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*
Department of Microbiology, College of Natural Sciences, Kangwon National University, Chunchon, Korea, and
Laboratory of Mucosal Immunology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093
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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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Cholera toxin (CT)3, an 86-kDa enterotoxin produced by Vibrio cholerae, consists of five B subunits of CT (CTB) that are arranged as a pentamer and are covalently linked to a single 33-kDa A subunit of CT (CTA) (11, 12). CTB can bind to GM1 gangliosides on the surface of intestinal epithelial cells, lymphocytes, and other cells, after which CTA can enter the cell (13, 14). In the case of intestinal epithelial cells, this induces ADP ribosylation of a Gs regulatory protein, which results in elevated levels of intracellular cAMP, increased Cl- secretion, and diarrhea (14), the hallmark of clinical cholera infection. CT is also a potent adjuvant. Thus, protein Ags, which themselves are not immunogenic by the enteric route, are rendered immunogenic when coadministered with CT (15, 16). In the intestine, the adjuvant effect of CT on mucosal immune responses results in marked up-regulation of local IgA responses and appears to be dependent on CD4 T cells, especially those belonging to the Th2 subset that produces IL-4 and IL-5 (17, 18). Moreover, CT is a potent mucosal immunogen and stimulates mucosal anti-CT IgA responses (19). In addition, CT in combination with IL-4 has been shown to increase the IgG1 response of LPS-activated spleen B cells in vitro (20).
CTB alone can also modulate mucosal immune responses. Thus, enteric administration of CTB, together with a protein Ag, increased murine Ag-specific mucosal IgA responses (21). In addition, oral or intranasal administration of CTB as a carrier conjugated to a protein resulted in the induction of local Ag-specific secretory IgA responses (22, 23). Moreover, enterically or intranasally administered proteins that were coupled to CTB were more effective than the proteins alone at inducing oral tolerance in mice (24, 25).
Several reports suggest CT and CTB can increase IgA isotype switching. Thus, CT and CTB increased the number of IgA-producing cells among populations of LPS-activated Peyer’s patch B cells (26), CT increased the frequency of IgA-secreting cells in cultures of CH12.LX (surface IgM+ (sIgM+)) murine B lymphoma cells (26, 27), and intraduodenal administration of CT increased the frequency of IgA precursor cells in Peyer’s patches (28). In the studies herein, we report that CT and CTB increase IgA isotype switching at the clonal level, independent of CTA. Moreover, CTB-stimulated IgA isotype switching is mediated through TGF-ß1 and requires IL-2 as a cofactor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice (8–12 wks old) were bred and maintained in our laboratory or purchased from Jackson Laboratories (Bar Harbor, ME).
Abs, cytokines, and other reagents
Anti-Thy-1.2 mAb HO13.4 (29) and anti-Lyt-2.2 mAb AD4.5 (30) were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). Anti-CD4 (L3T4) mAb RL172.4 (31) was provided by M. J. Bevan (University of Washington, Seattle, WA). Goat anti-mouse isotype-specific Abs were obtained from Southern Biotechnology Associates, Inc. (Birmingham, AL). LPS from Escherichia coli O127:B8 was obtained from Sigma Chemical Co. (St. Louis, MO). CT, CTA, CTB, and goat anti-CTB Ab were obtained from List Biologic Laboratories (Campbell, CA). rCTB was provided by J. Holmgren (Göteborg University, Göteborg, Sweden). Soluble TGF-ß1 type IIR, rabbit anti-TGF-ß Ab, and purified porcine TGF-ß1 were obtained from R&D Systems (Minneapolis, MN). Porcine TGF-ß1 differs from murine TGF-ß1 by a single amino acid (32), but is fully active on mouse cells. TGF-ß1 was reconstituted in 4 mM HCl containing 1 mg/ml BSA before use. Human rIL-2 was obtained from Cellular Products (Buffalo, NY).
B cell preparations and cultures
Mouse spleen cell suspensions were prepared as previously
described (5). Cell suspensions were treated with 0.83% ammonium
chloride to lyse RBCs. T cells were depleted from cell suspensions by
treatment with a mixture of anti-Thy-1.2, anti-Lyt-2.2, and
anti-L3T4 mAbs and low-toxicity rabbit complement (Accurate
Chemical Co., Westbury, NY) (33). Cells were washed three times with
HBSS and resuspended in RPMI 1640 medium supplemented with 10% FBS. B
cell preparations contained >90% surface Ig-expressing cells and
1% residual T cells, as assessed by flow cytometry.
To prepare surface IgA- (sIgA-) B cells, T cell-depleted spleen B cells (2.5 x 108 cells in 5 ml) were added to 60-mm plastic petri dishes precoated with goat anti-mouse IgA Ab (7 µg/ml), and incubated for 70 min at 4°C (33). Flow cytometric analysis showed that the nonadherent B cell population contained <0.1% sIgA+ cells.
B cells were cultured in 96-well tissue culture plates (flat bottom, 0.32-cm2 growth area) (Costar, Cambridge, MA) at 1 x 105 cells/well in a volume of 160 µl in RPMI 1640 medium containing 10% FBS, 50 µM 2-ME, 2 mM glutamine, and LPS (12.5 µg/ml). Unless indicated, other reagents were added in a volume of 40 µl/well 24 h after initiation of the cultures.
Isotype-specific ELISA
The level of specific isotypes produced in the B cell cultures were determined by ELISA as previously described (5). Isotype specificity of the ELISAs was confirmed by assaying a panel of myeloma proteins containing IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM. ELISAs were sensitive to Ab concentrations of 1 to 10 ng/ml.
Isotype-specific enzyme linked immunospot (ELISPOT) assays
Polystyrene 96-well plates (Millipore, Bedford, MA) containing nitrocellulose inserts were coated with 150 µl of affinity-purified goat anti-mouse isotype-specific Ab (1.2 µg/ml) in PBS (3). Control wells were incubated with PBS/0.05% Tween-20 (PBST) containing 1% BSA. After overnight incubation, the plates were washed with PBST and blocked with 200 µl of 1% BSA/PBST for 1 h at 37°C. Titrated numbers of B cells were added to triplicate wells and incubated for 2.5 h at 37°C. The plates were washed and then incubated with 150 µl/well biotin-labeled goat anti-mouse isotype-specific Ab in 1% BSA/PBST. After incubation for 2 h at room temperature, plates were washed and 150 µl/well of avidin-peroxidase (Boehringer Mannheim, Indianapolis, IN) at a 1:2000 dilution in PBST was added for 2 h. Spots were developed with 3-amino-9-ethylcarbazole and hydrogen peroxide, and counted using a dissecting microscope. Data are presented as numbers of spot-forming cells per well with backgrounds subtracted.
Determination of IgA secretion rates
sIgA- B cell cultures were stimulated with LPS and 24 h later with CT and CTB in the presence of IL-2. After 5 days in culture, cells were harvested, washed, and the number of IgA secreting cells was analyzed by ELISPOT assay. In parallel, at the end of the 5 day period, 4 x 105 cells/well were recultured for 6 h in 96-well plates in a 200-µl volume, and IgA levels in the supernatants were determined by ELISA. The IgA secretion rate for the 6-h period was calculated by dividing the amount of IgA secreted during this period by the number of IgA-secreting cells present in the culture as determined by the ELISPOT assay.
Limiting dilution analysis
sIgA- spleen B cells were cultured at various cell densities, ranging from 101 to 105/well, in 96-well tissue culture plates (Costar) in 200 µl/well. Forty eight replicate wells were set up for each cell density. Cultures were stimulated for 6 days, and wells were assayed individually for the number of isotype-specific Ig-secreting cells by ELISPOT assay. Wells containing spot-forming cells were scored as positive.
Calculations to determine the frequency of B cell precursors that develop into IgA- or IgM-secreting B cells were based on the Poisson distribution analysis as previously described (3). Briefly, the log10 of the frequency of negative cultures was plotted against the number of cultured B cells and the precursor frequency was derived graphically from the point at which 37% of cultures were negative. The average number of IgA- or IgM-secreting cells that developed from each precursor (i.e., the clonal burst size) was determined based on the input number of B cells that yielded 37% negative cultures, because positive cultures contain an average of one precursor per culture at this input number. Clonal burst size was calculated using data from 48 wells.
TGF-ß bioassay
TGF-ß activity in culture supernatants was determined by assaying the inhibition of proliferation of mink lung epithelial-like cells (ATCC CCL64) (34). Mink lung epithelial-like cells (5 x 104/well) were seeded in 96-well flat-bottom microtiter plates in 100 µl DMEM containing 2% FBS and incubated overnight. After plates were washed with HBSS, dilutions of test supernatants, or of porcine TGF-ß1 as a standard, were added in a final volume of 200 µl/well. After a 24-h incubation, [3H]-thymidine (2 µCi/well) was added, and then cultures were incubated for an additional 5 h and harvested onto glass fiber filters. Radioactivity was determined using a liquid scintillation counter. Values obtained from test supernatants represent active TGF-ß, because latent TGF-ß was not converted to the active form before bioassay. TGF-ß activity in culture supernatants was confirmed by depleting TGF-ß from supernatants with rabbit anti-TGF-ß Ab (5 µg/ml) before bioassay.
Quantitative RT-PCR analysis
Total cellular RNA was extracted using Trizol reagent (Life Technologies, Gaithersburg, MD). TGF-ß1 mRNA levels were determined by RT-PCR using standard RNAs under the conditions we have previously described (35). Data are expressed as number of mRNA molecules/µg of total cellular RNA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stimulation of LPS-activated murine total spleen B cells with CT
or CTB alone did not significantly increase IgA secretion in the
cultures (Table I
). However, because CT
was reported to increase IgA switching among Peyer’s patch B cells and
murine B lymphoma cells (26, 27), and because we and others have
previously demonstrated that IL-2 can promote IgA secretion by spleen B
cells (3, 33), we assessed whether IL-2 affected IgA production by CT-
or CTB-stimulated total B cell cultures. As shown in Table I, the
addition of combinations of IL-2 with either CT or CTB to total B cell
cultures increased IgA secretion by five- to ninefold. In contrast, CT
or CTB had little effect on IgM and IgG1 secretion by total spleen B
cells, irrespective of whether IL-2 was present (Table I).
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, CT and CTB increased IgA
secretion in IL-2-stimulated sIgA- B cells by 7- and
18-fold, respectively, while they had little effect in the absence of
IL-2 costimulation. The relative increase in IgA secretion after CT or
CTB stimulation was higher in cultures of sIgA- B cells
compared with total B cells (Table I), suggesting that the target cells
of CT and CTB were mostly among the sIgA- B cells.
Furthermore, as in the total B cell cultures, CT or CTB had little
effect on the IgM response, but CT and CTB in the presence of IL-2
increased IgG1 secretion by 1.7- to twofold in the sIgA- B
cell cultures (Table I). CT effects on IgA production are mediated by CTB but not CTA
The above studies show that CTB, like CT holotoxin, stimulates IgA
secretion. Because purified CTB preparations can contain small amounts
of CTA as a contaminant, we determined whether CTA played a role in
mediating the increased IgA response to CT holotoxin and CTB. As shown
in Figure 1, purified CTA had little or
no effect on IgA secretion when tested over a 5000-fold concentration
range, irrespective of whether IL-2 was added to the cultures. In
contrast, rCTB, which is completely free of CTA, markedly increased the
IgA response in a dose-dependent manner when IL-2 was present.
Furthermore, rCTB was as efficient as purified CTB and CT holotoxin in
inducing maximal IgA responses (Fig. 1), indicating that the CTB
component of CT alone is responsible for modulating the IgA
response.
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Next, we determined if CT and CTB increased IgA secretion by
increasing the number of IgA-secreting cells or, alternatively, by
increasing the secretion of IgA-committed B cells already present in
culture. sIgA- B cells were cultured for 6 days in
the absence or presence of CT or CTB, combined with IL-2, and the
number of IgA-producing cells was determined by ELISPOT assay. As shown
in Figure 2, the addition of CT or CTB to
B cell cultures supplemented with IL-2 increased the number of
IgA-secreting cells by 6- to 20-fold, an increase similar to that in
total IgA secretion (Table I). These data indicate that CT and CTB act
mainly to increase the number of IgA-secreting B cells. In contrast to
IgA, the number of IgG1- and IgM-secreting cells was not significantly
altered in B cell cultures stimulated with either CT or CTB, in the
presence of IL-2 (Fig. 2, and data not shown).
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CTB increases the IgA precursor frequency
To determine whether CT and CTB, either alone or in the presence
of IL-2, increase IgA switching, changes in IgA precursor frequency
were determined using limiting dilution analysis (3). As shown in Table II, stimulation of
sIgA- spleen B cells with CTB and IL-2, but not CTB
or IL-2 alone, increased the IgA precursor frequency by more than
sixfold. In contrast, CTB and IL-2 did not increase the number of
IgA-secreting cells that developed from each IgA precursor (i.e., the
clonal burst size). Stimulation with IL-2 alone, as a control,
increased the number of IgA-secreting cells per IgA precursor, but did
not increase IgA precursor frequency, which is consistent with
previously reported data (3). Thus, together with the findings above,
these data show that CTB increases IgA switching in the presence of
IL-2 but does not increase proliferation and secretion of IgA-committed
B cells.
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To further characterize the mechanisms that underlie the
CTB-induced increase in IgA secretion, we determined the effects of CTB
and IL-2 stimulation on B cell proliferation. As shown in Figure 3, CTB markedly inhibited the
proliferation of LPS-stimulated sIgA- B cells, and
this inhibition was not affected by the addition of IL-2.
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The above data show that CTB increases IgA switching and inhibits
B cell proliferation. These findings are reminiscent of those
previously reported for TGF-ß1 (3), which raised the possibility that
TGF-ß1 mediates the effects of CTB on sIgA- B
cells. To test this possibility, TGF-ß1 activity was neutralized in
cultures stimulated with either CT and IL-2 or CTB and IL-2 using an
anti-TGF-ß Ab or soluble TGF-ß1 type IIR. As shown in Table III, anti-TGF-ß Ab inhibited the
CT- or CTB-stimulated IgA response by >75% in sIgA- B
cell cultures, while an isotype-matched control Ab had no effect.
Anti-TGF-ß Ab did not decrease basal IgA secretion in control
cultures. Furthermore, addition of soluble TGF-ß1 type IIR, which
bind active TGF-ß1 and prevent it from activating cellular receptors,
markedly inhibited the CTB- and IL-2-activated IgA response. These data
suggest the CTB-induced increase in IgA secretion is mediated by
TGF-ß1.
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). Next,
we assayed the levels of active TGF-ß1 after CT and CTB stimulation.
Because we previously reported that TGF-ß1 is maximally active on IgA
switching when added early during the culture period (33), 24-h
supernatants from sIgA- B cell cultures stimulated with
either CT and IL-2, or CTB and IL-2, and control supernatants were
assayed for TGF-ß activity using the TGF-ß sensitive cell line
CCL64. In one experiment, supernatants from sIgA- B cell
cultures stimulated with either CT and IL-2 or CTB and IL-2 contained
200 pg/ml and 100 pg/ml of active TGF-ß, respectively, whereas <50
pg/ml of TGF-ß was detected in IL-2-stimulated or control cultures.
In two additional experiments, TGF-ß levels were below the lower
limit of sensitivity of the bioassay (i.e., 50 pg/ml). Nonetheless,
these findings are consistent with a role of TGF-ß1 in mediating the
CT- and CTB-induced IgA response, because levels of active TGF-ß1 as
low as 25 pg/ml were sufficient to increase IgA secretion by
sIgA- B cell cultures in the presence of IL-2 (Fig. 4). The inability to consistently detect
active TGF-ß in culture supernatants could reflect the low total
levels produced, with levels sufficient for activation of the IgA
response being present in the immediate microenvironment of
TGF-producing cells. Additionally, active TGF-ß could be utilized by
the TGF-ß responsive cells in culture.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTB and IL-2 increased the precursor frequency of IgA secreting-cells within the sIgA- B cell population, but they did not increase the burst size of IgA-secreting clones. Thus, CTB and IL-2 did not stimulate the growth of residual B cells in culture that were already committed to the IgA isotype. On the contrary, concentrations of CTB that significantly increased IgA production were shown to inhibit B cell growth. TGF-ß1 also markedly inhibits B cell growth (3), but whether the effect on B cell growth was a direct effect of CT and CTB or a secondary effect of TGF-ß1 is not known. However, cell cycle inhibitors, including hydroxyurea and thymidine, were shown to increase the number of IgA-secreting cells among populations of LPS-activated sIgA- spleen B cells (3), and the ability of CTB to promote IgA switching may be related in part to its inhibitory action on cell growth.
TGF-ß1 appeared to be responsible for IgA switching in response to CTB and CT stimulation because the addition of anti-TGF-ß Ab or soluble TGF-ß1 type IIR completely abrogated the increased IgA response induced by CTB and IL-2. This observation was further confirmed by showing that CTB and CT increased TGF-ß1 mRNA transcripts in LPS-stimulated sIgA- B cell cultures and by the presence of active TGF-ß, within the range shown to be active on IgA production, in CTB- or CT-stimulated culture supernatants. Taken together, these observations indicate that CTB or CT, in combination with IL-2, promotes IgA isotype switching via TGF-ß1. Nonetheless, the source of TGF-ß1 is not known. Based on flow cytometric analysis, the sIgA- B cell population used in the present study contained <0.1% of sIgA+ B cells and few, if any, Thy1+ T cells. Thus, based on the relative numbers of B cells, T cells, and macrophages, and based on the fact that B cells have been reported to produce TGF-ß1 (10, 37), sIgA- B cells are the likely source of TGF-ß1, but we cannot exclude the possibility that residual T cells or macrophages are the source of TGF-ß1, or that latent TGF-ß1 in the serum is either directly or indirectly activated by CT and CTB.
The CT and CTB effect on IgA switching and production by splenic sIgA- B cells required IL-2 as a cofactor, which was not the case in studies by others using Peyer’s patch sIgM+ B cells and sIgM+ B lymphoma cells (26, 27). Peyer’s patch B cells that are induced to the IgA isotype may be at a different developmental stage and have different growth requirements after switching than those in the spleen. Moreover, CH12.LX sIgM+ B lymphoma cells are known to spontaneously switch at a low frequency to the IgA isotype in the absence of any added stimuli (38). Because IL-2 also increases IgA secretion of B cells induced to undergo IgA isotype switching by TGF-ß1 (1, 33), IL-2 may be an essential cofactor for IgA switching because it enables the detection of B cells that have switched to the IgA class by promoting IgA secretion.
Our results have implications for understanding the role of CTB in inducing oral tolerance. This form of tolerance is induced by administering Ag by the enteric or intranasal mucosal route, and has been used to down-regulate systemic immune responses and to treat autoimmune disease in animal models (39). TGF-ß1 is one of the major mediators of oral tolerance. Consistent with this observation, enterically administered haptenized colon proteins inhibited 2,4,6-trinitrobenzene sulfonic acid-induced colitis via the induction of TGF-ß1, which was likely released from mucosal T cells (40). Proteins coupled to CTB have been shown to induce oral tolerance at doses significantly lower than those required by mucosal administration of the protein alone (24, 41). Based on the studies herein, we suggest that CTB, as a carrier, may prime mucosal lymphoid cells to secrete TGF-ß1, and therefore that this is the underlying mechanism responsible for the CTB effect on oral tolerance. More importantly, in the context of the present study, secreted TGF-ß1 may be involved in the concurrent IgA isotype commitment that often accompanies oral tolerance (42, 43, 44).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martin F. Kagnoff, Department of Medicine 0623D, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0623. E-mail address:
3 Abbreviations used in this paper: CT, cholera toxin; CTB, B subunit of CT; CTA, A subunit of CT; sIgM, surface IgM, sIgA, surface IgA; ELISPOT, enzyme linked immunospot; PBST, PBS/0.05% Tween-20.
Received for publication August 14, 1997. Accepted for publication October 22, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and C2 germ-line and mature mRNA transcripts in human peripheral blood B cells. J. Immunol. 153:1466.[Abstract]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or with
(•) 60 IU/ml of IL-2. Supernatants were harvested after 7 days of
culture, and IgA levels were assayed by ELISA. Data are means ±
SEM of six replicate cultures. Similar results were obtained in two
additional experiments, although, as shown in Tables I and III, CTB
generally was slightly more effective than CT in increasing IgA
responses.
), LPS + IL-2 (
), LPS + CTB (