Nature Immunology
3, 951 - 957 (2002)
Published online: 16 September 2002; | doi:10.1038/ni839
A GPI-linked isoform of the IgD receptor regulates resting B cell activationAkanksha Chaturvedi, Zaved Siddiqui, Fahri Bayiroglu
& Kanury V.S. RaoImmunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Correspondence should be addressed to Kanury V.S. Rao kanury@icgeb.res.inThe induction of a humoral response depends upon efficient cross-linking by antigen of surface immunoglobulin on primary B lymphocytes. We demonstrate here the presence of a glycosylphosphatidylinositol-linked isoform of membrane IgD (mIgD) receptors on murine resting B cells. This subset was constitutively localized to cell membrane raft microdomains. Its stimulation resulted in the activation of cAMP-dependent signaling pathways, which integrated with signals derived from the transmembrane mIgD receptors. This, in turn, provided a mechanism by which the activation status of the target cells could be variably regulated. Thus, by partitioning receptor activity, preimmune B cells can moderate the extent to which they are activated, depending upon the strength of the antigenic stimulus.Both the development and physiological function of B lymphocytes is tightly regulated by signals generated from the B cell antigen receptor complex (BCR)1. Whereas their maintenance in the periphery depends on low-intensity constitutive signaling—by an as yet unknown mechanism—through the BCR2, signaling induced upon cross-linking of the BCR by antigen stimulates a cellular response that can either be apoptotic or proliferative, depending on the maturation status of the cell1.
The BCR is a complex hetero-oligomeric structure in which the ligand binding and signal transduction properties are compartmentalized into distinct receptor subunits3,
4. The membrane immunoglobulin (mIg) unit binds ligand, whereas the associated disulfide-bonded heterodimer of CD79a (Ig ) and CD79b (Ig ) transduces signals. Cross-linking of mIg molecules induces phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) sequences within CD79a and CD79b by the Src family of protein tyrosine kinases (PTKs)3,
4. This results in the recruitment of at least four major intracellular signaling pathways, including those that are dependent upon phospholipase C- (PLC-), the Rho family of GTPases, the Ras GTPase and the phospholipid kinase phosphatidylinositol-3 kinase (PI3K)3,
4. Each of these pathways plays a critical role in defining B cell responsiveness to both activating and differentiating stimuli.
Although information on BCR-dependent signaling pathways and their regulation continues to accumulate1,
2,
3,
4,
5, the mechanisms by which these biochemical processes interact to modulate B cell fate and function are not clear. Indeed, how plasticity in BCR signaling is achieved with a limited number of downstream pathways remains a key confounding issue in B cell signaling.
We demonstrate here the presence of a glycosylphosphatidylinositol (GPI)-linked isoform of IgD (mIgD) receptors on murine resting B cells. Activation of GPI-linked mIgD induced intracellular cAMP accumulation, which triggered pathways that engaged in cross-talk with the known BCR-dependent signaling pathways. The extent of this cross-talk defined the spectrum of cell surface activation markers that were induced. Our results suggest that it is this segregation of receptor activities that facilitates the graded responsiveness of preimmune B cells, depending on the strength of the antigenic stimulus.
Results
Activation of resting B lymphocytes
Immunization with a T cell−dependent antigen induces B cells to follow two compartmentalized and distinct pathways of differentiation. After activation of B cells in the T cell−rich extrafollicular sites, foci of antigen-specific antibody-forming cells appear in the periphery of the periarteriolar lymphoid sheath. In a second pathway, some of these cells within the next few days move to the germinal center (GC) in the primary B cell follicles, where the processes of somatic mutation and differentiation of antigen-activated B cells are initiated6,
7.
To examine phenotypic alterations that characterize the above two pathways in resting B lymphocytes, we used the F(ab')2 fragment of a monoclonal anti-IgD as a surrogate antigen (referred to hereafter as anti-IgD). Purified resting B cells were cocultured with anti-IgD and monitored for cell-surface modulation of specific proteins associated with activation and GC B cells. We found that mIgD expression was down-regulated, whereas surface amounts of CD86 and major histocompatibility complex (MHC) class II molecules were enhanced8,
9,
10. Several additional markers—CD80, peanut agglutinin receptor (PNA-R), CD24 and GL7—that characterize murine GC B cells were also up-regulated (Web Fig. 1a online), which confirmed published data11,
12,
13,
14,
15,
16,
17,
18,
19. Thus, stimulation of murine resting B cells with anti-IgD induced phenotypic changes that characterized both the activated and the GC B cell.
In addition to phenotypic changes, optimal stimulation of resting B cells with anti-IgD also conferred upon these cells the ability to populate GCs in a T cell−dependent manner. Thus, stimulated BALB/c IgHa B cells, loaded with a synthetic T cell epitope peptide (peptide CT3)20 reconstituted GCs when transferred into CT3-primed BALB/c IgHb mice (Web Fig. 1b online). In contrast, peptide-loaded unstimulated B cells or stimulated B cells loaded with an irrelevant peptide were unable to reconstitute GCs. Similarly, no GCs were formed when stimulated CT3-loaded B cells were transferred into either naïve recipients or recipients primed with an irrelevant peptide (data not shown). Thus, stimulation with anti-IgD primed resting B lymphocytes for seeding GCs, although development of the latter occurs only in the presence of T cell help.
cAMP-dependent pathways regulate the GC phenotype
To determine which intracellular pathways are involved in the induction of GCs, we investigated the effect of a variety of pharmacological inhibitors on anti-IgD−dependent B cell activation. We found that stimulation in the presence of either EGTA (an inhibitor of Ca2+ influx) or calphostin C (a protein kinase C (PKC) inhibitor) completely suppressed up-regulation of all the surface molecules observed (data not shown). These findings were not surprising, given that Ca2+ and PKC represent downstream mediators in the PLC- pathway, which is required for BCR signaling1,
3,
4.
We also noticed that the pharmacological agent H89 (an inhibitor of the Ser-Thr kinase PKA21) also produced inhibitory effects on anti-IgD−dependent modulation of the B cell surface phenotype. The effects, however, were more selective than those of either EGTA or calphostin C because the extent of inhibition varied from partial to complete depending upon the marker examined (Fig. 1a). Stimulation of B cells with anti-IgD in the presence of H89 also attenuated the GC response (Fig. 1b), which suggested this inhibitor had a functional influence. Essentially similar results were also obtained with H9, an alternative inhibitor of PKA (data not shown). These results suggested, therefore, the involvement of PKA in defining the GC seeding competency of stimulated B cells.
 | | |
The implication that mIgD-dependent intracellular pathways are PKA-dependent was unexpected, because activation of PKA normally requires up-regulation of intracellular cAMP (cAMPi). An increase in cAMPi upon BCR-stimulation has not been reported. Therefore, we examined whether activation of resting murine B cells with anti-IgD affected cAMPi concentrations. Stimulation with anti-IgD caused a near tenfold increase in cAMPi concentrations (Fig. 1c), which was comparable to that obtained when MHC class II was cross-linked with anti−I-A. No effect on cAMPi concentrations was observed when B cells were treated with the F(ab')2 fragment of a nonspecific rat IgG (Fig. 1c). These data suggested that cross-linking of mIgD on murine resting B cells enhanced cAMPi concentrations. However, this response was completely insensitive to the PKC inhibitor calphostin C or inhibitors of intracellular Ca2+ flux (EGTA and TMB-8) (Fig. 1c), which suggested that mIgD-dependent enhancement of cAMPi is independent of downstream signaling molecules in the PLC- pathway.
Membrane distribution of mIgD
Receptor-mediated signaling in lymphocytes can occur in the context of discrete and specialized domains called glycosphingolipid-enriched membrane domains (GEMs) or lipid rafts22,
23,
24,
25. Lipid rafts may serve as platforms for signaling and membrane trafficking for a range of immune receptors, including the BCR, T cell antigen receptor (TCR), Fc RI and MHC class II23,
24,
25,
26,
27,
28,
29,
30,
31.
An examination of mIgD membrane distribution in our unstimulated B cell preparation showed that, as expected25,
32,
33, the majority of mIgD receptors were present outside lipid rafts in the soluble fraction of the membrane. Only a small component could be detected in the detergent-insoluble fraction or in lipid rafts (Web Fig. 2 online). Cross-linking with anti-IgD induced translocation of a large proportion of these receptors into the raft fraction (Web Fig. 2 online). Consistent with published observations26, the addition of either methyl--cyclodextrin (MCD) (Web Fig. 2 online) or nystatin (data not shown) to the stimulated B cells resulted in disruption of the raft domains. These effects, however, were reversible, as removal of these agents from the culture resulted in a reconstitution of the raft domains when monitored in membrane fractions prepared 1 h later. Also, stimulation of these B cells with anti-IgD again led to translocation of mIgD into the raft domains (data not shown).
Raft dependency of mIgD signaling
Stimulation of B cells with anti-IgD in the presence of optimal concentrations of MCD or nystatin had no effect on the Cai2+ response (Fig. 2a); this suggested that this response was independent of the raft domains and consistent with the earlier observations34. In contrast, one study reported that the lipid raft integrity domains were critical for the induction of mIgG-dependent Cai2+ responses in the B lymphoma cell line A2035. Differences in the nature of the B cells used or the isotype of the BCR examined may have accounted for this discrepancy.
| | Figure 2. Distinct mIgD isoforms with independent biochemical activities. | | |  | (a,b) Purified cells were stimulated with anti-IgD (10  g/ml) in the absence (profile 1 in a; cells in b) or presence of nystatin (profile 2 in a; Nys in b) or MCD (profile 3 in a; MCD in b). The effects on both Cai2+ (a) and cAMPi (b) were monitored. Profile 4 shows unstimulated cells. (c) Cells were treated with 1 U/108 cells of PI-PLC (PLC) at 37° C for 1 h, then surface proteins were biotinylated with sulfo-NHS-biotin. For comparison, untreated cells were also surface-biotinylated. Cells from both groups were then solubilized in Triton X-100, and detergent-soluble (DS) and detergent-insoluble (DIS) fractions were obtained. The presence of mIgD in these fractions was then assessed by incubating with fluorescein isothiocyanate (FITC)−labeled anti-IgD, followed by magnetic separation with streptavidin-coated beads. Mean  s.d. data from five experiments are shown. mIgD was undetectable in experiments in which FITC-labeled anti-IgD was substituted with labeled rat IgG or immunoprecipitations were done with anti-IgM. RFU, relative fluorescence units. (d) To determine the effect of PI-PLC treatment on the cAMP response, untreated (aIgD) and PI-PLC−treated (PLC + aIgD) cells were stimulated with anti-IgD (10 g/ml). As controls, untreated (aI-A) or PI-PLC−treated (PLC + aI-A) cells were stimulated with an anti−I-Ad (mAb AMS-32.1, 10 g/ml). Increases in cAMPi are shown; mean s.d. data from values from four determinations, in which each experiment was done in quadruplicate, are shown. (e) B cells were pulsed with radiolabeled analogs of glucosamine (GA), mannose (MN) or palmitic acid + myristic acid (FA). Anti-IgD immunoprecipitates from the detergent-soluble and -insoluble fractions were then de-glycosylated and analyzed for incorporated radioactivity. FA + PI-PLC denotes fatty acid−labeled cells were treated with PI-PLC before analysis. Data are mean s.e.m from four separate experiments. For aliquots of each fraction (before immunoprecipitation), immunoblots obtained with anti-IgD, are shown.
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In contrast to the effects on Cai2+ mobilization, mIgD-dependent recruitment of cAMPi was, however, highly dependent on the integrity of raft microdomains. Enhanced cAMPi concentrations induced by anti-IgD were abrogated when B cell stimulation was done in the presence of either nystatin or MCD (Fig. 2b). Thus the Cai2+ and cAMPi signaling pathways induced by mIgD cross-linking stemmed from different compartments of the resting B cell membrane.
In addition to mIgD association with CD79a-CD79b, it has been suggested36 that linkage to GPI may provide an additional mode for mIgD receptor expression. This was demonstrated by transfection of mIgD into CD79a-deficient cells36. However, whether this occurs in normal resting B cells is not known. We investigated whether the spatially distinct anti-IgD−dependent signaling activities were derived from distinct isoforms of mIgD. To investigate this, we used phosphatidylinositol-specific PLC (PI-PLC), an enzyme that specifically releases GPI-linked proteins from the cell surface, including GPI-linked sIgD36.
We found that treatment with PI-PLC had little effect on the amount of mIgD present in the detergent-soluble fraction of unstimulated cells. However, the minor population of mIgD present in the detergent-insoluble fraction was reduced by nearly 60% (Fig. 2c). In parallel experiments, treatment with PI-PLC did not alter surface I-A and B220 expression in the detergent-soluble or -insoluble fractions from resting B cell membranes (data not shown). Thus, these data suggested that a large proportion of the raft-associated mIgD receptors in unstimulated resting B cells exist in a form that is sensitive to PI-PLC treatment. Anti-IgD− but not anti−I-A−dependent cAMP responses were also completely abolished in PI-PLC−treated cells (Fig. 2d). Cai2+ mobilization by anti-IgD was, however, unaffected and similar profiles were obtained in both PI-PLC−treated and untreated cells (data not shown). Thus, the cAMPi response obtained upon anti-IgD stimulation of resting B cells was dependent on a PI-PLC−sensitive isoform of raft-associated sIgD.
To further establish the presence of a GPI-linked mIgD isoform, we independently pulsed purified resting B cells with radioactive analogs of glucosamine and mannose or a mixture of the fatty acids palmitic and myristic acid. These three chemical entities are present collectively only in GPI-anchored proteins23. Next we resolved the detergent-soluble and -insoluble membrane fractions and determined the amount of mIgD-associated radioactivity in each fraction. mIgD isolated from the raft fraction copurified with glucosamine, mannose and the lipid components (Fig. 2e). The association of fatty acids with this fraction also proved to be PI-PLC−sensitive (Fig. 2e). In contrast, no radiolabeling could be detected in immunoprecipitates from the detergent-soluble fractions of any of the groups pulsed with glucosamine, mannose or the fatty acid mixture. This was despite the higher amounts of mIgD that were present in these fractions (Fig. 2e). Thus, at least some of the mIgD that was constitutively localized in the rafts existed in a GPI-linked form.
mIgD-mediated cAMPi requires GPI biosynthesis
We examined next the effects of mannosamine, an inhibitor of intracellular GPI-biosynthesis37, on the expression of raft-associated mIgD. We pulsed B cells with the radiolabeled forms of glucosamine, mannose or a mixture of palmitic and myristic acid, in the absence or presence of varying concentrations of mannosamine. After labeling, raft-localized mIgD was isolated and the extent of associated radioactivity was determined. Inclusion of mannosamine resulted in dose-dependent inhibition of the incorporation of glucosamine, mannose and the fatty acid mixture (Fig. 3a); this further supported the case for a GPI-linked mIgD isoform. Mannosamine treatment of B cells also resulted in a near complete, but specific, abrogation of the anti-IgD−dependent cAMPi response (Fig. 3b). However, in contrast to the effects on cAMPi, mannosamine treatment had no effect on the anti-IgD−dependent Cai2+ mobilization (Fig. 3c). Thus, the cAMPi, but not the Cai2+, response to anti-IgD stimulation of resting B cells was dependent upon the integrity of the intracellular GPI-biosynthetic pathway.
| | Figure 3. Generation of raft-localized sIgD receptors and the anti-IgD−dependent cAMPi response. | | |  | (a) Tunicamycin-equilibrated cells were pre-equilibrated for 3 h, cultured in the absence or presence of mannosamine, then pulsed with radiochemical analogs as in Fig. 2e. Cells were then solubilized, the detergent-insoluble fraction obtained and the radioactivity incorporated into the deglycosylated IgD heavy chain determined. Mean s.d. data from three separate experiments are shown. Immunoblotting analyses of the corresponding detergent-soluble and -insoluble fractions from mannosamine-treated and untreated cells showed mannosamine had no effect on mIgD amounts in the detergent-soluble fraction. However, mIgD amounts in the detergent-insoluble fraction were reduced by 40−60%, as determined by densitometry (data not shown). (b) Mannosamine-treated (10 mM; groups 1, 3, 5 and 6) and untreated (groups 2 and 4) B cells were stimulated with anti-IgD (groups 2 and 3) or, as a control, with anti−I-A (groups 4 and 5). An additional group of mannosamine-treated cells was stimulated with 10 M forskolin (group 6). cAMPi induction was then assessed; mean s.d. data from three independent experiments are shown. (c) The Cai2+ response obtained upon stimulation of mannosamine-treated (10 mM, profile 1) and untreated (profile 2) cells with anti-IgD was measured. Profile 3 shows basal levels in mannosamine-treated unstimulated cells. Data are from one representative of four separate experiments.
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The GPI-anchored mIgD receptor mobilizes cAMPi
Although the cAMP signaling pathway was dependent upon GPI-linked mIgD, it was not clear whether this isoform acted independently of the transmembrane mIgD receptors. Thus, we examined whether signaling from transmembrane mIgD can influence the anti-IgD−dependent cAMP response. Genistein, a protein tyrosine kinase inhibitor38, completely abrogated anti-IgD−dependent tyrosine phosphorylation (data not shown) and the Cai2+ response in our B cell preparation (Fig. 4a). However, no concomitant effect could be observed on the anti-IgD−dependent accumulation of cAMPi (Fig. 4b); this suggested that the cAMPi response was independent of signals initiated by the transmembrane mIgD receptor.
| | Figure 4. cAMP-dependent signaling by GPI-linked mIgD is independent of transmembrane mIgD. | | |  | (a,b) Cells were pre-equilibrated at 37° C for 30 min in the presence or absence of 60 g/ml of genistein, then stimulated with 10 g/ml of anti-IgD. (a) Cai2+ responses and (b) the corresponding cAMPi responses in genistein-treated (profile 2) and untreated (profile 1) cells are shown. The various groups are as follows: 1, untreated cells; 2, genistein-treated cells; 3, cells treated with genistein and PI-PLC; 4, genistein-treated cells stimulated with forskolin as a positive control. Mean s.e.m data from (a) three and (b) four separate experiments are shown. (c,d) J558L cells were transiently transfected with a retroviral vector encoding the mIgD heavy chain48. Cells were analyzed for mIgD expression 48 h later by flow cytometry (c). Mock-transfected cells (thin line); transfected cells (thick line); PI-PLC−treated transfected cells (dashed line). Mannosamine treatment of transfected cells gave a profile similar to that obtained with PI-PLC. The transfected cells were also stimulated with anti-IgD and the cAMPi response measured (d). The groups were as follows: 1, untransfected cells; 2, cells mock-transfected with vector only; 3, transfected cells; 4, transfected cells treated with PI-PLC; 5, transfected cells treated with mannosamine. Mean s.e.m. data from four separate experiments are shown.
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To establish the origin of the cAMP response, we transfected J558L cells—which are CD79a-deficient36—with a construct that encoded sIgD. We found that the transfected sIgD was sensitive to treatment with either PI-PLC or mannosamine (Fig. 4c); this showed that the J558L cell line exclusively expressed GPI-anchored sIgD, confirming published data36. Stimulation of transfected J558L cells with anti-IgD, however, induced a vigorous cAMPi response that was sensitive to PI-PLC and mannosamine treatment (Fig. 4d). Therefore, these data demonstrated that GPI-anchored mIgD has the ability to mobilize cAMPi independent of transmembrane mIgD.
Stimulation thresholds and B cell responsiveness
The differential distribution of GPI-linked and transmembrane mIgD suggested that variations in the extent of antigen-BCR interactions may also influence the activation status of the target preimmune B cell. We stimulated, therefore, both PI-PLC−treated and untreated resting B cells with varying concentrations of anti-IgD and monitored the effects on capacitative Ca2+ influx and cAMPi mobilization. Ca2+ influx was highly responsive to anti-IgD stimulation, with peak amounts induced with low anti-IgD concentrations (1 g/ml) (Fig. 5a). Virtually identical dose-dependent profiles were obtained in both PI-PLC−treated and untreated cells, which suggested that this response was completely insensitive to the presence or absence of the GPI-linked subset of IgD receptors (Fig. 5a). In contrast, induction of a cAMP response in PI-PLC−untreated cells required higher concentrations of anti-IgD, with maximal amounts achieved at 5 g/ml (Fig. 5a). This latter response was completely inhibited in PI-PLC−treated cells, even at high concentrations of anti-IgD (Fig. 5a), which further supported the dependence of the cAMP response on the GPI-linked receptor.
| | |
Examination of CD86, I-A, PNA-R and GL7 expression at varying anti-IgD concentrations highlighted the physiological consequences of differential signaling responses to different anti-IgD concentrations. These markers were selected as representatives of activated (CD86, I-A) and GC (PNA-R, GL7) phenotypes. Stimulation of cells with low concentrations (1 g/ml) of anti-IgD induced maximal I-A and CD86 up-regulation, whereas induction of the GC markers occurred only at high anti-IgD concentrations (5 g/ml) (Fig. 5b). Thus, the activation markers were induced at anti-IgD concentrations that selectively generated a Ca2+ response, whereas induction of the GC phenotype required higher anti-IgD concentrations that activate cAMP-dependent pathways.
To determine the mechanism for differential signaling at different concentrations of anti-IgD, we examined next the effects of anti-IgD stimulation on the activation of NF- B and the cAMP-response element binding protein (CREB). Activation of NF-B was monitored in terms of its translocation from the cytoplasmic to the nucleic fraction of cell lysates and activation of CREB by its phosphorylation on Ser133 (refs. 39,40). Untreated cells or cells treated with PI-PLC were stimulated with anti-IgD (5 g/ml). Stimulation of untreated, but not PI-PLC−treated, cells induced nuclear translocation of RelA (also known as p65), a subunit of NF-B (Fig. 5c). In contrast, treatment of B cells with PI-PLC had no effect on the anti-IgM−dependent activation of RelA (Fig. 5c), thus demonstrating the specificity of anti-IgD−induced RelA translocation in untreated cells. Similarly, PI-PLC treatment inhibited anti-IgD−dependent phosphorylation of CREB at Ser133 by >70%, but had no effect on CREB phosphorylation induced by anti-IgM stimulation (Fig. 5d). Thus cooperative contributions from the cAMP signaling pathway were also evident during the activation of at least some of the BCR-responsive transcriptional activators. Although anti-IgM does induce a cAMPi response in B cells, the resulting CREB phosphorylation is mediated by PKC and at least some of the calmodulin-dependent protein kinases41.
We found also that the relative extents of CREB and NF-B activation and Ser133 phosphorylation were dependent upon the dose of anti-IgD used. Thus, stimulation of cells with 1.0 g/ml of anti-IgD yielded a ratios of 0.15 0.06 (n = 4) for nuclear:cytoplasmic RelA and 0.11 0.04 (n = 4) for phosphorylated:unphosphorylated CREB. In contrast, stimulation of cells with 5.0 g/ml of anti-IgD yielded corresponding ratios of 0.48 0.12 and 0.57 0.14, respectively (ratios for unstimulated cells were 0.08 0.03 and 0.03 0.01).
To confirm that anti-IgD dose-dependent activation of NF-B and CREB involves cAMP-dependent pathways, we performed additional experiments in both PI-PLC−treated and untreated cells. The anti-IgD−dependent activation of both RelA and CREB in PI-PLC−untreated cells was inhibited in the presence of the PKA inhibitor H89 (Fig. 5e). The extent of this inhibition was comparable to that obtained with anti-IgD−stimulated and PI-PLC−treated cells in the absence of this inhibitor (Fig. 5e). These data suggested that the GPI-linked mIgD isoform plays a role in RelA and CREB activation in anti-IgD−stimulated resting B cells.
PI-PLC−treated B cells were also stimulated with anti-IgD in the presence of dibutyryl cAMP (db-cAMP), which increases cAMPi concentrations41. The PI-PLC−induced inhibition could be overcome readily by stimulation of B cells with anti-IgD in the presence of 100 M db-cAMP. In addition, the effect of db-cAMP was neutralized by simultaneous addition of the PKA inhibitor H89 (Fig. 5e). Again, these results highlighted a distinct role for the GPI-linked mIgD receptors and further supported the idea that variations in mIgD occupancy—which lead to variable cAMPi mobilization—can markedly influence activation of at least some of the transcriptional activators involved.
Optimal GC responses require the GPI-linked mIgD
To determine directly the biological role of the GPI-linked mIgD receptor, PI-PLC−treated cells were stimulated in the presence or absence of 100 M db-cAMP with 5.0 g/ml of anti-IgD. B cells were either analyzed for the resulting phenotypic modifications or evaluated for GC seeding potential. With the exception of CD86, PI-PLC treatment led to varied but large inhibition of up-regulation of all the markers studied. Inhibition, however, could be overcome by the inclusion of db-cAMP during stimulation with anti-IgD (Fig. 6a).
Consistent with the effects observed on marker up-regulation, treatment of resting B cells with PI-PLC also attenuated the GC response (Fig. 6b). Optimal GC responses, however, could be restored upon stimulation of these cells in the presence of 100 M db-cAMP (Fig. 6b). These results validated the functional significance of the GPI-linked mIgD receptors and the cAMP-signaling pathway that they recruit.
Discussion
Our identification of a GPI-linked mIgD isoform in primary resting B cells has provided insights into how the development of a primary humoral response may be controlled. They provide a rationale for the findings that affinity for antigen, as well as the degree of receptor occupancy, play regulatory roles in defining the quality of intracellular signals generated and the potential of activated clonotypes to seed GCs20,
42,
43,
44.
We have shown here that the GPI-linked receptor isoform constitutes a minor fraction of the total sIgD pool and is constitutively localized within raft microdomains of the plasma membrane. A key distinction between this subset and the transmembrane pool of mIgD was its ability to mobilize cAMP-, but not Ca2+-dependent, signaling pathways. In other words, these two receptor isoforms also represent a segregation of mIgD signaling function, which provides a platform for regulating activation thresholds via cross-talk between signals generated from the two mIgD isoforms. Thus cAMP-dependent pathways, induced by GPI-linked mIgD stimulation, act in concert with signals generated from the transmembrane sIgD to further optimize GC responses by activated primary B cells. This is probably mediated by increasing the activation of at least some of the BCR-responsive transcriptional activators and subsequent up-regulation of the GC markers. We cannot rigorously exclude the possibility that expression of the GPI-anchored mIgD is restricted to only a subset of the preimmune B cell pool. Nevertheless, the fact that this receptor isoform plays a key role in optimizing GC responses indicates its physiological relevance. The mechanism through which the GPI-linked sIgD mobilizes cAMPi, however, remains to be determined.
Although B cell stimulation with anti-IgM does not induce a cAMPi response, signaling through mIgM is sensitive to cAMP-dependent signaling pathways41. Thus, simultaneous ligation of mIgM and CD54 on B cells leads to an increase both in CREB phosphorylation and the phenotypic response41. This augmentation is mediated by cooperative interactions between mIgM-dependent phosphoinositol signaling and CD54-activated cAMP-dependent pathways41. It is, therefore, possible that by mobilizing cAMPi the GPI-linked sIgD may also enhance mIgM-dependent signaling in resting B cells.
Although mIgM and mIgD are thought to be functionally equivalent in resting B cells, it is generally accepted that it is the mIgD class of receptors that serves as the principal sensor for antigen. This is because of the increased expression of this isotype compared to mIgM, and the more rapid down-regulation of mIgM by antigen, than mIgD45. It has been suggested that the mIgD receptors play a critical role in humoral defense against pathogens undergoing rapid expansion and mutational drift upon entry into the host46. This is supported by the delayed antibody production and affinity maturation, in response to model T cell−dependent antigens46, of IgD-deficient mice. In addition, we have observed that the number of GCs obtained from anti-IgM−stimulated resting B cells is reduced compared to those obtained from anti-IgD−stimulated cells (unpublished data). Thus, although the functional relevance of mIgD receptors has been well established, the data we present here add a new dimension by characterizing and highlighting the role played by the GPI-linked isoform in the efficient functioning of these receptors.
The transmembrane and GPI-linked isoforms of mIgD are distributed unequally: the former make up  95% of the total mIgD pool. This unequal distribution probably permits graded responsiveness of primary B cells, depending upon the strength of the antigenic stimulus. Thus high antigen concentrations or affinity would ensure saturating receptor occupancy so that both subsets are stimulated and an optimal GC response is induced. Conditions that limit receptor occupancy (that is, where antigen concentration or affinity are low) would selectively stimulate the dominant transmembrane subset, leading to B cells expressing only an activated phenotype with an attenuated GC seeding capacity. Thus, it is the simple arithmetic of receptor partitioning, which integrates into distinct signaling thresholds, that contributes towards the acumen of a preimmune B cell.
In summary, we have shown here that, in murine resting B cells, a minor fraction of the mIgD is GPI-linked. This isoform is constitutively localized within raft microdomains of the cell membrane and is distinguished from its transmembrane counterpart by its ability to activate cAMP-dependent signaling pathways. The cAMP signaling pathways activated through the GPI-linked mIgD contribute through cooperative interaction with intracellular signals generated from the transmembrane mIgD. This, in conjunction with the unequal distribution of these two mIgD isoforms, provides a mechanism by which the target preimmune B cells can be differentially activated, depending upon the strength of the antigenic stimulus that is experienced.
Methods
Materials.
All fluorescently labeled antibodies used in flow cytometric analysis and the allotype-specific antibodies mouse anti−mouse IgMa and IgG1a were from Pharmingen (San Diego, CA). EGTA and db-cAMP were from Sigma Chemical Co. (St. Louis, MO), whereas H89, calphostin C, TMB-8 and ionomycin were from Calbiochem (San Diego, CA). Biotinylated PNA, alkaline phosphatase−streptavidin and horseradish peroxidase−streptavidin were from Vector Laboratories (Burlingame, CA). Peptide CT3 (DIEKKIAKMEKASSVFNVVNS) was synthesized by solid-phase chemistry as described20. The anti-IgD hybridoma JA 12.5 (a gift of S. Rath, National Institute of Immunology, New Delhi) was used to produce ascites in irradiated BALB/c mice and the antibodies subsequently purified over an anti−rat IgG sepharose column. F(ab)2 fragments were generated by digestion with pepsin as described47, followed by purification over a Sephadex G-200 column.
All animal experiments and their protocols were approved by the Institutional Animal Ethics Committee.
Resting B cell purification.
Spleens from naïve BALB/c IgHa mice (6−8 weeks old) were first depleted of adherent cells by multiple cycles of panning on a plastic surface. From this, resting B cells were purified by magnetic sorting with a negative selection approach. This involved two rounds of depletion with a combination of magnetic beads coated with anti-CD4, anti-CD8 or anti-CD90 and two additional rounds of depletion with a mixture of anti-CD11b− and anti-CD11c−coated beads. Next, sIgG+ cells were also removed with beads coated with anti-IgG. The final step involved Percoll density-gradient centrifugation over steps of 70%, 66%, 60% and 50% Percoll. The high-density fraction of cells (at the interface of 66% and 70%) was collected and used. This fraction contained 96−98% sIgD+ B220+ cells, as determined by flow cytometry. CD3+ cells accounted for <2% of the population.
Stimulation of resting B cells.
Purified cells (1  106 cells/ml) were cultured in RPMI containing 10% fetal calf serum (FCS) + antibiotics and stimulated with 10 g/ml of anti-IgD for 30 h. Cells were then washed and the culture continued for an additional 30 h. Preliminary experiments established this schedule as being optimal for up-regulation of all the various cell surface markers studied. The viability of the cells remained >95% at the 60-h time point, as assessed by Trypan blue staining. The same culture conditions were used when alternative stimulants, such as db-cAMP, were utilized. The effects of inhibitors were examined by first pre-equilibrating with the appropriate inhibitor at 37 °C for 30 min before stimulant was added. The concentrations of the various inhibitors used were as follows: EGTA, 3 mM; TMB-8, 50 M; calphostin C, 0.1 M; and H89, 100 nM. These values were optimized in preliminary titration experiments and ranged from one to two times the individual IC50 values. The expression of various cell surface markers and the Cai2+ responses in FLUO-3-AM−loaded cells were monitored by flow cytometry as described41. Similarly, both intracellular cAMP concentrations and the effects of addition of exogenous db-cAMP were also determined as described41.
Reconstitution of GCs.
Resting B cells were stimulated with anti-IgD. Peptide CT3 (12 M) was added to the culture 48 h later and the culture continued for an additional 12 h. After this, cells were washed and injected intravenously (2 106 cells/mouse) into BALB/c IgHb mice that had been primed 4 days earlier with 50 g of CT3 emulsified in CFA that was injected into the tail base. Seven days later the spleens were removed and 6-m sections were immunohistochemically stained for IgHa-specific (IgG1a + IgMa) and PNA+ cells as described20. We established, in preliminary experiments, that loading of cells with peptide CT3 did not result in any further phenotypic alterations over those observed with anti-IgD stimulation alone (unpublished data).
Isolation of raft fractions.
Lipid rafts were isolated as described32,
33. Cells (1 108 per experimental group) were lysed in a Tris-EDTA buffer (pH 7.4) containing 1% Triton X-100 + protease inhibitors and sodium orthovanadate for 30 min on ice. After this, the supernatant was clarified at 900g for 10 min, then resolved by flotation on a discontinuous sucrose gradient of 85%, 35% and 5% in an SW41 rotor at 200,000g for 20 h at 4 °C. Fractions (1 ml each) were then collected from the top of the gradient. Fractions 4−6 were pooled and the raft-containing fractions and fractions 10−12 were pooled as the soluble fractions. Isolation of lipid rafts was confirmed by immunoblotting for GM1-ganglioside and CD45R. Where necessary, rafts were disrupted by incubating either with nystatin (50 g/ml) for 10 min at 37 °C or with MCD (10 mM) for 15 min at 37 °C.
Radiochemical labeling of GPI-linked sIgD.
Radiolabeling of cells with either glucosamine or mannose was done in the presence of tunicamycin, an inhibitor of NH2-linked glycosylation. Purified resting B cells were washed in Hank's balanced salt solution, resuspended in glucose-free RPMI-1640 containing 10% FCS (2.5 107 cells/ml) and first pre-equilibrated with tunicamycin (5 g/ml for 1 h at 37 °C). After this, they were pulsed with 25 Ci/ml of either [3H]glucosamine (49 Ci/mmol) or [3H]mannose (24 Ci/mmol) at 37 °C for 4 h. For fatty acid labeling, separate aliquots of cells resuspended in RPMI-1640 supplemented with 1% fatty acid−free bovine serum albumin (BSA) and tunicamycin were used. These cells were incubated with a mixture of 25 Ci/ml each of [14C]palmitic acid (850 mCi/mmol) and [3H]myristic acid (12.5 Ci/mmol) at 37 °C for 4 h.
Cells from each of the groups were then washed and the detergent-soluble and detergent-insoluble fractions of the plasma membrane isolated and internally pooled as described above. sIgD from the pooled detergent-soluble and -insoluble fractions from each group was immunoprecipitated by first incubating with biotinylated anti−mouse IgD (10 g/ml at 4 °C for 1 h in 10 mM Tris containing 1% Triton X-100, 150 mM NaCl, 1 mM each of EDTA and EGTA, 2.5 g/ml each of leupeptin, pepstatin and aprotinin, 1 mM PMSF and 1 mM orthovnandate at pH 7.5), followed by streptavidin-agarose (at 4 °C for 30 min). Samples were centrifuged, and the pellets washed extensively in the Tris buffer. All immunoprecipitates were then treated with endo-O-glycosidase (1 U/ml for 2 h at 37 °C in PBS) to remove O-linked glycosylation. Where necessary, immunoprecipitates from fatty acid−labeled cells were also treated with PI-PLC (1 U/ml at 37 °C for 1 h). Immunoprecipitated proteins were then resolved on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The regions corresponding to the IgH chain (as identified from immunoblotting) were excised and processed to determine incorporated radioactivity by liquid scintillation counting.
As negative controls, immunoprecipitations with either anti−mouse IgM or nonspecific rat IgG were done. However, no significant radioactivity (<150 cpm) was immunoprecipitated from any of the groups examined. The deglycosylation protocol was standardized against mIgM immunoprecipitations from resting B cells; an efficiency of >96% was achieved under these conditions.
Note: Supplementary information is available on the Nature Immunology website.
Received 2 July 2002; Accepted 20 August 2002; Published online: 16 September 2002.
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