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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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The CD19-negative  and -positive J558Lμm3CD45+  plasmacytoma variants mobilize  [Ca2+]i to different extent after  antigen stimulation. J558Lμm3CD45+CD19− and J558Lμm3CD45+CD19+ cells were loaded  with indo-1 AM, and analysis of  [Ca2+]i initiated before antigen  stimulation (250 ng NP9BSA/ 106cells/ml). Mean [Ca2+]i (top)  and cells responding (bottom) after  antigen stimulation of J558Lμm3CD45+CD19− and J558Lμm3CD45+CD19+. The analysis was  conducted under conditions of  60 nM (dotted line) or 1.3 mM  (solid line) extracellular free calcium concentration buffered as  calculated by the CalCalc program (74). [Ca2+]i was calculated  according to Grynkiewicz et al.  (75). Approximately 600 cells were  analyzed per second. [Ca2+]i in  resting cells is 100 nM.
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Figure 2: The CD19-negative and -positive J558Lμm3CD45+ plasmacytoma variants mobilize [Ca2+]i to different extent after antigen stimulation. J558Lμm3CD45+CD19− and J558Lμm3CD45+CD19+ cells were loaded with indo-1 AM, and analysis of [Ca2+]i initiated before antigen stimulation (250 ng NP9BSA/ 106cells/ml). Mean [Ca2+]i (top) and cells responding (bottom) after antigen stimulation of J558Lμm3CD45+CD19− and J558Lμm3CD45+CD19+. The analysis was conducted under conditions of 60 nM (dotted line) or 1.3 mM (solid line) extracellular free calcium concentration buffered as calculated by the CalCalc program (74). [Ca2+]i was calculated according to Grynkiewicz et al. (75). Approximately 600 cells were analyzed per second. [Ca2+]i in resting cells is 100 nM.

Mentions: To investigate the differences in BCR signaling in the CD19-negative and -positive contexts, we loaded the J558Lμm3 CD45+CD19-negative and CD19-positive variants with indo-1 AM and analyzed changes in intracellular free calcium concentration ([Ca2+]i) in response to antigen stimulation. Initially, this was done under conditions where the free calcium concentration in the medium ([Ca2+]o) was maintained at physiological levels of 1.3 mM. As shown in Fig. 2 (solid lines), antigen stimulation induced a rise in [Ca2+]i in both cell lines; in the CD19-positive cell line the maximal mean [Ca2+]i reached 430 nM while it was 260 nM in the CD19-negative cells. In the CD19-positive cell line, antigen stimulation also induced a much more prolonged increase in [Ca2+]i than in the CD19-negative clone. To determine whether intracellular or extracellular calcium was responsible for the respective [Ca2+]i increases, we added 3 mM EGTA at the time of initiation of analysis of [Ca2+]i. This results in buffering of [Ca2+]o to a concentration of 65 nM (which corresponds to the [Ca2+]i in resting cells) and prevents influx of extracellular calcium into the cell. Comparison of antigen-induced responses of CD19-negative and CD19-positive cells in low [Ca2+]o (Fig. 2, dotted lines) indicated that optimal receptor-mediated release of calcium from intracellular stores requires CD19. Comparison of responses in high [Ca2+]o revealed that influx is also dependent on the expression of CD19 (Fig. 2, solid lines).


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)

The CD19-negative  and -positive J558Lμm3CD45+  plasmacytoma variants mobilize  [Ca2+]i to different extent after  antigen stimulation. J558Lμm3CD45+CD19− and J558Lμm3CD45+CD19+ cells were loaded  with indo-1 AM, and analysis of  [Ca2+]i initiated before antigen  stimulation (250 ng NP9BSA/ 106cells/ml). Mean [Ca2+]i (top)  and cells responding (bottom) after  antigen stimulation of J558Lμm3CD45+CD19− and J558Lμm3CD45+CD19+. The analysis was  conducted under conditions of  60 nM (dotted line) or 1.3 mM  (solid line) extracellular free calcium concentration buffered as  calculated by the CalCalc program (74). [Ca2+]i was calculated  according to Grynkiewicz et al.  (75). Approximately 600 cells were  analyzed per second. [Ca2+]i in  resting cells is 100 nM.
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Related In: Results  -  Collection

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Figure 2: The CD19-negative and -positive J558Lμm3CD45+ plasmacytoma variants mobilize [Ca2+]i to different extent after antigen stimulation. J558Lμm3CD45+CD19− and J558Lμm3CD45+CD19+ cells were loaded with indo-1 AM, and analysis of [Ca2+]i initiated before antigen stimulation (250 ng NP9BSA/ 106cells/ml). Mean [Ca2+]i (top) and cells responding (bottom) after antigen stimulation of J558Lμm3CD45+CD19− and J558Lμm3CD45+CD19+. The analysis was conducted under conditions of 60 nM (dotted line) or 1.3 mM (solid line) extracellular free calcium concentration buffered as calculated by the CalCalc program (74). [Ca2+]i was calculated according to Grynkiewicz et al. (75). Approximately 600 cells were analyzed per second. [Ca2+]i in resting cells is 100 nM.
Mentions: To investigate the differences in BCR signaling in the CD19-negative and -positive contexts, we loaded the J558Lμm3 CD45+CD19-negative and CD19-positive variants with indo-1 AM and analyzed changes in intracellular free calcium concentration ([Ca2+]i) in response to antigen stimulation. Initially, this was done under conditions where the free calcium concentration in the medium ([Ca2+]o) was maintained at physiological levels of 1.3 mM. As shown in Fig. 2 (solid lines), antigen stimulation induced a rise in [Ca2+]i in both cell lines; in the CD19-positive cell line the maximal mean [Ca2+]i reached 430 nM while it was 260 nM in the CD19-negative cells. In the CD19-positive cell line, antigen stimulation also induced a much more prolonged increase in [Ca2+]i than in the CD19-negative clone. To determine whether intracellular or extracellular calcium was responsible for the respective [Ca2+]i increases, we added 3 mM EGTA at the time of initiation of analysis of [Ca2+]i. This results in buffering of [Ca2+]o to a concentration of 65 nM (which corresponds to the [Ca2+]i in resting cells) and prevents influx of extracellular calcium into the cell. Comparison of antigen-induced responses of CD19-negative and CD19-positive cells in low [Ca2+]o (Fig. 2, dotted lines) indicated that optimal receptor-mediated release of calcium from intracellular stores requires CD19. Comparison of responses in high [Ca2+]o revealed that influx is also dependent on the expression of CD19 (Fig. 2, solid lines).

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
Related in: MedlinePlus