freshly isolated sIgM+sIgA or sIgA+ B cells from i-LP, the receptor message was ∼30 fmol/1 ng synthesized cDNA in intestinal B-1 cells of both sIgM+sIgA and sIgA+ phenotypes. In contrast, much lower levels of IL-15Rα-specific mRNA were noted in intestinal and splenic B-2 cells (Fig. 7,A). These findings directly demonstrate that intestinal B-1 cells express much higher levels of IL-15R than those of B-2 cells. When both sIgM+sIgA and sIgA+ B-1 cells were cocultured with 100 ng/ml IL-15 for 24 h at 37°C, the mRNA expression of IL-15Rα was more pronounced (250∼350 fmol/1 ng cDNA) than in untreated and freshly isolated B-1 cells (Fig. 7,B). When identical culture conditions were provided to sIgM+sIgA and sIgA+ B-2 cells, only a slight increase of IL-15Rα-specific mRNA was noted (∼50 fmol/1 ng cDNA; Fig. 7 B). In contrast to IL-15, IL-2 did not induce IL-15Rα-specific mRNA expression in either B-1 or B-2 cells (data not shown). These results suggest the interesting possibility that the expression of IL-15R by B-1 cells could be autocrine-regulated with IL-15.

FIGURE 7.

Levels of IL-15Rα-specific mRNA in intestinal B-1 cells. For assessment of IL-15Rα mRNA levels in RNA freshly isolated from each cell fraction (A) or from fractions cocultured with IL-15 (B), quantitative RT-PCR was performed. Synthesized cDNA (1 ng) was applied to provide an external standard for comparing target-specific rDNA. The LightCycler technology combines rapid thermocycling with on-line fluorescence detection of PCR product formation by using hybridization probes. The results represent the mean ± SE of five separate experiments.

FIGURE 7.

Levels of IL-15Rα-specific mRNA in intestinal B-1 cells. For assessment of IL-15Rα mRNA levels in RNA freshly isolated from each cell fraction (A) or from fractions cocultured with IL-15 (B), quantitative RT-PCR was performed. Synthesized cDNA (1 ng) was applied to provide an external standard for comparing target-specific rDNA. The LightCycler technology combines rapid thermocycling with on-line fluorescence detection of PCR product formation by using hybridization probes. The results represent the mean ± SE of five separate experiments.

Close modal

It would be of interest to know whether IL-15 was involved in the IgA isotype-switching process of B-1 and B-2 cells. To this end, highly purified sIgM+sIgA B-1 and B-2 cells from i-LP and peritoneal cavity as well as splenic sIgM+sIgA B-2 cells were isolated by FACS for the analysis of the Cα germline transcript by RT-PCR. Surprisingly, the specific message for the Cα germline transcript was detected in de novo isolated intestinal and peritoneal sIgM+sIgA B-1 cells (Fig. 8). However, intestinal, peritoneal, and splenic sIgM+sIgA B-2 cells did not express the Cα germline transcript (Fig. 8). When these different subsets of B-1 and B-2 cells were cocultured with IL-15, the level of LPS-stimulated Cα germline transcript mRNA was enhanced in sIgM+sIgA B-1 cells (Fig. 8). However, IL-15 did not induce the Cα germline transcript for intestinal, peritoneal, and splenic sIgM+sIgA B-2 cells (Fig. 8). As positive controls, splenic and peritoneal sIgM+sIgA B-2 cells were incubated with TGF-β1, resulting in the induction of the Cα germline transcript. These results suggest that IL-15 is not directly involved in the IgA class-switching of B-1 and B-2 cells. An additional interesting finding of this series of experiments was the detection of the Cα germline transcript in de novo isolated intestinal sIgM+sIgA B-1 cells but not intestinal and splenic B-2 cells.

FIGURE 8.

Characterization of germline Cα transcript expression in freshly isolated and IL-15-treated intestinal sIgM+sIgA B-1 cells. The expression of Cα transcripts was determined by RT-PCR analysis of 10 ng cDNA synthesized from freshly isolated cell fraction (lane 1) or from the intestinal B-1 cell cultures containing LPS with IL-15 (lane 2) or TGF-β1 (lane 3), respectively. As control, an identical experiment was performed by sIgM+sIgA B-1 and B-2 cells isolated from peritoneal cavity and SP. The size of the expected PCR products for germline Cα transcripts and β-actin are mainly 379 and 239 bp, and 349 bp, respectively. The result represents from three separate experiments.

FIGURE 8.

Characterization of germline Cα transcript expression in freshly isolated and IL-15-treated intestinal sIgM+sIgA B-1 cells. The expression of Cα transcripts was determined by RT-PCR analysis of 10 ng cDNA synthesized from freshly isolated cell fraction (lane 1) or from the intestinal B-1 cell cultures containing LPS with IL-15 (lane 2) or TGF-β1 (lane 3), respectively. As control, an identical experiment was performed by sIgM+sIgA B-1 and B-2 cells isolated from peritoneal cavity and SP. The size of the expected PCR products for germline Cα transcripts and β-actin are mainly 379 and 239 bp, and 349 bp, respectively. The result represents from three separate experiments.

Close modal

In our final experiment for this study, subsets of B-1 or B-2 cells with varying expressions of sIgM and sIgA were isolated from i-LP and then cocultured with or without IL-2, IL-5, IL-6, and IL-15 in a LPS-stimulated in vitro system. When sIgMsIgA+ B-1 cells were incubated with IL-5 and IL-15 but not IL-2 and IL-6, high levels of IgA synthesis were noted (Fig. 9). Although both IL-5 and IL-15 enhanced IgA synthesis in sIgMsIgA+ B cells, the level of IgA was higher in the culture containing IL-5 than in that containing IL-15. However, it should be interesting to note that IL-15 induced high levels of IgA synthesis in sIgM+sIgA B-1 cells in addition to sIgMsIgA+ B-1 cells (Fig. 9). In contrast, such an effect was not seen in IL-15-treated sIgM+sIgA B-2 cells. When sIgMsIgA+ B-2 cells were incubated with IL-15, an increase of IgA synthesis was noted. However, the magnitude of the IgA-enhancing effect was lower in sIgMsIgA+ B-2 cells than in sIgMsIgA+ B-1 cells. As one might expect based on our previous study (18), both IL-5 and IL-6 supported IgA synthesis in LPS-stimulated sIgMsIgA+ B-2 cells (Fig. 9). These findings suggest that IL-15 is an important cytokine for the differentiation of both sIgM+sIgA and sIgMsIgA+ B-1 cells into IgA-producing plasma cells, though it has little effect on B-2 cells. Because sIgM+sIgA B-1 cells were considered to be already class switched based on their expression of the germline Cα transcript, these cells might be differentiated into sIgA+ B-1 cells and developed into IgA plasma cells by the direct influence of IL-15.

FIGURE 9.

Comparison of IgA production from intestinal sIgM+sIgA or sIgA+ B-1 and B-2 cells cocultured with IL-2, IL-5, IL-6, or IL-15. sIgM+sIgA (□) or sIgA+ (▪) B-1 and B-2 cells were isolated from i-LP by flow cytometry sorting. Purified B cells (1 × 104 cells) were then cultured in 100 μl complete medium containing 2 μg/ml LPS with or without 100 ng/ml IL-2, IL-5, or IL-15 or 500 pg/ml IL-6. After 3 days, culture supernatants were harvested and levels of IgA production were measured by isotype-specific ELISA. The results represent the mean ± SE of five separate experiments. ∗, p < 0.05; ∗∗, p < 0.01.

FIGURE 9.

Comparison of IgA production from intestinal sIgM+sIgA or sIgA+ B-1 and B-2 cells cocultured with IL-2, IL-5, IL-6, or IL-15. sIgM+sIgA (□) or sIgA+ (▪) B-1 and B-2 cells were isolated from i-LP by flow cytometry sorting. Purified B cells (1 × 104 cells) were then cultured in 100 μl complete medium containing 2 μg/ml LPS with or without 100 ng/ml IL-2, IL-5, or IL-15 or 500 pg/ml IL-6. After 3 days, culture supernatants were harvested and levels of IgA production were measured by isotype-specific ELISA. The results represent the mean ± SE of five separate experiments. ∗, p < 0.05; ∗∗, p < 0.01.

Close modal

A group of Th2 cytokines has been shown to be an essential signaling factor in the triggering of sIgA+ B cells to become IgA-secreting plasma cells. For example, IL-5, IL-6, and/or IL-10 were detected in vitro after sIgA+ B cells had differentiated into plasma cells, resulting in IgA synthesis (39, 40, 41, 42, 43). An important finding of the present study is that IL-15, like the above-mentioned IgA-enhancing Th2-type cytokines, affected IgA B cell development in mucosal but not in systemic compartments in vivo (Figs. 2 and 3). Further, it is interesting and important to note that the B-1, but not the B-2, lineage of sIgA+ B cells express a corresponding receptor for the new IgA-enhancing cytokine. Our present study builds on our previous research which suggested that B-1 cells could be a major source for CMIS-independent IgA plasma cells by suggesting that the IL-15 and IL-15R signaling cascade is an essential element for the differentiation of CMIS-independent sIgA+ B-1 cells into IgA plasma cells.

IL-2 and IL-15 have been shown to play a comparable biological role in the induction of B cell proliferation and differentiation (4). IL-15 possesses a synergistic effect for the proliferation of anti-μ or phorbol ester-activated, but not resting, B cells (4). Further, IL-15 uses the β-chain of IL-2R for signal transduction in B cells (4). Our present study sheds new light on IL-2- and IL-15-regulated B cell responses by showing that these two functionally redundant cytokines may act on two different subsets of B cells. The frequency of sIgA+ B-2 cells was shown to increase under the influence of IL-2 (Fig. 4). Further, cocultivation of anti-μ-treated B-2 cells with IL-2 resulted in a higher degree of proliferative response than with identically treated B-1 cells (data not shown). In the course of the early study investigating the IgA-enhancing effect of different cytokines, IL-2 caused a 2- to 3-fold enhancement in IgA secretion in cultures of LPS-stimulated B cell blasts (44). Thus, IL-2 might be capable of playing an important role in the terminal differentiation of sIgA+ B-2 cells in mucosal immunity. In contrast, IL-15 treatment led to the enhancement of sIgA+ B-1 but not B-2 cells (Fig. 4). A similar effect was also provided by IL-5 (Fig. 4). These findings demonstrate that IL-15 is an important stimulation cytokine for B-1 cells while IL-2 is more effective on B-2 cells. Thus, although IL-15 and IL-2 share a similar biological activity, these two cytokines regulate two distinct subsets of B cells, namely B-1 and B-2 cells, respectively.

In regard to the regulation of B-1 cells for the induction of IgA responses, IL-5 has been shown to be a key cytokine for inducing IgA-committed B-1 cells to differentiate into IgA-producing plasma cells (18, 45). Our own previous study provided evidence of this key role for IL-5 by directly demonstrating that the lack of the IL-5R gene resulted in the reduction of B-1 cell-derived IgA synthesis (18). Our present study provides new evidence that IL-15, like IL-5, plays a crucial role in the regulation of B-1 cell differentiation into IgA plasma cells. Our findings in this study lead us to suggest that IL-15 and IL-5 may act on two different differentiation stages of B-1 cells. Increased levels of IgA synthesis were noted in the culture containing sIgM+sIgA B-1 cells and IL-15 (Fig. 9), while, in contrast, IL-5 supported high levels of IgA production in sIgMsIgA+ B-1 cells. IL-15 may act on an earlier stage of differentiation of B-1 cells (e.g., sIgM+sIgA), while IL-5 may influence the process of final differentiation (e.g., sIgMsIgA+) into IgA plasma cells (Fig. 10). Two additional scenarios for IL-15 and IL-5 regulation of B-1 cells for IgA responses are plausible. Because IL-15 induced IgA synthesis in sIgMsIgA+ B-1 cells in addition to sIgM+sIgA B-1 cells, this soluble factor could be a compensatory cytokine for IL-5. Alternatively, IL-15 may function as an IgA isotype-switching factor for B-1 cells because the production of IgA was induced in sIgM+sIgA B-1 cells (Fig. 9).

FIGURE 10.

A schematic view of CMIS-independent B-1 cell differentiation into IgA-producing cells by IL-15 and IL-5. IL-15 may act on an earlier stage of the differentiation of B-1 cells (e.g., sIgM+sIgA), while IL-5 influence on the process of final differentiation of B-1 cells (e.g., sIgMsIgA+) into IgA plasma cells. In contrast, the process of CMIS-dependent B-2 cell development consists with TGF-β induced isotype switching and IL-5/IL-6 regulated B cell differentiation into IgA-producing cells.

FIGURE 10.

A schematic view of CMIS-independent B-1 cell differentiation into IgA-producing cells by IL-15 and IL-5. IL-15 may act on an earlier stage of the differentiation of B-1 cells (e.g., sIgM+sIgA), while IL-5 influence on the process of final differentiation of B-1 cells (e.g., sIgMsIgA+) into IgA plasma cells. In contrast, the process of CMIS-dependent B-2 cell development consists with TGF-β induced isotype switching and IL-5/IL-6 regulated B cell differentiation into IgA-producing cells.

Close modal

Inasmuch as IL-15 supported IgA synthesis in sIgM+sIgA B-1 cells, it was important to determine whether the cytokine was capable of inducing IgA class switching. Because TGF-β1 has been shown to be a key cytokine for the induction of IgA class switching (46, 47), we compared the ability of IL-15 to induce class-switching for IgA isotypes to the well-known IgA isotype-switching factor TGF-β1. According to the results described in this study, it is unlikely that IL-15 plays an important role in the induction of IgA isotype class-switching in B-1 cells. Our study showed that sIgM+sIgA B-1 cells isolated from the intestinal mucosa and the peritoneal cavity already expressed germline Cα transcript-specific mRNA (Fig. 8). Further, cocultivation of these sIgM+sIgA B-1 cells and IL-15 resulted in high levels of IgA synthesis (Fig. 9). However, it might be possible that additional factors, such as costimulation signals provided by CD40 ligand and/or B cell receptor, are necessary to induce IgA class-switching by IL-15. Thus, ligation of CD40 and CD40 ligand by direct B cell and T cell interaction has been shown to be an important costimulation signal for isotype-switching (48). Thus, the class-switching potential of IL-15 needs to be more carefully examined using B-1a, B-1b, and B-2 cells at varying stages of development.

Another new and interesting finding of this study is that intestinal sIgM+sIgA B-1 cells were already class-switched to IgA because Cα transcript-specific mRNA expression was detected (Fig. 8). IgA class-switching in sIgM+sIgA B cells has been shown to precede the synthesis of germline Cα mRNA transcripts (49). In contrast, it was found that intestinal and splenic sIgM+sIgA B-2 cells did not express Cα transcript-specific mRNA (Fig. 8), while peritoneal B-1 cells did as like intestinal B-1 cells. Further, peritoneal B-1 cells have been considered to be one of the major sources for intestinal IgA plasma cells (14, 19, 20, 30). Taken together, these findings provide new evidence for the existence of a CMIS-independent IgA B cell development pathway and suggest for the first time that it consists of two distinct steps regulated by IL-15/IL-15R and IL-5/IL-5R signaling cascades. The process of IgA class-switching for B-1 cells may initially occur in the peritoneal cavity before the migration of the B-1 cells to intestinal mucosa. Alternatively, such CMIS-independent B-1 cell IgA isotype class-switching may occur at an unidentified mucosal inductive site in the intestinal tract. To this end, it was recently shown that cryptopatches are sites for the development of thymus-independent intestinal T cells (50, 51). These class-switched sIgM+sIgA B-1 cells from peritoneal cavity or unidentified mucosal inductive site, especially those of the B-1b subset, will preferentially migrate to the intestinal tract and will become sIgMsIgA+ B cells under the influence of the IL-15 and IL-15R signaling cascades. These IL-15/IL-15R-stimulated sIgMsIgA+ B-1 cells will become responsible for the well-known IL-5- and IL-5R-derived stimulation signal for the final differentiation into IgA-producing plasma cells (Fig. 10).

In summary, our study provides new evidence that IL-15 is a critically important mucosal cytokine for the regulation of IgA responses. It has shown that intestinal B-1 cells preferentially express IL-15R. Further, it has demonstrated that IL-15 induced differentiation of not only sIgMsIgA+ B-1 cells but also of sIgM+sIgA B-1 cells into plasma cells for subsequent IgA production in mucosal effector tissues (Fig. 10). It is interesting to note that IL-15 acts on two different differentiation stages (sIgM+sIgA and sIgMsIgA+) of CMIS-independent B-1 cells while the well-known mucosal cytokine IL-5 only acts on the stage of sIgMsIgA+ B-1 cells (Fig. 10). In contrast, CMIS-dependent B-2 cells originating from IgA inductive sites (e.g., Payer’s patch) express IL-5R and IL-6R, but not IL-15R, and can respond to stimulation signals provided by both IL-5 and IL-6 to become IgA-producing cells (18). Taken together, these findings suggest that the induction of IgA-producing cells by intestinal B-1 and B-2 cells is regulated by two groups of cytokines, with IL-5 appearing in both groups. Along with IL-15, IL-5 induces CMIS-independent IgA responses, but it also shares with IL-6 a regulatory role for CMIS-dependent IgA responses.

We thank the members of the Mucosal Immunology Group of Osaka University for their critical comments. We also thank Noriko Kitagaki for her technical help and Dr. Kimbery K. McGhee (University of Alabama, Birmingham, AL) for editorial assistance.

1

This work is supported by grants from the Ministry of Education, Science, Sports, and Culture, the Ministry of Health and Welfare, and the Organization for Pharmaceutical Safety and Research, Japan.

3

Abbreviations used in this paper: i-EC, intestinal epithelial cells; AFC, Ab-forming cells; CMIS, common mucosal immune system; ELISPOT, enzyme-linked immunospot; i-IEL, intestinal intraepithelial lymphocytes; i-LP, intestinal lamina propria; s, surface; SMG, submandibular gland; SP, spleen.

1
Grabstein, K. H., J. Eisenman, K. Shanebeck, C. Rauch, S. Srinivasan, V. Fung, C. Beers, J. Richardson, M. A. Schoenborn, M. Ahdieh, et al
1994
. Cloning of a T cell growth factor that interacts with the β-chain of the interleukin-2 receptor.
Science
264
:
965
2
Giri, J. G., M. Ahdieh, J. Eisenman, K. Grabstein, S. Kumaki, A. Namen, L. S. Park, D. Cosman, D. Anderson.
1994
. Utilization of the β- and γ-chains of the IL-2 receptor by the novel cytokine IL-15.
EMBO J.
13
:
2822
3
Burton, J. D., R. N. Bamford, C. Peters, A. J. Grant, G. Kurys, C. K. Goldman, J. Brennan, E. Roessler, T. A. Waldmann.
1994
. A lymphokine, provisionally designated interleukin T and produced by a human.
Proc. Natl. Acad. Sci. USA
91
:
4935
4
Armitage, R. J., B. M. Macduff, J. Eisenman, R. Paxton, K. H. Grabstein.
1995
. IL-15 has stimulatory activity for the induction of B cell proliferation and differentiation.
J. Immunol.
154
:
483
5
Bulfone-Paus, S., D. Ungureanu, T. Pohl, G. Lindner, R. Paus, R. Ruckert, H. Krause, U. Kunzendorf.
1997
. Interleukin-15 protects from lethal apoptosis in vivo.
Nat. Med.
3
:
1124
6
Griebel, P., T. Beskorwayne, A. Van den Broeke, G. Ferrari.
1999
. CD40 signaling induces B cell responsiveness to multiple members of the γ-chain-common cytokine family.
Int. Immunol.
11
:
1139
7
Suzuki, H., G. S. Duncan, H. Takimoto, T. W. Mak.
1997
. Abnormal development of intestinal intraepithelial lymphocytes and peripheral natural killer cells in mice lacking the IL-2 receptor β-chain.
J. Exp. Med.
185
:
499
8
Carson, W. E., J. G. Giri, M. J. Lindemann, M. L. Linett, M. Ahdieh, R. Paxton, D. Anderson, J. Eisenmann, K. Grabstein, M. A. Caligiuri.
1994
. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor.
J. Exp. Med.
180
:
1395
9
Reinecker, H. C., R. P. MacDermott, S. Mirau, A. Dignass, D. K. Podolsky.
1996
. Intestinal epithelial cells both express and respond to interleukin 15.
Gastroenterology
111
:
1706
10
Inagaki-Ohara, K., H. Nishimura, A. Mitani, Y. Yoshikai.
1997
. Interleukin-15 preferentially promotes the growth of intestinal intraepithelial lymphocytes bearing γδ T cell receptor in mice.
Eur. J. Immunol.
27
:
2885
11
Hirose, K., H. Suzuki, H. Nishimura, A. Mitani, J. Washizu, T. Matsuguchi, Y. Yoshikai.
1998
. Interleukin-15 may be responsible for early activation of intestinal intraepithelial lymphocytes after oral infection with Listeria monocytogenes in rats.
Infect. Immun.
66
:
5677
12
Ohteki, T., H. Yoshida, T. Matsuyama, G. S. Duncan, T. W. Mak, P. S. Ohashi.
1998
. The transcription factor interferon regulatory factor 1 (IRF-1) is important during the maturation of natural killer 1.1+ T cell receptor-α/β+ (NK1+ T) cells, natural killer cells, and intestinal intraepithelial T cells.
J. Exp. Med.
187
:
967
13
Yamada, K., Y. Kimura, H. Nishimura, Y. Namii, M. Murase, and Y. Yoshikai. Characterization of CD4+CD8αα+ and CD4CD8αα+ intestinal intraepithelial lymphocytes in rats. Int. Immunol. 11:21.
14
Murakami, M., H. Yoshioka, T. Shirai, T. Tsubata, T. Honjo.
1995
. Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells.
Int. Immunol.
7
:
877
15
Coffman, R. L., I. L. Weissman.
1981
. B220, a B cell specific member of the T200 glycoprotein family.
Nature
289
:
681
16
Kantor, A. B., L. A. Herzenberg.
1993
. Origin of murine B cell lineages.
Annu. Rev. Immunol.
11
:
501
17
Kantor, A. B., A. M. Stall, S. Adams, L. A. Herzenberg, L. A. Herzenberg.
1992
. Differential development of progenitor activity for three B-cell lineages.
Proc. Natl. Acad. Sci. USA
89
:
3320
18
Hiroi, T., M. Yanagita, H. Iijima, K. Iwatani, T. Yoshida, K. Takatsu, H. Kiyono.
1999
. Deficiency of IL-5 receptor α-chain selectively influences the development of the common mucosal immune system-independent IgA-producing B-1 cell in mucosa-associated tissues.
J. Immunol.
162
:
821
19
Kroese, F. G., E. C. Butcher, A. M. Stall, P. A. Lalor, S. Adams, L. A. Herzenberg.
1989
. Many of the IgA-producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity.
Int. Immunol.
1
:
75
20
Kroese, F. G., W. A. Ammerlaan, A. B. Kantor.
1993
. Evidence that intestinal IgA plasma cells in μ-, κ-transgenic mice are derived from B-1 (Ly-1 B) cells.
Int. Immunol.
5
:
1317
21
Solvason, N., A. Lehuen, J. F. Kearney.
1991
. An embryonic source of Ly1 but not conventional B cells.
Int. Immunol.
3
:
543
22
McGhee, J. R., J. Mestecky, C. O. Elson, H. Kiyono.
1989
. Regulation of IgA synthesis and immune response by T cells and interleukins.
J. Clin. Immunol.
9
:
175
23
Fujihashi, K., J. R. McGhee, M.-N. Kweon, M. D. Cooper, S. Tonegawa, I. Takahashi, T. Hiroi, J. Mestecky, H. Kiyono.
1996
. γ/δ T cell-deficient mice have impaired mucosal immunoglobulin A responses.
J. Exp. Med.
183
:
1929
24
Hiroi, T., K. Fuihashi, J. R. McGhee, H. Kiyono.
1995
. Polarized Th2 cytokine expression by both mucosal γδ and αβ T cells.
Eur. J. Immunol.
25
:
2743
25
Hiroi, T., K. Fujihashi, J. R. McGhee, H. Kiyono.
1994
. Characterization of cytokine-producing cells in mucosal effector sites: CD3+ T cells of Th1 and Th2 type in salivary gland-associated tissues.
Eur. J. Immunol.
24
:
2653
26
Jackson, R. J., K. Fujihashi, J. Xu-Amano, H. Kiyono, C. O. Elson, J. R. McGhee.
1993
. Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant.
Infect. Immun.
61
:
4272
27
Mega, J., J. R. McGhee, H. Kiyono.
1992
. Cytokine and Ig-producing T cells in mucosal effector tissues: analysis of IL-5 and IFN-γ-producing T cells, TCR expression and IgA plasma cells from mouse salivary gland-associated tissues.
J. Immunol.
148
:
2030
28
Kantor, A. B., C. E. Merrill, L. A. Herzenberg, J. L. Hillson.
1997
. An unbiased analysis of VH-D-JH sequences from B-1a, B-1b and conventional B cells.
J. Immunol.
158
:
1175
29
Peters, M. G., H. Secrist, K. R. Anders, G. S. Nash, S. R. Rich, R. P. MacDermott.
1989
. Normal human intestinal B lymphocytes: increased activation compared with peripheral blood.
J. Clin. Invest.
83
:
1827
30
Murakami, M., T. Tsubata, R. Shinkura, S. Nishitani, M. Okamoto, H. Yoshioka, T. Usui, S. Miyawaki, T. Honjo.
1994
. Oral administration of lipopolysaccharides activates B-1 cells in the peritoneal cavity and lamina propria of the gut and induces autoimmune symptoms in an autoantibody transgenic mouse.
J. Exp. Med.
180
:
111
31
Wittwer, C. T., K. M. Ririe, R. V. Andrew, D. A. David, R. A. Gundry, U. J. Balis.
1997
. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control.
BioTechniques
22
:
176
32
Wittwer, C. T., M. G. Herrmann, A. A. Moss, R. P. Rasmussen.
1997
. Continuous fluorescence monitoring of rapid cycle DNA amplification.
BioTechniques
22
:
130
33
Yanagita, M., T. Hiroi, N. Kitagaki, S. Hamada, H. Ito, H. Shimauch, S. Murakami, H. Okada, H. Kiyono.
1999
. Nasopharyngel-associated lymphoreticular tissue (NALT) immunity: fimbriae-specific Th1 and Th2 cell-regulated IgA responses for the inhibition of bacterial attachment to epithelial cells and subsequent inflammatory cytokine production.
J. Immunol.
162
:
3559
34
De Silva, D., M. Herrmann, K. Tabiti, C. Wittwer.
1998
. Rapid genotyping and quantification on the LightCycler with hybridization probes.
Biochemica
2
:
12
35
de Waard, R., P. M. Dammers, J. W. Tung, A. B. Kantor, J. A. Wilshire, N. A. Bos, L. A. Herzenberg, F. G. Kroese.
1998
. Presence of germline and full-length IgA RNA transcripts among peritoneal B-1 cells.
Dev. Immunol.
6
:
81
36
Harriman, G. R., A. Bradley, S. Das, P. Rogers-Fani, A. C. Davis.
1996
. IgA class switch in I α exon-deficient mice: role of germline transcription in class switch recombination.
J. Clin. Invest.
97
:
477
37
Bromander, A. K., L. Ekman, M. Kopf, J. G. Nedrud, N. Y. Lycke.
1996
. IL-6-deficient mice exhibit normal mucosal IgA responses to local immunizations and Helicobacterfelis infection.
J. Immunol.
156
:
4290
38
Wells, S. M., A. B. Kantor, A. M. Stall.
1994
. CD43 (S7) expression identifies peripheral B cell subsets.
J. Immunol.
153
:
5503
39
Beagley, K. W., J. H. Eldridge, H. Kiyono, M. P. Everson, W. J. Koopman, T. Honjo, J. R. McGhee.
1988
. Recombinant IL-5 induces high rate of IgA synthesis in cycling IgA-positive Peyer’s patch B cells.
J. Immunol.
141
:
2035
40
Lebman, D. A., R. L. Coffman.
1988
. The effects of IL-4 and IL-5 on the IgA response by murine Peyer’s patch B cell subpopulations.
J. Immunol.
141
:
2050
41
Kunimoto, D. Y., R. P. Nordan, W. Strober.
1989
. IL-6 is a potent cofactor of IL-1 in IgM synthesis and of IL-5 in IgA synthesis.
J. Immunol.
143
:
2230
42
Sonoda, E., R. Matsumoto, Y. Hitoshi, S. Mita, T. Ishii, M. Sugimoto, S. Araki, A. Tominaga, N. Yamaguchi, K. Takatsu.
1989
. Transforming growth factor-β induces IgA production and acts additively with IL-5 for IgA production.
J. Exp. Med.
170
:
1415
43
Beagley, K. W., J. H. Eldridge, F. Lee, H. Kiyono, M. P. Everson, W. J. Koopman, T. Hirano, T. Kishimoto, J. R. McGhee.
1989
. Interleukins and IgA synthesis: human and murine IL-6 induce high rate of IgA secretion in IgA-committed B cells.
J. Exp. Med.
169
:
2133
44
Coffman, R. L., B. Shrader, J. Carty, T. R. Mosmann, M. W. Bond.
1987
. A mouse T cell product that preferentially enhances IgA production. I. Biologic characterization.
J. Immunol.
139
:
3685
45
Bao, S., K. W. Beagley, A. M. Murray, V. Caristo, K. I. Matthaei, I. G. Young, A. J. Husband.
1998
. Intestinal IgA plasma cells of the B1 lineage are IL-5 dependent.
Immunology
94
:
181
46
Iwasato, T., H. Arakawa, A. Shimizu, T. Honjo, H. Yamagishi.
1992
. Biased distribution of recombination sites within S regions upon immunoglobulin class switch recombination induced by transforming growth factor β and lipopolysaccharide.
J. Exp. Med.
175
:
539
47
Harriman, G. R., D. Y. Kunimoto, J. F. Elliott, V. Paetkau, W. Strober.
1988
. The role of IL-5 in IgA B cell differentiation.
J. Immunol.
140
:
3033
48
Jumper, M. D., J. B. Splawski, P. E. Lipsky, K. Meek.
1994
. Ligation of CD40 induces sterile transcripts of multiple Ig H chain isotypes in human B cells.
J. Immunol.
152
:
438
49
Lebman, D. A., D. Y. Nomura, R. L. Coffman, F. L. Lee.
1990
. Molecular characterization of germline immunoglobulin A transcript produced during transforming growth factor type β-induced isotype switching.
Proc. Natl. Acad. Sci. USA
87
:
3962
50
Kanamori, Y., K. Ishimaru, M. Nanno, K. Maki, K. Ikuta, H. Nariuchi, H. Ishikawa.
1996
. Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop.
J. Exp. Med.
184
:
1449
51
Saito, H., Y. Kanamori, T. Takemori, H. Nariuchi, E. Kubota, H. Takahashi-Iwanaga, T. Iwanaga, H. Ishikawa.
1998
. Generation of intestinal T cells from progenitors residing in gut cryptopatches.
Science
280
:
275