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*
Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and
Department of Pathology, Harvard Medical School, Boston, MA 02135
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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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To clarify the ambiguities associated with the use of exogenous IgM and to examine the role of endogenous IgM in the Ab response, we have constructed a novel mouse strain that is deficient in secreted IgM. In these mutant mice, B cells are still capable of expressing surface IgM and IgD and secreting other Ig isotypes. Unexpectedly, mutant mice have elevated levels of B-1 cells in both the peritoneum and spleen. B-1 cells differ from the conventional B (B-2) cells in their differentiation during fetal and neonatal development, in a capacity for self renewal, and in their characteristic localization in lamina propria and pleural and peritoneal cavities in adult mice (6, 7, 8). Because B-1 cells secrete the major portion of natural IgM, our finding in the mutant mice suggests a physiologic role of natural IgM in the feedback regulation of B-1 cell differentiation and/or maintenance. In addition, our data demonstrate that natural IgM secreted by B-1 cells promotes the T cell-dependent (TD)3 Ab response by conventional B cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A mutant mouse strain that does not secrete IgM but still
expresses membrane-bound IgM was made by targeted mutagenesis in
embryonic stem (ES) cells. In the targeting vector, the
µs exon and its three downstream polyadenylation
(poly(A)) sites were replaced by a cDNA fragment encoding the Cµ4 and
µm exons (Fig. 1
). The
positive selectable marker neomycin phosphotransferase
(neo) gene was inserted at the SphI
site 200 bp downstream of the µm poly(A) site so that the
µm RNA processing was not affected by the inserted
neo gene. The vector contained 2 kb and 1.5 kb of homologous
sequences at the 5' and 3' end of the neo gene,
respectively. The negative selectable marker thymidine kinase
(tk) gene was placed just outside of the 3'
homologous region (Fig. 1). After homologous recombination, only
µm heavy chain can be expressed from the Cµ locus.
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Flow cytometry analyses
Lymphocyte development in the mutant mice was analyzed by flow
cytometry. Fluorescein- and phycoerythrin-conjugated mAbs specific for
cell surface markers, including CD45R (B220), CD40, CD19, CD5, CD11b
(Mac-1), CD23, MHC IA, CD4, CD8, CD90 (Thy-1.2), TCR
ß, and
TCR
, were from PharMingen (San Diego, CA). Conjugated
anti-IgM and -IgD were generously provided by Dr. Leonore
Herzenberg (Stanford University, Stanford, CA). Single-cell suspensions
were prepared from the spleen, lymph node, and thymus. Bone marrow
cells were obtained by flushing femurs with cold PBS plus 2% FCS.
Peritoneal cells were obtained by lavage. Erythrocytes in splenocyte
and bone marrow cell suspensions were removed by lysis with 0.14 M
NH4Cl and 17 mM Tris · Cl, pH 7.4. Cells (5
x 105) were stained with conjugated Abs and analyzed by
FACScan (Becton Dickinson, Mountain View, CA). Ten thousand live cells
were collected for each sample.
B cell stimulation assay
Splenocytes prepared as described above were incubated with rat
mAbs specific for CD90 (Thy-1.2), CD4, and CD8. The bound T cells were
removed by magnetic immunoselection using goat anti-rat IgG-coated
beads (Dynal, Oslo, Norway). The resulting B cells (>95%) were
cultured in quadruplicates at 4 x 106/ml in RPMI
1640 plus 5% FCS, 10 mM HEPES, pH 7.2, 50 µM 2-ME, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin. B cells were stimulated with 1 µg/ml or 10 µg/ml of
F(ab')2 fragments of goat anti-mouse IgM (Jackson
ImmunoResearch Laboratories, West Grove, PA); soluble CD40L-CD8
fusion proteins (as developed by Peter Lane (24)) cross-linked by 1
µg/ml anti-CD8 Ab; the combination of anti-IgM and
cross-linked CD40L-CD8 fusion proteins; or different concentrations
of LPS (Sigma, St. Louis, MO). After 48 h, cultures were pulsed
with 0.5 µCi/ml of [3H]thymidine, and cell
proliferation as indicated by [3H]thymidine incorporation
was determined 6 h later.
Immunization
Eight- to twelve-week-old homozygous mutant mice and wild-type or heterozygous controls were used for immunizations with both TD and TI (T cell-independent) Ags. For TD Ag, different amounts of hapten (4-hydroxyl-3-nitrophenyl)acetyl-conjugated keyhole limpet hemocyanin (NP-KLH) in 0.1 or 0.2 ml of HBSS were injected i.v. through the tail vein on day 0 and again on day 22. Mice were bled from the tail vein at day 7, 14, 21, and 29 after primary immunization. For TI Ag, different amounts of NP-Ficoll in 0.2 ml of HBSS were injected i.p. Mice were bled on day 7 and 14 after immunization.
ELISA assay
Serum Ig levels were measured by ELISA (Southern Biotechnology Associates, Birminghan, AL). Plates were coated with goat anti-mouse Ig(M+G+A) and developed with horseradish peroxidase (HRP)-conjugated goat Abs specific for each mouse Ig isotype. The concentrations were calculated using the linear ranges of the dilution and purified mouse Ig isotypes as standards. NP-specific IgG Abs were measured by ELISA with NP-BSA-coated assay plates and developed with HRP-conjugated anti-IgG Abs. Ab affinity maturation was also determined by ELISA. Briefly, plates were coated with either NP5-BSA or NP15-BSA (generously provided by Dr. Garnett Kelsoe, University of Maryland, College Park, MD). Serial dilutions of serum were added to the coated plates, and the relative binding of total IgG Abs was determined using HRP-conjugated anti-IgG. The ratios of binding to NP5-BSA and NP15-BSA were calculated for individual mice using OD405 in the linear ranges of the assays.
Immunohistochemistry
Spleens were harvested 7 days after secondary immunization and snap frozen in OCT embedding media. Serial, 4-µm-thick frozen sections were cut in a cryostat microtome, thaw mounted onto polyL-lysine-coated slides, air dried, fixed in ice-cold acetone for 10 min, and stored at -80°C. For two-color immunolabeling of germinal centers, spleen sections were incubated with biotin-conjugated anti-CD45R (B220) and HRP-conjugated peanut agglutinin (HRP-PNA, E-Y Laboratories, San Mateo, CA), followed by incubation with streptavidin-alkaline phosphatase (ExtrAvidin-AP, Sigma). Bound AP and HRP were visualized using naphthol AS-MX phosphate/Fast blue BB base and 3-aminoethylcarbazole, respectively (25). For Ag trapping, mice were immunized with either 1 or 10 µg of NP-KLH on day 0 and were then injected with 50 µg of NP-BSA-biotin on day 12. Spleens were harvested 18 h later. To visualize the trapped NP-BSA-biotin on the surface of follicular dendritic cells (FDCs), spleen sections were incubated with streptavidin-AP and HRP-PNA. Bound AP and HRP were visualized as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ig µ heavy chains are expressed in both membrane-bound
(µm) and secreted (µs) forms from the
same Cµ gene during B cell development (26). In association with
either surrogate light chains 
5 and VpreB or 
and
light chains, the µm heavy chain is required for the
stepwise progression from pro-B cells to Ab-secreting plasma cells
(27). Therefore, to construct a mutant mouse strain in which B cells
are rendered incapable of expressing µs, µm
expression has to be preserved. The expression of µs and
µm results from the differential utilization of poly(A)
sites and alternative splicing of the same precursor RNA (Fig. 1
) (28, 29). Utilization of poly(A) sites downstream of the µs
exon produces a mature RNA that will be translated into the
µs heavy chain. Utilization of the poly(A) site
downstream of the µm exon leads to the deletion of the
µs exon by splicing and to production of a mature RNA
encoding the µm heavy chain. To engineer the locus so
that only µm heavy chain is expressed, we employed
homologous recombination to replace the µs exon and its
three downstream poly(A) sites with a cDNA fragment encoding the
µm exons already spliced to the Cµ4 exon (Fig. 1; see
Materials and Methods for details). Two properly targeted ES
cell clones were obtained and used to derive germline mutant mice.
Mutant mice were identified by Southern blot analysis of tail DNA after
EcoRI digestion and hybridization with an 0.9-kb
XhoI-EcoRI probe (Fig. 1C). The
normal allele gives rise to a 13-kb fragment, and the targeted allele
gives rise to a 2-kb fragment (Fig. 1E).
Targeted mutation specifically blocks IgM secretion
To determine the effect of the targeted mutation on IgM secretion,
sera were collected from wild-type and homozygous mutant mice at 4, 8,
and 12 wk of age, and the concentrations of IgM, IgA, IgG1, -2a, -2b,
and -3 were determined by ELISA. An average of 250 µg/ml of IgM was
detected in the sera of wild-type mice at all three ages, whereas in
the sera of mutant mice, IgM was only 0.1 µg/ml (Fig. 2). This level of IgM was <10-fold above
the background in the recombination activating gene
(RAG)-2 deficient mice (data not shown).
Furthermore, the level of IgM did not change in mutant mice as they
aged (up to 1 yr, data not shown). The residual serum IgM detected in
mutant mice probably represents membrane IgM released from the B cell
surface as a result of proteolytic degradation and/or B cell death.
Thus, in practical terms, the targeted mutation renders mutant mice
deficient in secreted IgM.
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A), but the differences disappeared when
mice matured (Fig. 2B). Mutant mice also had slightly
elevated levels of IgA. The elevated levels of serum IgG3 and IgA at
early ages in mutant mice are probably due to the increased numbers of
B-1 cells, while the elevated levels of IgG2a are probably due to other
effects of the absence of natural IgM on the immune system (see below
and Discussion). Nevertheless, in adult mice, the
targeted mutation specifically abolishes IgM secretion without
significantly affecting the overall levels of serum Ig. Enhanced B-1 cell differentiation in mutant mice
The normal levels of serum IgG and IgA in adult mutant mice
suggest a relatively normal B cell development in these mice. To
determine the effect of the absence of natural IgM on lymphocyte
development and to rule out any aberrant effect of the targeted
mutation, the cell numbers in spleen were enumerated and the phenotypes
of splenocytes were extensively characterized by flow cytometry for the
expression of various surface markers. While the total numbers of cells
recovered from wild-type and mutant mice were very similar, the
percentages of B (B220+, IgM+) cells
showed some variation from mouse to mouse (Table I). However, the differences in the
percentages of B cells in the mutant and wild-type mice were not
significant (p = 0.1). Similarly to wild-type
mice, almost all B220+ splenic B cells from mutant mice
expressed IgM (Fig. 3A), suggesting that
targeted mutation did not grossly affect membrane-bound IgM expression.
Furthermore, almost all IgM+ B cells also expressed IgD
(Fig. 3A), indicating that the introduced
neo gene did not block significantly the transcription and
alternative splicing to the locus. In addition, B cells from mutant
mice expressed normal levels of CD40, CD19, CD23, CD16, and MHC IA
(data not shown). Finally, histologic staining of spleen sections of
mutant mice yielded normal numbers and morphologies of lymphoid
follicles (data not shown). However, there was a clear difference in
the B cell compartment in the spleen between mutant and wild-type mice.
The percentage of CD5+ B-1 cells was significantly
increased in the spleen of mutant mice (Fig. 3A),
resulting in an 
2.6-fold increase of B-1 cell numbers (Table I,
p < 0.01). Consistently, more splenic B cells in the
mutant mice were of B-1 cell phenotype: B220low,
IgMhigh, IgDlow, and Mac-1+ (Fig. 3A and data not shown). The elevated level of B-1 cells in
mutant mice was already established by 4 wk of age (data not
shown).
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B and Table I, 0.05<p < 0.01), but
no difference was detected in mice at 1 mo of age (data not shown).
Staining of bone marrow B cells for IgM and IgD failed to detect any
consistent changes in the percentages of immature B cells
(IgM+, IgD-) and mature B cells
(IgM+, IgD+) (data not shown). The relatively
normal number of B cells in the spleen of mutant mice suggests that the
absence of natural IgM does not significantly affect conventional B
cell differentiation in the bone marrow.
The most apparent effect of the absence of natural IgM on B cell
development was on B-1 cells in the peritoneum. More cells were
recovered from peritoneal lavage from mutant mice than from wild-type
mice (Table I). Significantly higher percentages of these peritoneal
cells from the mutant mice were B-1 cells (B220+,
IgM+, IgD+, CD5+/-,
Mac-1low, and CD23-) (Fig. 3C and
data not shown). Quantitation revealed that the numbers of B-1 cells in
the peritoneum were increased approximately threefold in the mutant
mice (Table I, p < 0.01), which was similar to the
increase of B-1 cells in the spleen. The elevated levels of B-1 cells
were already detectable at 4 wk of age and were stably maintained up to
12 mo of age, in the oldest mice analyzed (data not shown). The
increased numbers of B-1 cells in the peritoneum (and spleen) were
observed in mutant mice of both the 129 background and the mixed
background of C57BL/6 and 129. Together, these observations in the
mutant mice suggest a physiologic role of natural IgM in B-1 cell
development.
T cells in the spleen, lymph node, thymus, and peritoneum were
characterized by surface expression of CD4, CD8, TCRß, TCR,
CD90 (Thy-1.2), and CD5. In addition to the slight difference in the
percentages of T cells in the spleen, no phenotypic difference in T
cells was detected in all lymphoid organs analyzed. Thus, the absence
of natural IgM has no effect on T cell development.
Enhanced IgG2a response to a TI Ag in mutant mice
In the mutant mice, the elevated levels of serum IgG2a, IgG3, and
IgA were correlated with the increased numbers of B-1 cells. B-1 cells
are known to secrete IgM, IgG3, and IgA but not IgG2a (30, 31). The
increased IgG2a level in the mutant mice suggests that natural IgM may
regulate IgG2a production in normal mice. This notion was further
supported by the Ab response to TI Ag NP-Ficoll in the mutant mice.
Homozygous mutant mice and heterozygous controls were immunized i.p.
with 1, 10, or 100 µg of NP-Ficoll. Sera were collected at day 7 and
14 after immunization, and the levels of NP-specific IgG, individual
IgG isotypes, and IgA were assayed by ELISA. While the total
NP-specific IgG was relatively normal, the levels of NP-specific IgG2a
were three- to fourfold higher in the homozygous mutant mice than in
the heterozygous controls at all three Ag doses (Fig. 4). In contrast, NP-specific IgG2b levels
were reduced in the homozygous mutant mice (Fig. 4). The levels of
NP-specific IgG1, IgG3, and IgA were similar in both types of mice
(data not shown). Thus, IgG2a response to TI Ag appears to be enhanced
in the absence of natural IgM.
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To determine the effect of the absence of natural IgM in Ab
response to TD Ag, homozygous mutant mice and heterozygous controls
were immunized i.v. with 0.1, 1, 10, and 100 µg of the TD Ag NP-KLH
on day 0 and rechallenged with the same doses of Ag on day 22. Sera
were collected on day 7, 14, 21, and 29 and assayed for NP-specific IgG
Abs by ELISA. Figure 5 shows the relative
levels of NP-specific IgG at day 14 (primary response) and 29
(secondary response). When the Ag dose was 1 µg (suboptimal), the
levels of NP-specific IgG Abs were 25-fold lower for both the
primary and secondary responses in homozygous mutant mice than in
heterozygous controls. Further analyses of the levels of NP-specific
IgG1, -2a, -2b, and -3 Abs showed that IgG1 was the most severely
reduced (>100-fold; data not shown). In contrast, when Ag doses were
10 and 100 µg, homozygous mutant mice generated similar levels of
NP-specific IgG Ab responses. Thus, in the absence of natural and/or
immune IgM, the IgG Ab response to limiting doses of TD Ag was
impaired.
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To distinguish whether the impaired TD Ab response in mutant
mice is due to the absence of endogenous IgM or to an intrinsic defect
of B cells, B cell activation and proliferation to various stimulations
were assayed in vitro. Splenic B cells were purified from homozygous
mutant mice and heterozygous controls and stimulated in vitro with
different doses of anti-IgM, cross-linked CD40L-CD8 fusion
protein, or both. B cells from homozygous mutant mice proliferated to
the same extent as those from control mice in a dose-dependent manner
to anti-IgM or CD40L stimulation individually and synergistically
to the combination of both (Fig. 6A). Similarly, B cells
from mutant mice proliferated normally in response to different
concentrations of LPS (Fig. 6B). Thus, B cells
differentiated in the absence of natural IgM do not appear to have any
intrinsic defect, suggesting that the impaired Ab response is likely
due to the absence of natural and/or immune IgM.
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Natural IgM is thought to enhance the Ab response by promoting the
formation of immune complexes containing activated C3 fragments (13, 15, 32). The immune complexes can be trapped through complement
receptors (CR) on FDC, promoting efficient germinal center reactions
(16, 33). To determine the underlying cause of the diminished Ab
response to suboptimal doses of NP-KLH in the mutant mice, germinal
centers in the spleen were assayed 7 days after secondary immunization
by immunohistochemical staining of frozen sections with PNA coupled to
HRP (Fig. 7A).
Consistent with the diminished Ab response to 1 µg of NP-KLH, the
number and size of germinal centers were dramatically reduced in
splenic sections of homozygous mutant mice compared with heterozygous
controls. Quantitation of many spleen sections revealed that the
reduction of the number of germinal centers was approximately fourfold.
In contrast, similar numbers of germinal centers were detected in
spleen sections of homozygous mutant mice as in heterozygous controls
when 10 and 100 µg of NP-KLH were used (Fig. 7A and data
not shown), consistent with the relatively normal IgG Ab response at
these Ag doses.
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C). On average, 5% of B cells were
PNAhigh germinal center B cells in heterozygous controls
compared with 1.5% in homozygous mutant mice. When 10 µg of NP-KLH
was used, the percentages of B220+PNAhigh
germinal center B cells were similar (10%). Together, these
findings suggest that the impaired IgG Ab response in the absence of
secreted IgM is associated with an impaired germinal center
reaction. Impaired Ag trapping on FDC in mutant mice
The impaired germinal center reaction in mutant mice could be due
to a defect in Ag trapping on FDC. To directly demonstrate this
mechanism, Ag trapped on FDC was assayed by immunohistochemical
staining of spleen sections (16, 34, 35, 36). Homozygous mutant mice and
heterozygous controls were immunized with either 1 or 10 µg of NP-KLH
on day 0 and then were given 50 µg of NP-BSA-biotin on day 12.
Spleens were harvested 18 h later, and NP-BSA-biotin trapped on
FDC was visualized by staining with AP-streptavidin in combination with
HRP-PNA staining for germinal centers. NP-BSA-biotin trapped on FDC in
the germinal center was clearly detectable in spleen sections of
control mice after immunization with 10 µg of NP-KLH (Fig. 7B). In contrast, no trapped Ag was detected on FDC
in the spleen sections of similarly immunized homozygous mutant mice.
Thus, Ag trapping was impaired in the mutant mice after immunization
with 10 µg of NP-KLH, although the titers of NP-specific IgG Abs to
this dose of Ag were similar in homozygous and heterozygous mutant mice
(Fig. 5). As shown below, Ab affinity maturation was also impaired in
the homozygous mutant mice after immunization with 10 µg of NP-KLH.
No Ag trapping was detected in spleen sections after immunization with
1 µg of NP-KLH in either type of mice, probably due to the low dose
of the Ag used (data not shown). Thus, in the absence of natural IgM,
Ag appears to be less efficiently trapped by FDC.
Impaired Ab affinity maturation in mutant mice
The germinal center is thought to be the major site at which Ab
affinity maturation occurs (37, 38, 39). The impaired germinal center
reaction and Ag trapping on FDC in the mutant mice could compromise Ab
affinity maturation. To assay for Ab affinity maturation in the absence
of IgM, a plate-binding assay was used (40, 41). Briefly, plates were
coated with either NP5-BSA or NP15-BSA,
and the relative binding of NP-specific IgG Abs was determined by
ELISA. The ratios of Ab binding to NP5-BSA and
NP15-BSA were calculated. These ratios give a relative
measure of Ab affinities, because Abs with high affinities will bind to
both NP5-BSA and NP15-BSA, whereas Abs with low
affinities may not bind to NP5-BSA. In both heterozygous
and homozygous mutant mice, the ratios increased from primary to
secondary responses (Fig. 8), indicating
the occurrence of Ab affinity maturation. However, the ratios of both
the primary and secondary responses were significantly lower in
homozygous mutant mice after immunization with 1 or 10 µg of NP-KLH.
At 100 µg of NP-KLH, the ratios were similar to those of the control
mice. In heterozygous controls, the ratios of the primary or secondary
responses were similar at all three Ag doses, whereas in homozygous
mutant mice, the ratios increased with increasing doses of Ags.
Together, these data suggest that in the absence of natural and/or
immune IgM, Ab affinity maturation is compromised in response to
suboptimal doses of Ag.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A mutant mouse strain deficient in secreted IgM
The expression of µm and µs
chains is mediated by the differential polyadenylation and alternative
RNA splicing. To construct a mutant mouse strain that lacks the
secreted IgM, the targeted mutation has to preserve the expression of
µm. We replaced the µs exon and its
downstream poly(A) sites with a cDNA fragment encoding the
µm exons already spliced to the Cµ4 exon (Fig. 1). The
constructed mutant mouse strain exhibited the expected phenotype. B
cells in the mutant mice expressed IgM on their surface at a level that
is at least as high as that on B cells from normal mice (Fig. 3). In
contrast, the level of serum IgM in mutant mice was reduced 2,500-fold
to a level that is <10-fold above the background (Fig. 2). The
residual serum IgM is probably released from the B cell surface and is
therefore monomeric. It is unlikely to be effective in binding to the
normal endogenous and/or exogenous Ags. Thus, the mutant mice are
functionally deficient in secreted IgM.
Multiple molecular events occur at the IgH locus during B cell
development, including VDJ rearrangement, transcription, and class
switching. In our targeted mutation, the neo gene was
introduced 200 bp downstream of the poly(A) site of the
µm exons. Considering that replacement of Eµ or E
enhancer with a neo gene resulted in a more severe block of
recombination at the IgH or Ig locus, respectively, than the clean
deletions (42, 43, 44, 45), it was a concern that the presence of the
neo gene at the IgH locus in our targeted mutation may have
artificially biased the observed phenotype. However, findings in the
mutant mice do not substantiate the concern. In our mutant mice, the
introduced neo gene is >10 kb downstream of the Eµ
enhancer. Flow cytometry analyses revealed a relatively normal early B
cell differentiation in the bone marrow and a relatively normal number
of conventional B cells in the spleen (Fig. 3 and Table I), indicating
that the neo gene did not significantly affect Ig heavy
chain gene rearrangement. The presence of the neo gene is
unlikely to have altered the transcription of the locus because the
level of IgM on B cells from mutant mice is at least as high as that on
B cells from normal mice (Fig. 3). Similarly, the neo gene
is unlikely to have affected class switching because mutant mice have
normal levels of serum IgG1 and IgG2b. Although the levels of IgG2a,
IgG3, and IgA are elevated at early ages in mutant mice, in adults the
levels are the same as in wild-type controls (Fig. 2). After class
switching, the neo gene is deleted from the locus and
therefore can not affect the expression of the switched Ig. Thus, if
the presence of the neo gene affected class switching to
IgG2a, IgG3, and IgA, it would have to exert the effect in an
age-dependent manner. As discussed below, the elevated levels of IgG2a,
IgG3, and IgA at earlier ages were probably due to the increased
numbers of B-1 cells and other effects of the absence of natural IgM in
the mutant mice.
Our targeted mutation is very similar to the targeted replacement of
the Ig locus in which the neo gene is inserted 2.1 kb
downstream of the µm exons (41). In contrast to our
mutant mice, replacement of the Ig locus by the neo gene
did not result in elevated levels of B-1 cells or serum IgG2a, IgG3,
and IgA, indicating that introduction of the neo gene
downstream of Cµ exons did not affect the recombination,
transcription, and class switching of the locus. In addition,
IgD-deficient mice had almost normal TD and TI Ab responses except that
the affinity maturation is delayed in the early primary response (41),
indicating that the neo gene did not grossly affect Ab
response. In our mutant mice, TD Ab response is impaired to suboptimal
dose of Ag but not to optimal doses of Ag. It is unlikely that the
introduced neo gene interfered with the Ab response in an Ag
dosage-dependent manner.
In our mutant mice, the level of IgD on B cells is slightly lower than
that on B cells from normal mice, indicating that the inserted
neo gene may interfere with the µ- RNA splicing.
Alternatively, the observed changes could be due to the increased
numbers of B-1 cells in the spleen, which are IgMhigh and
IgDlow (6, 7, 8). Nevertheless, the subtle change in IgD level
on B cells did not grossly affect conventional B cell differentiation
and function as indicated by the normal number of B-2 cells in the
spleen and their normal activation and proliferation in in vitro
stimulation assays (Fig. 6). Further supporting this notion, studies
using IgD-deficient mice have shown that IgD is dispensable for B cell
development and Ab response (41).
Role of natural IgM in B-1 cell development
The most dramatic effect of the absence of natural IgM on B cell
development is the elevated levels of B-1 cells in the mutant mice. At
4 wk, an average of threefold more B-1 cells was already detectable in
the peritoneum of mutant mice than in wild-type mice (Table I and data
not shown). The increase was also evident in the spleen, where B-1
cells are just barely detectable in normal mice (Fig. 3 and Table I).
The elevated levels of B-1 cells were stably maintained when mice
became older (1 yr). B-1 cells are known to secrete IgG3 and IgA (30, 31). Correlating with the increased numbers of B-1 cells, mutant mice
had elevated levels of serum IgG3 and IgA at early ages (<8 wk old),
but the differences disappeared as mice matured (>12 wk old) (Fig. 2).
Natural IgM has been postulated to play important roles in the
regulation of the immune system (1, 2). Our findings in the mutant mice
are probably the first unequivocal demonstration that natural IgM
regulates B cell development under physiologic conditions.
The differentiation pathways of B-1 cells are still controversial. The "lineage" hypothesis postulates distinct precursors for B-1 cells and conventional B (B-2) cells (6, 46). B-1 cells are differentiated predominantly from precursors in fetal omentum and liver but rarely from B cell precursors in adult bone marrow, which give rise to B-2 cells (31, 47, 48). The differentiation of B-1 cells in the bone marrow of adult mice is inhibited by the existing B-1 cells through an undefined feedback mechanism (49, 50). In the absence of continuous differentiation, the relatively constant level of the B-1 cell population is thought to be maintained by the capacity for self-renewal of B-1 cells (47, 51). In contrast, the "specificity or differentiation pathway" hypothesis proposes that both B-1 and B-2 cells are differentiated from a common precursor pool. The acquisition of the B-1 cell phenotype is due to the cross-linking of surface Ig receptors in the absence of a cognate T cell help (52, 53, 54). Fetally derived B cells are preferentially selected into the long-lived B-1 cell compartment on the basis of germline-encoded self-reactivity (7, 8). Various cell surface receptors and signaling molecules that mediate B cell activation have been implicated in B-1 cell differentiation and maintenance (55). For example, mice deficient in B cell co-receptor components CD19 or CD21 or signal transduction molecules Btk or Vav have reduced levels of B-1 cells (56, 57, 58, 59). Transgenic mice overexpressing CD19 or mice deficient in CD22 or SHP-1 have elevated levels of B-1 cells (30, 60, 61, 62). Considering that B-1 cells secrete the major portion of natural IgM, and given our findings that mutant mice lacking natural IgM have increased numbers of B-1 cells, we propose that natural IgM is involved in the feedback regulation of B-1 cell differentiation and/or maintenance.
In mutant mice, the effect of the absence of natural IgM on the levels
of B-1 cells is already established by 4 wk of age, suggesting that
natural IgM may regulate B-1 cell differentiation at fetal and neonatal
stages. Potentially, in the absence of natural IgM as a competitor,
fetally derived B cells have an increased probability to bind to the
normal self-Ag through surface Ig receptors, leading to an enhanced B-1
cell differentiation (52, 53, 54). Cytokines such as IL-5 and IL-10 can
promote B-1 cell proliferation, while IFN- inhibits B-1 cell growth
(63, 64, 65, 66, 67). Natural IgM may exert its effect on B-1 cell differentiation
through regulating the secretion and/or function of these cytokines.
Consistent with this notion, natural Abs have been shown to be capable
of binding to cytokines and their receptors (2, 68, 69). IL-5 also
augments IgA secretion (64), and the IgA level was elevated in the
mutant mice (Fig. 2). It is also possible that natural IgM may mediate
the maintenance of B-1 cell levels in adults including the feedback
inhibition of further B-1 cell differentiation in adult bone marrow by
the existing B-1 cells (49, 50). For example, natural IgM tends to be
self-reactive (9, 10, 70), so it is possible that natural IgM may
directly interact with Ag receptor or other receptors on B-1 cells and
inhibit B-1 cell proliferation. Supporting this notion, cross-linking
of surface IgM on B-1 cells usually leads to apoptosis rather than
proliferation, as in conventional B cells (71, 72, 73). In addition,
evidence from Ig transgenic mice suggests that B-1 cells can be
activated by gut bacterial Ags such as LPS in the lamina propria and
are able to traffic between the lamina propria and peritoneum (74, 75).
IgM is secreted into the lumen of the gut and may bind to bacterial
and/or food Ags, thereby neutralizing their ability to stimulate B-1
cell proliferation. The involvement of natural IgM in the feedback
regulation of B-1 cell homeostasis is consistent with the close
association of B-1 cell levels and serum IgM levels. The expression of
µm has been shown to be required for the stepwise
progression of B cells (27, 76). Our observation in mutant mice
demonstrates that the secreted IgM is also utilized for regulating B-1
cell differentiation and/or maintenance.
Role of natural IgM in IgG2a production
The elevated level of serum IgG2a in the mutant mice was not
expected (Fig. 2). It is unlikely that the targeted insertion of the
neo gene specifically affected class switching to IgG2a but
not to other isotypes. Neither is it due to the increased numbers of
B-1 cells, because B-1 cells are not known to secrete a large portion
of serum IgG2a (30, 31). Furthermore, IgG2a response to the TI Ag
NP-Ficoll was also elevated in mutant mice (Fig. 4), whereas IgG3 and
IgA, which are known to be secreted by B-1 cells, were not affected
(data not shown). Thus, the increased IgG2a production in the mutant
mice represents a specific effect due to the absence of natural IgM.
One possible mechanism by which natural IgM may modulate IgG2a
production may be through its regulation of cytokine secretion and/or
function, which in turn affects class switching to IgG2a (2, 68, 69, 77, 78). Consistent with this notion, IFN- induces class switching
to IgG2a and IgG3 (78), both of which were elevated in mutant mice at
early ages (Fig. 2). Investigation of cytokine profiles in the mutant
mice at early ages or after challenge with a TI Ag may help to
elucidate the mechanisms underlying the increased IgG2a production.
Role of natural IgM in B cell Ab response
The mutant mice had an impaired IgG Ab response to a
limiting dose of the TD Ag NP-KLH (Fig. 5). This effect is not due to
an intrinsic defect of the B cells differentiated in the absence of
natural IgM. First, mutant mice have relatively normal numbers of B-2
cells, and these B cells respond normally to in vitro stimulation by
anti-IgM, CD40L, and LPS (Table I and Fig. 6). Second, at high
doses of Ag, IgG Ab responses are normal in the mutant mice (Fig. 5),
suggesting that B cells are intrinsically capable of mounting a normal
Ab response. Thus, the impaired IgG response is likely caused by the
absence of natural and/or immune IgM. IgM is the first class of Ab
produced during a primary Ab response. The absence of the immune IgM in
the mutant mice could also contribute to the observed defect. While the
resolution of the two possibilities awaits further experimentation in
mutant mice that have been reconstituted with normal serum IgM,
existing evidence supports a more important role of natural IgM in the
process. Natural IgM that binds KLH is readily detectable in normal
human sera (79). KLH-binding natural IgM is probably also present in
normal mouse serum. Coinjection of Ag and Ag-specific IgM usually
results in augmented Ab responses (17, 18, 20, 21, 80). However,
injection of IgM just 1 or 2 h after the Ag suppresses rather than
augments the Ab response (81), indicating that the immune IgM may not
play an important role in enhancing the ongoing Ab response. Similarly,
since adult mutant mice have normal levels of IgGs (Fig. 2), natural
IgG Abs are clearly not sufficient to compensate for the IgM deficiency
in promoting the Ab response. Together, these findings suggest that
natural IgM is most likely required for efficient Ab responses to
limiting doses of TD Ags.
Similar to mice and guinea pigs deficient in C3, C4, or CD21
(CR2) (82, 83, 84, 85, 86), IgM-deficient mice had impaired TD IgG responses to
limiting doses but not to optimal doses of Ags (Fig. 5), suggesting
that natural IgM promotes Ab response through the same pathways as C3,
C4, and CD21. C3, C4, and CD21 can potentially augment Ab responses
through two common pathways. First, C3-containing immune complexes can
cross-link B cell Ag receptor (BCR) and CD19/CD21 complex and reduce
the threshold dose of Ag required to activate B cells (56, 57, 87, 88).
Second, immune complexes are more efficiently trapped on FDC through
the expressed complement receptors (CD21 and CD35 in mice), leading to
an effective germinal center reaction during the TD Ab response (13, 32, 89). Natural IgM could augment the Ab response through the same
pathways by promoting an efficient formation of immune complexes.
Supporting this notion, Ag trapping on FDC was impaired in the mutant
mice (Fig. 7) indicating a role of natural IgM in immune complex
formation. Furthermore, we found that germinal center formation and Ab
affinity maturation were impaired in the mutant mice (Fig. 7 and 8).
Thus, natural IgM, through its associated immune complexes, stimulates
germinal center reactions and Ab affinity maturation (16, 34, 35, 90).
In addition, natural IgM may also be involved in the initial B cell
activation by promoting immune complex formation.
Increasing evidence suggests a critical role of the innate immune system in the priming of adaptive immune responses (12, 33). One mechanism that promotes efficient Ab response is the formation of immune complexes containing activated C3 fragment (87). Under physiologic conditions, complement can be activated by the classical, alternative, and lectin pathways (91). Both the alternative and lectin pathways require specific structures of the Ag such as bacterial surface and carbohydrates. Most protein Ags such as KLH do not contain such structural determinants. They have to rely on the classical pathway to activate complement and generate C3-containing immune complexes. However, before B cell activation and the Ab response, where does the Ab that can bind to an Ag come from? Natural IgM is ideally suited for this purpose due to its natural presence, polyreactivities with high avidities, and exquisite ability to activate complement. Our findings in mutant mice demonstrate a physiologic role of endogenous natural IgM in this process. Through natural IgM secreted by B-1 cells, the adaptive immune system appears to have evolved a built-in mechanism that interacts with the innate immune system to promote efficient adaptive Ab responses by conventional B cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jianzhu Chen, Center for Cancer Research, Massachusetts Institute of Technology, E17-128, 40 Ames Street, Cambridge, MA 02139. E-mail address:
3 Abbreviations used in this paper: TD, T cell-dependent; ES, embryonic stem; poly(A), polyadenylation; CD40L, CD40 ligand; µm, membrane-bound form of µ heavy chain; µs, secreted form of µ heavy chain; FDC, follicular dendritic cells; AP, alkaline phosphatase; HRP, horseradish peroxidase; PNA, peanut agglutinin; TI, T cell-independent; NP-KLH, (4-hydroxyl-3-nitrophenyl)acetyl-keyhole limpet hemocyanin; neo, neomycin phosphotransferase gene; tk, thymidine kinase gene.
Received for publication November 3, 1997. Accepted for publication January 21, 1998.
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