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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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BCR-mediated  stimulation of PI3-kinase activation is diminished in splenic B  cells from CD19−/− mice compared with CD19+/+ littermates. Splenic B cells (ρ ⩾1.066)  were purified from spleens of  CD19−/− or CD19+/+ mice, prewarmed, stimulated with F(ab′)2  RAMIG (26.4 μg/5 × 106/ml)  for 2 min, lysed, and then immunoprecipitated with an anti-p85  antibody. Immunoprecipitates  were washed and assayed for PI3-kinase activity. Fold increase in  PI3-kinase activity after stimulation was determined using a  PhosphorImager. (A) Primary  autoradiographic data from a representative experiment. (B) Mean fold increase over basal in three independent experiments ± standard error of the mean.  Statistical significance was determined by JMP version 3.16 statistical software (SAS Institute).
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Figure 10: BCR-mediated stimulation of PI3-kinase activation is diminished in splenic B cells from CD19−/− mice compared with CD19+/+ littermates. Splenic B cells (ρ ⩾1.066) were purified from spleens of CD19−/− or CD19+/+ mice, prewarmed, stimulated with F(ab′)2 RAMIG (26.4 μg/5 × 106/ml) for 2 min, lysed, and then immunoprecipitated with an anti-p85 antibody. Immunoprecipitates were washed and assayed for PI3-kinase activity. Fold increase in PI3-kinase activity after stimulation was determined using a PhosphorImager. (A) Primary autoradiographic data from a representative experiment. (B) Mean fold increase over basal in three independent experiments ± standard error of the mean. Statistical significance was determined by JMP version 3.16 statistical software (SAS Institute).

Mentions: To extend our observations regarding CD19 dependence of BCR-mediated signaling in B lymphocytes we compared BCR-mediated PI3-kinase activation and calcium mobilization responses of B cells from CD19 knockout mice and normal mice. It has previously been reported that no distinguishable difference can be seen between CD19-deficient and normal B cells in the pattern or the degree of substrate phosphorylation after surface immunoglobulin stimulation (27). We obtained the same results when comparing whole cell lysates from CD19−/− and CD19+/+ cells (results not shown). However, careful investigation of several signaling events revealed that ρ ⩾1.066 resting splenic B lymphocytes from CD19−/− mice are impaired in their response to surface immunoglobulin crosslinking when compared with CD19+/+ littermates. F(ab′)2RAMIG-mediated PI3-kinase activation was severely diminished in the CD19−/− B cells (Fig. 10), reminiscent of the situation in the plasmacytoma cell lines. As shown in Fig. 11, absence of CD19 also clearly affects BCR-mediated calcium mobilization. A decrease in calcium mobilization was seen in B lymphocytes from the CD19−/− mice in response to stimulation with an optimal dose of antibody (Fig. 11 A). At suboptimal concentrations of antibody (1.3 μg F(ab′)2RAMIG) almost no BCR-mediated calcium mobilization was seen in cells from CD19−/− mice, while cells from the CD19+/+ littermates still responded well (Fig. 11 B). We obtained equivalent results in 10 independent experiments. Our results appear to conflict with those of Sato et al.(34), who observed enhancement of later phase Ca2+ mobilization in CD19−/− cells. These contrasting findings may result from technical differences in the respective studies.


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)

BCR-mediated  stimulation of PI3-kinase activation is diminished in splenic B  cells from CD19−/− mice compared with CD19+/+ littermates. Splenic B cells (ρ ⩾1.066)  were purified from spleens of  CD19−/− or CD19+/+ mice, prewarmed, stimulated with F(ab′)2  RAMIG (26.4 μg/5 × 106/ml)  for 2 min, lysed, and then immunoprecipitated with an anti-p85  antibody. Immunoprecipitates  were washed and assayed for PI3-kinase activity. Fold increase in  PI3-kinase activity after stimulation was determined using a  PhosphorImager. (A) Primary  autoradiographic data from a representative experiment. (B) Mean fold increase over basal in three independent experiments ± standard error of the mean.  Statistical significance was determined by JMP version 3.16 statistical software (SAS Institute).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199152&req=5

Figure 10: BCR-mediated stimulation of PI3-kinase activation is diminished in splenic B cells from CD19−/− mice compared with CD19+/+ littermates. Splenic B cells (ρ ⩾1.066) were purified from spleens of CD19−/− or CD19+/+ mice, prewarmed, stimulated with F(ab′)2 RAMIG (26.4 μg/5 × 106/ml) for 2 min, lysed, and then immunoprecipitated with an anti-p85 antibody. Immunoprecipitates were washed and assayed for PI3-kinase activity. Fold increase in PI3-kinase activity after stimulation was determined using a PhosphorImager. (A) Primary autoradiographic data from a representative experiment. (B) Mean fold increase over basal in three independent experiments ± standard error of the mean. Statistical significance was determined by JMP version 3.16 statistical software (SAS Institute).
Mentions: To extend our observations regarding CD19 dependence of BCR-mediated signaling in B lymphocytes we compared BCR-mediated PI3-kinase activation and calcium mobilization responses of B cells from CD19 knockout mice and normal mice. It has previously been reported that no distinguishable difference can be seen between CD19-deficient and normal B cells in the pattern or the degree of substrate phosphorylation after surface immunoglobulin stimulation (27). We obtained the same results when comparing whole cell lysates from CD19−/− and CD19+/+ cells (results not shown). However, careful investigation of several signaling events revealed that ρ ⩾1.066 resting splenic B lymphocytes from CD19−/− mice are impaired in their response to surface immunoglobulin crosslinking when compared with CD19+/+ littermates. F(ab′)2RAMIG-mediated PI3-kinase activation was severely diminished in the CD19−/− B cells (Fig. 10), reminiscent of the situation in the plasmacytoma cell lines. As shown in Fig. 11, absence of CD19 also clearly affects BCR-mediated calcium mobilization. A decrease in calcium mobilization was seen in B lymphocytes from the CD19−/− mice in response to stimulation with an optimal dose of antibody (Fig. 11 A). At suboptimal concentrations of antibody (1.3 μg F(ab′)2RAMIG) almost no BCR-mediated calcium mobilization was seen in cells from CD19−/− mice, while cells from the CD19+/+ littermates still responded well (Fig. 11 B). We obtained equivalent results in 10 independent experiments. Our results appear to conflict with those of Sato et al.(34), who observed enhancement of later phase Ca2+ mobilization in CD19−/− cells. These contrasting findings may result from technical differences in the respective studies.

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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