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Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Buhl AM, Pleiman CM, Rickert RC, Cambier JC - J. Exp. Med. (1997)

Bottom Line: PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515.Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses.Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA.

ABSTRACT
Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

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Antigen-induced  IP3 and total inositol phosphate  release is greater in the CD19-positive than in CD19-negative  J558Lμm3CD45+ cells. (A)  Cells (10 × 106/ml) were stimulated with 2.5 μg NP9BSA for  various times and the responses  terminated by addition of 100%  TCA (20% TCA final). After organic extraction the aqueous  phase was assayed for IP3 content  in a [3H]IP3 receptor binding inhibition assay (NEN-DuPont).  The results are expressed as fold  increase of IP3 release over basal  (3 pmol IP3/2.5 × 106 cell  equivalents). Shown is mean fold  increase from three independent  experiments ± standard error.  (B) Cells were labeled with Myo- [2-3H(N)]-inositol (21.0 Ci/mmol; 2 μCi/ml) in inositol-free medium containing 5% FCS at 106 cells/ml for 18 h and the generation of inositol phosphates was measured after stimulation of cells for 25 min with 2.5 μg NP9BSA/107 cells/ml. The fold increase in inositol phosphate release was calculated;  the two cell lines had comparable basal rates of inositol turnover as determined by the basal inositol phosphate release. Shown are the mean fold increases from  three independent experiments ± standard error. Statistical significance was determined by JMP version 3.16 statistical software (SAS Institute, Cary, NC).
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Figure 3: Antigen-induced IP3 and total inositol phosphate release is greater in the CD19-positive than in CD19-negative J558Lμm3CD45+ cells. (A) Cells (10 × 106/ml) were stimulated with 2.5 μg NP9BSA for various times and the responses terminated by addition of 100% TCA (20% TCA final). After organic extraction the aqueous phase was assayed for IP3 content in a [3H]IP3 receptor binding inhibition assay (NEN-DuPont). The results are expressed as fold increase of IP3 release over basal (3 pmol IP3/2.5 × 106 cell equivalents). Shown is mean fold increase from three independent experiments ± standard error. (B) Cells were labeled with Myo- [2-3H(N)]-inositol (21.0 Ci/mmol; 2 μCi/ml) in inositol-free medium containing 5% FCS at 106 cells/ml for 18 h and the generation of inositol phosphates was measured after stimulation of cells for 25 min with 2.5 μg NP9BSA/107 cells/ml. The fold increase in inositol phosphate release was calculated; the two cell lines had comparable basal rates of inositol turnover as determined by the basal inositol phosphate release. Shown are the mean fold increases from three independent experiments ± standard error. Statistical significance was determined by JMP version 3.16 statistical software (SAS Institute, Cary, NC).

Mentions: To study CD19 dependence of signaling events upstream from calcium mobilization, we first compared antigen stimulation of IP3 production in CD19-positive and -negative cells. IP3 binds to its receptors on the ER leading to release of Ca2+ into the cytoplasm. Emptying of the ER stores triggers entry of calcium from the extracellular space by a mechanism known as capacitative Ca2+ entry (36). Thus, CD19 dependence of IP3 generation-induced response by antigen could explain the dependence of both intracellular calcium release and extracellular calcium influx on CD19 expression. Stimulation of the cell lines led to a very rapid and sustained rise in IP3 production in the CD19-positive clone as measured using an IP3-receptor binding inhibition assay (Fig. 3 A). Antigen stimulation of the CD19-negative clone led to rapid, but much more modest and only transient generation of IP3. The kinetics and magnitude of IP3 generation was consistent with those of calcium mobilization responses shown in Fig. 2 supporting the possibility that the attenuated calcium mobilization response may be caused by the attenuated BCR-mediated hydrolysis of PIP2. We also measured the generation of [3H]-inositol mono- and polyphosphates after antigen stimulation of myo-[2-3H(N)]-inositol–labeled cell lines. As shown in Fig. 3 B the fold increase in total inositol mono- and polyphosphates generated during a 25-min stimulation was consistent with the mass measurement of IP3 production (Fig. 3 A); stimulation of the CD19-positive clone led to an ∼250% increase in inositol phosphate generation, while stimulation of the CD19-negative cell line induced an ∼30% increase.


Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Buhl AM, Pleiman CM, Rickert RC, Cambier JC - J. Exp. Med. (1997)

Antigen-induced  IP3 and total inositol phosphate  release is greater in the CD19-positive than in CD19-negative  J558Lμm3CD45+ cells. (A)  Cells (10 × 106/ml) were stimulated with 2.5 μg NP9BSA for  various times and the responses  terminated by addition of 100%  TCA (20% TCA final). After organic extraction the aqueous  phase was assayed for IP3 content  in a [3H]IP3 receptor binding inhibition assay (NEN-DuPont).  The results are expressed as fold  increase of IP3 release over basal  (3 pmol IP3/2.5 × 106 cell  equivalents). Shown is mean fold  increase from three independent  experiments ± standard error.  (B) Cells were labeled with Myo- [2-3H(N)]-inositol (21.0 Ci/mmol; 2 μCi/ml) in inositol-free medium containing 5% FCS at 106 cells/ml for 18 h and the generation of inositol phosphates was measured after stimulation of cells for 25 min with 2.5 μg NP9BSA/107 cells/ml. The fold increase in inositol phosphate release was calculated;  the two cell lines had comparable basal rates of inositol turnover as determined by the basal inositol phosphate release. Shown are the mean fold increases from  three independent experiments ± standard error. Statistical significance was determined by JMP version 3.16 statistical software (SAS Institute, Cary, NC).
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Related In: Results  -  Collection

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Figure 3: Antigen-induced IP3 and total inositol phosphate release is greater in the CD19-positive than in CD19-negative J558Lμm3CD45+ cells. (A) Cells (10 × 106/ml) were stimulated with 2.5 μg NP9BSA for various times and the responses terminated by addition of 100% TCA (20% TCA final). After organic extraction the aqueous phase was assayed for IP3 content in a [3H]IP3 receptor binding inhibition assay (NEN-DuPont). The results are expressed as fold increase of IP3 release over basal (3 pmol IP3/2.5 × 106 cell equivalents). Shown is mean fold increase from three independent experiments ± standard error. (B) Cells were labeled with Myo- [2-3H(N)]-inositol (21.0 Ci/mmol; 2 μCi/ml) in inositol-free medium containing 5% FCS at 106 cells/ml for 18 h and the generation of inositol phosphates was measured after stimulation of cells for 25 min with 2.5 μg NP9BSA/107 cells/ml. The fold increase in inositol phosphate release was calculated; the two cell lines had comparable basal rates of inositol turnover as determined by the basal inositol phosphate release. Shown are the mean fold increases from three independent experiments ± standard error. Statistical significance was determined by JMP version 3.16 statistical software (SAS Institute, Cary, NC).
Mentions: To study CD19 dependence of signaling events upstream from calcium mobilization, we first compared antigen stimulation of IP3 production in CD19-positive and -negative cells. IP3 binds to its receptors on the ER leading to release of Ca2+ into the cytoplasm. Emptying of the ER stores triggers entry of calcium from the extracellular space by a mechanism known as capacitative Ca2+ entry (36). Thus, CD19 dependence of IP3 generation-induced response by antigen could explain the dependence of both intracellular calcium release and extracellular calcium influx on CD19 expression. Stimulation of the cell lines led to a very rapid and sustained rise in IP3 production in the CD19-positive clone as measured using an IP3-receptor binding inhibition assay (Fig. 3 A). Antigen stimulation of the CD19-negative clone led to rapid, but much more modest and only transient generation of IP3. The kinetics and magnitude of IP3 generation was consistent with those of calcium mobilization responses shown in Fig. 2 supporting the possibility that the attenuated calcium mobilization response may be caused by the attenuated BCR-mediated hydrolysis of PIP2. We also measured the generation of [3H]-inositol mono- and polyphosphates after antigen stimulation of myo-[2-3H(N)]-inositol–labeled cell lines. As shown in Fig. 3 B the fold increase in total inositol mono- and polyphosphates generated during a 25-min stimulation was consistent with the mass measurement of IP3 production (Fig. 3 A); stimulation of the CD19-positive clone led to an ∼250% increase in inositol phosphate generation, while stimulation of the CD19-negative cell line induced an ∼30% increase.

Bottom Line: PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515.Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses.Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA.

ABSTRACT
Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

Show MeSH