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The Journal of Immunology, 2008, 181, 5350 -5359
Copyright © 2008 by The American Association of Immunologists, Inc.
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Activation of Human Peripheral IgM+ B Cells Is Transiently Inhibited by BCR-Independent Aggregation of Fc{gamma}RIIB1

Emilie M. Fournier*,{dagger},{ddagger}, Sophie Sibéril2,*,{dagger},{ddagger}, Anne Costes*,{dagger},{ddagger}, Audrey Varin*,{dagger},{ddagger}, Wolf-Herman Fridman*,{dagger},{ddagger}, Jean-Luc Teillaud3,*,{dagger},{ddagger} and Catherine Sautès-Fridman3,4,*,{dagger},{ddagger}

* Institut National de la Santé et de la Recherche Médicale, Unité 872, Paris, France; {dagger} Centre de Recherche des Cordeliers, Université Pierre et Marie Curie-Paris 6, Unité Mixte de Recherche Scientifique 872, Paris, France; and {ddagger} Université Paris Descartes, Unité Mixte de Recherche Scientifique 872, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immune complexes can trigger a SHIP-1-independent proapoptotic signal in mouse class-switched IgG+ B cells and plasma cells by binding to Fc{gamma}RIIB, in the absence of concomitant coaggregation with BCR, hence regulating plasma cell survival and participating in the selection of B cells producing high affinity Abs during secondary Ab responses. By contrast, we demonstrate in the present study that the unique aggregation of Fc{gamma}RIIB on human peripheral IgM+ B cells does not induce apoptosis but transiently inhibits B cell proliferation and calcium influx triggered by BCR cross-linking. Using human peripheral B cells and IIA1.6 lymphoma B cells expressing wild-type human Fc{gamma}RIIB (IIA1.6-Fc{gamma}RIIB), we also show that the unique aggregation of human Fc{gamma}RIIB induces ITIM phosphorylation. This aggregation provokes the recruitment of phosphorylated SHIP-1 by Fc{gamma}RIIB and inhibits the constitutive phosphorylation of Akt in human IIA1.6-Fc{gamma}RIIB cells. This inhibitory signaling pathway is abrogated in IIA1.6 cells expressing ITIM-mutated Fc{gamma}RIIB (Fc{gamma}RIIBY292G), suggesting that ITIM phosphorylation is necessary for Fc{gamma}RIIB-induced B cell blockade. Overall, we demonstrate that the unique aggregation of Fc{gamma}RIIB on human peripheral IgM+ B cells is sufficient to transiently down-regulate their activation without inducing apoptosis. Our results suggest that Fc{gamma}RIIB could negatively regulate IgM+ B cells before class-switch occurrence and that its unique engagement by immune complexes represents a reversible checkpoint for peripheral IgM+ B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
During an immune response, B cell Ab production is initiated by Ag-binding to the BCR. After BCR activation, naive IgM+ B cells proliferate and differentiate rapidly into IgM-secreting plasma cells. Some of them mature into class-switched IgG+ B cells, which differentiate into IgG-secreting plasma cells or join the memory B cell compartment (1). The Ag-driven expansion and differentiation of B cells is tightly controlled, preventing inadequate levels of circulating Ab, plasma cells, and memory B cells. The inhibitory Fc receptor type IIB, Fc{gamma}RIIB, with low affinity for the Fc portion of monomeric IgG Abs, plays a major role in the negative feedback regulation of B cell responses (2, 3). IgG immune complexes that are formed can bind simultaneously to the BCR and Fc{gamma}RIIB leading to the inhibition of the IgG+ B cell response (4). This mechanism results in the control of IgG+ B cell expansion and IgG Ab production. In addition, it is assumed that when sufficient levels of IgG Abs are reached after class-switch, immune complexes can cross-link the BCR of IgM+ B cells to Fc{gamma}RIIB, inhibiting the production of IgM Abs. A various line of evidence suggests that the coaggregation of BCR and Fc{gamma}RIIB induces the phosphorylation of Fc{gamma}RIIB by the Src family kinase Lyn on the tyrosine of the Fc{gamma}RIIB ITIM (5, 6, 7, 8). This response results in the recruitment and phosphorylation of SHIP-1, the Src homology 2 domain-containing inositide phosphatase 1 (9, 10, 11, 12, 13). SHIP-1 dephosphorylates phosphatidylinositol 3,4,5-trisphosphate, thus preventing the recruitment and activation of Btk, PDK1/2, and Akt kinases (14, 15, 16, 17) and leading, therefore, to the inhibition of both phospholipase C{gamma}-dependent calcium influx (18) and Akt-dependent antiapoptotic and proliferative responses (19).

In addition to the inhibitory signal induced by Fc{gamma}RIIB coaggregation with the BCR, several reports have suggested that the unique engagement of Fc{gamma}RIIB triggers apoptosis of mouse class-switched B cells, and of human and mouse plasma cells (20, 21, 22). This proapoptotic signal induced by the sole triggering of mouse Fc{gamma}RIIB is ITIM- and SHIP-1-independent (21). It activates a signaling pathway dependent on the phosphorylation of a tyrosine residue located outside the ITIM by the c-Abl kinase (21). It was suggested that this scenario might play a role in the negative selection of centrocytes exhibiting low affinity for Ag in germinal centers and in the homeostasis of plasma cells (23). Thus, IgG+ B cells exhibiting a low affinity for an Ag embedded into immune complexes will receive Fc{gamma}RIIB signal only and will be deleted, whereas IgG+ B cells with hypermutated BCR exhibiting a high affinity for the Ag will integrate both BCR and Fc{gamma}RIIB signals and, hence, will survive. Similarly, it has been argued that terminally differentiated plasma cells, which express virtually no BCR but high levels of Fc{gamma}RIIB, might be deleted upon Fc{gamma}RIIB proapoptotic signal, allowing the access of newly differentiated plasma cells to the limited number of niches in the bone marrow (22).

However, immune complexes can also encounter irrelevant IgM+ B cells before their differentiation into IgG+ cells and, hence, activate Fc{gamma}RIIB independently of the BCR. Strikingly, the role of Fc{gamma}RIIB has not been elucidated in this situation. Interestingly, Ab responses to T-independent Ags that do not induce class-switch are up-regulated in Fc{gamma}RIIB-deficient mice, suggesting that Fc{gamma}RIIB could negatively regulate mature IgM+ B cells even in the absence of specific IgG (24).

The present work has investigated the consequences of the unique aggregation of Fc{gamma}RIIB in human IgM+ peripheral B cells and the signaling pathways involved. We show in this study that the aggregation of Fc{gamma}RIIB transiently inhibits IgM+ B cell proliferation and calcium influx triggered by BCR cross-linking, without inducing apoptosis. We demonstrate also that human Fc{gamma}RIIB aggregation induces tyrosine phosphorylation of the ITIM, recruitment of SHIP-1 and down-regulates the activation of Akt. Altogether, these data suggest that immune complexes can control peripheral IgM+ B cell activation and proliferation. They extend the BCR-independent inhibitory role of Fc{gamma}RIIB to peripheral IgM+ cells before class-switch occurrence and indicate that its unique engagement by immune complexes represents a reversible checkpoint for peripheral IgM+ B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of IIA1.6 expressing wild-type Fc{gamma}RIIB and Fc{gamma}RIIBY292G cells

Tyr292 in the ITIM of the human Fc{gamma}RIIB was replaced by glycine (Y292G) by site-directed mutagenesis (QuikChange Site-Directed Mutagenesis kit; Stratagene). Briefly, PCR was performed on the human Fc{gamma}RIIB cDNA cloned into pBR322, provided by Dr. P. Bruhns (Institut Pasteur, Paris, France) with the following specific primers: 5'-CTGAGAACACAATCACCGGTTCACTTCTCATGCACC-3' and 5'-GGTGCATGAGAAGTGACCGGTGATTGTGTTCTCAG-3'. The cDNA encoding Fc{gamma}RIIBY292G was then subcloned into the pNT mammalian expression vector (25). Mouse B lymphoma IIA1.6 cells (26) were transfected with the expression vectors encoding Fc{gamma}RIIB or Fc{gamma}RIIBY292G as previously described (27, 28). Stable IIA1.6 transfectants, expressing Fc{gamma}RIIBY292G, were selected with 1 mg/ml Zeocin (Invitrogen) and cloned by limiting dilution. Expression of the receptor was detected using the FITC-labeled mouse anti-human Fc{gamma}RII (CD32) mAb AT10 (AT10-FITC), purchased from Serotec. IIA1.6 cells from clone 16 expressing a high level of Fc{gamma}RIIBY292G were used in the study.

Cells

IIA1.6-Fc{gamma}RIIB and IIA1.6-Fc{gamma}RIIBY292G cells were cultured in RPMI 1640 plus Glutamax (Life Technologies) supplemented with 10% heat-inactivated FCS (Biowest), 100 U/ml penicillin/100 µg/ml streptomycin (Life Technologies), 0.5 µM 2-ME (Life Technologies), 2 mM sodium pyruvate (Life Technologies), and 0.5 mg/ml G418 (Life Technologies) (IIA1.6-Fc{gamma}RIIB) or 1 mg/ml Zeocin (Invitrogen) (IIA1.6-Fc{gamma}RIIBY292G). PBMC were isolated from peripheral blood of healthy human volunteers by Ficoll density gradient centrifugation. CD19+ B cells were positively selected by immunomagnetic CD19-microbeads isolation kit (CD19 MicroBeads; Miltenyi Biotec). CD19+ B lymphocytes were cultured in RPMI 1640 plus Glutamax supplemented with 10% heat-inactivated FCS and 100 U/ml penicillin/100 µg/ml streptomycin (complete medium). Cells were maintained at 37°C in a 5% CO2 humid atmosphere.

Ab analysis

The AT10 and the mouse anti-phosphotyrosine 4G10 mAbs as well as the rabbit polyclonal Abs directed against the intracellular region of human Fc{gamma}RIIB (29, 30, 31) were affinity-purified on protein G-Sepharose. IgG and F(ab')2 rabbit anti-human (RAH)5 Fc5mu (RAH-IgM), IgG and F(ab')2 goat anti-mouse IgG (H+L) (GAM), HRP-conjugated mouse anti-rabbit mAb specific to nondenaturated rabbit L chain, streptavidin, and mouse anti-biotin mAb were purchased from Jackson ImmunoResearch Laboratories. Rabbit polyclonal anti-human phospho-Fc{gamma}RIIB (Tyr292), polyclonal anti-SHIP-1, polyclonal anti-phospho-Akt (Ser473), and polyclonal anti-phospho-SHIP-1 (Tyr1020) Abs were purchased from Cell Signaling Technology. HRP-conjugated goat anti-rabbit IgG (GAR-HRP), HRP-conjugated GAM IgG (GAM-HRP), HRP-conjugated rabbit anti-goat IgG, and goat anti-Akt1 polyclonal Abs were purchased from Santa Cruz Biotechnology. Intravenous human IgG (Tegeline) were a gift from the Laboratoire Français du Fractionnement et des Biotechnologies (LFB, les Ulis, France) and human Fc fragments of IgG were either purchased from Calbiochem or were a gift from Dr. M.-C. Bonnet (Pasteur Mérieux, Marcy l’Etoile, France) (32).

Biotin labeling of purified AT10 Ab

Purified AT10 mAb (1–2 mg/ml) was dialyzed against 0.1 M NaHCO3 (pH 8.3) for 3 h at 4°C. Biotin N-succinimide ester (Sigma-Aldrich) was dissolved in DMSO at the concentration of 1 mg/ml. The 120 µl of biotin solution was added to 1–2 mg of AT10 IgG, and the mixture was incubated at room temperature for 2 h under agitation. The biotinylation reaction was stopped by dialysis against PBS overnight at 4°C.

Immobilized and heat-aggregated human IgG or Fc fragments

Polyclonal human IgG or Fc fragments were immobilized on tissue culture plates to mimic the effect of complexes IgG. Briefly, polyclonal human IgG or Fc fragments at 1 mg/ml in PBS (Life Technologies) were incubated for 24 h at 4°C in 96-well round-bottom plates. Plates were washed twice in PBS before use in B cell proliferation assays. Heat-aggregated IgG and Fc fragments used in B cell proliferation assays and in Fc{gamma}RIIB transfected IIA1.6 cells stimulation were obtained by incubation for 30 min at 65°C.

Whole cell lysate and immunoprecipitation

To aggregate Fc{gamma}RIIB, IIA1.6 transfected cells (3 x 106 cells/0.3 ml) or purified human peripheral B lymphocytes (1 x 106 cells/0.3 ml) were incubated with AT10-biotin (10 µg/ml) and then washed, and incubated either with streptavidin (0.5 or 2 µg/ml) or mouse anti-biotin mAb (10 µg/ml). Coaggregation between BCR and Fc{gamma}RIIB was achieved by incubating cells with IgG GAM (15 µg/ml). All reagents were incubated for 20 min on ice followed by 2 min at 37°C. Cells were lysed in cold lysis buffer containing 10 mM Tris (pH 7.4), 50 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 50 mM NaF, 25 mM sodium pyrophosphate, 5 mM EDTA, 10 µg/ml Pepstatin and Protease Inhibitor Cocktail tablets (Complete, Mini EDTA-free; Roche Diagnostics). Cell lysates were then centrifuged for 15 min at 15,000 x g, and proteins were quantified using a Bradford assay (Bio-Rad). Cell lysates (corresponding to 20–30 µg of protein) were then mixed with NuPAGE LDS sample buffer (Invitrogen) containing 0.1 M DTT (Sigma-Aldrich) and incubated for 10 min at 75°C before SDS-PAGE and Western blotting. Immunoprecipitation of Fc{gamma}RIIB or SHIP-1 from IIA1.6 cell lysates was conducted either with AT10 mAb or with anti-SHIP-1 polyclonal Abs bound to protein G-Sepharose (GE Healthcare). After overnight incubation, protein G-Sepharose beads were washed three times in cold lysis buffer, and proteins were eluated with NuPAGE LDS sample buffer containing DTT as well as incubated for 10 min at 75°C before SDS-PAGE and Western blotting.

Western blot analysis

Proteins were fractionated by 10% SDS-PAGE (NuPAGE Novex 10% Bis-Tris gel; Invitrogen) and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore). Membranes were saturated with blocking buffer containing 5% BSA (Sigma-Aldrich), 0.1% Tween 20 (Merck) in TBS (150 mM NaCl and 10 mM Tris) and incubated with the relevant Abs overnight at 4°C in the same buffer. Membranes were washed with TBS-0.1% Tween and incubated with polyclonal Abs GAR-HRP, GAM-HRP, HRP-conjugated rabbit anti-goat, or HRP-conjugated mouse anti-rabbit for 1 h at room temperature. After washing with TBS-0.1% Tween, peroxidase-labeled Abs were detected by chemiluminescence using an ECL kit (Amersham Biosciences).

Apoptosis assays

To aggregate Fc{gamma}RIIB, IIA1.6-RFc{gamma}IIB cells or purified human peripheral B lymphocytes (106 cells/ml) were incubated with biotinylated AT10 IgG (AT10-biotin, 10 µg/ml) and streptavidin (0.5 µg/ml) or left untreated during the indicated period of time. Apoptotic cells used as control were obtained by incubating cells with 50 µM staurosporine (Sigma-Aldrich) or etoposide (Sigma-Aldrich).

Measurement of B cell apoptosis by Annexin V/propidium iodide (PI) staining

B cell apoptosis was measured by Annexin V/PI staining. After culture, IIA1.6-Fc{gamma}RIIB cells or purified peripheral human B lymphocytes were washed with cold PBS (Life Technologies) and resuspended in 100 µl of binding buffer from an Annexin V- Allophycocyanin/Propidium Iodide kit (BD Pharmingen), containing 2 µl from Annexin V-allophycocyanin solution stock and 2 µl from 50 µg/ml PI staining solution. After 15 min of incubation at room temperature in a light-protected area, the reaction was stopped by adding 400 µl of binding buffer before analysis by flow cytometry using a FACSCalibur cytometer (BD Biosciences) and the BD CellQuest Pro software. Data were expressed as a percentage of double positive Annexin V+/PI+ cells.

Measurement of B cell apoptosis by counting apoptotic nuclei with hypodiploid DNA

B cell apoptosis was measured by hypodiploid DNA analysis as previously described (33) with minor modifications. Briefly, after culture, IIA1.6-Fc{gamma}RIIB cells or purified human peripheral B lymphocytes were washed with cold PBS (Life Technologies) and analyzed for hypodiploid DNA content. Cell pellets were fixed in 70% ethanol at 4°C for at least 2 h. After ethanol removal, cells were permeabilized with 0.5% Tween PBS. Cells were then centrifuged and resuspended with PBS containing 100 µg/ml RNase A (Sigma-Aldrich) and 50 µg/ml PI (Sigma-Aldrich) before analysis by flow cytometry using a FACSCalibur cytometer and the BD CellQuest Pro software. Data were expressed as a percentage of apoptotic nuclei containing degraded hypodiploid DNA.

B cell proliferation assay

Purified human peripheral B lymphocytes were then cultured in 96-well round-bottom plates (105 cells/well in 200 µl of complete medium) in the presence or absence of Abs to aggregate Fc{gamma}RIIB (AT10-biotin, 10 µg/ml) plus streptavidin (0.5 or 1 µg/ml) or mouse anti-biotin mAb (10 µg/ml) or to coaggregate Fc{gamma}RIIB and BCR (IgG RAH-IgM, 15 µg/ml). B cell proliferation was induced by adding 10 µg/ml F(ab')2 RAH-IgM. A 1 µCi/well of [3H]thymidine (Amersham Radiochemicals; GE Healthcare) was added after 72 h of incubation and cells were harvested at 96 h and incorporated radioactivity was measured by scintillation counting.

Flow cytometry analysis of calcium mobilization

B cells were isolated from purified PBMCs using the Dynal B Cell Negative Isolation kit (Dynal). B cells were loaded with 5 mM Fluo-3-AM (Molecular Probes) in RPMI 1640 containing 0.2% pluronic acid F-127 (Sigma-Aldrich) for 30 min at room temperature, washed three times in RPMI 1640, and resuspended with complete medium. Cells were then incubated with AT10-biotin plus streptavidin preformed complexes (at 10/0.5 µg/ml) for 20 min on ice and for 2 min at 37°C before stimulation with F(ab')2 RAH-IgM Abs (50 µg/ml). BCR and Fc{gamma}RIIB were coaggregated using IgG RAH-IgM (75 µg/ml). Mean intracellular Ca2+ concentration was determined by flow cytometry using a FACSCalibur cytometer. Data analysis was performed using the BD FCS Assistant 1.2.9.β software.

Statistical analysis

Paired and unpaired Student’s t tests were used to perform statistical analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Aggregation of Fc{gamma}RIIB transiently inhibits BCR-induced proliferation of human peripheral IgM+ B cells and calcium influx

First, we investigated whether the unique aggregation of Fc{gamma}RIIB modifies BCR-induced proliferation of human peripheral IgM+ B cells purified from healthy donors. CD19+ B cells were purified from blood from healthy donors and the aggregation of Fc{gamma}RIIB was obtained by AT10-biotin followed by a mouse anti-biotin mAb or streptavidin. Immobilized or heat-aggregated IgG or immobilized Fc were also tested. In a first set of experiments, cells were activated with specific F(ab')2 RAH-IgM to target IgM+ cells only, and proliferation was evaluated by measuring [3H]thymidine incorporation. The F(ab')2 RAH-IgM used in these experiments were Fc5mu fragment-specific and did not cross-react with human or mouse IgG, as shown both by flow cytometry analyses using human and mouse IgG+ cell lines (see supplemental Fig. S1 and Fig. S2, respectively), by isotype-specific ELISA (see supplemental Fig. S3), and by a proliferation assay performed on purified IgG+ B cells (see supplemental Fig. S4).6 Fig. 1A shows the results obtained after 4 days of culture Fc{gamma}RIIB aggregation with AT10-biotin followed by mouse anti-biotin mAb or streptavidin inhibits up to 45% of B cell proliferation induced by independent BCR cross-linking, whereas addition of streptavidin or mouse anti-biotin mAb alone has no significant effect (p > 0.05). The same is found with the addition of AT10 alone (p > 0.05), indicating in the latter case that the observed inhibition was not due to a nonspecific binding of the F(ab')2 RAH-IgM to the mouse IgG mAb. This inhibition could be observed also after 3 days of culture (see supplemental Fig. S5).6 Culture of B cells with IgG RAH-IgM, which triggers the coaggregation of BCR with Fc{gamma}RIIB, resulted to an almost complete blockade of [3H]thymidine incorporation (Fig. 1A).


Figure 1
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  		FIGURE 1. Inhibition of BCR-mediated B cell proliferation by the unique aggregation of Fc{gamma}RIIB. A and B, Purified peripheral human B cells were untreated (medium) or first incubated with AT10-biotin (AT10-b) and streptavidin (SA) (A and B) or AT10-biotin and mouse anti-biotin (MAB) (A) to aggregate Fc{gamma}RIIB. Alternatively, B cells were incubated with heat-aggregated or immobilized human polyclonal IgG or in the presence of immobilized human Fc fragments to mimic engagement of Fc{gamma}RIIB by immune complexes (B). Human B cells were stimulated at the same time with F(ab')2 RAH-IgM Abs to cross-link the BCR or with IgG RAH-IgM to coaggregate BCR and Fc{gamma}RIIB (A and B). C, Purified peripheral human B cells were left untreated or incubated with AT10-biotin and streptavidin to aggregate Fc{gamma}RIIB, and F(ab')2 RAH-IgM Abs were then added after various times. D, B cells were incubated with F(ab')2 RAH-IgM Abs, and Fc{gamma}RIIB was aggregated or not at different time points after BCR stimulation using AT10-biotin and streptavidin. In all experiments, B cell proliferation was measured as described in Materials and Methods (8646 ± 3600 cpm in presence of F(ab')2 RAH-IgM alone). Results are mean percentage ± SD of inhibition from six (A) or three (B–D) experiments performed in triplicate each with different healthy donors. The statistical analysis was performed using paired Student’s t test. *, p < 0.05 and **, p < 0.005 vs medium.

 
Addition of immobilized or aggregated IgG or of immobilized Fc exerts an inhibition level of [3H]thymidine incorporation similar to anti-Fc{gamma}RII Abs (Fig. 1B). Heat-aggregated and immobilized IgG inhibited up to 80% of B cell proliferation (Fig. 1B).

Kinetics experiments were then performed to further extend these findings. Firstly, (Fab')2 RAH-IgM were added at different time points following Fc{gamma}RIIB aggregation with anti-Fc{gamma}RII. Results illustrated in Fig. 1C indicate that the inhibitory signal is transient (lasting ~15 min) and reappears after 12 h of culture. In a second set of experiments, aggregation of Fc{gamma}RIIB with anti-Fc{gamma}RII was performed at different time points after BCR stimulation with F(ab')2 RAH-IgM. Results illustrated in Fig. 1D indicate that B cells remained sensitive to the inhibitory signal at least 12 h after BCR activation.

An early event that follows BCR interaction with Ag is calcium mobilization. We investigated whether Fc{gamma}RIIB aggregation modulates calcium influx induced by BCR cross-linking in human IgM+ B cells. Purified peripheral human B cells were preincubated with AT10-biotin in combination with either streptavidin or mouse anti-biotin mAb. Calcium influx was then induced with F(ab')2 RAH-IgM. Fig. 2A shows that the aggregation of Fc{gamma}RIIB induced either by streptavidin (Fig. 2A, left) or mouse anti-biotin mAb (Fig. 2A, right) results in a significant inhibition of calcium influx as compared with F(ab')2 RAH-IgM alone (p = 0.007 and p = 0.005, respectively) (Fig. 2B). Therefore, the unique aggregation of human Fc{gamma}RIIB inhibits calcium mobilization triggered by BCR cross-linking in human peripheral IgM+ B cells.


Figure 2
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  		FIGURE 2. Fc{gamma}RIIB aggregation inhibits calcium influx induced by independent BCR activation. A, Purified peripheral human B cells were loaded with fluo-3-AM and incubated first either with AT10-biotin and streptavidin (SA) (left) or AT10-biotin and mouse anti-biotin (MABiotin) (right) and then stimulated with F(ab')2 RAH-IgM (thin black line). Cells incubated with F(ab')2 RAH-IgM alone are indicated (thick black line). Cells incubated with IgG RAH-IgM to coaggregate BCR and Fc{gamma}RIIB are also shown (gray line). Intracellular Ca2+ concentration was measured by flow cytometry at different time points. Results are representative of three independent healthy donors. B, Histograms represent mean relative fluorescence intensity ± SD, calculated at the plateau of calcium responses (from 150 to 170 s). Three independent experiments were performed with different healthy donors. Statistical analysis was performed using paired Student’s t test. *, p < 0.05 and **, p < 0.01 vs F(ab')2 RAH-IgM.

 
Aggregation of Fc{gamma}RIIB in human B cells does not induce apoptosis

Fc{gamma}RIIB aggregation induces apoptosis in class-switched mouse IgG+ cells (20), in human plasma cells, and in myeloma cell lines (22). We therefore investigated whether human Fc{gamma}RIIB aggregation triggers apoptosis in human peripheral B lymphocytes. Purified peripheral human B cells were incubated for 24 or 96 h in presence of AT10-biotin and streptavidin to aggregate Fc{gamma}RIIB or in control medium, and the percentage of Annexin V+/PI+ cells (Fig. 3A) or hypodiploid cells (Fig. 3B) was analyzed by flow cytometry. Fig. 3C illustrates FACS profiles of hypodiploid cell detection for one representative donor. Etoposide or staurosporine were used as positive controls for early (Fig. 3A) and late (Fig. 3, B and C) apoptosis detection. No significant difference was observed after a 24-h incubation of B cells with medium and AT10-biotin and streptavidin in the percentage of Annexin V+/PI+ cells (Fig. 3A) or hypodiploid cells (Fig. 3B). Indeed, a large percentage of Annexin V+/PI+ B cells were detected after 96 h of incubation with control medium and is similar to that observed in AT10-biotin- and streptavidin-treated cells. However, the percentage of hypodiploid cells remained low at this time point and did not differ significantly between B cells incubated in culture medium or with AT10-biotin and streptavidin (Fig. 3B). Thus, these results indicate that the unique aggregation of human Fc{gamma}RIIB does not induce detectable apoptosis in peripheral human B cells. To confirm these results, we investigated whether Fc{gamma}RIIB aggregation triggers apoptosis in IIA1.6 mouse B lymphoma cells that express the wild-type human Fc{gamma}RIIB (IIA1.6-Fc{gamma}RIIB cells). IIA1.6-Fc{gamma}RIIB cells were incubated for 15, 24, or 48 h in presence of AT10-biotin and streptavidin to aggregate Fc{gamma}RIIB, and the percentage of Annexin V+/PI+ cells (Fig. 3D) and hypodiploid cells (Fig. 3E) were analyzed by flow cytometry. Again, no difference in Annexin V+/PI+ and hypodiploid cells was observed between control cells and cells whose Fc{gamma}RIIB had been aggregated, indicating that the unique aggregation of human Fc{gamma}RIIB at the surface of IIA1.6 cells does not trigger detectable apoptosis.


Figure 3
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  		FIGURE 3. Fc{gamma}RIIB aggregation does not induce apoptosis. Purified peripheral human B cells from healthy donors (A–C) or IIA1.6-Fc{gamma}RIIB cells (D and E) were incubated for the indicated period of time with AT10-biotin and streptavidin (SA), with the proapoptotic drugs etoposide or staurosporine, or in complete medium alone. Cells were analyzed by flow cytometry and the percentage of apoptotic cells was assessed as double positive cells for Annexin V and PI (AnV+/PI+) (A and D) or as hypodiploid cells using PI staining (B, C, and E). Results represent the mean percentage ± SD of Annexin V+/PI+ and hypodiploid cells from n = 3 (A) and n = 4 (B) healthy donors. Statistical analysis was performed using paired Student’s t test. *, p < 0.05 and **, p < 0.001 vs medium. C, Analysis of hypodiploid cells by flow cytometry for one healthy donor.

 
Human Fc{gamma}RIIB aggregation induces tyrosine phosphorylation of the ITIM

To delineate the signaling cascade triggered by human Fc{gamma}RIIB aggregation, we first investigated the phosphorylation state of the Fc{gamma}RIIB following aggregation. Purified peripheral human B cells (Fig. 4A) or IIA1.6-Fc{gamma}RIIB cells (Fig. 4B) were incubated with AT10-biotin followed by streptavidin or mouse anti-biotin mAb to aggregate Fc{gamma}RIIB. Tyrosine phosphorylation of ITIM was analyzed by Western blotting using rabbit polyclonal Abs that specifically bind phosphorylated Tyr292 of Fc{gamma}RIIB ITIM. As shown in Fig. 4, A and B, the aggregation of human Fc{gamma}RIIB results in ITIM tyrosine phosphorylation upon cross-linking in both conditions. As expected, Fc{gamma}RIIB became also tyrosyl-phosphorylated following coaggregation with BCR induced by IgG RAH (Fig. 4A) or IgG GAM (Fig. 4B). Following Fc{gamma}RIIB aggregation, kinetic experiments showed that phosphorylation of Fc{gamma}RIIB peaks at 2 min and decreases thereafter, being still detectable after 1 h (Fig. 4C). To insure that the tyrosyl-phosphorylation of Fc{gamma}RIIB occurred on Tyr292 and was not due to cross-reactivity of the polyclonal Abs directed against Fc{gamma}RIIB Tyr292 with other phosphorylated tyrosines, IIA1.6-Fc{gamma}RIIBY292G cells were established. Wild-type and mutant Fc{gamma}RIIB were aggregated using AT10-biotin followed by streptavidin or mouse anti-biotin mAb. No phosphorylation was observed following Fc{gamma}RIIBY292G mutant aggregation, demonstrating that the phosphorylation observed in the IIA1.6-Fc{gamma}RIIB cells does correspond to ITIM phosphorylation of human Fc{gamma}RIIB (Fig. 4D).


Figure 4
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  		FIGURE 4. Human Fc{gamma}RIIB aggregation results in ITIM tyrosine phosphorylation. Purified peripheral human B cells (A), IIA1.6-Fc{gamma}RIIB cells (B), or IIA1.6-Fc{gamma}RIIBY292G cells (D) were first incubated with AT10-biotin or AT10 as control, or not treated (Medium). Cells were then incubated with streptavidin (SA) or mouse anti-biotin (MABiotin) to aggregate Fc{gamma}RIIB, or with IgG RAH-IgM (A) or IgG GAM (B and D) to coaggregate BCR and Fc{gamma}RIIB for 2 min at 37°C. C, IIA1.6-Fc{gamma}RIIB were treated with AT10-biotin followed by streptavidin during 20 min on ice and incubated at 37°C during different periods of time. In all experiments, Fc{gamma}RIIB phosphorylation on Tyr292 of ITIM (pITIM Fc{gamma}RIIB) was analyzed by Western blotting (top). Blot was probed again with RAH Fc{gamma}RIIB Abs (bottom). Each result is representative of three independent experiments.

 
We then investigated whether human IgG and Fc fragments induce ITIM phosphorylation after engagement of the low affinity Fc{gamma}RIIB similarly to AT10-biotin-streptavidin complexes. Human polyclonal IgG and human Fc fragments were heat-aggregated to mimic IgG complexes and incubated with IIA1.6-Fc{gamma}RIIB cells for 2 min, a time point corresponding to a strong ITIM phosphorylation induced by AT10-biotin. Incubation of the cells with heat-aggregated IgG (Fig. 5A) or with heat-aggregated Fc fragments (Fig. 5B) induced ITIM Fc{gamma}RIIB phosphorylation in a dose-dependent manner. As expected, due to the low affinity of Fc{gamma}RIIB (2), monomeric IgG had no effect (Fig. 5A).


Figure 5
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  		FIGURE 5. Engagement of Fc{gamma}RIIB with aggregated IgG or Fc fragments induces Tyr292 phosphorylation. IIA1.6-Fc{gamma}RIIB cells were incubated with either monomeric (A, left), heat-aggregated human IgG (A, right), or heat-aggregated human Fc fragment (B) at different doses for 2 min at 37°C. After cell lysis, total proteins were fractionated by SDS-PAGE and blotted with anti-human phospho-Fc{gamma}RIIB Tyr292 (pITIM Fc{gamma}RIIB) (top). Blots were probed again with RAH Fc{gamma}RIIB Abs (bottom). Relative fluorescence intensity was calculated as the ratio of pITIM-Fc{gamma}RIIB to total Fc{gamma}RIIB. Results are representative of three independent experiments.

 
Mutated Y292G Fc{gamma}RIIB is not tyrosyl-phosphorylated upon aggregation

In addition to the ITIM tyrosine at position 309, mouse Fc{gamma}RIIB contains three tyrosine residues within its cytoplasmic tail at positions 264, 290, and 326. Cross-linking of mouse Fc{gamma}RIIB induces the phosphorylation of Tyr264 located outside of the ITIM (21). The cytoplasmic region of human Fc{gamma}RIIB contains one additional tyrosine residue outside of the ITIM, at position 258. Thus, we investigated whether human Fc{gamma}RIIB aggregation induces the phosphorylation of Tyr258, in addition to the ITIM Tyr292. The total phosphotyrosine profile of Fc{gamma}RIIB was assessed in IIA1.6-Fc{gamma}RIIB and IIA1.6-Fc{gamma}RIIBY292G cells after Fc{gamma}RIIB aggregation. Then, Fc{gamma}RIIB was immunoprecipitated with AT10 mAb and Western blots were performed using the anti-phosphotyrosine 4G10 mAb. No phosphorylation of Fc{gamma}RIIBY292G (Fig. 6B) could be detected even after 1 h of aggregation, by contrast to wild-type Fc{gamma}RIIB (Fig. 6A). It indicates that the additional Tyr258 is not phosphorylated or may require the phosphorylation of Tyr292 to be phosphorylated upon Fc{gamma}RIIB aggregation.


Figure 6
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  		FIGURE 6. Lack of Fc{gamma}RIIB phosphorylation in IIA1.6-Fc{gamma}RIIBY292G cells following Fc{gamma}RIIB aggregation. IIA1.6-Fc{gamma}RIIB (A) and IIA1.6-Fc{gamma}RIIBY292G (B) cells were first incubated with AT10-biotin or not stimulated (NS), then incubated with streptavidin (SA) or not stimulated (NS) to aggregate Fc{gamma}RIIB for various periods of time at 37°C. After cell lysis, Fc{gamma}RIIB was immunoprecipitated (IP) using AT10 mAb as described in Materials and Methods. Immunoprecipitates were then fractionated by SDS-PAGE and blotted with anti-phosphotyrosine (pTyr) 4G10 mAb (top). Blot was probed again with RAH Fc{gamma}RIIB Abs (bottom). Relative intensity was calculated as the ratio of phosphotyrosine to total Fc{gamma}RIIB. Results are representative of three independent experiments performed in parallel.

 
Aggregated Fc{gamma}RIIB recruits SHIP-1 and down-modulates Akt activation

Several studies demonstrated that the coaggregation of mouse BCR and Fc{gamma}RIIB results in the recruitment of SHIP-1 by the phosphorylated ITIM (10, 13, 34, 35). Thus, to determine whether SHIP-1 is associated with human Fc{gamma}RIIB following aggregation of the receptor, IIA1.6-Fc{gamma}RIIB and IIA1.6-Fc{gamma}RIIBY292G cells were incubated with AT10-biotin followed by mouse anti-biotin mAb to aggregate Fc{gamma}RIIB. Wild-type and mutant Fc{gamma}RIIB were immunoprecipitated from cell lysates using AT10, and SHIP-1 coprecipitation was analyzed by Western blotting. As shown in Fig. 7A, SHIP-1 is phosphorylated and is associated with the wild-type but not with the Fc{gamma}RIIBY292G mutant upon aggregation. Conversely, phosphorylated Fc{gamma}RIIB coprecipitates with SHIP-1 from wild-type but not from mutant IIA1.6 cells (Fig. 7B). In agreement with previous studies, the coaggregation of BCR and Fc{gamma}RIIB induced by IgG GAM has similar effects (Fig. 7). Altogether, these results indicate that the aggregation of human Fc{gamma}RIIB allows the phosphorylation and the recruitment of SHIP-1 by phosphorylated Fc{gamma}RIIB.


Figure 7
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  		FIGURE 7. Aggregated Fc{gamma}RIIB recruits SHIP-1. A and B, IIA1.6-Fc{gamma}RIIB and IIA1.6-Fc{gamma}RIIBY292G cells were first incubated with AT10-biotin or not stimulated (NS), then incubated with either mouse anti-biotin (MABiotin) to aggregate Fc{gamma}RIIB or with IgG GAM to coaggregate BCR and Fc{gamma}RIIB for 2 min at 37°C. After cell lysis, Fc{gamma}RIIB (A) or SHIP-1 (B) was immunoprecipitated (IP) using AT10 mAb or anti-SHIP-1 rabbit Abs, respectively. Immunoprecipitates were fractionated by SDS-PAGE and blotted with anti-SHIP-1 and blotted again with anti-phospho-SHIP-1 (pSHIP1) (A), or blotted with anti-human phospho-Fc{gamma}RIIB Tyr292 (pITIM Fc{gamma}RIIB) (B), respectively. Immunoprecipitation of Fc{gamma}RIIB (A) or SHIP-1 (B) was assessed by blotting again with the relevant Abs. Results are representative of three independent experiments.

 
Activation of the serine/threonine kinase Akt induced by BCR cross-linking is inhibited when BCR is coaggregated with Fc{gamma}RIIB, its phosphorylation on Ser473 being markedly affected (36, 37, 38). We took advantage of the constitutive phosphorylation of Akt in cycling IIA1.6-Fc{gamma}RIIB cells to investigate whether Fc{gamma}RIIB aggregation affects its phosphorylation. Cell lysates were fractionated by 10% SDS-PAGE, and Akt phosphorylation on Ser473 was detected using specific rabbit polyclonal Abs. As illustrated in Fig. 8A, a decrease of Akt phosphorylation in a time-dependent manner was observed 5 and 10 min following Fc{gamma}RIIB aggregation (AT10-biotin plus mouse anti-biotin mAb) but not in its absence (HBSS). Strikingly, Akt phosphorylation did not decrease after 5 and 10 min of Fc{gamma}RIIB aggregation in IIA1.6-Fc{gamma}RIIBY292G cells (Fig. 8B). These results were confirmed in three independent experiments. The relative phosphorylation of Akt decreases significantly upon Fc{gamma}RIIB aggregation in IIA1.6-Fc{gamma}RIIB (p = 0.04 and p = 0.03 after 5 and 10 min of incubation, respectively) (Fig. 8C) but not in IIA1.6-Fc{gamma}RIIBY292G cells (p = 0.77 and p = 0.2 after 5 and 10 min of incubation, respectively) (Fig. 8D). Thus, these data suggest that the engagement of Fc{gamma}RIIB alone down-regulates the Akt-dependent signaling cascade.


Figure 8
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  		FIGURE 8. Fc{gamma}RIIB aggregation affects Akt phosphorylation on Ser473. IIA1.6-Fc{gamma}RIIB (A) and IIA1.6-Fc{gamma}RIIBY292G (B) cells were treated or not stimulated (NS) with AT10-biotin and mouse anti-biotin (MABiotin) for the indicated periods of time (top). The same experience was monitored with IIA1.6-Fc{gamma}RIIB and IIA1.6-Fc{gamma}RIIBY292G cells in HBSS in absence of Abs (bottom). Cells were lysed and Akt phosphorylation (pAkt (Ser473)) was analyzed by Western blot as described in Materials and Methods. Data are representative of three independent experiments. Relative intensity was expressed as the ratio of pAkt to total Akt. C and D, Histograms represent the mean relative intensity ± S.D. of Akt phosphorylation in IIA1.6-Fc{gamma}RIIB (C) and IIA1.6-Fc{gamma}RIIBY292G (D) cells at different time points after Fc{gamma}RIIB aggregation. Results are from three independent experiments. Statistical analyses were performed using unpaired Student’s t test. *, p < 0.05 vs nonstimulated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The feedback inhibition of Ab responses by immune complexes was first described in the late 1970s (39). Later, it was demonstrated that the coaggregation of BCR and Fc{gamma}RIIB by immune complexes down-regulates naive B cell proliferation and Ig secretion (4, 40). This inhibitory effect is triggered by the phosphorylation of the ITIM tyrosine of Fc{gamma}RIIB (5, 6), and the recruitment and phosphorylation of the phosphatase SHIP-1 (9, 10, 11, 12, 13, 34). It has also been shown that the engagement of mouse Fc{gamma}RIIB, in absence of coaggregation with the BCR, transduces an ITIM- and SHIP-independent proapoptotic signal (20, 21). The present study shows that the unique aggregation of human Fc{gamma}RIIB leads to the ITIM tyrosine 292 phosphorylation resulting in the transient inhibition of peripheral human IgM+ B cell proliferation and inhibition of calcium influx activated via the BCR, without inducing apoptosis. No phosphorylation of the Fc{gamma}RIIBY292G could be detected in IIA1.6 cells, indicating that the aggregation of human Fc{gamma}RIIB triggers the phosphorylation of the ITIM only, or at least represents the required first step before any phosphorylation of the other tyrosine at position 258 in the intracellular domain of human Fc{gamma}RIIB. In addition, SHIP-1 was found phosphorylated and associated with human Fc{gamma}RIIB upon aggregation as observed upon its coaggregation with BCR (Fig. 7).

The pioneering experiments of Pearse et al. (20) showed that the proapoptotic signal induced by mouse Fc{gamma}RIIB aggregation is partially blocked by the recruitment of SHIP-1. In accordance with these data, the present work shows that the aggregation of human Fc{gamma}RIIB, which induces the recruitment of SHIP-1 on phosphorylated ITIM, does not induce apoptosis in either IIA1.6 cells or human B cells (Fig. 3). Altogether, these results suggest that aggregation of human Fc{gamma}RIIB triggers an ITIM- and SHIP-1-dependent inhibitory signal, whereas the unique cross-linking of mouse Fc{gamma}RIIB has been shown to induce an apoptotic signal that is ITIM- and SHIP-1-independent. The different signaling pathways triggered upon human and mouse Fc{gamma}RIIB aggregation could be due to cytoplasmic regions differences between these receptors, as reflected by the low sequence homology between the two receptors (i.e., below 50%). Notably, two tyrosines residues are present in human Fc{gamma}RIIB intracytoplasmic domain, as compared with four in the mouse receptor. In addition, the regulatory role of Fc{gamma}RIIB is complex and could depend on the stage of B cell differentiation. In terminally differentiated B cells, such as centrocytes or plasma cells, the proapoptotic role of Fc{gamma}RIIB requires high levels of the receptor and was found to be SHIP-1-independent (22, 41).

It is known that dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate by SHIP-1 prevents the activation of the serine/threonine Akt and of the Btk kinase by the phosphatidylinositol (17, 18, 36). Akt plays an important role in the control of proliferation and survival signaling, whereas Btk is responsible for the activation of phospholipase C{gamma}, which is involved in calcium mobilization. In accordance with these observations, our results show that Fc{gamma}RIIB aggregation leads to the recruitment of SHIP-1, down-regulates constitutive Akt phosphorylation, decreases calcium influx, and inhibits BCR-activated B cell proliferation. Kinetics experiments showed that the Fc{gamma}RIIB inhibitory signal was reversed 15 min after induction and for a period of 8 h of culture. This effect is not due to the down-regulation of Fc{gamma}RIIB surface expression (data not shown). Furthermore, only a transient blockade of B cell proliferation triggered by Fc{gamma}RIIB engagement was observed. BCR-dependent proliferation could be inhibited when Fc{gamma}RIIB was aggregated not only at the initiation of the culture, but during at least a 12-h period after BCR stimulation, whereas this inhibition resumed afterward (Fig. 1E). This observation is reminiscent of a previous study showing that any interruption of PI3K activity during the first 12 h following T cell activation prevents their proliferation (42). Thus, these data demonstrate that Fc{gamma}RIIB and BCR can mediate, without any coaggregation, opposite signals that can be mutually reversed.

From our data, we propose that the transient inhibitory signal triggered by Fc{gamma}RIIB engagement upon encounter of IgM+ B cells with bystander immune complexes could regulate IgM+ B cell proliferation and differentiation. It should be stressed that this inhibition of IgM+ B cell proliferation was observed even in absence of IgG+ B cells using CD19+ cell suspensions depleted in IgG+ B cells by IgG-microbead isolation kit (data not shown).

Aggregation of Fc{gamma}RIIB on B cells could occur in vivo independently from concomitant BCR coaggregation, when IgM+ B cells encounter immune complexes containing Ag with no affinity for their BCR. These B cells might become therefore transiently refractory to other activating signals. In accordance with this hypothesis, the increase of primary IgM responses to T-dependent and T-independent Ags, observed in Fc{gamma}RIIB-deficient mice, suggests that Fc{gamma}RIIB can control the activation of naive B cells (24, 43, 44). Interestingly, Fc{gamma}RIIB-deficient mice develop an autoimmune syndrome (45), although it was later shown that mice from a congenic line carrying a 129-derived chromosome 1 interval on a C57BL/6 background also develop humoral autoimmunity, suggesting that the lack of Fc{gamma}RIIB expression is not the only parameter responsible for the autoimmune phenotype (46). However, overexpression of Fc{gamma}RIIB, independent from this 129 region, suppresses the autoimmune syndrome, an observation that excludes a relationship between the autoimmune syndrome and the adjacent area of 129 DNA (47).

Overall, our work demonstrates for the first time to our knowledge that the unique Fc{gamma}RIIB engagement is sufficient to transiently inhibit human IgM+ B cell activation and suggests that Fc{gamma}RIIB could regulate IgM+ B cell responses before switch occurrence following irrelevant immune complexes binding.


   Acknowledgments
 
We thank Dr. Catherine Régnier for comments on the manuscript, for help in the mutagenesis experiments, and for helpful discussions and Dr. Alexis Mathian for comments on the manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Institut National de la Santé et de la Recherche Médicale, Universités Paris Descartes-Paris 5, and Université Pierre et Marie Curie-Paris 6, and Canceropôle Île-de-France. E.M.F. was a recipient of a fellowship from the French Ministère de l’Enseignement Supérieur et de la Recherche and then from Association pour la Recherche sur le Cancer. S.S. was supported by Université Pierre et Marie Curie-Paris 6. Back

2 Current address: Institut National de la Santé et de la Recherche Médicale, Unité 768, Laboratoire du Développement Normal et Pathologique du Système Immunitaire, Université Paris Descartes, Paris F-75015, France. Back

3 J.-L.T. and C.S.-F. are senior coauthors. Back

4 Address correspondence and reprint requests to Dr. Catherine Sautès-Fridman, Team 13, Centre de Recherche des Cordeliers, Unité Mixte de Recherche Scientifique 872, 15 rue de l’Ecole de Médecine, 75006 Paris, France. E-mail address: catherine.fridman{at}crc.jussieu.fr Back

5 Abbreviations used in this paper: RAH, rabbit anti-human; PI, propidium iodide; GAM, goat anti-mouse; GAR, goat anti-rabbit. Back

6 The online version of this article contains supplemental material. Back

Received for publication January 29, 2008. Accepted for publication August 11, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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