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Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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transcript was detected
and was then further enhanced by IL-15. IL-15 also supported
differentiation of both sIgM+sIgA- and
sIgM-sIgA+ 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
sIgM-sIgA+ B-1 cells expressing IL-15R into
IgA-producing cells in mucosal tissues. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-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- (11). IL-15 has also
been shown to control the development of CD4-
CD8+ 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
In vivo treatment with neutralizing anti-cytokine mAb and recombinant cytokine
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 x 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).
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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).
ELISA analysis of Ig isotype
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, , or 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).
Enumeration of Ig-producing cells by ELISPOT
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-, -, or
-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.
Analysis and purification of B cell subsets by flow cytometry
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 C 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.
Cell culture conditions for B cells
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 x 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-ß1 (R&D Systems, Minneapolis, MN; 1 ng/ml) for 1 and 3 days to characterize isotype switching and IL-15R expression, respectively.
Quantitative RT-PCR for measurement of IL-15R-chain mRNA
For quantitation of IL-15R-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 1x LightCycler-DNA master hybridization probes mix (Roche
Diagnostics). The oligonucleotide primers specific for the IL-15R
(sense, 5'-ATGGCCTCGCCGCAGCTCCG-3'; antisense,
5'-CCTGAGGGAGGTGGAGGCTG-3'; Ref. 10), IL-15R 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-15R-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).
Detection of C-specific mRNA
For detection of C 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 1x PCR buffer II (Perkin-Elmer
Cetus). The oligonucleotide primers specific for germline C
transcript (I-leader, 5'-GACATGATCACAGGCACAAGGC-3'; C,
5'-TTCCCCAGGTCACATTCATCGT-3') were prepared (36). The
sequence of ß-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 C transcript or 35 cycles
in ß-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.
Cell culture conditions for analysis of IgA production
Highly purified murine
sIgM+sIgA- or
sIgM-sIgA+ 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 x 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-ß1 or 100 ng/ml IL-15 to analyze germline
C transcript as described above.
Statistics
The results were statistically analyzed by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 2A). 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. 2A). 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. 2B). In contrast, the
numbers of IgM- and IgG-producing cells in SP were significantly
decreased in the anti-IL-2 mAb-treated mice (Fig. 2B). 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.
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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. 3A). 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. 3B). 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.
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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.
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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.
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mRNA expressions on B-1 cells
When IL-15R 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-15R-specific mRNA were noted in intestinal and splenic B-2
cells (Fig. 7A). 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-15R was
more pronounced (250350 fmol/1 ng cDNA) than in untreated and
freshly isolated B-1 cells (Fig. 7B). When identical culture
conditions were provided to
sIgM+sIgA- and
sIgA+ B-2 cells, only a slight increase of
IL-15R-specific mRNA was noted (50 fmol/1 ng cDNA; Fig. 7B). In contrast to IL-15, IL-2 did not induce
IL-15R-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.
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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 C germline transcript by
RT-PCR. Surprisingly, the specific message for the C 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 C 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 C germline transcript mRNA was enhanced in
sIgM+sIgA- B-1 cells (Fig. 8). However, IL-15 did not induce the C 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-ß1, resulting in the
induction of the C 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 C germline transcript in de
novo isolated intestinal
sIgM+sIgA- B-1 cells but
not intestinal and splenic B-2 cells.
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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
sIgM-sIgA+ 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
sIgM-sIgA+ 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 sIgM-sIgA+ B-1
cells (Fig. 9). In contrast, such an effect was not seen in
IL-15-treated sIgM+sIgA-
B-2 cells. When sIgM-sIgA+
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
sIgM-sIgA+ B-2 cells than
in sIgM-sIgA+ 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
sIgM-sIgA+ B-2 cells (Fig. 9). These findings suggest that IL-15 is an important cytokine for the
differentiation of both
sIgM+sIgA- and
sIgM-sIgA+ 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 C transcript, these cells might be
differentiated into sIgA+ B-1 cells and developed
into IgA plasma cells by the direct influence of IL-15.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 ß-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 sIgM-sIgA+
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.,
sIgM-sIgA+) 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
sIgM-sIgA+ 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).
|
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 C 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 C mRNA transcripts
(49). In contrast, it was found that intestinal and
splenic sIgM+sIgA- B-2
cells did not express C 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
sIgM-sIgA+ B cells under
the influence of the IL-15 and IL-15R signaling cascades. These
IL-15/IL-15R-stimulated
sIgM-sIgA+ 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
sIgM-sIgA+ 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
sIgM-sIgA+) of
CMIS-independent B-1 cells while the well-known mucosal cytokine IL-5
only acts on the stage of
sIgM-sIgA+ 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.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Hiroshi Kiyono, Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, 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.
Received for publication November 5, 1999. Accepted for publication July 26, 2000.
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) 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.
), IL-5 (
