Involvement of stress-activated protein kinase and p38 mitogen-activated protein kinase in mIgM-induced apoptosis of human B lymphocytes

Abstract

Despite intensive efforts, the intracellular signaling pathways that mediate apoptosis remain unclear. The human B lymphoma cell line, B104, possesses characteristics that make it an attractive model for analysis of receptor-mediated apoptosis. Although these cells express both membrane IgM (mIgM) and membrane IgD (mIgD) crosslinking mIgM results in significant apoptosis while crosslinking mIgD does not. Our results show that crosslinking mIgM but not mIgD induced a delayed and sustained activation of the mitogen-activated protein kinase (MAPK) family members stress-activated protein kinase (SAPK) and p38 MAPK. The calcium ionophore ionomycin, which also induces apoptosis in B104 cells, stimulated a similar SAPK and p38 MAPK response. Cyclosporin A, a potent inhibitor of apoptosis induced by either mIgM or ionomycin, inhibited activation of both SAPK and p38 MAPK, suggesting that stimulation of these kinases may be required for induction of apoptosis. Collectively, our results indicate that SAPK and p38 MAPK may be downstream targets during mIgM-induced, calcium-mediated, apoptosis in human B lymphocytes.

Figure 1

Characteristics of the mIgM+IgD+ human B cell lymphoma line, B104. (A) Flow cytometric analysis of B104 cells with mAbs specific for IgM (4B8), IgD (δTA-1), or CD8 (G10-1). (B) Effect of treatment with anti-IgM (•) (1 μg/ml), anti-IgD (▵) (1 μg/ml), IgG (○) (1 μg/ml), or ionomycin (▪) (200 mg/ml) on B104 cell viability. (C and D) Effect of graded doses of anti-IgM (solid symbols) or anti-IgD (open symbols) on B104 [Ca²⁺]_i levels as measured by changes in Indo-1 ratio. Antibody doses were 1.0 μg/ml (circles), 0.1 μg/ml (squares), and 0.01 μg/ml (triangles).

Figure 2

Crosslinking of mIgM or mIgD stimulates ERK activity. (A) Effect of treatment of B104 cells with anti-IgM (•) or anti-IgD (○) (1 μg/ml) for the indicated times on ERK activity. (B) B104 cells were treated with either 0.5 μg/ml anti-IgM or 1.0 μg/ml anti-IgD. (Left) ERK activity measured at 3 min. (Right) Cell viability measured at 24 hr.

Figure 3

Crosslinking of mIgM or treatment with ionomycin stimulates activation of SAPK and p38 MAPK. B104 cells were treated for the indicated time with either anti-IgM (•; 1 μg/ml), anti-IgD (○; 1 μg/ml), or ionomycin (▪; 200 ng/ml). SAPK (A) or p38 MAPK (B) activities were measured by immune complex kinase assay. (Left) Autoradiograms. (Right) Activities, as determined by densitometric quantification. The effect of treatment with anisomycin (20 μg/ml) (A) or sorbitol (500 mM) (B) for 2 hr are also shown.

Figure 4

Comparison of the kinetics of induction of ERK, SAPK, p38 MAPK activities, and with apoptosis induced by crosslinking of mIgM. The effect of anti-IgM on induction of ERK (□), SAPK (•), and p38 MAPK (○) activities and apoptosis (▵), expressed as percentages of the maximum response at the indicated times.

Figure 5

Effect of CsA on induction of apoptosis, SAPK, and p38 MAPK activities by crosslinking of mIgM or ionomycin treatment. B104 cells were treated with anti-IgM (squares) (1 μg/ml) or ionomycin (circles) (200 ng/ml) for the indicated times in the presence (solid symbols) or absence (open symbols) of CsA (150 ng/ml). At the indicated times, the following characteristics were measured: (A) Cell viability; (B) SAPK activity; and (C) p38 MAPK activity.