B1 Cells Contribute to Serum IgM, But Not
to Intestinal IgA, Production in Gnotobiotic
Ig Allotype Chimeric Mice
This information is current as
of July 12, 2017.
M. Christine Thurnheer, Adrian W. Zuercher, John J. Cebra
and Nicolaas A. Bos
J Immunol 2003; 170:4564-4571; ;
doi: 10.4049/jimmunol.170.9.4564
http://www.jimmunol.org/content/170/9/4564
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References
The Journal of Immunology
B1 Cells Contribute to Serum IgM, But Not to Intestinal IgA,
Production in Gnotobiotic Ig Allotype Chimeric Mice1
M. Christine Thurnheer,* Adrian W. Zuercher,* John J. Cebra,* and Nicolaas A. Bos2†
B
1 cells comprise a distinct B cell population within the
immune system of mice. They differ from conventional
B2 cells in localization, expression of phenotypic markers, Ab repertoire, and reaction to proliferative stimuli (1–3). They
contribute a significant amount of natural serum IgM, thereby serving as a first line of defense against systemic viral and bacterial
infections (4 –7). IgA is thought to play a similar role in protecting
mucosal surfaces from infection with potentially pathogenic microorganisms (8, 9). Upon specific interaction with APC and T
cells in Peyer’s patches (PP),3 B2 cells can relocate to the intestinal
lamina propria, differentiate into plasma cells, and produce Agspecific secretory IgA (10, 11). However, a large amount of intestinal IgA is either nonspecific or of low affinity for a broad range
of Ag, and the source of this natural IgA as well as its functional
significance remain to be clarified. Production of secretory IgA is
virtually absent in germfree animals, but can be induced upon bacterial colonization (12, 13). B1 cells have the ability to switch Ig
class and can migrate to the intestinal lamina propria and differentiate into IgA-secreting plasma cells (14 –16). Furthermore, it
has been shown that B1 cells can be activated by either LPS or the
microbial flora, and that some B1-derived IgA is reactive with
surface Ag of intestinal bacteria (17, 18). These features led to the
*Department of Biology, University of Pennsylvania, Philadelphia, PA 19104; and
†
Department of Cell Biology, Section Histology and Immunology, Faculty of Medical
Sciences, University of Groningen, Groningen, The Netherlands
Received for publication July 10, 2002. Accepted for publication March 5, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AI37108 (to J.J.C.).
M.C.T. was supported by a fellowship from the Swiss National Science Foundation.
A.W.Z. is the recipient of a fellowship from the Swiss Foundation for MedicalBiological Grants. The Flow Cytometry Facility of the Cancer Center at University of
Pennsylvania is supported by The Lucille P. Markey Trust.
2
Address correspondence and reprint requests to Dr. Nicolaas A. Bos, Department of
Cell Biology, Section of Histology and Immunology, Faculty of Medical Sciences,
University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail address: [email protected]
3
Abbreviations used in this paper: PP, Peyer’s patch(es); GC, germinal center; PeC,
peritoneal cavity cells; PNA, peanut agglutinin; SFB, segmented filamentous
bacterium.
Copyright © 2003 by The American Association of Immunologists, Inc.
hypothesis that B1 cells might be a major source of natural, polyreactive, low affinity IgA in the gut. Indeed, several studies in
either SCID mice or irradiated animals reconstituted with bone
marrow and peritoneal cavity cells (PeC) suggested that a significant number of IgA-producing plasma cells in the gut originate
from B1 cells (14 –19). However, it is not known whether B1 cells
contribute a similarly large proportion of intestinal IgA in normal,
immunocompetent mice.
To address this issue, we generated Ig allotype chimeric mice
under germfree conditions, using a protocol first described by Lalor et al. (20, 21) and applied in several studies by Baumgarth et al.
(4, 5) under conventional conditions. In this model B1 and B2
cell-derived Abs can be distinguished based on different allotypes.
Germfree mice were used to establish a balanced B1/B2 cell chimerism before microbial stimulation. Systemic and local intestinal
immune responses were studied after colonization with Morganella morganii, Bacteroides distasonis, or segmented filamentous bacterium (SFB) and compared with conventionally reared
chimeric mice. We show that B1 cells contribute a large amount of
natural serum IgM under germfree conditions, which can be further induced by bacterial colonization. While B2 cells respond to
intestinal colonization with the production of total and specific
IgA, B1 cells contribute only a minimal amount of intestinal IgA
in this model.
Materials and Methods
Ig allotype chimeric mice
Germfree BALB/c mice were bred and housed in the germfree facility at
University of Pennsylvania (Philadelphia, PA) under sterile conditions in
Trexler isolators (Standard Safety, McHenry, IL). Conventionally reared
BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor,
ME), C.B-17 (C.B-Igh1b/IcrTac) mice were obtained from Taconic Farms
(Germantown, NY) and housed in the animal facility of University of
Pennsylvania.
To generate germfree Ig allotype chimeric mice, newborn germfree
BALB/c mice (a allotype) were treated with 200 ␮g of anti-IgMa (clone
DS-1) twice a week starting from day 1 after birth for a total of 10 injections in 32 days. On day 3 after birth, 2 ⫻ 106 PeC isolated by peritoneal
lavage from conventionally reared, 8- to 12-wk-old C.B-17 donors (b allotype) were transferred by i.p. injection. Conventionally reared, Ig allotype chimeric animals were generated without anti-IgMa treatment by
0022-1767/03/$02.00
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B1 cells are a significant source of natural serum IgM, thereby serving as a first line of defense against systemic bacterial and viral
infections. They can migrate to the intestinal lamina propria and differentiate into IgA-producing plasma cells and thus might play
a similar role in mucosal immunity. To investigate the contribution of B1 cells to the intestinal IgA response induced by the
commensal flora in immunocompetent animals, we generated gnotobiotic and conventionally reared Ig allotype chimeric mice. In
this system B1- and B2-derived Abs can be distinguished based on different allotypes. FACS analysis of peritoneal cavity cells and
analysis of B1- and B2-derived serum IgM indicated stable B1/B2 chimerism and the establishment of a functional B1 population.
Monoassociation with either Morganella morganii, Bacteroides distasonis, or segmented filamentous bacteria induced germinal
center reactions in Peyer’s patches and led to the production of intestinal IgA, partially reactive with bacterial Ag. A considerable
amount of serum IgM was B1 cell derived in both monoassociated and conventionally reared mice. However, most of the total as
well as bacteria-specific intestinal IgA was produced by B2 cells. These data suggest that intestinal IgA production induced by
commensal bacteria is mainly performed by B2, not B1, cells. The Journal of Immunology, 2003, 170: 4564 – 4571.
The Journal of Immunology
transferring 2 ⫻ 106 CB.17-derived PeC on days 1, 3, 5, and 28 after birth
into BALB/c recipients.
Bacteria and monoassociation
Flow cytometry
Single-cell suspensions (2 ⫻ 105–106/sample) of PeC were stained for 20
min at 4°C with FITC-conjugated anti-IgDb (217-170; BD PharMingen,
San Diego, CA), biotinylated anti-IgMb (AF6-78; BD PharMingen), FITCconjugated anti-IgDa (AM89.1; BD PharMingen), and biotinylated antiIgMa (DS-1; BD PharMingen). Cells were washed, and biotinylated Abs
were revealed by PE-conjugated streptavidin (BD PharMingen). Singlecell suspensions of PP were stained for 20 min at 4°C with FITC-conjugated peanut agglutinin (PNA; Pierce, Rockford, IL; coupled to FITC in
our laboratories as described previously (22)) and PE-conjugated anti-␬
L chain (Southern Biotechnology Associates, Birmingham, AL). Cells
were washed and fixed in 1% paraformaldehyde in PBS and analyzed on a
FACScan flow cytometer (BD Biosciences, Sunnyvale, CA). Germinal
center (GC) B cells are defined as ␬ L chainlow/PNA⫹ cells, while memory
B cells are excluded as ␬ L chainhigh/PNA⫺, as described previously (12).
WinMDI2.8 software was used for evaluation (The Scripps Research
Institute).
Analysis of Ab production in organ fragment cultures and serum
Conventionally reared, Ig allotype chimeric mice were sacrificed at 10 and
14 wk of age (n ⫽ 4/time point). Monoassociated mice were sacrificed on
days 0, 7, 14, 28, 42, 56, and 70 after colonization (n ⫽ 2–3/time point),
and blood was collected by heart puncture for serum analysis. The entire
intestinal tract was surgically removed. For organ fragment culture (23),
tissues were sterilized by sequential washes as described in detail previously (24). Pieces (3 ⫻ 3 mm) of duodenum, jejunum, ileum, cecum, and
colon were incubated in 1 ml of Kennett’s HY medium, supplemented with
10% FBS, L-glutamine, penicillin, streptomycin, and gentamicin (all reagents from Life Technologies) for 7 days under a 90% O2/10% CO2
atmosphere at 37°C.
Total serum IgM was measured by RIA. Flexible polyvinyl plates (Serocluster; Costar, Cambridge, MA) were coated with 50 ␮l/well of 20
␮g/ml goat anti-mouse F(ab⬘)2 (Jackson ImmunoResearch Laboratories,
West Grove, PA) and blocked with PBS containing 1% BSA (SigmaAldrich, St. Louis, MO), and serum was incubated overnight at 4°C. Bound
IgM was detected using 125I-labeled anti-IgM (Southern Biotechnology
Associates). Radioactivity of individual wells was measured using a 1272
Clini␥ gamma counter (LKB-Wallac, Gaithersburg, MD). A standard curve
of monoclonal IgM was used to convert counts per minute to micrograms
per milliliter.
To determine levels of IgMa or IgMb, 50 ␮l/well of 10 ␮g/ml anti-IgMa
(DS-1; BD PharMingen) in PBS or 50 ␮l/well of 10 ␮g/ml anti-IgMb
(AF6-78; BD PharMingen) in PBS was used for coating. Blocking and
incubation were performed as described above, and bound Ab was detected
with 125I-labeled anti-IgM. To convert counts per minute to nanograms per
milliliter, standard curves were established for each allotype using purified
monoclonal IgMa or IgMb, respectively.
Total IgA was measured by RIA as described previously (23), and a
standard curve of purified, monoclonal IgA was used to convert counts per
minute to nanograms per milliliter. To assess total IgAa and IgAb levels,
plates were coated with 50 ␮l/well of 10 ␮g/ml anti-IgAa (HY15, a gift of
Dr. M. Pawlita, originally generated and described by Potter and Lieberman (25), produced and purified in our laboratory) in PBS or 50 ␮l/well of
10 ␮g/ml anti-IgAb (HISM2, originally generated and produced in our
laboratory, currently available at BD PharMingen) in PBS, blocking and
incubation were performed as described, and bound Ab was detected with
125
I-labeled anti-IgA (Southern Biotechnology Associates). Standard
curves of purified IgAa or IgAb were used to convert counts per minute to
nanograms per milliliter.
Bacteria-specific IgA was measured by RIA. Plates were coated with
either 50 ␮l of 10 ␮g/ml bacterial sonicate (B. distasonis, SFB) overnight
at 4°C or 100 ␮l/well freshly cultured bacteria in PBS (OD600 ⫽ 2) for 24 h
at 37°C (M. morganii) and were blocked with 1% BSA in PBS. Incubation
with organ fragment culture supernatant fluid was performed overnight at
4°C. Thereafter, plates were incubated for 4 h at room temperature with
125
I-labeled anti-IgA. To assess levels of bacteria-specific IgAa or IgAb,
plates were coated, blocked, and incubated with organ fragment culture
supernatant fluid as described above. HY15 (anti-IgAa) and HISM2 (antiIgAb) were used for allotype-specific IgA detection, and 125I-labeled antiIgG1 and anti-IgG2a (Southern Biotechnology Associates) were used to
detect bound HY15 and HISM2, respectively.
Results
Generation of neonatal allotype chimeric mice under germfree
and conventional conditions
To study the contributions of B1 and B2 cells to the immune response against commensal bacteria, Ig allotype chimeric mice
were generated under germfree and conventional conditions.
Treatment of newborn germfree BALB/c mice (a allotype; recipient) with anti-IgMa for 32 days and transfer of PeC cells from
C.B-17 mice (b allotype; donor) on day 3 after birth resulted in Ig
allotype chimeric animals. As shown in Fig. 1, BALB/c- and C.B17-derived PeC cells were specifically stained with Abs against
surface IgMa and IgDa or IgMb and IgDb, respectively. In Ig allotype chimeric mice, most B1 cells in the PeC were of the donor
allotype (IgMb high IgDb low), and importantly, no donor-derived
B2 cells (IgMb low IgDb high) were detectable (Fig. 1). Most recipient-derived B cells were of the B2 phenotype (IgMa low IgDa high)
even though some endogenous B1 cells (IgMa high IgDa low) were
observed. Taken together these results show that most B1 cells in
Ig allotype chimeric mice were donor derived, while all B2 cells
were recipient derived. The chimerism (Table I) and cell numbers
in the peritoneal cavity (data not shown) remained stable over the
entire experimental period in both germfree and conventionally
FIGURE 1. Chimerism of B1 and B2 cells in Ig allotype chimeric mice
compared with BALB/c (recipients) and C.B-17 (donor) mice. PeC were
isolated from BALB/c, C.B-17, or Ig allotype chimeric animals by peritoneal lavage. Single-cell suspensions were stained with fluorescently labeled, allotype-specific anti-IgM and anti-IgD Abs. Numbers are the percentages of B1 cells (IgMhigh, IgDlow) and B2 cells (IgMlow, IgDhigh)
among gated lymphocytes. One example representative of 17 experiments
is shown.
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M. morganii, a Gram-negative aerobic rod, related to the Proteus species,
was originally isolated by Potter from mouse feces and was provided by A.
Feeny (The Scripps Research Institute, La Jolla, CA). B. distasonis (TAC:
ASF 519), a Gram-negative, anaerobic rod, was purchased from Taconic
Farms. SFB, an obligate anaerobic, spore-forming, Gram-positive, segmented bacterium, related to Clostridia sp., was provided by H. Snel (University of Nijmegen, Nijmegen, The Netherlands). M. morganii was cultured in vitro in Brain Heart Infusion medium (Difco, Fisher Scientific,
Pittsburgh, PA) under aerobic conditions. B. distasonis was grown in altered Schaedler broth (Difco), supplemented with 5% sterile FCS (Life
Technologies, Grand Island, NY) under anaerobic conditions using the
Aerogen system (OXOID, Basingstoke, U.K.). No in vitro culture method
for SFB has been established to date. Therefore, spore-containing intestinal
contents were isolated from SCID mice previously monoassociated with
SFB, and fecal suspensions in PBS were used for further monoassociation.
The purity of cultures or spore-containing fecal suspensions was verified
microscopically in Gram-stained smears.
Eight-week-old germfree Ig allotype chimeric mice were transferred
into sterile experimental Trexler isolators and monoassociated with M.
morganii, B. distasonis, or SFB by oral inoculation with 200 ␮l (5 ⫻ 107
CFU) of in vitro cultured bacteria (M. morganii, B. distasonis) or sporecontaining fecal suspensions (SFB). The expansion of bacteria in the gut
and the persistence of intestinal colonization were monitored by in vitro
culture of fecal contents (M. morganii, B. distasonis) or analysis of Gramstained swabs from the luminal side of the intestinal tract (SFB).
4565
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CONTRIBUTION OF B1 CELLS TO INTESTINAL IgA PRODUCTION
Table I. B1/B2 chimerism in PeC of neonatal chimeric mice
Ig Allotype b
(donor)
Intestinal
Floraa
Ig Allotype a
(recipient)
Dayb
% B1c
% B2d
% B1c
% B2d
M. morganii
3
2
2
3
3
2
0
14
28
42
56
70
40e
34
42
38
42
51
0
0
0
0
0
0
11
15
25
17
10
18
23
13
19
26
25
30
B. distasonis
3
3
3
3
3
3
0
14
28
42
56
70
38
28
61
31
43
50
0
0
0
0
0
0
10
16
11
15
21
19
25
24
34
23
38
32
SFB
5
3
2
3
2
3
0
7
14
28
42
56
35
29
41
37
42
55
0
0
0
0
0
0
12
8
15
18
18
19
28
18
13
26
29
29
Conventional
4
4
70
102
30
38
0
1
18
24
21
12
a
Germ-free neonatal chimeric mice were monoassociated with M. morganii, B.
distasonis, or SFB.
b
Days after monoassociation or days after birth (for conventional).
c
Defined as IgMhigh IgDlow cells among all lymphocytes in the PeC.
d
Defined as IgMlow IgDhigh cells among all lymphocytes in the PeC.
e
Mean percentage per time point.
reared Ig allotype chimeric mice. The germfree chimeras were
constructed identically to the method used by Lalor et al. (20) and
can be compared directly to the original Lalor data as published.
The reason why we went to the technically very difficult task of
producing mice under germfree conditions is because of the comments on the original papers by Lalor et al. that in that instance B1
cells have an advantage over B2 cells because the latter have to
FIGURE 2. Production of serum IgM in Ig allotype chimeric mice after monoassociation with
M. morganii, B. distasonis, or SFB. Ig allotype chimeric mice were sacrificed at the indicated time
points after monoassociation and conventionally
reared, Ig allotype chimeric mouse (CNV) chimeras were sacrificed at the age of 70 days. Blood was
collected by heart puncture. A, Total serum IgM
(micrograms per milliter) was measured by RIA
using anti-Fab-coated plates and radiolabeled antiIgM for detection. B, Total serum IgMa (recipientderived) and IgMb (donor-derived; micrograms per
milliter) were measured by RIA. Plates were coated
with anti-IgMa or anti-IgMb, respectively, and
bound IgMa or IgMb was detected by radiolabeled
anti-IgM Ab. C, Relative contributions of B1 and
B2 cells to production of serum IgM. CNV, Conventionally reared Ig allotype chimeric mice.
B1 cells contribute to the production of natural serum IgM
Germfree Ig allotype chimeric animals showed levels of serum
IgM comparable to those in conventional naive mice, and monoassociation stimulated additional IgM production (Fig. 2A). M.
morganii induced a protracted IgM response with a peak on day 28
and continuously elevated levels until day 70. After monoassociation with B. distasonis serum IgM increased over the initial 14
days and thereafter waned. Colonization with SFB induced maximal production of serum IgM between days 14 –28, followed by
a return to baseline levels during the later phase of the experiment.
To assess the contributions of B1 (b allotype) and B2 (a allotype) cells, IgM levels were measured for each allotype. As shown
in Fig. 2B, monoassociation with M. morganii induced an early
increase in B1 cell-derived IgMb that peaked around day 28 and
thereafter waned to baseline levels. In contrast, B2 cell-derived
IgMa reached maximal levels around day 56 after only a slight
initial increase. Colonization with B. distasonis mainly stimulated
the production of IgMa by B2 cells, while B1 cell derived IgMb
remained at levels similar to those found in germfree animals. SFB
induced an increase in both IgMb and IgMa with similar kinetics
for both allotypes during the first 4 wk of colonization. While
production of IgMb waned after day 28, levels of IgMa remained
elevated for a longer period and reached baseline levels around day
70. These distinct kinetics of IgM production resulted in different
relative contributions of B1- and B2-derived IgM over time (Fig.
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n
develop from bone marrow precursors, while the antigenic stimulation by gut organisms is already present during that time for
donor B1 cells. To avoid the same drawback, we tried a variation
of the original Lalor method in conventional mice by continuously
giving B2 cells the opportunity to react to the gut flora by making
IgA. Using multiple injections and higher doses of B1 cells, we
tried to overcome the described feedback inhibition (20). In fact,
the transferred cells did establish donor B1 cells in the PeC in
conventional mice at an ⬃2:1 ratio with respect to recipient B1
cells compared with the 6:1 ratio in germfree neonatal chimeras
(Table I).
The Journal of Immunology
2C). In conventionally reared Ig allotype chimeric mice the proportions of B1- and B2-derived IgM were similar to those found at
late time points after monoassociation in gnotobiotic animals (Fig.
2, B and C). These data show that B1 cells contribute an appreciable amount to the total natural serum IgM in germfree animals
and mount an early IgM Ab response after colonization with commensal microorganisms.
Monoassociation with M. morganii, B. distasonis, or SFB
induces GC reactions
Colonization with M. morganii, B. distasonis, or SFB induces
the production of intestinal IgA
To assess mucosal Ab responses induced by intestinal colonization
with M. morganii, B. distasonis, or SFB, IgA levels in organ frag-
ment culture supernatant fluid were measured by RIA (Fig. 4). The
mucosal immune system in germfree animals was quiescent, with
barely detectable levels of intestinal IgA, but secretion of IgA was
readily induced after monoassociation with any of the three commensals. Interestingly, the kinetics as well as the localization of
maximal IgA production varied depending on the microorganism
used for monoassociation. M. morganii stimulated IgA production
mainly in the lower gastrointestinal tract (cecum, colon). Nevertheless, some stimulation of IgA secretion in the small intestine
was observed. Colonization with B. distasonis induced an early
IgA response, most pronounced in the small intestine and cecum,
peaking on day 14 postinoculation. SFB stimulated an early IgA
response in the upper gastrointestinal tract, with maximal levels
around days 14 –28. However, a slightly delayed response in cecum and colon followed around days 28 – 42. These data demonstrate that colonization with M. morganii, B. distasonis, or SFB
stimulated intestinal IgA production at distinct sites and different
times for each bacterium.
Intestinal IgA secreted in response to colonization with M.
morganii, B. distasonis, or SFB is reactive with bacterial Ag
Supernatant fluid of organ fragment cultures was analyzed for the
presence of bacteria-specific IgA by RIA (Fig. 5). M. morganiispecific IgA was produced in all intestinal tissues, but in accordance with the findings for total IgA production, maximal levels of
specific IgA were found in the colon (note the different scale). The
production of specific IgA in the small intestine after colonization
with B. distasonis reflected the data obtained for total IgA. Surprisingly, the highest level of B. distasonis-specific IgA was found
on day 28 in the cecum. Monoassociation with SFB led to the rapid
secretion of bacteria-specific IgA in the small intestine, followed
by a specific response in the lower gastrointestinal tract. These data
show that monoassociation with M. morganii, B. distasonis, or
SFB not only stimulated the secretion of total IgA, but led to the
production of Ag-specific IgA with distinct kinetics and localization of production for each bacterium.
Contributions of B1 and B2 cells to the production of total and
bacteria-specific intestinal IgA
FIGURE 3. Induction of GC reactions in PP lymphocytes after monoassociation with M. morganii, B. distasonis, or SFB. Monoassociated Ig
allotype chimeric mice were sacrificed at the indicated time points after
colonization, PP were isolated, and single-cell suspensions were stained
with fluorescently labeled Ab specific for ␬ L chain and fluorescently labeled PNA. Numbers indicate the percentage of GC B cells (␬ L chainlow,
PNA-binding) among total B cells (␬ L chain⫹).
Next we analyzed the contributions of B1 and B2 cells to intestinal
IgA production by determining levels of total and bacteria-specific
IgAa and IgAb in organ fragment culture supernatant fluids. As
shown in Fig. 6A, most of the total as well as bacteria-specific IgA
induced by colonization was produced by B2 cells, with only a
minor contribution of B1 cells, probably due to the paucity or even
the absence of donor-derived B cells in the gut lamina propria.
Similarly, analyses by immunofluorescence revealed only negligible numbers of IgAb-expressing cells in the gut lamina propria of
chimeric mice (data not shown). Fig. 6B depicts the relative contribution of IgAa or IgAb, respectively, to total and bacteria-specific intestinal IgA. Germfree animals produced a low amount of
IgA reactive with bacterial Ag compared with maximal levels of
specific IgA after colonization, and B1 cells contributed significantly (45–73%) to this minute amount of naturally occurring bacteria-specific IgA (Fig. 6B). Monoassociation with M. morganii, B.
distasonis, or SFB induced intestinal IgA production by B2 cells
with very low contribution of B1 cells. Similar ratios of B1 and B2
cell-derived total IgA were found in conventionally reared animals. Thus, monoassociation with M. morganii, B. distasonis, or
SFB as well as the presence of conventional microflora locally
stimulated B2 cells to produce total and bacteria-specific intestinal
IgA. The contribution of B1 cells to total intestinal IgA appeared
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Next we tested the stimulatory potentials of the different commensal bacteria on B1 and B2 cells in the gut. All three commensals
led to activation of the intestinal mucosal immune system with
marked GC reaction in PP, characterized by the appearance of
PNA-binding B cells (␬ L chain low PNA binding, as defined by
Lebman et al. (26); Fig. 3). While the GC reaction induced by B.
distasonis peaked around day 28, monoassociation with M. morganii led to a prolonged activation of PNA-binding B cells in PP
over 70 days. Colonization with SFB induced maximal GC reaction on day 14 after monoassociation. These results show that inoculation of germfree Ig allotype chimeric mice with each of the
three commensals induced activation of a previously quiescent gut
mucosal immune system.
4567
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CONTRIBUTION OF B1 CELLS TO INTESTINAL IgA PRODUCTION
to be limited (1–15%); however, some bacteria-specific B1 cellderived IgA was detectable at late time points after monoassociation (15–20%).
Discussion
To try to establish a mouse model that would be informative about
the physiologically normal contributions of cells of the B1 lineage
to gut IgA plasmablasts and the role(s) of commensal enteric microbes in stimulating the development of these cells, we generated
Ig allotype chimeras using germfree neonatal mice. Our analysis of
B1 and B2 cell-derived IgA in these monoassociated and conventionally reared Ig allotype chimeric mice revealed that most of the
intestinal IgA was produced by B2 cells. Other models, using irradiated, formerly immunocompetent mice and conventionally
reared SCID mice showed that cells of the B1 lineage could contribute ⬎50% of the IgA plasmablasts to the gut lamina propria
(14 –18, 24). Using conventionally reared TCR-␤/␦⫺/⫺ mice, it has
been suggested that the production of most, if not all, of the natural
IgA in the gut was specifically driven by particular microbial Ags
and that it probably was the product of B1 cells developing in a T
cell-independent fashion (19). While data obtained from adoptive
transfer models clearly demonstrated that transferred B1 cells can
establish a self-renewing population from which IgA-secreting
plasma cells arise (14 –18), these models might only partially reflect the normal physiological behavior of B1 cells and might not
be applicable for quantitative analysis of B1 cell function in nor-
mal immunocompetent animals. Ig allotype chimeric animals, either conventionally reared or germfree and having a fully functional immune system, might more accurately reflect normal
physiological conditions.
Cotransfer of self-renewing B1 cells and a source of B2 cells,
such as bone marrow, into conventionally reared recipients might
favor the outgrowth and differentiation of B1 cells by exposing
them to a stimulatory environment before the establishment of a
mature B2 population. We therefore generated Ig allotype chimeric
mice neonatally under germfree conditions, allowing a potentially
more balanced establishment of functional populations of both B1
and B2 cells. The newborns were treated repeatedly with an antiIgM allotype against the host allotype while being transferred with
PeC cells at 3 days of age. After 8 wk we found a predominantly
donor B1 cell population in the PeC, presumably normal populations of B2 cells derived from host bone marrow and a balanced
donor/host contribution to circulating IgM.
Monoassociation of germfree Ig allotype chimeric animals with
either M. morganii or SFB induced a rapid increase in B1-derived
IgM with either simultaneous or subsequent stimulation of IgM
production by B2 cells. Likewise, in our conventionally reared,
neonatally developed, Ig allotype chimeric mice, 27% of total serum IgM was produced by B1 cells, a ratio similar to the proportions found at late time points after monoassociation of gnotobiotic
animals. These data support the idea that B1 cells are a major
source of natural IgM and might function as a link between innate
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FIGURE 4. Induction of total intestinal
IgA production after monoassociation with
M. morganii, B. distasonis, or SFB. Levels
of total IgA (micrograms per milliliter ⫾
SD) in supernatant fluid of organ fragment
cultures were determined by RIA using
plates coated with anti-Fab Ab and radiolabeled anti IgA Ab for detection.
The Journal of Immunology
4569
and adaptive immune systems providing early humoral defense
against microbial invasion (4, 5). This first line of defense can
provide early systemic protection from potentially dangerous microorganisms, a vital precaution considering the importance of
central organ systems for immediate survival. Interestingly, B. distasonis stimulated mainly B2 cells, while B1 cell-derived IgM remained at levels comparable to those in germfree animals. These
results suggest that B1 cells need specific stimulation to mount
additional IgM and that this stimulation depends on the colonizing
microorganism.
We used unfractionated PeC cells from conventionally reared
donors as a source of B1 cells. Such unfractionated populations
also contain donor B2 cells as well as some activated T cells. Also,
since these cells derived from conventionally reared donors, they
could have been previously selected for reactivity with a vast array
of environmental Ags different from the microbe used to monoassociate, thereby precluding an effective response against the microbes selected for our study. Our arguments to minimize these
potentially confusing aspects of our model are that 1) the donorderived cells were of the B1 phenotype, as shown by FACS analysis of PeC isolated from Ig allotype chimeric mice, and did establish in PeC, but contributed little to gut IgA production; and 2)
donor-derived B1 cells were functional, i.e., they contributed to the
natural serum IgM in germfree animals as well as to the IgM response after monoassociation. Thus, a rapid decay of transferred
B1 cells with the specificities to respond to any particular microbe
seems improbable, particularly since their product is often rather
close to a germline specificity.
However, monoassociation of formerly germfree, adult mice
with commensal bacteria is different from the naturally occurring
codevelopment of the immune system and flora in conventionally
reared animals. The induction of GC reactions upon monoassociation of adult germfree mice might favor stimulation and differentiation of B2 cells and therefore lead to the predominance of B2derived IgA in our system. Conventionally reared Ig allotype
chimeric mice were generated by repeated injection of donor-derived PeC into newborn mice without additional anti-IgMa treatment. This modification allowed undisturbed development of both
donor-derived and endogenous B cells during natural colonization.
The resulting chimerism was comparable to that of antiIgMa-treated germfree Ig allotype chimeric mice with a majority of
donor-derived B1 cells in the PeC. However, in conventionally
reared Ig allotype chimeric mice the contribution of B1 cells to the
intestinal IgA production was only marginal, and most gut IgA was
produced by B2 cells.
Monoassociation with each of the three commensals also induced GC reactions in PP of the small intestine and led to the
production of intestinal IgA with distinct kinetics and particular
main sites of IgA secretion for each bacterium. These data are in
accordance with a recent report by Jiang et al. (27) demonstrating
a clear correlation between the localization of SFB in the intestine
and the IgA response by the host. Taken together, our results show
that all three microorganisms interact with their host in a way that
leads to activation of the formerly quiescent gut mucosal immune
system.
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FIGURE 5. Induction of bacteriaspecific intestinal IgA production after
monoassociation with M. morganii, B.
distasonis, or SFB. Levels of bacteriaspecific IgA (counts per minute ⫾ SD)
in supernatant fluid of organ fragment
cultures were assessed by RIA using
plates coated with either bacterial sonicates (B. distasonis, SFB) or whole bacteria (M. morganii) and radiolabeled antiIgA Ab for detection. Note the different
scales.
4570
CONTRIBUTION OF B1 CELLS TO INTESTINAL IgA PRODUCTION
Until recently the mechanisms involved in recruiting IgA-committed B cells to the intestinal lamina propria have been only
poorly understood. It has been established that ␣4␤7 integrin mediates binding of lymphocytes to mucosal addressin cell adhesion
molecule-1 and is therefore crucial for lymphocyte homing to the
gut (28). Work by Bowman et al. (29) has shown that TECK/
CCL25, expressed by intestinal epithelial cells, is a potent and
selective chemoattractant for IgA-secreting B cells. Their work
focused on B cell populations obtained from mesenteric lymph
nodes, PP, and spleen. As B2 cells represent the major B cell
population in these organs, it might well be that the mechanisms
described by these authors, while true for the recruitment of B2
cells, only marginally apply for B1 cells. Several mechanisms involved in the accumulation of B1 cells in the peritoneal cavity, Ig
class switching, and migration to the lamina propria have been
described (30 –32), and Fagarasan et al. (33) have demonstrated
the potential of B220⫹IgM⫹ lamina propria cells, presumably B1
cells, for in situ class switching and differentiation to IgA-producing
cells. However, they also showed that the mechanism described
applies for a very small fraction of lamina propria B cells compared
with PP B cells. Taken together, these studies suggest that different
mechanisms are involved in the attraction of B1 and B2 cells to the
lamina propria, and that the quantitative importance of these mechanisms might vary. We cannot completely exclude that some of the
intestinal IgAa is B1 derived in our model, as the Ig allotype chimeric
mice showed the presence of some endogenous B1 cells. However, if
B1 cells contribute a major proportion of the intestinal IgA production
in our model, we would expect the donor-derived B1 cells to
participate in a quantitatively significant way.
How can we reconcile our observations with respect to the findings in other systems? The potential of B1 cells to produce intestinal IgA has been shown in transfer models (14 –18). However,
the immunologic environment in these settings is greatly altered.
The function of B2 cells is impaired either by delayed development of a functional B2 population (transfer models) or by attenuated GC reaction in PP and disturbed cognate B-T cell interaction
(TCR-␤/␦⫺/⫺ mice). The SCID mouse lacks PP GC reactions, and
the TCR-␤/␦⫺/⫺ mouse has only minimal gut PP GC reactions,
probably due to B2 cells, and they do not lead to appreciable affinity maturation (point mutations) (34). Such B2 responses may
account for the observed microbial Ag dependence of natural IgA
production in TCR-␤/␦⫺/⫺ mice (19). Possibly the cytokines
present in these vestigial GC reactions are sufficient to allow minimal clonal expansion and switching to IgA expression. Our model
system includes normal GC reactions in PP, favoring specific B2
cell development. Thus, we believe our proffered model is likely to
be more physiologically normal and relevant to the immunocompetent mouse than other models.
In summation, our data demonstrate that B1 cells are a major
source of natural IgM and can mount an early serum IgM response
given the right antigenic stimuli, but that Ig class switching and
migration to the gut after colonization with commensal bacteria are
dominated by B2 cells and not B1 cells. Our model suggests that
the physiologic contribution of B1 cells to IgA production in response to colonization with commensal organisms is limited, and
most of the total as well as specific intestinal IgA is produced by
B2 cells.
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FIGURE 6. Intestinal IgA in gnotobiotic or
conventionally reared (CNV) Ig allotype chimeric mice is predominantly produced by B2,
not B1, cells. Levels of B1 and B2 cell-derived
intestinal IgA were measured by RIA. A, To assess total IgAa (donor-derived) and IgAb (recipient-derived), plates were coated with anti-IgAa
(HY15) or anti-IgAb (HISM2); pooled organ
fragment culture supernatant fluid from duodenum, jejunum, ileum, cecum, and colon was incubated in serial dilutions; and bound IgAa or
IgAb (micrograms per milliliter) was detected
with radiolabeled anti-IgA Ab. To determine the
amount of bacteria-specific IgAa or IgAb, plates
were coated with either bacterial sonicates (B.
distasonis, SFB) or whole microorganisms (M.
morganii); pooled organ fragment culture supernatant fluid from duodenum, jejunum, ileum, cecum, and colon was incubated; and bound IgAa
or IgAb (counts per minute) was detected with
HY15 or HISM2, respectively. B, Relative contributions of B1 and B2 cells to total and specific
intestinal IgA production.
The Journal of Immunology
Acknowledgments
We thank Alec McKay for running the FACS and preparing radiolabeled
Abs, and Michelle Albright for maintaining the germfree facility. We also
thank Dr. Han-Qing Jiang for technical and intellectual support.
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