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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 induction of [Ca2+]i mobilization in splenic B cells  from normal mice is sensitive to wortmannin. Splenic B cells (ρ ⩾1.066)  were isolated from spleens of normal mice, and loaded with Indo-1 AM.  The cells were preincubated with DMSO or increasing concentrations of  wortmannin for 25 min at 25°C. They were then warmed to 37°C for 4  min and stimulated with 13.2 μg F(ab′)2RAMIG/106/ml (at arrow).  Shown is the mean [Ca2+]i over time with analysis of ∼600 cells/s.  [Ca2+]i in resting cells is 70 nM.
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Figure 9: Antigen induction of [Ca2+]i mobilization in splenic B cells from normal mice is sensitive to wortmannin. Splenic B cells (ρ ⩾1.066) were isolated from spleens of normal mice, and loaded with Indo-1 AM. The cells were preincubated with DMSO or increasing concentrations of wortmannin for 25 min at 25°C. They were then warmed to 37°C for 4 min and stimulated with 13.2 μg F(ab′)2RAMIG/106/ml (at arrow). Shown is the mean [Ca2+]i over time with analysis of ∼600 cells/s. [Ca2+]i in resting cells is 70 nM.

Mentions: To investigate the role of PI3-kinase in antigen-induced signaling in a more physiological system, we purified splenic B cells (ρ ⩾1.066) from spleens of normal mice and assessed the effects of wortmannin on BCR signaling. After preincubation for 30 min with DMSO or wortmannin, splenic B cells were stimulated with 13.2 μg/106 cells F(ab′)2RAMIG. As observed in the J558L μm3CD45+CD19+ cell line, wortmannin inhibited, as a function of its concentration, the antigen-mediated calcium mobilization in these primary B cells (Fig. 9). Wortmannin also inhibited the residual antibody-mediated Ca2+ mobilization in CD19−/− splenic B cells (data not shown). Thus, BCR signaling in J558Lμm3CD45+CD19+ and normal B cells is similarly sensitive to PI3-kinase inhibition and CD19 function (see below).


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 induction of [Ca2+]i mobilization in splenic B cells  from normal mice is sensitive to wortmannin. Splenic B cells (ρ ⩾1.066)  were isolated from spleens of normal mice, and loaded with Indo-1 AM.  The cells were preincubated with DMSO or increasing concentrations of  wortmannin for 25 min at 25°C. They were then warmed to 37°C for 4  min and stimulated with 13.2 μg F(ab′)2RAMIG/106/ml (at arrow).  Shown is the mean [Ca2+]i over time with analysis of ∼600 cells/s.  [Ca2+]i in resting cells is 70 nM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199152&req=5

Figure 9: Antigen induction of [Ca2+]i mobilization in splenic B cells from normal mice is sensitive to wortmannin. Splenic B cells (ρ ⩾1.066) were isolated from spleens of normal mice, and loaded with Indo-1 AM. The cells were preincubated with DMSO or increasing concentrations of wortmannin for 25 min at 25°C. They were then warmed to 37°C for 4 min and stimulated with 13.2 μg F(ab′)2RAMIG/106/ml (at arrow). Shown is the mean [Ca2+]i over time with analysis of ∼600 cells/s. [Ca2+]i in resting cells is 70 nM.
Mentions: To investigate the role of PI3-kinase in antigen-induced signaling in a more physiological system, we purified splenic B cells (ρ ⩾1.066) from spleens of normal mice and assessed the effects of wortmannin on BCR signaling. After preincubation for 30 min with DMSO or wortmannin, splenic B cells were stimulated with 13.2 μg/106 cells F(ab′)2RAMIG. As observed in the J558L μm3CD45+CD19+ cell line, wortmannin inhibited, as a function of its concentration, the antigen-mediated calcium mobilization in these primary B cells (Fig. 9). Wortmannin also inhibited the residual antibody-mediated Ca2+ mobilization in CD19−/− splenic B cells (data not shown). Thus, BCR signaling in J558Lμm3CD45+CD19+ and normal B cells is similarly sensitive to PI3-kinase inhibition and CD19 function (see below).

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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