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Abstract
Regulation of B cell receptor (BCR)-induced Ca2+ signaling by CD40 co-stimulation was compared in long-term BCR-stimulated immature (WEHI-231) and mature (Bal-17) B cells. In response to long-term pre-stimulation of immature WEHI-231 cells to α-IgM antibody (0.5 ∼ 48 hr), the initial transient decrease in BCR-induced [Ca2+]i was followed by spontaneous recovery to control level within 24 hr. The recovery of Ca2+ signaling in WEHI-231 cells was not due to restoration of internalized receptor but instead to an increase in the levels of PLCγ2 and IP3R-3. CD40 co-stimulation of WEHI-231 cells prevented BCR-induced cell cycle arrest and apoptosis, and it strongly inhibited the recovery of BCR-induced Ca2+ signaling. CD40 co-stimulation also enhanced BCR internalization and reduced expression of PLCγ2 and IP3R-3. Pre-treatment of WEHI-231 cells with the antioxidant N-acetyl-L-cysteine (NAC) strongly inhibited CD40-mediated prevention of the recovery of Ca2+ signaling. In contrast to immature WEHI-231 cells, identical long-term α-IgM pre-stimulation of mature Bal-17 cells abolished the increase in BCR-induced [Ca2+]i, regardless of CD40 co-stimulation. These results suggest that CD40-mediated signaling prevents antigen-induced cell cycle arrest and apoptosis of immature B cells through inhibition of sustained BCR-induced Ca2+ signaling.
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Fig. 1.
Differential BCR-induced Ca2+ responses and its modulation by CD40 co-stimulation in Bal-17 and WEHI-231 cells. B cells were pretreated with 5 μg/ml of α-IgM (Aa, Ba) or α-IgM + CD40L (0.5 μg/ml) (Ab, Bb) for 0.5, 2, 4, 8, 24, and 48 hr. After harvesting the cells, the pre-treatment agents were removed, and α-IgM (5 μg/ml)-triggered [Ca2+]i increases were measured in 1.5 mM [Ca2+]o containing normal Tyrode solution. Representative [Ca2+]i traces obtained from each B cell type are drawn, and the duration of α-IgM pre-stimulation (0, 2, 8, and 24 hr) are indicated inside the figure. (Ac, Bc) Δ[Ca2+]i values (difference between the resting and peak [Ca2+]i levels) were calculated and normalized against Δ[Ca2+]i values of un-stimulated control cells. Changes in Δ[Ca2+]i values are plotted against the duration of pre-stimulation. All experiments were repeated at least three times, and the detailed values are described in the text. ∗p<0.05.
Fig. 2.
Contribution of cell membrane potential, amounts of intracellular Ca2+ release and store-operated Ca2+ entry (SOC) on sustained BCR-induced Ca2+ responses in immature WEHI-231 cells. WEHI-231 cells were pretreated with 5 μg/ ml of α-IgM or α-IgM+CD40L (0.5 μg/ml) for 24 hr. After harvesting the cells, the pre-treatment agents were removed, and α-IgM (5 μg/ml)-triggered [Ca2+]i increases were measured in 130 mM [KCl]o and 1.5 mM [Ca2+]o containing solution (A) or extracellular Ca2+-free Tyrode solution (B). The amounts of store-operated Ca2+ entry in response to the stimulation of the cells with α-IgM (B) or thapsigargin (C) were measured by the re-addition of 1.5 mM [Ca2+]o after depleting the stores in Ca2+-free Tyrode solution. (D) Δ[Ca2+]i values (difference between the resting and peak [Ca2+]i levels) from (A), (B) and (C) were calculated and plotted. Mean values±S.E.M. from at least 3 repeats were shown. #p<0.05.
Fig. 3.
Inhibition of α-IgM-induced cell cycle arrest and apoptosis of immature WEHI-231 cells by CD40 co-stimulation. WEHI-231 and Bal-17 cells were stimulated with α-IgM or α-IgM+CD40L for 24 and 48 hr, after which FACS analysis was performed to quantify the degree of cell cycle arrest (A∼C) and apoptosis (D∼F). Cell cycle and apoptosis were analyzed on linear FL2-area (FL2-A, total cell PI fluorescence) and logarithmic FL2-height (FL2-H, maximum PI fluorescence emission) scales, respectively. All experiments were repeated three times. Mean±S.E.M. ∗,#p<0.05, ∗∗,##p<0.01, N.S., no significance.
Fig. 4.
Effects of CD40 co-stimulation on internalization of surface BCR triggered by α-IgM pre-stimulation. WEHI-231 (A) and Bal-17 (B) cells were stimulated with α-IgM alone or α-IgM + CD40L for the indicated times, followed by surface BCR measurement indicated by PE fluorescence emission (FL2-H) in FACS machine. Effects of CD40 co-stimulation on α-IgM-triggered internalization of BCR were quantified and normalized (% surface BCR) to that of un-treated cells and then plotted against the indicated times of stimulation in WEHI-231 (C) and Bal-17 (D) cells. Mean±S.E.M. values of three repeats are plotted. ∗p<0.05.
Fig. 5.
Effects of CD40 co-stimulation on expression of PLCγ2 and IP3R-3. WEHI-231 (A) and Bal-17 (B) cells were treated long-term with α-IgM in the absence or presence of CD40 co-stimulation. Extracted total protein content was subjected to Western blotting to measure PLCγ2 and IP3R-3 expression. β-Actin was used as a protein loading control. The quantified intensities of PLCγ2 (PLC32/β-actin) and IP3R-3 (IP3R-3/β-actin) were normalized (% expression) to those of un-treated cells and then plotted against the indicated times of stimulation. Mean±S.E.M. of three independent experiments are shown in A and B. ∗p<0.05 compared to untreated cells. #p<0.05 compared to α-IgM-treated cells. (C) WEHI-231 cells were treated for 24 hr with α-IgM or α-IgM + CD40L. The harvested cells were re-suspended in NT solution and stimulated with α-IgM (5 μg/ml) for 3 min at 37°C. The α-IgM-stimulated cells were immunoprecipitated to measure the amount of tyrosine-phosphorylated PLCγ2 (pY-PLCγ2). Densities of pY-PLCγ2 were compared against those of total PLCγ2 and β-actin. (D) The amounts of pY-PLCγ2 in response to 3 min of BCR ligation in C were clearly determined based on the amplitudes of α-IgM-triggered [Ca2+]i increases in WEHI-231 cells.
Fig. 6.
An antioxidant, NAC, inhibits the effect of CD40 co-stimulation in WEHI-231 cells. An antioxidant, NAC (10 mM), was treated 30 min before stimulation of WEHI-231 cells with α-IgM or α-IgM + CD40L. After 24 hr of stimulation in the absence or presence of NAC, the cells were harvested and subjected to measurement of BCR-induced [Ca2+]i increase in NT solution (A), analysis of surface BCR level (B), and Western blotting for PLCγ2 and IP3R-3 expression (C). Mean±S.E.M. ∗p<0.05.
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