A double-edged kinase Lyn: a positive and negative regulator for antigen receptor-mediated signals

Abstract

B cells from young lyn-/- mice are hyperresponsive to anti-IgM-induced proliferation, suggesting involvement of Lyn in negative regulation of B cell antigen receptor (BCR)-mediated signaling. Here we show that tyrosine phosphorylation of FcgammaRIIB and CD22 coreceptors, which are important for feedback suppression of BCR-induced signaling, was severely impaired in lyn-/- B cells upon their coligation with the BCR. Hypophosphorylation on tyrosine residues of these molecules resulted in failure of recruiting the tyrosine phosphatase SHP-1 and inositol phosphatase SHIP, SH2-containing potent inhibitors of BCR-induced B cell activation, to the coreceptors. Consequently, lyn-/- B cells exhibited defects in suppressing BCR-induced Ca2+ influx and proliferation. Thus, Lyn is critically important in tyrosine phosphorylation of the coreceptors, which is required for feedback suppression of B cell activation.

Figure 1

(A) Serum levels of IgM and IgA in unimmunized infant mice. Wild-type mice (closed circles) and lyn ^−/− mice (open circles) were bled at 0–4 wk of age. Concentrations of IgM and IgA in their serum were determined by isotype-specific ELISA (13). Averages of the results from three experiments are shown. (B) Relative proliferative responses to BCR cross-linking. Splenic B cells from wild-type (left) or lyn ^−/− (right) mice were cultured with goat F(ab′)₂ anti-IgM (5 μg/ml) or goat intact anti-IgM (1 or 10 μg/ml). After 42 h of incubation, cells were pulse labeled for 6 h with [³H]thymidine, and [³H]thymidine incorporation was determined (13). All assays were performed in triplicate. The proliferative responses were presented with [³H]thymidine incorporation expressed as a percentage of the response against F(ab′)₂ anti-IgM stimulation.

Figure 2

(A) Anti-IgM–induced tyrosine phosphorylation of CD22. Splenic B cells were stimulated with F(ab′)₂ anti-IgM (30 μg/ml) (lanes 3 and 6) or intact anti-IgM (50 μg/ml) (lanes 2 and 5). Anti-CD22 immunoprecipitates from the cell lysates were probed with α-PY (phosphotyrosine antibody; top) or anti-CD22 antibody (bottom) by immunoblotting. (B) Anti-IgM–induced tyrosine phosphorylation of SHP-1 and its associated molecules. Splenic B cells were stimulated with intact anti-IgM (50 μg/ml) for indicated times. Anti–SHP-1 immunoprecipitates from the cell lysates were probed with antiphosphotyrosine monoclonal antibody 4G10 (α-PY) by immunoblotting. About 150-kD proteins (upper arrowhead) coimmunoprecipitated with SHP-1 (lower arrowhead) corresponded to CD22.

Figure 2

(A) Anti-IgM–induced tyrosine phosphorylation of CD22. Splenic B cells were stimulated with F(ab′)₂ anti-IgM (30 μg/ml) (lanes 3 and 6) or intact anti-IgM (50 μg/ml) (lanes 2 and 5). Anti-CD22 immunoprecipitates from the cell lysates were probed with α-PY (phosphotyrosine antibody; top) or anti-CD22 antibody (bottom) by immunoblotting. (B) Anti-IgM–induced tyrosine phosphorylation of SHP-1 and its associated molecules. Splenic B cells were stimulated with intact anti-IgM (50 μg/ml) for indicated times. Anti–SHP-1 immunoprecipitates from the cell lysates were probed with antiphosphotyrosine monoclonal antibody 4G10 (α-PY) by immunoblotting. About 150-kD proteins (upper arrowhead) coimmunoprecipitated with SHP-1 (lower arrowhead) corresponded to CD22.

Figure 3

(A) Anti-IgM–induced tyrosine phosphorylation of FcγRIIB. Splenic B cells from wild-type (lanes 1–3), lyn ^−/− (lanes 4–6), or fyn ^−/− (lanes 7–9) mice were stimulated with F(ab′)₂ anti-IgM (30 μg/ml; lanes 3, 6, and 9) or intact anti-IgM (50 μg/ml; lanes 2, 5, and 8) for 2 min. Proteins were immunoprecipitated with 2.4G2 anti-FcγRIIB monoclonal antibody from the cell lysates, and the precipitates probed with α-PY (top) or anti-FcγRIIB antibody (α-mβ1; bottom) by immunoblotting. (B) Recruitment of SHIP to FcγRIIB. Splenic B cells from wild-type (lanes 1–3) or lyn ^−/− (lanes 4–6) mice were stimulated with F(ab′)₂ anti-IgM (30 μg/ml; lanes 3 and 6) or intact anti-IgM (50 μg/ml; lanes 2 and 5) for 2 min. Anti-FcγRIIB (2.4G2) immunoprecipitates from the cell lysates were probed with anti-SHIP antibodies by immunoblotting. (C) Time course of Ca²⁺ flux in splenic B cells upon BCR coligation to FcγRIIB. Panels show real time Fura-2 ratios (340/380) for splenic B cells of wild-type mice (top) and lyn ^−/− mice (bottom). Splenic B cells were stimulated with F(ab′)₂ anti-IgM (5 μg/ml; line), intact anti-IgM (10 μg/ ml; dotted line), or intact anti-IgM (10 μg/ml) in the presence of 1.5 mM EGTA (perforated line).

Figure 3

(A) Anti-IgM–induced tyrosine phosphorylation of FcγRIIB. Splenic B cells from wild-type (lanes 1–3), lyn ^−/− (lanes 4–6), or fyn ^−/− (lanes 7–9) mice were stimulated with F(ab′)₂ anti-IgM (30 μg/ml; lanes 3, 6, and 9) or intact anti-IgM (50 μg/ml; lanes 2, 5, and 8) for 2 min. Proteins were immunoprecipitated with 2.4G2 anti-FcγRIIB monoclonal antibody from the cell lysates, and the precipitates probed with α-PY (top) or anti-FcγRIIB antibody (α-mβ1; bottom) by immunoblotting. (B) Recruitment of SHIP to FcγRIIB. Splenic B cells from wild-type (lanes 1–3) or lyn ^−/− (lanes 4–6) mice were stimulated with F(ab′)₂ anti-IgM (30 μg/ml; lanes 3 and 6) or intact anti-IgM (50 μg/ml; lanes 2 and 5) for 2 min. Anti-FcγRIIB (2.4G2) immunoprecipitates from the cell lysates were probed with anti-SHIP antibodies by immunoblotting. (C) Time course of Ca²⁺ flux in splenic B cells upon BCR coligation to FcγRIIB. Panels show real time Fura-2 ratios (340/380) for splenic B cells of wild-type mice (top) and lyn ^−/− mice (bottom). Splenic B cells were stimulated with F(ab′)₂ anti-IgM (5 μg/ml; line), intact anti-IgM (10 μg/ ml; dotted line), or intact anti-IgM (10 μg/ml) in the presence of 1.5 mM EGTA (perforated line).

Figure 3

(A) Anti-IgM–induced tyrosine phosphorylation of FcγRIIB. Splenic B cells from wild-type (lanes 1–3), lyn ^−/− (lanes 4–6), or fyn ^−/− (lanes 7–9) mice were stimulated with F(ab′)₂ anti-IgM (30 μg/ml; lanes 3, 6, and 9) or intact anti-IgM (50 μg/ml; lanes 2, 5, and 8) for 2 min. Proteins were immunoprecipitated with 2.4G2 anti-FcγRIIB monoclonal antibody from the cell lysates, and the precipitates probed with α-PY (top) or anti-FcγRIIB antibody (α-mβ1; bottom) by immunoblotting. (B) Recruitment of SHIP to FcγRIIB. Splenic B cells from wild-type (lanes 1–3) or lyn ^−/− (lanes 4–6) mice were stimulated with F(ab′)₂ anti-IgM (30 μg/ml; lanes 3 and 6) or intact anti-IgM (50 μg/ml; lanes 2 and 5) for 2 min. Anti-FcγRIIB (2.4G2) immunoprecipitates from the cell lysates were probed with anti-SHIP antibodies by immunoblotting. (C) Time course of Ca²⁺ flux in splenic B cells upon BCR coligation to FcγRIIB. Panels show real time Fura-2 ratios (340/380) for splenic B cells of wild-type mice (top) and lyn ^−/− mice (bottom). Splenic B cells were stimulated with F(ab′)₂ anti-IgM (5 μg/ml; line), intact anti-IgM (10 μg/ ml; dotted line), or intact anti-IgM (10 μg/ml) in the presence of 1.5 mM EGTA (perforated line).

Figure 4

(A) Tyrosine phosphorylation of FcγRIIB upon FcεRI and FcγRIIB coligation. BMMCs from wild-type (lanes 1–4), lyn ^−/− (lanes 5–8) mice were sensitized with anti-DNP monoclonal IgE (10 μg/ml) followed by stimulation for 2 min at 37°C with 30 ng/ml DNP-HSA (FcεRI cross-linking; lanes 2 and 6), or DNP-HSA/rabbit anti-HSA IgG immune complexes (FcγRII coligation to FcεRI; lanes 3 and 7). BMMCs were stimulated with DNP-HSA/rabbit anti-HSA IgG immune complexes without IgE sensitization (lanes 4 and 8). Anti-FcγRIIB (2.4G2) immunoprecipitates from the cell lysates were probed with α-PY (top) or anti-FcγRIIB antibody (α-mβ1) (bottom) by immunoblotting. (B) Inhibitory effect of FcγRIIB coligation to FcεRI in lyn ^−/− BMMCs. The mast cell was sensitized with biotinylated mouse IgE, followed by cross-linking with streptavidin (no FcγRII coligation). FcγRIIB was coligated to FcεRI by adding biotinylated anti-FcγRII monoclonal antibody (biotin-2.4G2) at the sensitization step (100% FcγRII coligation). A mixture of biotinylated/nonbiotinylated 2.4G2 (1/9 for 10% or 1/99 for 1% coligation) was used to vary the extent of FcγRII coligation. The degree of degranulation was determined by measuring the release of β-hexosaminidase as described (12). Closed columns, the results of wild-type mice; open columns, the results of lyn ^−/− mice. Standard errors of triplicate samples are indicated on each column.

Figure 4

(A) Tyrosine phosphorylation of FcγRIIB upon FcεRI and FcγRIIB coligation. BMMCs from wild-type (lanes 1–4), lyn ^−/− (lanes 5–8) mice were sensitized with anti-DNP monoclonal IgE (10 μg/ml) followed by stimulation for 2 min at 37°C with 30 ng/ml DNP-HSA (FcεRI cross-linking; lanes 2 and 6), or DNP-HSA/rabbit anti-HSA IgG immune complexes (FcγRII coligation to FcεRI; lanes 3 and 7). BMMCs were stimulated with DNP-HSA/rabbit anti-HSA IgG immune complexes without IgE sensitization (lanes 4 and 8). Anti-FcγRIIB (2.4G2) immunoprecipitates from the cell lysates were probed with α-PY (top) or anti-FcγRIIB antibody (α-mβ1) (bottom) by immunoblotting. (B) Inhibitory effect of FcγRIIB coligation to FcεRI in lyn ^−/− BMMCs. The mast cell was sensitized with biotinylated mouse IgE, followed by cross-linking with streptavidin (no FcγRII coligation). FcγRIIB was coligated to FcεRI by adding biotinylated anti-FcγRII monoclonal antibody (biotin-2.4G2) at the sensitization step (100% FcγRII coligation). A mixture of biotinylated/nonbiotinylated 2.4G2 (1/9 for 10% or 1/99 for 1% coligation) was used to vary the extent of FcγRII coligation. The degree of degranulation was determined by measuring the release of β-hexosaminidase as described (12). Closed columns, the results of wild-type mice; open columns, the results of lyn ^−/− mice. Standard errors of triplicate samples are indicated on each column.