RIIb inhibits both B cell receptor-and CD 19-induced Ca 2 F mobilization in Fc γ R-transfected human B cells

FcγRIIb (CD32) controls antibody production by down-regulating cell activation, when co-clustered with B cell antigen receptors (BCR) in vivo , via immune complexes consisting of secreted IgG and antigen. Fc γRIIb–BCR co-ligation in vitro was shown to inhibit the Ca 2F influx from the extracellular space, the mechanism of which is not fully understood. Human B cells express Fc γRIIb1 and FcγRIIb2, differing only in a 19 amino acid long insert in the cytoplasmic tail of the former. To elucidate whether Fc γRIIb1 and Fc γRIIb2 isoforms show any difference in the down-regulation of B cells, we have studied the effect of co-clustering of BCR and Fc γRIIb1 or Fc γRIIb2 on the Ca 2F signaling in a Burkitt’s lymphoma cell line, ST486, transfected with the two isoforms respectively. We have shown here, for the first time, that co-aggregation of BCR and Fc γRIIb may also inhibit Ca2F release from the endoplasmic reticulum pool of human B cells. Both isoforms mediated this inhibition and the inhibitory effect depended on the ratio of BCR to Fc γRIIb cross-linking. In contrast to Fc γRIIb, the CD21/CD19 complex was shown to up-regulate B cell response by lowering the activation threshold. We have shown here that co-clustering of Fc γRIIb with CD19 inhibited the CD19-induced Ca 2F influx. Furthermore, the three party co-aggregation of Fc γRIIb with BCR and CD19 resulted in a decreased Ca 2F response, as compared to the BCRplus CD19-induced one, indicating that Fc γRIIb may inhibit CD19-induced enhancement of B cell activation. On the basis of these data we suggest that IgG-containing and C3d-fixing immune complexes may down-regulate the B cell response by interfering with both BCRand CD19-mediated Ca 2F mobilization. Co-clustering of the B cell antigen receptor (BCR) and FcγRIIb induces tyrosyl phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) of the latter and, consequently, translocation of negative regulator molecules from the cytosol to the proximity of BCR signal transduction complex (1–3). This leads to the inhibition of the early steps of signaling as well as to the reduction of antibody synthesis. The inhibition of [Ca21] influx is one of the earliest signs of FcγRIIb-mediated down-regulation, the mechanism of which is not fully understood. SH2 domain-containing protein tyrosine phosphatases (SHP-1 and SHP-2) as well as polyphosphoinositol 5-phosphatase (SHIP) were found to bind in vitro to the phosphorylated ITIM (P-ITIM) peptide of murine FcγRIIb (1–3), while SHIP and SHP-1 were shown to associate with the BCR coCorrespondence to: G. Sármay Transmitting editor: I. Pecht Received 8 September 1997, accepted 21 October 1997 ligated FcγRIIb in intact murine B cells (1,3). Furthermore, selective in vivo recruitment of SHIP was demonstrated by the phosphorylated FcγRIIb in mast cells (4). Nevertheless, the target molecules on which these phosphatases exert their effect in vivo and the influence of phosphatases on Ca21 signaling are not yet known. Human B cells express both FcγRIIb1 and FcγRIIb2, two isoforms of the same receptor differing in a 19 amino acid long insert in the cytoplasmic tail of the former, while murine B cells express only FcγRIIb1. Both FcγRIIb1 and FcγRIIb2 possess ITIM, which is not fully identical with the murine homolog and contains a Lys instead of a Met near to the C-terminal end. We have reported previously that the adapter protein Shc and several Tyr-phosphorylated molecules were associated with human FcγRIIb when co142 Ca21 signaling in FcγRIIb-transfected human B cells ligated with BCR, and that the Tyr phosphorylation of Shc is reduced in the FcγRIIb–BCR co-clustered samples, resulting in decreased activity of p21ras and of mitogenactivated protein kinases (MAPK) (5,6). Furthermore, we found that a synthetic peptide corresponding to the P-ITIM of human FcγRIIb bound SHIP and SHP-2 but not SHP-1 (7). SHP1 was recently shown to be dispensable for FcγRIIb-mediated inhibition of B cell activation, while enhanced phosphorylation of SHIP, and its association with Shc, was demonstrated during negative signaling (8,9). Membrane localization of SHIP by BCR co-aggregated FcγRIIb provides its access to lipid substrates, such as phosphatidylinositol 3,4,5-trisphosphate (PIP3) (10), and hydrolysis of the 59 phosphate may terminate its function as second messenger (11). SHIP may also hydrolyze inositol 1,3,4,5-tetrakisphosphate (InsP4) and hence may influence the InsP4-gated Ca21 channel (12), as well as InsP4induced mobilization of Ca21 from intracellular stores (13). In order to test FcγRIIb1and FcγRIIb2-mediated functions in the same cell type, the FcγRIIb-negative human Burkitt’s lymphoma cell line (ST486) was transfected with FcγRIIb1 and FcγRIIb2 cDNA respectively. To obtain various degrees of co-aggregation of FcγRIIb and BCR, the cells were treated with a constant amount of CD32 mAb, previously found to saturate FcγRIIb, followed by the addition of F(ab9)2 fragments of anti-murine IgG and different amounts of anti-human κ light chain specific mAb. Co-clustering of both FcγRIIb1 and FcγRIIb2 with BCR inhibited the rise of intracellular Ca21 induced by low-dose anti-κ stimulation, while the inhibition was gradually diminished with higher anti-κ doses (Fig. 1). The response of control, nontransfected cells was not affected by CD32 mAb pretreatment. Since the shape of the curves showing a lower Ca21 response in the FcγRIIb–BCR co-clustered samples suggested that not only the Ca21 influx was inhibited, the experiments were repeated in the presence of EGTA. As expected, depleting the extracellular Ca21 with EGTA blocked only the late phase of Ca21 response, representing Ca21 influx from the extracellular space. However, FcγRIIb– BCR co-ligation inhibited the immediate Ca21 release from the endoplasmic reticulum pool in EGTA pretreated samples as well, while EGTA further diminished the late phase of this response (Fig. 2). These data suggest that co-clustering of FcγRIIb1 or FcγRIIb2 with BCR in transfected human Burkitt’s lymphoma cells inhibits Ca21 mobilization from the intracellular pool. Fig. 1. FcγRIIb-mediated inhibition of Ca21 mobilization in ST486 cells, transfected with FcγRIIb1 and FcγRIIb2 respectively, depends on the degree of BCR co-clustering. ST486 human Burkitt’s lymphoma line, negative for FcγRIIb, was transfected with the pRCCMV vector containing FcγRIIb1 and FcγRIIb2 cDNA respectively. Cells (53106) were loaded with 5 μM fluo-3/AM indicator and 30 μg/ml Pluronic F-127 for 30 min at 37°C in 1 ml medium. The cells were diluted 10 times and incubated for another 30 min at 37°C, then washed, resuspended in 53105 cells/ml and labeled with 7-AAD to exclude the dead cells. All studies were carried out in RPMI 1640 culture medium. The cells were preincubated with 2 μg/ml CD32 mAb for 10 min followed by 8 μg/ml F(ab9)2 fragment of anti-mouse IgG (closed symbols), then stimulated with 1 (n), 0.3 (j) or 0.1 (d) μg/ml human κ-chain-specific mAb; or the cells were activated with 1 (∆), 0.3 (u ) or 0.1 (s) μg/ml κchain-specific mAb alone, followed by anti-mouse IgG F(ab’)2. Kinetics of the change of mean fluorescence are shown, as calculated by Lysys II software (Becton Dickinson, San Jose, CA). Ca21 signaling in FcγRIIb-transfected human B cells 143 Fig. 2. Co-clustering FcγRIIb and BCR inhibits Ca21 release from the intracellular pool. The cells were stimulated with 0.3 μg/ml κ-chainspecific mAb followed by 8 μg/ml anti-mouse IgG F(ab’)2 (open symbols) or were preincubated with 2 μg/ml CD32 mAb for 10 min, then triggered with anti-mouse IgG F(ab9)2 and anti-κ mAb (closed symbols), in the presence (squares, bold line) or in the absence (circles) of EGTA . The cells were pretreated with 0.5 mM EGTA at the beginning of the measurements. One of the earliest changes reported on FcγRIIb–BCR co-ligated cells, using intact IgG anti-Ig antibodies, was the inhibition of capacitive Ca21 entry from the extracellular space (14). Triggering B cells, via BCR, induces an elevation of intracellular free Ca21 level, originating in part from the pool of the endoplasmic reticulum and in part from the extracellular Ca21 sources. BCR-associated protein tyrosine kinases (PTK) activate phopholipase C (PLC)γ which converts phosphatidylinositol (4,5) bisphosphate into Ins(1,4,5)P3 and diacyl glycerol. InsP3 binding to its receptor triggers the release of Ca21 from the endoplasmic reticulum. This transient release is followed by capacitive Ca21 entry through an ion-selective channel, prolonging the increase of cytoplasmic Ca21. Thus the Ca21-dependent processes in the cells (Ca21 waves, activity of Ca21-dependent kinases, etc.) are accelerated (15). Here we show for the first time that co-clustering of FcγRIIb and BCR partially inhibits the release of Ca21 from the intracellular pool in human Burkitt’s lymphoma cells transfected with the b1 and b2 isoforms of FcγRIIb respectively. The degree of inhibition depends on the extent of BCR co-aggregation with FcγRIIb saturated with mAb. We did not find any significant difference between FcγRIIb1 and FcγRIIb2 in the transfected cells—both receptor isoforms mediated a similar decrease in Ca21 mobilization at a suboptimal BCR stimulation, while a greater extent of BCR engagement abrogated FcγRIIb-mediated inhibition. This might be the case in vivo, when antigen-specific IgG antibodies have already been secreted and involvement of additional B cells into the response is no longer necessary. The FcγRIIb-mediated inhibition of B cell activation might be overcome by a high dose of antigen, triggering more