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Brief Definitive Reports |
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
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Abstract |
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RIIB 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.
The Src family protein-tyrosine kinase, Lyn, is highly expressed in hematopoietic cells. Lyn physically associates with the BCR in B cells and with Fc
Recently, however, two groups reported that B cells from young lyn–/– mice were hyperresponsive to anti-IgM–induced proliferation due in part to the impairment of Fc
Proliferative Responses.
Qualification of Total Ig by ELISA.
Immunoprecipitation and Immunoblotting.
FcR Cross-linking and Degranulation Assay.
Measurement of Internal Ca2+ Concentration.
RI in mast cells, and is rapidly activated upon cross-linking of the antigen receptors (1–3). Lyn interacts with and phosphorylates a number of substrates, such as the Syk kinase (4, 5), HS1 protein (6, 7), and Cbl protooncogene product (8). Taken together, Lyn is thought to play important roles in the antigen receptor–mediated positive signaling. RIIB-mediated feedback suppression of the B cell antigen receptor (BCR) signaling (9, 10). Therefore, it is suggested that Lyn may play some roles in the antigen receptor–mediated negative signaling, too. In this study, using splenic B cells or bone marrow–derived mast cells (BMMCs) from lyn–/– mice, we addressed molecular mechanisms by which the Lyn kinase would act as a key regulator of antigen receptor signaling.
Materials and Methods
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Abstract
Materials and Methods
Results and Discussion
References
Cells and Cell Culture.
All lyn–/– mice had been back-crossed at least six generations to C57BL/6J. Splenic B cells were isolated from 6–8-wk-old mice by T cell depletion with anti-Thy 1.2 mAb and with rabbit complement, followed by Percoll gradient purification (11). The resulting cells were >85% B220+ as determined by FACS® (Becton Dickinson, Mountain View, CA) analysis. BMMCs were obtained from bone marrow cells cultured with IL-3 for at least 4 wk as described (12).
For proliferation assay, B cells (105/ well) were cultured in 96-well flat-bottomed tissue culture plates either alone or in the presence of goat F(ab')2 anti-IgM (Cappel, Durham, NC) or intact anti-IgM (Southern Biotechnology Assoc., Inc., Birmingham, AL). Cultured cells were pulse-labeled and assayed as described (13). All assays were performed in triplicate with <20% variation among assays.
Amounts of each Ig isotype in sera and in culture supernatants were determined by ELISA with antibodies specific for each membrane-bound Ig (mIg) isotype (13).
For the activation of B cells, splenic B cells were treated with goat F(ab')2 anti-IgM (Cappel) or intact anti-IgM (Southern Biotechnology Assoc., Inc.) for 2 min at 37°C. For the activation of mast cells, BMMCs were sensitized for 1 h with antidinitrophenyl (anti-DNP) monoclonal IgE (10 µg/ml; Sigma Chemical Co., St. Louis, MO) followed by stimulation for 2 min at 37°C with 30 ng/ml DNP-conjugated human serum albumin (DNP-HSA; resulting in FcRI cross-linking; Sigma Chemical Co.), or DNP-HSA/rabbit anti-HSA IgG immune complexes (resulting in FcRII coligation to FcRI). The stimulated splenic B cells (107) or BMMCs (107) were lysed in TNE (1% [vol/vol] Nonident P-40, 50 mM Tris-HCl [pH 8], 20 mM EDTA, and 0.2 mM sodium orthovanadate with aprotinin at 10 µg/ml) buffer and subjected to immunoprecipitation/immunoblotting using SDS-7.5% PAGE as described (14). Antibodies used in these experiments were 2.4G2 mAb (PharMingen, San Diego, CA), anti-FcRIIB (
-mβ1, gift from J.V. Ravetch, The Rockefeller University, New York), anti-CD22 (gift from E.A. Clark, University of Washington, Seattle, WA), anti–SHP-1 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-SHIP (gift from M.N. Lioubin, Fred Hutchinson Cancer Research Center, Seattle, WA). Biotinylated 4G10 antiphosphotyrosine antibody (Upstate Biotechnology Inc., Lake Placid, NY) was used to detect phosphorylated proteins in the immunoblotting experiments. The blots were treated with horseradish peroxidase (HRPO)-streptavidin, and then antibody-reacted bands were visualized by the use of enhanced chemiluminescence detection system (Amersham, Co., Arlington Heights, IL).
FcRI-triggered mast cell activation was induced by 1 h of sensitization with 10 µg/ml biotinylated mouse IgE, followed by cross-linking with 10 µg/ml streptavidin (0% of FcRII coligation). FcRII was coligated to FcRI by adding biotinylated anti-FcRII monoclonal antibody (biotin-2.4G2) at the sensitization step (100% of FcRII 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 FcRII coligation. The degree of degranulation was determined by measuring the release of β-hexosaminidase as described (12).
Splenic B cells and IgE-sensitized BMMCs from lyn+/+ and lyn–/– mice were incubated with 3 µM Fura-2/acetoxymethyl (AM; Dojindo, Osaka, Japan) in PBS containing 20 mM Hepes (pH 7.2), 5 mM glucose, 0.025% BSA, and 1 mM CaCl2 at 37°C for 45 min. After the reaction, the cells were resuspended in 500 µl of the same buffer at 2 x 106 cells/ml in a stirring cuvette. Then, by using CAF-110 fluorescence spectrophotometer (JASCO, Osaka, Japan), emission at 500 nm was monitored after excitation of the sample with two different wavelengths (340 and 380 nm).
Results and Discussion
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Abstract
Materials and Methods
Results and Discussion
References
lyn–/– mice, generated by gene targeting, exhibit splenomegaly as they age. The enlarged spleen accumulates unusual lymphoblast-like cells and plasma cells (13, 15). lyn–/– mice with splenomegaly contain high concentrations of serum IgM and IgA, and often develop glomerulonephritis due to the production of autoreactive antibodies (13, 15). The lymphoblast-like cells are IgM+/–, IgD+/–, CD5–, Mac-1+, CD23–, B220+/–, and IL-5R+/–, and are therefore B-1b–like cells. These cells produce spontaneously a large amount of the antibodies. However, we found that serum levels of IgM and IgA were extremely high in lyn–/– mice compared to wild-type mice even at their early ages (<1 mo old) when the lymphoblast-like cells did not yet accumulate (Fig. 1 A). This suggests that Lyn is involved in negative regulation of antibody production by the normal B cells.
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RIIB, a low-affinity immunoglobulin G receptor (16, 17). In contrast to wild-type B cells, lyn–/– B cells from young mice (<6 wk old) were highly responsive to the intact anti-IgM, exhibiting a proliferation level similar to that with F(ab')2 anti-IgM. The data suggest that the lyn–/– B cells are defective in FcRIIB-mediated suppression of BCR signaling (9, 10, and Fig. 1 B). Furthermore, the disorders of lyn–/– mice, such as the hyperresponse to proliferation after BCR ligation and high concentrations of serum Igs, are found in mice lacking FcRIIB (18). Moreover, the phenotypes of mice deficient in SHP-1, an SH2-containing protein tyrosine phosphatase (19, 20), or CD22, a B-lineage specific surface molecule (21–24), overlap those of lyn–/– mice. Note that both SHP-1 and CD22 participate in the feedback suppression of BCR signaling, and that tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs; reference 25) of CD22 and FcRIIB is involved in the suppression (26–28). The expression levels of SHP-1, CD22, and FcRII in lyn–/– mice did not differ from those in wild-type mice (data not shown). Therefore, the inhibitory signalings through SHP-1, CD22, and FcRIIB are likely related to Lyn kinase activity. Upon stimulation of the BCR, CD22 and SHP-1 become rapidly tyrosine-phosphorylated (29) and associate with one another (30–32). To determine whether Lyn is involved in the tyrosine phosphorylation of CD22 and SHP-1, we immunoprecipitated these proteins from the lysates of BCR–cross-linked splenic B cells and probed the immunoprecipitates with antiphosphotyrosine antibody. As shown in Fig. 2 A, tyrosine phosphorylation of CD22 was extremely impaired in lyn–/– B cells (lanes 5 and 6), as compared with that in wild-type B cells (lanes 2 and 3). A level of tyrosine phosphorylation of SHP-1 was also low, and association of CD22 with SHP-1 was poor in the absence of Lyn (Fig. 2 B and data not shown).
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RIIB immunoprecipitates prepared from the lysates of wild-type and lyn–/– splenic B cells on which FcRIIB and BCR had been cross-linked. Immunoblot analysis of the precipitates with antiphosphotyrosine antibody (Fig. 3 A) showed a very low level of tyrosine phosphorylation of FcRIIB in the absence of Lyn. The recruitment of SHIP, an inositol phosphatase important for the feedback suppression (26–28), from cytoplasm to membrane was also impaired in lyn–/– B cells (Fig. 3 B). Accordingly, the magnitude of BCR-induced calcium influx was less affected by coligated FcRIIB in lyn–/– B cells than in wild-type B cells (Fig. 3 C). Moreover, as mentioned above, the proliferative response to BCR cross-linking was not suppressed by coligated FcRIIB in lyn–/– B cells (Fig. 1 B). Thus, in the case of B cells, Lyn is directly involved in the feedback suppression mechanisms as well as in the BCR positive signaling. The controversial observation that B cells from relatively older lyn–/– mice are hyporeactive to BCR stimulation (13, 15) may suggest that negative signaling pathways are differently tuned with age.
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RI-mediated signaling prompted us to examine whether tyrosine phosphorylation of FcRIIB coligated with FcRI was impaired in lyn–/– mast cells. As shown in Fig. 4 A, tyrosine phosphorylation of coligated FcRIIB was suppressed in the lyn–/– BMMCs. This likely results in the failure of recruitment of SHIP and/or SHP-1 to the ITIM of FcRIIB, leading to the lack of suppression of FcRI signaling by the inhibitory coreceptor. However, in both wild-type and lyn–/– BMMCs, the calcium response after IgE cross-linking was similarly suppressed by coligation of FcRIIB with FcRI–IgE complexes (data not shown). Furthermore, coligation of FcRIIB with FcRI–IgE complexes with increasing amounts of the cross-linking antibody resulted in increasing inhibition of degranulation, as measured by β-hexosaminidase release, in both wild-type and lyn–/– BMMCs (Fig. 4 B). Coligation of FcRIIB with FcRIII on both wild-type and lyn–/– BMMCs also inhibited receptor-triggered degranulation (the pair columns at the foremost right in Fig. 4 B). These results suggest that, unlike the case with the B cells, the negative signaling pathways appear to function similarly in wild-type and lyn–/– BMMCs.
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RIIB feedback function may be due to differences in their FcRIIB-mediated signalings. Such differences could produce the cell-type specific balance between the positive and negative signalings. Present data together with previous observations (9, 10, 12, 13, 15) indicate that positive signaling is less affected than negative signaling in lyn–/– B cells, and vice versa in lyn–/– mast cells. The balance of the positive and negative signalings in lyn–/– cells could well be influenced by some other Src-like kinases and Syk kinase expressed in the cells. In B cells, the imbalance induced by lyn deficiency seems to severely affect the immune responses. As a consequence, lyn–/– mice, which have lost the control of positive and negative signalings, exhibit abnormal phenotypes such as hyperactivation of B cells (9, 10) and elevated serum levels of IgM and IgA (13, 15), resulting in development of autoimmune disease (13, 15).
Our data show that Lyn is critically involved in tyrosine phosphorylation of particular proteins such as CD22 and FcRIIB that are involved in the feedback regulation in B cells and/or BMMCs (for review see references 33–35). Lyn is also important in tyrosine phosphorylation of immunoreceptor tyrosine-based activation motif (ITAM) family of proteins and the proteins involved in positive regulation of the BCR and FcRI signaling events (4, 5, 12, 13, 15, 36). The other membrane molecules with the putative ITIMs have been described (37, 38). These include KIR on NK cells, glycoprotein 49B and MAFA-1 on mast cells, and CD23 and CD72 on B cells. To understand the molecular basis of the balance of the positive and negative signalings, it is important to address what kinases preferentially tyrosine phosphorylate and regulate these molecules. The candidates include the Src family kinases, JAK kinases, Tec family kinases, and Syk/ZAP-70 kinases. The balance, of course, could be affected by the absence of a critical kinase(s).
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Acknowledgments |
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This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, Human Frontier Science Program Organization, and Japan Society for the Promotion of Science.
Submitted: 22 October 1997
Revised: 28 January 1998
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References |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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