Truncation of the µ heavy chain alters BCR signalling and allows recruitment of CD5+ B cells
Xiangang Zou,
Christine Ayling,
Jian Xian,
Tony A. Piper,
Patrick J. Barker and
Marianne Brüggemann
Laboratory of Developmental Immunology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
Correspondence to:
M. Brüggemann
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Abstract
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Ig are multifunctional molecules with distinct properties assigned to individual domains. To assess the importance of IgM domain assembly in B cell development we generated two transgenic mouse lines with truncated µH chains by homologous integration of the neomycin resistance gene (neor) into exons Cµ1 and Cµ2. Upon DNA rearrangement shortened µH chain transcripts, VHDJHCµ3Cµ4, are produced independent of the transcriptional orientation and termination signals provided by neor. The truncated µH chain of ~52 kDa associates non-covalently with the L chain to form a monovalent HL heterodimer. Surface IgM is assembled into a defective BCR complex which has lost important signalling capacity. In immunizations with T-dependent and T-independent antigens, specific IgM antibodies cannot be detected, whilst IgG responses remain normal. B cell development in the bone marrow is characterized by an increase in early B cells, but a decrease of B220+ cells from the stage when µH chain rearrangement is completed. The peritoneal lymphocyte population has elevated levels of CD5+ B cells and their expansion may be the result of a negative feedback mechanism. The results show that antigenic stimulation is compromised by truncated monovalent IgM and that this deficit in stimulation leads to reduced levels of conventional B-2 lymphocytes, but dramatically increased levels of B-1 cells.
Keywords: µH chain truncation, BCR signalling, B-1 cell expansion, monovalent IgM, CD5+ B cells
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Introduction
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The high degree of structural similarity of Ig has led to the conclusion that H and L chain exons have evolved by various duplication processes with subsequent diversification of individual domains having produced the different isotypes (1). The structure of the µH chain has been highly conserved during vertebrate evolution from elasmobranchs (e.g. shark) through teleosts (e.g. trout), amphibia (e.g. Xenopus), reptiles (e.g. turtle), birds (including chickens) to mammals and therefore probably most closely resembles the original archaic Ig (2). Nevertheless, a remarkable diversity in alternative RNA processing has arisen, e.g. secreted and membrane-bound forms of IgM, obtained from differentially spliced µH chain transcripts, can comprise the secretory tailpiece at the 3' end of Cµ4 or the addition of two membrane exons and the use of a cryptic donor splice site in Cµ4. In catfish, but perhaps all teleosts, unusual spliced products join Cµ3 with the membrane exons and maintain Cµ4 only in the secreted form (3). In mammalian IgM, Cµ3, Cµ4 and the transmembrane exons are regarded essential for assembly of the BCR complex permitting association of Ig
and Igß (4). B cell development is determined by signalling through the BCR as demonstrated in mice with defective BCR signalling components where B cell development is blocked or altered (reviewed in 5). C-terminal cysteines in Cµ4 and the tailpiece allow H chain and J chain anchoring, whilst cysteine residues in Cµ1 and Cµ2 are critical in securing the structure of IgM via interchain disulphide linkage (summarized in 6). Multivalent IgM, the first antibody produced, is generally of low affinity but highly effective in mediating complement-dependent lysis, opsonization, aggregation and phagocytosis. After antigen binding, a single multimeric IgM can activate complement via the C1q binding sites located in Cµ3/4. The broad reactivity of IgM, which can be a problem in autoimmunity, is generally regarded as a template for affinity maturation of class-switched antibodies. Despite the variability in the assembly of µH exons, vertebrates expressing Ig with one or two Cµ domains have not been reported (2, 7) and single Ig domain proteins found in invertebrates do not appear to carry out the function of antibodies (8). To allow optimal function of µH the presence of all domains appears to be necessary and the reason for this may be that domain combinations are essential for extensive developmental processes in signal transduction and immune recognition.
Transgenic mice deficient in IgM, obtained by targeted deletion of Cµ, show normal B cell development and maturation as IgD expression appears to substitute for the lack of IgM (9). Although there appears to be isotype redundancy, the importance of IgM is not disputed. Experiments involving targeted germline mutation of Cµ demonstrated that the membrane form of µH is required for B cell development (10). The mechanism by which surface-bound µH chain influences subsequent differentiation events is not well understood, but it appears that the association with Vpre-B/
5-encoded surrogate L chain and formation of the pre-BCR are crucial to the progress of B cell development. In transgenic mice, a truncated µH chain, Dµ lacking VH, associates with a surrogate L chain and can be expressed in pre-B cells. However, Dµ does not assemble with
L chain and mature B cells fail to express Dµ (11). Although it has been shown that
5 is covalently bound to Cµ1 (12, 13), it was surprising to find that µH chains lacking Cµ1 or Cµ2 can still associate with surrogate L chains (14). Expression of a shortened µH construct, µ
M lacking VH and Cµ1, on the surface of Abelson murine leukemia-transformed Ig- pre-B cells did not associate with
5 and Vpre-B polypeptides, and these cells failed to develop further (15). Similarly, transfected µH chain constructs lacking Cµ1 or Cµ2 were unable to induce L chain rearrangement (14). Nevertheless, progression in development was obtained by cross-linking of µ
M which induced
L chain rearrangement (15). The importance of Cµ1 in development has been further analysed in transgenic mice which show a maturation block of µ
1 lymphocytes at the stage of L chain rearrangement. H chains lacking Cµ1 appear neither to aggregate with surrogate L chain nor to be associated with Ig
/Igß but are displayed on the surface via glucosyl-phosphatidylinositol (GPI) linkage (16). It is discussed that removal of Cµ1 which contains one possible association site for the IgH binding protein BiP (1720), an endoplasmic reticulum retention protein which binds free µH chain, may result in altered transit through the endoplasmic reticulum which allows surface deposition via GPI (16).
Here we have analysed BCR signalling events at important checkpoints in lymphocyte development. We show that truncation of µH results in the accumulation of cells at the pre-B cell stage, whilst cell reduction is obtained when BCR assembly is initiated and monomeric IgM fails to transmit an efficient proliferation signal. Furthermore, immunization does not elicit specific IgM responses, but the newly acquired signalling capacity allows the expansion of IgM+ CD5+ B cells. These results identify an important role of IgM in mediating positive and negative feedback signals to assure controlled B cell development and proliferation.
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Methods
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Targeting constructs and generation of mice
A
phage library, obtained from E14 embryonic stem (ES) cell DNA, was hybridized with a Cµ probe (21) and several positive clones were mapped. The targeting construct was then assembled by subcloning of BamHIBglII fragments into pUC19. The ~6.5-kb fragment from JH3/4 to Cµ1 and the ~5.5-kb fragment from Cµ2 to C
were linked with neor (Stratagene, La Jolla, CA) in either orientation which removed the BamHI and BglII sites in Cµ. The construct, shown in Fig. 1
, was linearized using BamHI and BglII. About 10 µg purified fragment was mixed with ~107 ZX3 ES cells and subjected to electroporation and selection as described (22). ZX3 ES cells were obtained from the 129/sv mouse strain as described (23). DNA was prepared from G418 resistant clones and analysed by Southern blotting with the hybridization probes indicated in Fig. 1
(A). A ~1.4-kb HindIII fragment comprised Cµ34 and the 5' external probe was a ~0.5-kb BamHIBglII fragment. A 335-bp 3' external probe was obtained by PCR with the following oligonucleotides: forward 5'-AACCTGACATGTTCCTCC-3' and reverse 5'-GGGATTAGCTGAGTGTGG-3'. PCR conditions were 95°C 1 min, 58°C 1 min and 72°C 30 s for 35 cycles. Several different ES cell clones with targeting events were injected into BALB/c blastocysts and transplanted into (C57BL/6xCBA)F1 foster mothers. Germline transmission was obtained and two lines for each neor orientation, NR3 (ES cell clones 30 and 36) and NR5 (ES cell clones 89 and 140) were used for analyses. The mice have been bred and investigated under a project licence granted by the Home Office, UK. In the mice, Cµ12 with (~1.5 kb) or without (0.6 kb) neor insertion was identified by PCR with the following oligonucleotides: forward 5'-CCATTTCCTTCACCTGGAACTACC-3' and reverse 5'-CATGGTGGAGGACACGTTCTTCAAG-3'. PCR conditions were 95°C 1 min, 58°C 1 min and 72°C 1 min for 30 cycles.

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Fig. 1. Targeted mutagenesis of the Cµ gene. (A) The germline Cµ region with the targeting construct is indicated by the hatched line. The neomycin resistance gene (Neo) in either orientation (pµNR5 in the same transcriptional orientation as Cµ and pµNR3 in opposite transcriptional orientation) was inserted into Cµ1Cµ2 after BamHIBglII digest which destroyed these sites. Internal (Cµ12, Cµ34) and external (5'ex, 3'ex) probes are indicated by underlining. Restriction sites are: B, BamHI; Bg, BglII; RI, EcoRI; H, HindIII; K, KpnI; Xh, XhoI. (B) Southern blot of BamHI digested DNA from targeted NR3 and NR5 ES cells and heterozygous (NR5+/) and homozygous (NR3/, NR5/) µ truncation mice. Hybridization with the 3' external probe shows a germline band of ~11 kb, the endogenous BamHI site is ~5.5 kb downstream of the 3' BglII site and a ~19 kb targeting band. Normal mouse (NM) DNA served as control. (C) Northern hybridization with the Cµ34 probe of spleen RNA from homozygous NR3 mice derived from ES cell clones 30 and 36, NR5 mice from clones 89 and 140, and µMT (10) and normal mice as control. Normal mice show a hybridization band of ~2.4 kb which is reduced to ~1.7 kb in NR3 and NR5 mice. (D) Partial sequence of the truncated µH chain, obtained by RT-PCR, which shows VDJ rearrangement and splicing to Cµ34.
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RNA and protein analysis
Spleen cell RNA was prepared using Tri reagent as described by the manufacturer (Sigma, St Louis, MO). RNA (~5 µg) was size fractionated on 1% formaldehyde gels and blotted. For RT-PCR of µH a mixture of VH forward primers (24) and the Cµ4 reverse oligonucleotide 5'-CCCTTCACCAAGCACGTGACTGTGGC-3' were used. RT-PCR was performed with the One-Step system (Gibco/BRL, Gaithersburg, MD) under the following conditions: 50°C for 30 min followed by 94°C for 2 min for cDNA synthesis, followed by 30 PCR cycles (15 s 92°C, 30 s 55°C and 30 s 72°C) and 5 min at 72°C to complete the reaction. The sequence of the PCR products, using the Cµ4 oligonucleotide, was performed on the ABI 373 automated sequencer (Applied Biosystems, Foster City, CA) in the Babraham Institute Microchemical Facility.
IgM mol. wt analyses were carried out by fractionation of 100500 µl serum on a Superose 6 prep grade FPLC column (Amersham Pharmacia, Little Chalfont, UK) in 40 mM Tris pH8, 200 mM NaCl. A calibration curve was established with the following mol. wt markers: thyroglobulin (669 kDa), apoferritin (443 kDa), ß-amylase (200 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa) and carbonic anhydrase (29 kDa). Fractions (12.5 ml) excluding the void volume were collected and analysed by ELISA. For Ig analysis Novex pre-cast 38% Trisacetate polyacrylamide gels were run under non-reducing conditions in a Novex Xcell II PAGE electrophoresis system (Invitrogen, Groningen, Netherlands). Size markers were ECL Western blotting mol. wt markers (RPN2107; Amersham Life Sciences). Samples were diluted 1:10 and 10 µl in Trisglycine SDS loading buffer was applied. Running conditions were as recommended by the system manufacturer (Invitrogen) at 150 mV for 2 h. Blotting of proteins onto Immobilin P membranes (IPVH00010; Millipore, Bedford, MA) was carried out at 50 mA (~cm2 gel area x0.8) for 1 h. Staining was carried out with biotinylated anti-mouse µ chain (04-6840; Zymed, San Francisco, CA) and, separately, anti-mouse L chain (02142D; PharMingen, San Diego, CA and RPN1178, Amersham) in PBS/0.1% Tween. The reaction was developed with streptavidinhorseradish peroxidase (RPN1051; Amersham).
Immunizations and ELISA
Hapten-coupled antigens (Biosearch Technologies, Novato, CA) were NP40-CGG (4-hydroxy-3-nitrophenylacetyl-chicken
-globulin), FlTC6-OVA (carboxy-fluorescein ovalbumin) and NP32-FITC3-Ficoll (4-hydroxy-3-nitrophenylacetyl-aminoethylcarboxymethyl Ficoll-fluoresceinyl). Immunizations were carried out with alum-precipitated antigens (100 µg/mouse) and 109 heat-inactivated Bacillus pertussis (a kind gift from Dr C. Rada, Medical Research Council, Cambridge, UK). Groups of five animals were primed i.p. and boosted 30 days later. Preimmune serum and primary serum was collected before the first and second immunization respectively. The mice were sacrificed 10 days after the second immunization with spleen and serum taken for further analysis.
Serum antibodies were assayed by ELISA as described (22) on plates coated with NP23-BSA (Biosearch Technologies) or chicken IgG, OVA, Ficoll 400, anti-mouse IgM (µ chain specific) and anti-mouse Ig (Sigma-Aldrich, Gillingham, UK). The biotinylated detection reagents were anti-mouse IgM (Sigma), anti-mouse
L chain (Rockland, Gilbertsville, PA) and anti-mouse Ig (Amersham). To determine the antibody concentration purified monoclonal mouse IgM,
(M7394; Sigma), IgG1,
(M9269; Sigma), NP-specific mouse IgM,
(B1-8) and NP-specific mouse IgG1,
(B1-48) (25) of known concentration were used as standards.
Flow cytometry analysis
For the analysis of B cell subpopulations by flow cytometry, bone marrow, spleen and peritoneal B cells were isolated. Multicolor staining was carried out with the following reagents in combinations shown in Figs 3, 5, 6 and 7


, and Table 1
: phycoerythrin (PE)-conjugated anti-mouse CD25 (P3317; Sigma), PE- or allophycocyanin (APC)-conjugated anti-mouse CD45R (B220) (01125A, 01129A; PharMingen, San Diego, CA), biotinylated anti-mouse IgM (02082D; PharMingen), PE-conjugated anti-mouse c-kit (CD117) (09995B; PharMingen), FITC-conjugated anti-mouse IgD (02214D; PharMingen), biotinylated anti-mouse CD43 (01602D; PharMingen), biotinylated goat anti-mouse IgG (6640; Zymed), FITC-conjugated anti-mouse CD11b (Mac-1
chain) (01714A; PharMingen), PE-conjugated anti-mouse CD5 (Ly-1) (01035A; PharMingen), FITC-conjugated anti-mouse CD79a (Ig
) (28164A; PharMingen) and FITC-conjugated monoclonal rat anti-mouse IgM (04-6811; Zymed). Binding of biotinylated antibody was developed with streptavidinQuantum Red (S2899; Sigma) or streptavidin PerCP (340130; Becton Dickinson, San Jose, CA).

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Fig. 3. Serum titers and levels of Ig+ spleen cells in normal (NM), NR3 and NR5 mice after immunization. (A) Mice (five per group) were immunized with NP-CGG and boosted 30 days later. Serum was collected prior to immunization (pre), before the second immunization (indicated as 1) and 10 days after the second immunization (indicated as 2). The anti-NP specific response and total Ig and IgM concentrations were determined by ELISA. Purified antibodies were used to calculate Ig concentrations. (B) Representative flow cytometry analysis of splenocytes from mice after two immunizations and from non-immunized mice. Cells were stained with biotinylated anti-IgM or biotinylated anti-IgG, FITC-labelled anti-IgD and PE-labelled anti-B220 antibodies. Lymphocytes and B220+ cells were gated, and populations of IgM+ or IgD+ or IgG+ cells plotted against forward scatter. Very similar reduction levels of IgM+ and IgD+ cells were found for all immunizations using T-dependent or T-independent antigens.
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Fig. 5. Surface IgM is insensitive to PI-PLC treatment and associates as BCR. (A) Spleen cells from normal (NM), NR3 and NR5 mice were stained with biotinylated anti-µ antibodies, and incubated with 0.1 or 1 U PI-PLC/ml. (B) Flow cytometry analysis of splenic lymphocytes stained with biotinylated anti-IgM and FITC-labelled anti-CD79a (Ig ).
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Fig. 6. Flow cytometry analysis of bone marrow B cell populations. The profiles show staining of gated lymphocytes with PE-conjugated anti-B220, biotinylated anti-CD43 or biotinylated anti-IgM and FITC-labelled IgD, and are representative for results obtained from at least five normal (NM), NR3 and NR5 mice per group.
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Fig. 7. Flow cytometry analysis of CD5+ B-1 cells. Representative profiles of (A) peritoneal, (B) bone marrow and (C) spleen cells from normal (NM), NR3 and NR5 mice stained with PE-labelled anti-CD5 and APC-labelled anti-B220 antibodies. Analysis of peritoneal cells from NR3 and NR5 mice was carried out on two gated cell populations (R1, the normal lymphocyte gate, and R2) obtained by conventional scatter parameters, side scatter plotted against forward scatter, as shown.
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B cell proliferation and calcium mobilization assay
Isolation of splenic B lymphocytes has been described (26). Briefly, spleen tissue was disaggregated using a 40 µm nylon cell strainer and cells were taken up in DMEM (Life Technologies, Paisley, UK)/2% FCS. Erythrocytes were depleted by resuspension in 0.8% NH4Cl before layering onto Lympholyte-M (Cedarlane, Ontario, Canada) and centrifugation for 20 min at 1000 g. Cells recovered from the interface were depleted of T lymphocytes by incubation with anti-Thy-1.2 (Sigma) for 20 min on ice prior to the addition of Low-Tox rabbit complement (Cedarlane) and incubation for 30 min at 37°C. The cell suspension was then again layered onto Lympholyte-M, centrifuged for 20 min at 1000 g and the enriched B cell fraction recovered. B cells (4x105) in 200 µl DMEM containing 10% FCS, 2 mM glutamine, 10 mM HEPES, pH 7.2, 50 µM 2-mercaptoethanol and 50 µg/ml gentamycin were seeded in triplicates into 96-well round-bottom plates. Cells were left unstimulated or stimulated with various concentrations of goat anti-IgM (µ chain specific), F(ab')2 (Jackson Laboratories, West Grove, PA) or lipopolysaccharide (LPS; Sigma) for 48 h before the overnight addition of 0.37µCi/well of [methyl-3H]thymidine (Amersham). Plates were harvested by a semi-automated cell harvester (Packard Biosciences, Meriden, CT) and the [3H]thymidine incorporation was determined using a Top Count scintillation counter (Packard).
Measurements of intracellular Ca2+ mobilization were performed as described (27). Cells were incubated with 5 µM Indo-1AM (Calbiochem, CN Biosciences, Nottingham, UK) for 30 min, washed with PBS/1% BSA, stained for 30 min with FITC-labelled anti-CD43 (01604D; PharMingen), washed again and stored in RPMI/10 µM EGTA until analysed. Cells were stimulated with 10 µg/ml goat anti-mouse IgM F(ab')2 and immediately analyzed by flow cytometry for the relative blue:violet colour ratio.
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Results
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Mice with truncated µH chain
The µH chain was disrupted by neor insertion into Cµ1 and Cµ2 which removed part of both exons on a BamHIBglII fragment. The targeting construct and Southern blot analysis of homologous integration are shown in Fig. 1
. The resulting mouse strains, NR3 (with neor in opposite transcriptional orientation to Cµ) and NR5 (with neor in the same orientation as Cµ), were each derived from two different ES cell clones. As can be seen in Fig. 1
(B), NR3 has a slightly smaller BamHI fragment than NR5. Different digests and hybridization with the 5' external probe showed that in contrast to wild-type mice, both NR3 clones have a somewhat reduced length of the µ switch region, whilst the NR5 clones appear to have maintained the expected size. A reason for this may be imprecision in homologous integration due to repetitiveness and instability of this region. Analysis of the targeting event of the different ES cell clones in various digests using a 3' external probe showed the expected fragment size for the region 3' of Cµ2 (data not shown). Effects of the targeted integration can be seen in Northern blot analysis of spleen RNA from homozygous mice (Fig. 1C
). In normal mice µH chain transcripts are ~2.4 kb, whilst NR3 and NR5 mice show a reduction in size to ~1.7 kb. This size reduction of µ mRNA suggested a lack of two exons as found in mutant hybridoma lines (28). Truncation of µH chain was independent of the orientation of the neor insertion, suggesting that neither its promoter nor poly(A) site are sufficient to block alternative splicing of Cµ exons. In RT-PCR using spleen RNA µH truncation was confirmed. Amplification with VH and Cµ4 oligonucleotides resulted in ~600-bp fragments for NR3 and NR5, whilst normal mice produced an ~1200-bp product. Sequence analysis of the PCR products showed diverse VHDJH linkage to Cµ3Cµ4 (Fig. 1D
).
Cµ12 splice deletions produce non-covalently linked HL monomers
Naturally occuring secreted IgM is a pentameric glycoprotein of ~900 kDa with individual H chains of ~75 kDa. To determine configuration and mol. wt of the truncated µH chain we fractionated serum Ig on a Superose gel filtration column and titrated the Ig content of 1.5 ml fractions by ELISA (Fig. 2A
). Binding on anti-µ-coated ELISA plates was developed with biotinylated anti-mouse IgM or anti-mouse
L chain. Column separation according to size allowed normal IgM to peak at fractions 913 (47 ml flow) which agrees with a mol. wt of 900 kDa, whilst truncated IgM appeared considerably later with a maximum at fractions 26 and 27 (70 ml flow). H and L chain association was identified by ELISA, and the graph obtained from the size standards determined a mol. wt of ~75 kDa which suggested that the truncated IgM is in monomeric configuration.
To determine the size of the H chain and its possible covalent association with L chain, sera from NR3, NR5 and normal mice were analysed by PAGE and Western blotting. Staining with biotinylated anti-µH and, separately, anti-
and anti-
L chain-specific antibodies (Fig. 2B
) shows that under non-reducing conditions Ig H and L chain from NR3 and NR5 mice do not co-migrate. Normal Ig scarcely enters the gel under these conditions, but L chain dimers and monomers are apparent in the normal mouse serum control lane. The truncated µH chain is ~52 kDa and non-covalently associated with L chain which runs separately and has the expected mol. wt of 2225 kDa. With the loss of Cµ1 and Cµ2 and subsequent lack of carbohydrate moieties the truncated H chain separates considerably better than normal µ chain which yields a diffuse band on PAGE due to extensive glycosylation.
Lack of antigen-specific monovalent IgM
To determine whether IgM association as HL monomer has an effect on the antibody concentration we analysed Ig levels in serum. ELISA assays showed that the serum IgM concentration in adult µH truncation mice is considerably lower than that found in normal mice kept in isolators under the same conditions. Antibody concentrations were determined by binding comparison with purified standards including IgM Fab to verify detection sensitivity. The results are illustrated in Fig. 3
(A) which shows IgM and total Ig titres from groups of five mice before and after immunization with NP-CGG. Upon immunization the IgM levels in NR3 and NR5 mice are increased, but are still only ~10% of what is seen in normal immunized mice. In contrast, there seems to be no significant difference in the levels of total Ig or IgG found in groups of NR3, NR5 or normal mice, although after immunization the titer can vary in individual mice. Immunization with NP-CGG, FITC-OVA or NP-FITC-Ficoll did not yield an antigen-specific IgM response in µH truncation mice and separate analysis of hapten and carrier responses in ELISA showed the same result: in T cell-dependent and T cell-independent immune responses NR3 and NR5 mice did not produce any detectable antigen-specific IgM antibodies. A likely reason for the lack of antigen-specific IgM, which could not be detected by ELISA, is that antigen binding constants of complete multimeric antibodies can be 100- to 1000-fold higher than those of, for example, Fab polypeptides (29, 30). However, total Ig responses, with ~1 mg/ml anti-NP titres, are in the expected range. The production of monovalent IgM is accompanied by extensive reduction in the number of spleen cells and spleen lymphocytes (Table 1
). Diminished B220+ populations comprise IgM+ and IgD+ cells which after immunization show a further drop in the levels of IgM+ cells which can be as low as half that found in normal immunized mice (Fig. 3B
). In addition, cells from NR3 and NR5 mice stained for sIgM show a reduction in fluorescence intensity which could be the result of differences in sIgM density compared to normal mice. However, such reduction was not seen when staining in parallel for the co-receptor component Ig
which suggests similar sIgM levels but a less efficient recognition of the truncated IgM monomer by monoclonal anti-IgM (see Figs 3B and 5
). Interestingly, there appears to be a remarkable recovery, in that the numbers of IgG+ cells in NR3 and NR5 mice are very similar to those found in normal mice, which shows that the low level of IgM+/IgD+ cells does not prevent or reduce proliferation of plasma cells producing isotypes other than IgM.
Reduction in BCR-mediated proliferative responses
The diminished capacity to activate cells carrying a truncated µH chain by antigen encounter and cross-linking was further analysed by culturing splenic B cells in the presence of bacterial LPS or goat anti-µ F(ab')2 (Fig. 4A
). Proliferative responses of NR3 and NR5 B cells to LPS were reduced by up to 50% compared to normal mice, whilst incubation with anti-µ F(ab')2 resulted in ~5-fold reduction of [3H]thymidine incorporation relative to control animals. In BCR-mediated calcium mobilization assays a similarly striking reduction in the transmission of cell-surface activation signals was found (Fig. 4B
). Staining of NR3 and NR5 spleen cells with Indo-1 and flow cytometry analysis according to Rabinovitch et al. (31) showed poor and delayed Ca2+ mobilization rates in comparison to those obtained from normal mouse cells. The results suggest that binding of F(ab')2 anti-µ to mutated µH chain provides only a weak stimulation signal. To ascertain that the reduced proliferation rates of B cells from the NR3/NR5 mice was not simply due to a loss of antibody binding we stained the cells with FITC-conjugated anti-µ F(ab')2. FACS analysis showed that B220+ cells from NR3/NR5 and normal mice indeed have similar staining profiles (data not shown). Whilst the truncated IgM is certainly secreted as monovalent heterodimer, the reduced activation capacity suggests that it may also be predominantly assembled in the HL configuration on the cell surface.

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Fig. 4. BCR mediated proliferation and stimulation. (A) Proliferative responses of splenic B cells from normal (NM), NR3 and NR5 mice to goat anti-µ F(ab')2 or LPS at indicated concentrations were determined by monitoring the incorporation of [3H]thymidine after 48 h in culture. Representative results from one of four experiments are shown with each bar depicting the mean incorporation of three individual mice with their cells seeded in triplicates. (B) Intracellular Ca2+ mobilization of Indo-1-treated CD43- splenocytes from normal (NM), NR3 and NR5 mice stimulated with 10 µg/ml goat anti-µ F(ab')2 (addition marked by arrow). The x-axis indicates real-time Ca2+ release over a 300 s interval. For the dashed line the right-hand y-axis indicates the percentage of cells showing Ca2+ mobilization. The solid line shows the increase of intracellular Ca2+ concentration indicated on the left-hand y-axis.
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Truncated IgM is associated as BCR
To test whether exon deletion of the µH chain and formation of single HL polypeptides prevents BCR assembly we asked whether IgM anchoring to the cell surface is accomplished by association with the Ig
/Igß co-receptor or, alternatively, by GPI linkage as shown for other Ig (32, 33). IgM association with the
/ß heterodimer appears to be crucial for transport and maintenance of surface IgM, and may also be essential to allow progression in B cell development (33, 34). To identify the association of truncated IgM we incubated spleen cells with phosphatidylinositol-specific phospholipase C (PI-PLC) which can specifically release GPI-linked proteins from the cell surface but has no effect on Ig
/Igß-associated surface Ig (32). Incubation with various amounts of PI-PLC prior to staining with anti-IgM antibodies had no detectable effect on the level of surface Ig suggesting correct BCR assembly (Fig. 5A
). To ascertain that the PI-PLC inefficiency did not simply reflect inactivity or resistance of GPI anchors (33) we stained the cells in parallel with anti-Thy-1. Thy-1 molecules on the cell surface are GPI linked (35) and PI-PLC treatment prior to staining showed the expected reduction in staining intensity (data not shown). To obtain further evidence of correct BCR assembly we analysed cells for the presence of the BCR component Ig
(CD79a). Staining of IgM+ B cells from NR3, NR5 and normal mice with anti-Ig
antibodies showed very similar profiles (Fig. 5B
), and precipitation of the solubilized receptor complex (digitonin lysates) with anti-IgM-coupled beads and analysis on SDSPAGE identified the expected band of ~31 kDa (data not shown). This and the insensitivity to PI-PLC implies that truncated IgM heterodimers can associate with the necessary co-receptor components to allow BCR expression.
Reduction of IgM+ B cells in spleen and bone marrow
To determine the effect of µH chain truncation on B cell development we analysed the proportion of lymphocyte subset populations in bone marrow, spleen and the peritoneal cavity (Table 1
). Comparison of the total cell numbers in normal and mutant animals showed around one-third reduction of cells in spleen and bone marrow from NR3 and NR5 mice, whilst a >3-fold increase in the numbers of peritoneal cells was found. The levels of conventional B220+ IgM+ B cells are significantly reduced in NR3 and NR5 mice, whilst the levels of CD5+ T and B cell populations in spleen, and c-kit+ and CD43+ pro/pre-B cells in bone marrow remained very similar to those found in normal mice. In developing B cells the earliest reduction in cell numbers was found for B220+ CD25+ populations. However, these reduced levels show a similar percentage distribution of CD25+ large and small pre-B-II cells to those found in normal mice. Flow cytometry analysis of bone marrow cells (Fig. 6
) illustrates the developmental changes of the B cell population by comparing the contribution of B220+ CD43+ cells, with increased ratios in NR3/NR5 mice, and the contribution of IgM+ and IgD+ cells, with decreased ratios in the mutant mice. The results show that in the IgM mutant mice, despite an expanded precursor cell pool, only a fraction of mature IgM+ and IgD+ B cells is generated. This indicates impaired but not abolished B cell development from the stage when H chain rearrangement is completed and µH is expressed (pre-B-I to pre-B-II) up to the mature IgM/IgD B cell stage.
Increased levels of CD5+ B cells
Unexpectedly, NR3 and NR5 mice show a dramatic increase in the number of peritoneal cells which appears to affect all subsets analysed (Table 1
, bottom). The B cell lineage predominantly found in the peritoneal cavity has been designated B-1 (36 and references therein). B-1 cells (which are B220+/Mac-1low as opposed to B220+/Mac-1- B-2 cells) are further classified into two subsets: B-1a, which is CD5+, and B-1b, which is CD5- (37,38 and references therein). Figure 7
illustrates the increased levels of CD5+ B-1 cells in peritoneum, bone marrow and spleen. The increase in CD5+ peritoneal cells appears to be independent of the genetic background, and homozygous NR3 and NR5 mice, obtained from breeding with BALB/c, C57Bl/6 and 129 mouse strains, show very similar elevations. For the cell analysis by flow cytometry the parameters to gate the lymphocyte population are based on forward and side scatter (indicated as R1). Apart from increased lymphocyte populations, NR3 and NR5 mice also show a distinct second population of large cells (indicated as R2). The presence and distinct nature of these large single cells was confirmed by sorting and microscopic analysis. Figure 7
(A) illustrates the contribution of CD5+ B-1a and CD5- B-1b cells in these populations. In a representative normal mouse ~34% B-1a and 41% B-1b cells are found, whilst in NR3 and NR5 mice the ratio was considerably elevated for B-1a cells to 75 and 70%, and reduced for B-1b cells to 14 and 21% respectively. In cells from gate R2 the ratio is further increased to 90% and 84% for B-1a cells, and decreased to 4 and 8% for B-1b cells. Staining with anti-IgM showed that all gated cells were >90% IgM+ (data not shown). B-1a cells are normally rare in bone marrow and spleen (39); however, in NR3 and NR5 mice levels in bone marrow are increased from 1.6 to 4.1 and 4.9%, and also the levels in spleen show a considerable increase from 3.5 up to 12% (Fig. 7B and C
). These results identified elevated levels of CD5+ B-1a lymphocytes with a dominant cell population in the peritoneal cavity of NR3/NR5 mice. This dominance may be established in the bone marrow from where the B-1a cells migrate and expand further, whilst the accompanying reduced B-1b subset may be arrested in B cell development.
 |
Discussion
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Truncated µH chain is produced by alternative splicing and gene deletion
Splicing is a complicated process which involves a diverse array of proteins including the spliceosome complex which brings together gene exons and removes the intervening sequence (reviewed in 40). In vertebrates the µH chain is highly conserved and is a template for the production of diverse RNA transcripts (2, 41). The various full-length splice products can be expressed as membrane or secreted polypeptides and exon deletion in µH does not prevent the correct splicing of subsequent exons (28). In the NR3/NR5 mice, correctly spliced products are obtained regardless of the remaining donor and acceptor splice sites of partial exons and neither PCR nor Northern blot analysis of NR3 and NR5 RNA showed any unusual transcripts. It has been proposed that a surveillance system exists that destroys RNAs if they contain an open reading frame interrupted by a translational termination signal (42). For this reason it was unexpected that homologous integration of neor in Cµ has essentially no detrimental effect on transcription levels of rearranged µH chains. For µH and also other IgH genes the use of separate termination signals and translation from different splice products concurs with defined stages in B cell development. This suggests that accurate processing of the µH chain exons is determined by intrinsic genetic factors which are essential for correct progression in development. However, what prevents association of multimeric IgM in the NR3 and NR5 mice? Conventional IgM can be split easily by mild reduction into µ2L2 subunits, a configuration which is maintained by strong intersulphide bonds in Cµ1 and Cµ2 (6). The inability to form covalently linked µ2L2 subunits may prevent the formation of multimeric IgM because of a lack of support to maintain such a large structure.
IgM expression controls B cell development
Germline modifications of Cµ showed that disruption of a membrane exon arrests B cell development at the pre-B cell stage (10), whilst removal of the secretory terminus delays antigen responses and affinity maturation (43). In the NR3 and NR5 mice, secreted IgM and possibly surface IgM is produced in truncated monovalent form which allows us to evaluate the importance of the IgM multimer in development and recognition. In the mutant mice the number of bone marrow and spleen cells is reduced to ~40% µ+ cells. However, whilst IgM concentration in serum is considerably lower than that of normal wild-type littermates, the levels of truncated Cµ RNA/cell are similar to full-length RNA levels in normal mice (see Fig. 1
). The reduced cell numbers and low IgM concentration in serum appear to be triggered by the aberrant µ polypeptide produced at the pre-B-I to pre-B-II stage of development, when H chain rearrangement should be completed successfully. Truncated monovalent IgM cannot provide feedback stimulation necessary for cellular interaction and antigen recognition, and diminished ability of IgM to mediate cross-linking signals has been identified in processes that block development (15). A reason for the importance of IgM must be its valency which allows binding and oligomerization of antigen. The interaction of a single pentameric IgM with antigen can lead to activation of the complement cascade. Recruitment of complement allows effective B cell activation through co-signalling with the antigen receptor in conjunction with the CD19/CD21 complex (44, 45). This lack of stimulation in the mutant mice may prevent rapid expansion of the IgM+ cell pool. The loss in valency prohibits the production of antigen-specific IgM but not IgG and does not delay or reduce the total Ig response. At present it is not clear if antigen recognition via monovalent IgM, with the predicted loss in its binding capacity (29, 30) is accomplished. A possible reason that switching and maturation of the response appears little affected could be that IgD largely substitutes for the loss of IgM function as an antigen uptake receptor to drive T cell-mediated class switching (9).
Negative and positive signalling recruits different B cell subsets
The BCR consists of membrane-bound IgM associated with the Ig
/Igß heterodimer, a configuration essential for antigen recognition and signalling. Recently the stoichiometric association of one membrane IgM (H2L2) with one Ig
/Igß polypeptide has been identified (46 and References therein). Schamel and Reth speculate that the associated membrane molecule is not completely symmetrical and, as sIgM provides two H chains for a possible association with Ig
/Igß, this could bias the choice of the co-receptor attachment side. In non-lymphoid cells Ig
/Igß is necessary and sufficient to allow surface expression and Ig association (47), perhaps similar in configuration to that of monovalent IgM in the µH truncation mice. B cell-specific activation signals are initiated by BCR cross-linkage which can be accomplished by multimeric antigens but not by monomers or Fab fragments. Conversely, monovalent IgM expression in NR3/NR5 mice does not allow efficient antigen recognition and this lack of stimulation prohibits proliferative expansion. Reth and co-workers propose that the BCR has alternative forms of an ordered oligomeric structure which may be altered after antigen contact such that associated signalling elements become active (48). This does not affect the pro-B cell pool in bone marrow and in NR3/NR5 mice levels remain similar to those found in normal mice despite an extensive reduction in the total cell number. The BCR also generates signals independent of encounter with antigen which are important for survival and possibly expansion of mature B cell subsets. It has been shown in mice with defects in their antigen receptor that signals from the BCR complex control differentiation events that determine quantity and quality of the various B cell subpopulations (34, 49). Efficient antigen-mediated receptor recognition appears to be important to determine death or survival, proliferation and differentiation (50), whilst anti-self antibodies and the lack of secreted IgM results in accumulation of the CD5+ B cell subset B-1a (43, 51, 52). Normally B-1 cells are absent from the bone marrow and there are only low levels in spleen, although in the peritoneal and pleural cavities they are the main self-replenishing population (36 and references therein). B-1 cells do participate in antigen-stimulated responses, but there appear to be recognition differences which may be advantageous simply because of the anatomical location of these cells. B-1 cell antibodies tend to have lower affinities and broader specificities than conventional B-2 cell antibodies. These differences in antigen recognition may initiate CD5 expression, perhaps important for BCR signalling to avoid apoptosis (53), and may allow the selection and accumulation of B-1a cells. The notion that CD5 may have an important role in regulating BCR signals via recruitment of the tyrosine phosphatase SHP-1 to the CD5BCR complex is supported by the finding that motheaten mice, which carry a mutation in the SHP-1 gene, have increased numbers of B-1 cells (54). The dramatic increase in peritoneal B-1a cells in the mutant mice, illustrated in Fig. 7
, may be the result of a lack of feedback regulation indicating that the BCR complex transmits a powerful signal in the absence of antigen. BCR specificity, determined by surface density and V gene choice, which we will analyse in NR3/5 mice in future experiments, appears to be important in the development of B-1 cells (55). The increased levels of B-1a cells in different tissues of the mutant mice suggest that they are generated in the bone marrow, from where they migrate to the spleen and peritoneum where they expand. Migration and expansion of precursor B-1a cells, which has not been identified in normal adult mice, could be tested in the µH truncation mice which, upon splenectomy, may not be able to maintain their CD5+ B cell pool.
 |
Acknowledgments
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This work was supported by the Biotechnology and Biological Sciences Research Council, and the Babraham Institute. We thank Drs G. Warr, M. Neuberger, L. Mårtensson and M. Turner for advice and helpful discussions. We are grateful to Drs A. Smith and T. Rabbitts for providing the ES cell library, J. Stevens for gel filtration analysis, L. Howes for help with Western blotting, G. Doody and J. Mead for help with the proliferation assay, S. Bell for help with receptor analysis, and N. Miller for helping with the flow cytometry.
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Abbreviations
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APC allophycocyanin |
CGG chicken -globulin |
ES embryonic stem |
GPI glucosyl-phosphatidylinositol |
LPS lipopolysaccharide |
neor neomycin resistance gene |
NP 4-hydroxy-3-nitrophenylacetyl |
OVA ovalbumin |
PE phycoerythrin |
PI-PLC phosphatidylinositol-specific phospholipase C |
 |
Notes
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Transmitting editor: E. Simpson
Received 27 April 2001,
accepted 28 August 2001.
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