IgG1 production by sIgD+ splenic B cells and peritoneal B-1 cells in response to IL-5 and CD38 ligation

Tokutaro Yasue, Masashi Baba, Shigeo Mori1, Chieko Mizoguchi, Shoji Uehara and Kiyoshi Takatsu

Departments of Immunology and
1 Pathology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Correspondence to: K. Takatsu


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induces proliferation, IL-5 receptor {alpha} chain expression and tyrosine phosphorylation of Bruton's tyrosine kinase. Furthermore, stimulation of splenic B cells with IL-5 together with CS/2 induces Blimp-1 expression and differentiation into Ig-producing cells. Here we examined the role of IL-5 in IgG1 and IgA production by B cells isolated from the spleen and peritoneal cavity. CD38 recognized by CS/2 was expressed in the follicular mantle B cells surrounding the germinal center, sIgD+ splenic B cells and peritoneal B cells. IL-5 induced IgG1 production in splenic sIgD+ B cells stimulated with CS/2, while it was ineffective to induce IgA production. Among the various cytokines tested, only IL-5 had a synergistic effect on IgG1 production with CS/2. IL-5 could induce the generation of Sµ–S{gamma}1 reciprocal recombination DNA products in CS/2-stimulated B cells. IL-4 was ineffective to induce either µ–{gamma}1 switch recombination or IgG1 secretion with CS/2, demonstrating that IL-5 promotes both µ–{gamma}1 switch recombination and IgG1 secretion in an IL-4-independent manner. The peritoneal B-2 cells exhibited both IgG1 and IgA production in response to IL-5 plus CS/2, while B-1 cells produced IgG1. These results imply that the pattern of differentiation to Ig-producing cells seen with peritoneal B cells is not identical to the pattern seen with splenic B cells and that peritoneal B-2 cells contain precursors of IgA-producing cells responding to IL-5 plus CS/2.

Keywords: B-1 cells, CD38, IgA, IgG1, IL-5, switch recombination


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The process of Ig isotype or class switching is critical for the generation of functional diversity in humoral immune responses and is highly regulated by cytokines, B cell activators or both (13). We have been particularly interested in understanding the role of IL-5 and CD38 in Ig class switching. IL-5 acts on activated B cells to induce terminal differentiation to Ig-secreting cells (4,5). CD38 is widely expressed in B-lineage cells and a role for CD38 in regulation of B-cell activation has been suggested by analysis using agonistic mAb to CD38 (610). Many investigators have begun characterizing some of the functional properties of CD38 using anti-CD38 antibodies to activate lymphocytes. Human CD38 is highly expressed in the germinal center B cells and is thought to play a key role in the signaling events involved in B cell development (11,12). Mouse CD38, on the other hand, is expressed in follicular B cells but is down-regulated in the germinal center B cells (13,14). Nevertheless, in both humans and mice, stimulation of CD38+ B cells with anti-CD38 mAb has profound effects on the cells' viability, activation, proliferation and differentiation (1322). The role of CD38 in Ig isotype switching remains unknown.

We previously reported that CD38 ligation of murine B cells by CS/2, an anti-mouse CD38 mAb, induces a potent proliferative response and expression of IL-5 receptor {alpha} chain (IL-5R{alpha}), and prevents B cell apoptosis (19). The proliferative response of CS/2-activated B cells can be augmented by addition of IL-5 which induces Blimp-1 expression and differentiation into IgM- and IgG1-producing cells (20,21). We also demonstrated that B cell differentiation induced by IL-5 and CS/2 is regulated by at least Bruton's tyrosine kinase and Lyn (20,21). While IL-5 has been shown to act on activated mouse B cells and enhance Ig production, limited information is available regarding the role of IL-5 in µ–{gamma}1 switch recombination.

B cells can be separated into at least two subsets, B-1 and B-2, based on the expression of CD5 and CD45/B220 (23), the intensity of sIgM and sIgD (24), as well as the differences in anatomical localization and functional characteristics (24). An intriguing observation is that up to 40% of IgA-producing cells in the murine intestinal lamina propria arise from a pool of B-1 precursors derived from the peritoneal cavity (25). B-1 cells isolated from peritoneal cavity of normal mice have been shown to be unresponsiveness to both anti-IgM and anti-CD38 stimulation even in the presence of cytokine (22). As we reported (26), all B-1 cells in the peritoneal cavity express IL-5R{alpha} and respond to IL-5 for terminal differentiation to IgM-producing cells. Taking this information together, peritoneal B cells may respond to CS/2 plus IL-5 for differentiation to IgG1- or IgA-producing cells.

In the present study, we characterized the responsiveness of splenic and peritoneal B cells to IL-5 plus CS/2 for proliferation and production of IgG1 and IgA in order to better understand the role of IL-5 in differentiation of the discrete stages or a subset of B cells. We demonstrate that stimulation of sIgD+ B cells in spleen with CS/2 plus IL-5 induces IgG1, but not IgA, production. We also present evidence that IL-5 is capable of inducing µ–{gamma}1 switch recombination in CS/2-stimulated splenic B cells. Although peritoneal B cells show a basal level of proliferation in response to IL-5 plus CS/2, they produce significant amounts of IgG1 and IgA.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
BALB/c and C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). IL-5R{alpha}–/– mice were generated as previously described (27). All mice were maintained on a diet of laboratory chow and water available ad libitum in the animal facility of the Institute of Medical Science, University of Tokyo. Mice were used for experimentation at 8–12 weeks of age. In some experiments, mice were immunized with 50 µg of DNP–keyhole limpet hemocyanin (KLH) in incomplete Freund's adjuvant by i.p. injection (28) and boosted with 100 µg of DNP–KLH in saline on day 28. Spleens were removed on the days indicated.

Antibodies and reagents
The following mAb and reagents were obtained from ATCC (Rockville, MD): RA3-6B2 (anti-B220), 2.4G2 [anti-mouse (m) Fc{gamma}R] and M1/70 (anti-Mac-1). CS/2 (anti-mCD38; 19), and T21 and H7 (anti-mIL-5R{alpha}; 26) were prepared as described. HB (anti-mouse ßc; 29), LB429 (anti-mCD40; 30) and Jo-2 (anti-mFas; 31) mAb were kindly provided by S. Yonehara (Kyoto University), N. Sakaguchi (Kumamoto University) and S. Nagata (Osaka University) respectively. Purified mAb were coupled with biotin (Pierce, Rockford, IL), FITC (Sigma, St Louis, MO) or phycoerythrin (PE; Pierce). FITC-conjugated and biotinylated AMS9.1 (anti-mIgDa) and biotinylated RB6-8C5 (anti-Gr-1) mAb were purchased from PharMingen (San Diego, CA). FITC-conjugated and biotinylated peanut agglutinin (PNA) was purchased from Vector (Burlingame, CA). Streptavidin (SA)–PE was purchased from Life Technologies (Tokyo, Japan). SA–allophycocyanin was purchased from Leinco Technologies (Manchester, UK). PE–goat F(ab')2 anti-rat IgG was purchased from Cedarlane Laboratories (Ontario, Canada). SA indirect microbeads and anti-FITC microbeads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Biotinylated goat anti-mIgG1 was purchased from Southern Biotechnology Associates (Birmingham, AL). Horseradish peroxidase-conjugated SA was purchased from Zymed (San Francisco, CA). Mouse IL-4 was purchased from R & D Systems (Minneapolis, MN). IL-5 was purified according to previously described procedures (28).

Immunohistochemistry
Frozen sections from spleens were prepared as described (32). Briefly, serial 4-µm thick frozen sections were fixed in ice-cold acetone for 15 min and blocked with normal horse serum. The sections were incubated with biotinylated or FITC-conjugated mAb or PNA; anti-Fc{gamma}R mAb was added to avoid non-specific binding of the labeled mAb. The sections were then incubated with SA–PE, as required, when using biotinylated mAb. Antibody incubations were carried out for 30 min in a moist chamber at 37°C and after each incubation the sections were washed 6 times with PBS. The sections were mounted with 50% glycerin in PBS and were inspected using a MD 2000 AZ confocal microscope equipped with an argon laser (Molecular Dynamics, Sunnyvale, CA). Green (FITC) and red (PE) signals were collected separately on the confocal system, and photographs were taken of the electronic overlays.

Preparation of B cells
Splenic B cells were isolated from 8-week-old mice after T cell depletion by treatment with anti-Thy-1.2 mAb and guinea pig complement as described previously (28). To purify sIgD+ cells, they were stained with FITC-labeled anti-B220 mAb and biotinylated anti-IgD mAb plus SA–PE; sIgD+ B220+ cells were then isolated by sorting using a FACS Vantage (Becton Dickinson, Mountain View, CA). The purified B cell population contained >99% sIgD+ cells, as assessed by fluorescence analysis using the FACS Vantage (Becton Dickinson). To enrich B cells in the peritoneal cavity, peritoneal exudate cells were collected by washing the peritoneal cavity with Hank's medium according to procedures previously described (33) and treated with anti-Thy-1.2 mAb and guinea pig complement as described above. The peritoneal B cells were stained with biotinylated-anti-Ly-1 mAb plus SA–PE and FITC-conjugated anti-B220 mAb. After washing, cells stained were sorted. We used the Ly-1+B220dull cells and Ly-1B220bright cells as a source of B-1 and B-2 cells respectively, and the purity of each population was >98%.

Flow cytometric analysis
Analyses of the expression of surface molecules on freshly isolated and cultured sIgD+ cells were carried out using flow cytometry (FACScan; Becton Dickinson). Cells were stained with FITC–anti-mIgDa, biotinylated mAb and unlabeled-anti-mFc{gamma}R, followed by SA–PE. After being stained, cells were suspended in buffer containing propidium iodide (2 mg/ml) to exclude dead cells from the analysis. Fluorescence intensity was measured on a FACScan.

Assays for Ig secretion
Total IgM, IgG1 and IgA present in culture supernatants was quantified by ELISA according to previous described procedures (34). Briefly, splenic B cells were cultured in RPMI 1640 medium supplemented with 8% FCS, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, penicillin at 50 units/ml and streptomycin at 50 µg/ml in 96-well flat-bottom microtiter plates at a concentration of 4–10x104 cells/200 µl/well with or without stimulants. Cultures were set up in triplicate. The amounts of total IgM, IgG1 and IgA present in the culture supernatants were calibrated using standard curves generated using myeloma proteins (Miles Scientific, Naperville, IL); concentrations of Ig were determined using Delta-Soft software (Biometallicus, Princeton, NJ). Each experiment was repeated at least 3 times and a single representative result is shown. Results are expressed as mean ± SE (ng/ml).

PCR Analysis of {gamma}1–µ reciprocal DNA recombination products
Total DNA (genomic and circular) was extracted using a QIAamp Blood Kit (Qiagen, Hilden, Germany) as described (35). For amplification of {gamma}1–µ recombination products, 200 ng of total DNA from cultured or freshly isolated B cells were subjected to PCR amplification in 50 µl volumes containing LA PCR Buffer II (Takara, Kyoto, Japan), 2.5 mM MgCl2, 0.4 mM dNTP, 2.5 U LA Taq (Takara), 1 mM sense-strand 5' S{gamma}1 primer (5'-CACTCCTGGGTATGGAAACACATCCTAC-3'; nucleotides 262–289 in the region 5' of the S{gamma}1 repeats; MUSIGHANB) in combination with an antisense 3' Sµ primer (5'-AGCCTAACTTATCTGAGCCTAGTTCAAC-3'; nucleotides 1347–1319 in the region 3' of the Sµ repeats; MUSIGCD09). Reciprocal {gamma}1–µ products were amplified using 35 cycles of 1 min of melting at 94°C, and 8 min of annealing and extension at 69°C. PCR products were transferred onto nylon membranes (GeneScreen; NEN, Boston, MA) and then hybridized with 32P-labeled DNA fragments. As an S{gamma}1 probe, we used the 1.1 kb BamHI–BglII fragment (nucleotides 537–1674; MUSIGHANB) in the 5' region of the S{gamma}1 repeats from p{gamma}1E/H10.0 (36); the Sµ probe was a 99 bp PCR-amplified fragment (nucleotides 1210–1308, MUSIGCD09) starting directly downstream of the Sµ region. Blots were analyzed with a Fujix BAS1000 Bioimaging Analyzer (Fuji Photo Film, Tokyo, Japan).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Bright expression of CD38 by the B cells in the follicular mantle zone
Using the various anti-mouse CD38 mAb, it has been shown that B cells around germinal centers in spleen and Peyer's patches express CD38 (13). To assess whether CS/2 also shows the similar staining pattern to other anti-CD38 mAb, the CD38 expression was analyzed in mouse splenic B cells using confocal imaging of immunostained frozen spleen sections. Figure 1Go shows immunohistochemical staining of spleen sections 7 days after secondary immunization with DNP–KLH; Fig. 1Go(A–C) depicts the same microscope field in a series of serial sections. The germinal centers were stained with PNA (red) and anti-B220 mAb (green; Fig. 1AGo), and cells with overlapping expression stained orange to yellow. Staining with anti-CD40 mAb was primarily restricted to the lymphoid follicles of spleens, and showed that B cells in the germinal centers and the mantle and marginal zones express CD40 (green; Fig. 1CGo), which is consistent with earlier studies (24). The B cells expressing CD38 (green) resided in the follicular mantle zone and were intensely stained (Fig. 1BGo). On the other hand, most B cells in the light zone of the germinal centers expressed CD38 only at low levels (CD38dim/–) and cells in the dark zone did not appear to express CD38 at all (Fig. 1BGo). CD38dim/–B220+ germinal center B cells were observed in immunized mice, but not in saline-injected mice (data not shown), indicating that this population arose in response to antigenic stimulation. The staining pattern of CS/2 was essentially identical to those reported by Oliver et al. (13).



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Fig. 1. Expression of B220, PNA, CD38 and CD40 in spleens of wild-type mice. Serial sections (A–E; x400) were stained with the indicated reagents. The zones within a secondary follicle from a DNP–KLH immunized wild-type mouse are identified in frozen sections stained with PNA, and anti-B220 mAb (A) and anti-CD38 mAb (B). The zones of a secondary follicle was stained with anti-CD40 mAb (C).

 
Expression of CD38 in mouse splenic B cells was examined by FACS analysis. As described (19), the majority of B220+ B cells in the spleen were CD38+, but expression of CD38 was substantially lower in the germinal center B cells than in sIgD+ non-germinal center B cells (28). Stimulation of sIgD+ splenic B cells with CS/2 for 3 days down-regulated the CD38 expression, but it did not enhance expression of CD95/Fas or PNA. The B cells stimulated with CS/2 exhibited enhanced expression of both IL-5R{alpha} and ßc (data not shown).

Induction of IgG1 production upon stimulation of sIgD+ splenic B cells with IL-5 and CS/2
We isolated splenic B cells from non-immunized mice and cultured them with CS/2, LB429, IL-4, IL-5 or their combination for 7 days; the concentration of IgG1 in culture supernatants were then measured. Stimulation of splenic B cells from wild-type mice with both CS/2 plus IL-5 and LB429 plus IL-5 induced remarkable IgG1 production (Table 1Go). Neither CS/2 plus IL-5 nor LB429 plus IL-5 induced IgG1 production in splenic B cells from IL-5R{alpha}–/– mice (data not shown). Stimulation with CS/2 plus IL-4 induced little, if any, IgG1 production, whereas stimulation with LB429 plus IL-4 induced significant IgG1 production. These results indicate that IL-5 efficiently induces IgG1 production in B cells stimulated with either anti-CD40 or anti-CD38 mAb. In contrast, IL-4 induces IgG1 production by anti-CD40-stimulated B cells, but not by anti-CD38-stimulated B cells.


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Table 1. Effects of IL-5 and CD38 ligation on IgM and IgG1 production
 
Because of the dual effects of IL-5 on B cell proliferation and differentiation, IgG1 secretion induced by CS/2 plus IL-5 could result from either isotype switch recombination or expansion of a pool of IgG1-committed B cells driven to differentiate towards plasma cells. To investigate in more detail the role of IL-5 in IgG1 secretion, we purified sIgD+ cells as naive B cells from spleen. The purity of this population was >99% and it contained <0.1% sIgG1+ B cells. The cells thus obtained were cultured for 7 days with either CS/2 or CS/2 plus various cytokines. After the culture period, the quantities of antibodies secreted into the culture supernatants were estimated by ELISA. Stimulation of sIgD+ B cells with either CS/2 or a cytokine alone did not induce IgG1 production (Fig. 2Go). Consistent with earlier results, sIgD+ B cells stimulated with CS/2 plus IL-5 produced IgG1. IL-5 had by far the most potent synergistic effect when administered together with CS/2. Splenic B cells from IL-4–/– mice responded to CS/2 plus IL-5 and produced IgG1, suggesting the existence of an IL-4-independent pathway for IgG1 production. We could not detect IgA in supernatants of culture of sIgD+ B cells stimulated with CS/2 plus IL-5 (data not shown).



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Fig. 2. Synergistic effects of CS/2 and various cytokines on IgG1 secretion by splenic B cells. T-cell-depleted splenic B cells (1x105/well) were cultured for 7 days in the presence or absence of CS/2, CS/2 plus cytokines or LPS plus IL-4. After the culture period, IgG1 levels in the culture supernatant were determined by ELISA.

 
IL-5 induces {gamma}1–µ switch recombination in CS/2-stimulated sIgD+ B cells
The above result suggests that IgG1 production elicited by culturing sIgD+ B cells in the presence of CS/2 plus IL-5 does not simply reflect the expansion and differentiation of a pre-existing pool of sIgG1+ B cells. Rather IL-5 appears to induce µ–{gamma}1 isotype switching in B cells. Isotype switching in B cells is preceded by cell proliferation and transcription of the corresponding unrearranged or constant region gene of the germline heavy chain (37,38). The looping-out and deletion model of switch recombination predicts that during the course of joining the 5' segment of Sµ to the 3' segment of S{gamma}1, intervening DNA between switch regions is excised as a circle (3942). In the absence of replication origins, these circles would not be replicated during cell division. Thus, the content of reciprocal S{gamma}1–Sµ junctions should reflect the frequency of switch recombination events regardless of subsequent proliferation.

We devised a method to detect {gamma}1–µ circular DNA by PCR (35). DNA from cells was obtained after 3 day of culture in the presence of LB429 plus IL-4. Total cellular DNA was amplified by PCR and subjected to hybridization with the 5' S{gamma}1 probe and thereafter with the 3' Sµ probe. The PCR products digested by BglII or HindIII were hybridized to our 5' S{gamma}1 probe and then to the 3' Sµ probe (35). These results reveal that all of the PCR products hybridized with our 5' S{gamma}1 and 3' Sµ probes have the expected 5' S{gamma}1–3' Sµ structure. Using this primer pair and the S{gamma}1 probe, we analyzed the generation of S{gamma}1–Sµ reciprocal products by sIgD+ B cells cultured for 3 days in the presence of CS/2, CS/2 plus IL-4, CS/2 plus IL-5 or CS/2, IL-5 and IL-4. Total DNA was amplified by PCR and hybridized with the 5' S{gamma}1 probe. Three independent amplifications were performed on identical aliquots of DNA template to improve detection of rare events and assess reproducibility. As shown in Fig. 3Go(A), few if any, reciprocal S{gamma}1–Sµ junctions were amplified in unstimulated (day 0) sIgD+ B cells, and only small numbers of amplified products were detected from cells cultured with CS/2 and CS/2 plus IL-4. On the other hand, the quantity of {gamma}1–µ switch circles (ranging from 2 to 10 kb) was substantially increased in cells cultured in the presence of CS/2 plus IL-5, indicating that IL-5 induces µ–{gamma}1 switch recombination in B cells simulated with CS/2. When the blots were stripped and reprobed with the 3' Sµ probe, the resultant images were virtually identical to those previously obtained using the 5' S{gamma}1 probe (data not shown), indicating that virtually all of the amplified products contain both the 5' S{gamma}1 and 3' Sµ segments.



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Fig. 3. IL-5 induces the generation of S{gamma}1–Sµ switch circles in CS/2-stimulated B cells. Samples (200 ng) of total DNA from (A) either uncultured cells or cells cultured for 3 days with CS/2, CS/2 plus IL-4, CS/2 plus IL-5 or the combination of CS/2, IL-4 and IL-5, or (B) uncultured cells or cells cultured for 3 days with LB429, LB429 plus IL-4, LB429 plus IL-5 or the combination of LB429, IL-4 and IL-5 were amplified using 5' S{gamma}1 and 3' Sµ primers and LA-Taq polymerase. Each PCR product was hybridized with 5' S{gamma}1 probes.

 
We cloned the amplified DNA segments prepared from B cells cultured for 3 days with CS/2 plus IL-5 and performed a sequence analysis on randomly selected 5' S{gamma}1-positive clones. The clones all contained the 5' S{gamma}1 and 3' Sµ sequences in a 5'–3' orientation (data not shown). We also examined effect of IL-5 on the generation of S{gamma}1–Sµ reciprocal products by sIgD+ B cells cultured for 3 days in the presence of LB429 plus IL-5 in comparison to LB429 plus IL-4. Results revealed that IL-5 induced µ–{gamma}1 switch recombination in B cells stimulated with LB429 to a lesser extent to that observed in B cells stimulated with IL-4 plus CS/2 (Fig. 3BGo).

IgG1 and IgA production by B cells in the peritoneal cavity upon stimulation with CS/2 and IL-5
As we reported (33), at least one out of four B cells in the peritoneal cavity responds to IL-5 for a significant proliferation and terminal differentiation to IgM-producing cells. This raised a possibility that CS/2 and IL-5 stimulation of peritoneal B cells may enhance proliferative response and IgM production. To address this issue, we examined expression of CD38 in B cells isolated from the peritoneal cavity by flow cytometry using CS/2, almost all of peritoneal B cells expressed CD38 (Fig. 4AGo). Then, we isolated B cells from the peritoneal cavity of normal mice and cultured them with CS/2, IL-5 or the combination of CS/2 and IL-5 for 3 days to determine proliferative response. The results revealed that peritoneal B cells did not show a significant proliferative response upon stimulation with either CS/2 alone or CS/2 plus IL-5 (Fig. 4BGo). This result was in good agreement with that of published data (22).



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Fig. 4. CD38 expression on and proliferative response of B cells isolated from peritoneal cavity. (A) Peritoneal washouts were treated with anti-Thy-1.2 plus complement to obtain purified peritoneal B cells. The B cells were stained with biotinylated CS/2 mAb followed by SA–PE and the cells stained were analyzed using a FACScan. Histograms corresponding to the number of cell binding each mAb (open profile) are superimposed on the histograms depicting numbers of unstained cells (solid profile). Horizontal and vertical axes express the log of the fluorescence intensity and relative cell number respectively. (B) Purified peritoneal B cells (1x105/ml) were cultured with stimuli for 3 days and proliferative response was monitored by incorporation of [3H]thymidine. Data represent mean c.p.m. ± SD of triplicate culture.

 
To examine IgG1 and IgA production by peritoneal B cells, we cultured peritoneal B cells for 7 days with CS/2 plus IL-5 or LPS plus IL-4 and titrated amounts of antibodies in cultured supernatants. Results revealed that stimulation of peritoneal B cells with CS/2 plus IL-5 enhanced both IgG1 and IgA production (Table 2Go). Stimulation of LPS plus IL-4 was ineffective in either IgG1 or IgA production. To further extend these observations, we enriched B-1 and B-2 cells from peritoneal washouts by cell sorting and stimulated them with CS/2, IL-5 or both for 7 days. Purity of B-1 and B-2 cells was >98%. Contamination of Mac-1+ cells in the B-2 population was <2%. B-1 cells stimulated with CS/2 plus IL-5 showed a significant IgG1, but not IgA, production (Table 3Go). Purified B-2 cells produced IgG1 and significant IgA antibodies in response to CS/2 plus IL-5 (Table 3Go).


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Table 2. Effects of IL-5 and CD38 ligation on IgM, IgG1, and IgA production by peritoneal B cells.
 

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Table 3. Effects of IL-5 and CD38 ligation on IgM, IgG1 and IgA production by peritoneal B-1 cells and B-2 cells
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We presented three major findings in this study. First, stimulation of naive (sIgD+) splenic B cells with CS/2 plus IL-5 induces IgG1 production. Among the various cytokines tested, only IL-5 has this synergistic effect on IgG1 production with CS/2. Second, in CS/2-stimulated sIgD+ B cells, IL-5 enhances the cellular content of reciprocal products, forming DNA circles as byproducts of µ–{gamma}1 recombination. Third, peritoneal B cells, almost all of which express CD38, respond to CS/2 plus IL-5 for enhanced production of IgG1 and IgA, although they do not proliferate. Among peritoneal B cells, B-2 cells are the major source of IgG1- and IgA-producing cells.

As we reported (17,19,21), CS/2 recognizes mouse CD38, enhances the proliferative response of mouse B cells and rescue them from apoptosis (19). The molecular mechanism(s) by which it contributes to B cell activation, proliferation and differentiation, however, remains unclear. We examined the expression pattern of CD38 using CS/2 in mouse B cells. Most mouse B cells in bone marrow and spleen expressed CD38. CD38+ B cells were found in both the mantle and marginal zones of secondary follicles in the immunized spleen (Fig. 1BGo). There was a CD38dim/– B cell population in the germinal centers of spleens from DNP–KLH-immunized mice. These results are consistent with an earlier report by Oliver et al. (13) and further suggest that after antigenic stimulation, germinal center formation is accompanied by down-regulation of the surface expression of CD38 in mice. CD38 stimulation in sIgD+ naive B cells did not lead to distinctive phenotypic modulation, although down-regulation of CD38 and up-regulation of IL-5R{alpha} and ßc expression were observed (data not shown).

The sIgD+ splenic B cells stimulated with CS/2 plus IL-5 became IgG1-producing cells (Table 1Go), while they did not become IgA-producing cells (data not shown). Splenic B cells from either STAT6–/– (35) or IL-4–/– mice (data not shown) could differentiate to IgG1-producing cells in response to CS/2 plus IL-5. It was obvious that sIgD+ splenic B cells stimulated with CS/2 plus IL-4 mounted little if any IgG1, whereas they become IgG1-producing cells in response to LB429 plus IL-4 (Table 1Go). These results indicate that IgG1 production by B cells in response to CS/2 plus IL-5 is IL-4 independent. There may be at least two different pathways to induce IgG1 production; IL-4-dependent and IL-4-independent pathways. Isotype switching has been clearly demonstrated to take place within the germinal center (43), in which CD40 and IL-4 are involved. The germinal center reaction may not be necessary for switching to occur (44). Stimulation of follicular B cells with CS/2, even in the absence of IgM stimulation, may mimic B cell differentiation via an extra-germinal center pathway together with IL-5.

The purity of sIgD+ cells in our B cell population was >99% and contamination by sIgG1+ B cells in the population was <0.1%. These data strongly suggest that stimulation of splenic sIgD+ cells through CD38, IL-5R or both elicits effects at the level of the DNA which induce switch recombination from µ to {gamma}1. DNA synthesis, expression of sterile transcripts of a particular Ig isotype and switch recombination are all known to be required for the Ig isotype switch. At the molecular level, the predominant mode of Ig isotype switching consists of a recombination event that includes looping-out and deletion of all heavy chain genes with the exception of the one that is to be expressed (3942). By itself, CS/2 stimulated sIgD+ splenic B cells to synthesize DNA. In our preliminary results, CS/2 was able to induce germline {gamma}1 transcription in cultured sIgD+ B cells, whereas IL-5 did not induce or enhance detectable germline {gamma}1 expression (35). We amplified deleted circular DNA fragments containing reciprocal S{gamma}1–Sµ junctions in order to detect switch recombination, since the cellular content of reciprocal S{gamma}1–Sµ junctions should reflect the frequency of switch recombination events regardless of subsequent proliferation. Results revealed that CS/2 stimulation did not show corresponding increases in the percentage of sIgG1+ cells or S{gamma}1–Sµ rearrangement events in B cells (Fig. 3Go). Surprisingly, further addition of IL-5 strongly induced S{gamma}1–Sµ rearrangement as well as the appearance of sIgG1+ cells (Fig. 3AGo). In addition, in the absence of CS/2-mediated targeting of the {gamma}1 gene, IL-5 failed to induce switching to IgG1. These results demonstrate that while the signals mediated by CD38 ligation are essential, they are not sufficient to evoke substantial Sµ–S{gamma}1 rearrangement. Furthermore, results support the notion that IL-5 induces Sµ–S{gamma}1 switch recombination. It still remains unclear whether IL-5 induces Ig isotype switching directly or indirectly. Further analysis will be required to address these issues.

A study using transgenic mice has provided supportive evidence that intestinal IgA plasma cells are derived from B-1 cells (45). In addition, up to 50% of intestinal B cells are CD5+, a large number of which secrete IgA in humans (46). Moreover, we recently demonstrated using IL-5R-deficient mice that the IL-5/IL-5R signaling pathway is critically important for the common mucosal immune system-independent sIgA+ B-1 cell development and for IgA responses in mucosal effector tissues in vivo (47). Taken together, B-1 cells could be an important source for IgA-producing cells in mucosal tissues. IL-5 was the first Th2 cytokine recognized as an IgA-enhancing cytokine capable of stimulating Peyer's patches and mitogen-stimulated splenic sIgA+ B cells to differentiate into IgA plasma cells (4851). As shown in Fig. 4Go, peritoneal B cells were incapable of responding to CS/2 and IL-5 for proliferation. Stimulation of peritoneal B cells with CS/2 and IL-5 induced IgA as well as IgG1 and IgM production (Table 2Go). This was in sharp contrast to splenic B cells which did not produce IgA antibody in response to CS/2 plus IL-5. We used CD5+B220dull B cells as a source of B-1 cells, also called B-1a cells, and CD5B220bright cells as a source of B-2 cells. Contamination of CD5Mac-1+ B cells, so-called B-1b cells, was <2%. Among peritoneal B cells, purified B-2 cells and a lesser extent of B-1 cells became IgG1-producing cells in response to IL-5 plus CS/2 (Table 3Go), suggesting that the major source of precursors for IgG1-producing cells is B-2 cells. We infer from results that peritoneal B-1 cells are capable of responding to CD38 ligation in combination with IL-5 for differentiation to IgG1-forming cells, although we cannot completely rule out the possibility that contaminated B-2 cells in the B-1 cell population might differentiate to IgG1-producing cells. Only B-2 cells became IgA-producing cells, suggesting the major source of precursors for IgA-producing cells. We should analyze in future whether switch recombination from µ to {gamma}1 is induced in peritoneal B-2 and B-1 cells. Our results will give us a new clue to understand molecular mechanisms of mucosal IgA production by peritoneal B cells upon stimulation with a cytokine and a B cell activator. We carried out several experiments to ask whether IL-5 induces µ to {alpha} switch recombination in DNA level, but we have no reliable data at this moment.

In conclusion, mouse CD38 is highly expressed and functional in follicular B cells but is down-regulated in the germinal center B cells. Furthermore, CD38 cross-linking with CS/2 in splenic sIgD+ B cells induces IgG1 production and {gamma}1–µ switch recombination in an IL-4-independent manner. Peritoneal B cells, particularly B-1 cells, are incapable of responding to CS/2 plus IL-5 for proliferation; however, they are capable of responding for IgG1 production. Further study of the molecular mechanisms underlying µ to {gamma}1 and µ to {alpha} class switching induced by CS/2 plus IL-5 in peritoneal B-1 and B-2 cells respectively should provide us with important additional information on the roles of CD38 ligation and IL-5 in B cell differentiation and isotype switching.


    Acknowledgments
 
We are indebted to Drs S. Nagata, S. Yonehara and N. Sakaguchi for Jo-2, HB and LB429 respectively, and to Dr A. Kariyone for her excellent support in operating a FACS Vantage. We also thank Drs S. Takaki and T. Kinashi for their encouragement and valuable suggestions throughout this study, and acknowledge Dr W. Goldman for critical review of this manuscript. This work was supported in part by a Grant-in-Aid for Advanced Research on Immunoregulation from the Ministry of Education, Science, Sports and Culture, Japan, by Research Fund from Meiji Milk Products Co. Ltd, and by Special Support for Germinal Center B Cell Research from Ajinomoto Co. Ltd.


    Abbreviations
 
IL-5R{alpha}IL-5 receptor {alpha} chain
KLHkeyhole limpet hemocyanin
PNApeanut agglutinin
SAstreptavidin

    Notes
 
The first two authors contributed equally to this work Back

Transmitting editor: M. Miyasaka Back

Received 26 October 1998, accepted 23 February 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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