IL-2, IL-4, IFN-γ or TNF-α enhances BAFF-stimulated cell viability and survival by activating Erk1/2 and S6K1 pathways in neoplastic B-lymphoid cells

Abstract

B-cell activating factor of the TNF family (BAFF) has been documented to act as a critical factor in the development of aggressive B lymphocytes and autoimmune diseases. However, the effect of various cytokines on BAFF-elicited neoplastic B-lymphoid cells is not known. In this study, we exhibited that administration of human soluble BAFF (hsBAFF), IL-2, IL-4, IFN-γ, or TNF-α alone increased cell viability and survival in Raji cells concentration-dependently, yet a more robust viability/survival was seen in the cells co-treatment of IL-2, IL-4, IFN-γ, or TNF-α with hsBAFF, respectively. Further research revealed that both Erk1/2 and S6K1 signaling pathways were essential for IL-2, IL-4, IFN-γ, or TNF-α enhancement of the viability/survival in the hsBAFF-stimulated cells, as inhibition of Erk1/2 with U0126 or down-regulation of Erk1/2, or blockage of S6K1 with rapamycin or silencing S6K1, or silencing S6K1/Erk1/2, respectively, reduced the cell viability/survival in the cells treated with/without hsBAFF±IL-2, IL-4, IFN-γ, or TNF-α. These findings indicate that IL-2, IL-4, IFN-γ or TNF-α enhances BAFF-stimulated cell viability/survival by activating Erk1/2 and S6K1 signaling in neoplastic B-lymphoid cells. Our data suggest that modulation of IL-2, IL-4, IFN-γ and/or TNF-α levels, or inhibitors of Erk1/2 or S6K1 may be a new approach to prevent BAFF-induced aggressive B-cell malignancies.

Keywords: B cell; BAFF; Cytokine; Erk1/2; S6K1.

Fig. 1

hsBAFF stimulates B-cell proliferation and survival in a concentration dependent manner. Raji cells were treated with 0–0.25 μg/ml hsBAFF for 48 h. (A) Cell proliferation was evaluated by cell counting. (B) Cell viability was monitored by MTS assay. (C) Relative number of live cells was estimated by trypan blue exclusion assay. (D) Quantitative live cells were calculated by FACS using annexin-V-FITC/PI staining. Results are presented as mean ± S.E. (n = 3–5). * p<0.05, ** p<0.01, difference with control group.

Fig. 2

IL-2, IL-4, IFN-γ, or TNF-α promotes B-cell viability/survival. Raji cells were treated with 0–100 ng/ml of IL-2, IL-4, IFN-γ, or TNF-α for 48 h, respectively. (A) Cell viability was monitored by MTS assay. (B) Relative number of live cells was estimated by trypan blue exclusion assay. Results are presented as mean ± S.E. (n = 5). *p<0.05, **p<0.01, difference with control group.

Fig. 3

IL-2, IL-4, IFN-γ, or TNF-α strengthens hsBAFF-stimulated B-cell viability/survival. Raji cells were treated with/without hsBAFF (0.25 μg/ml) in the presence or absence of IL-2 (5 and 50 ng/ml), IL-4 (5 and 25 ng/ml), IFN-γ (10 and 100 ng/ml), or TNF-α (5 and 50 ng/ml) for 48 h. (A) Cell viability was monitored by MTS assay. (B) Relative number of live cells was estimated by trypan blue exclusion assay. Results are presented as mean ± S.E. (n = 5). ^ap<0.05, difference with control group. ^bp<0.05, difference with 0.25 μg/ml hsBAFF group.

Fig. 4

IL-2, IL-4, IFN-γ, or TNF-α reinforces hsBAFF-induced activation of Erk1/2 and S6K1 pathways in B cells. Raji cells were treated with hsBAFF (0–0.25 μg/ml) for 12 h, or with/without hsBAFF (0.25 μg/ml) in the presence or absence of IL-2 (5 and 50 ng/ml), IL-4 (5 and 25 ng/ml), IFN-γ (10 and 100 ng/ml), or TNF-α (5 and 50 ng/ml) for 12 h. (A and C) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments (A and C), and the blots for p-Erk1/2, p-S6K1, and p-S6 were semi-quantified (B and D). Results are presented as mean ± S.E. (n = 3). ^ap<0.05, difference with control group. ^bp<0.05, difference with 0.25 μg/ml hsBAFF group.

Fig. 5

Pharmacological inhibition of Erk1/2 or down-regulation of Erk1/2 prevents hsBAFF-stimulated B-cell viability/survival enhanced by IL-2, IL-4, IFN-γ, or TNF-α. Raji cells, or Raji cells infected with lentiviral shRNAs to Erk1/2 and GFP, respectively, were pre-incubated with/without U0126 (5 μM) for 1 h and then treated with/without hsBAFF (0.25 μg/ml) in the presence or absence of IL-2 (50 ng/ml), IL-4 (25 ng/ml), IFN-γ (100 ng/ml) or TNF-α (50 ng/ml) for 12 h (for Western blotting) or 48 h (for cell viability/survival assay). (A and D) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. (B and E) Cell viability was monitored by MTS assay. (C and F) Relative number of live cells was estimated by trypan blue exclusion assay. Results are presented as mean ± S.E. (n = 5). ^ap < 0.05 difference with control group; ^bp < 0.05, difference with 0.25 μg/ml hsBAFF group; ^cp < 0.05 − U0126 group vs + U0126 group Erk1/2 shRNA group vs GFP shRNA group.

Fig. 6

Pharmacological inhibition of S6K1 or down-regulation of S6K1 blocks hsBAFF-stimulated B-cell viability/survival enhanced by IL-2, IL-4, IFN-γ, or TNF-α. Raji cells, or Raji cells infected with lentiviral shRNAs to S6K1 and GFP, respectively, were pre-incubated with/without rapamycin (0.2 μg/ml) for 2 h and then treated with/without hsBAFF (0.25 μg/ml) in the presence or absence of IL-2 (50 ng/ml), IL-4 (25 ng/ml), IFN-γ (100 ng/ml) or TNF-α (50 ng/ml) for 12 h (for Western blotting) or 48 h (for cell viability/survival assay). (A and D) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. (B and E) Cell viability was monitored by MTS assay. (C and F) Relative number of live cells was estimated by trypan blue exclusion assay. Results are presented as mean ± S.E. (n = 5). ^ap < 0.05 difference with control group; ^bp < 0.05, difference with 0.25 μg/ml hsBAFF group; ^cp < 0.05 − Rapamycin group vs + Rapamycin group or S6K1 shRNA group vs GFP shRNA group.

Fig. 7

Double silencing of Erk1/2 and S6K1 powerfully inhibits hsBAFF-stimulated B-cell viability/survival enhanced by IL-2, IL-4, IFN-γ, or TNF-α. Raji cells were infected with lentiviral shRNAs to S6K1/Erk1/2 and GFP, respectively. (A) The cells were subjected to Western blotting. The blots were probed for β-actin as a loading control. Similar results were observed in at least three independent experiments. (B and C) The cells were treated with/without hsBAFF (0.25 μg/ml) in the presence or absence of IL-2 (50 ng/ml), IL-4 (25 ng/ml), IFN-γ (100 ng/ml) or TNF-α (50 ng/ml) for 48 h, followed by cell viability using MTS assay (B) and relative number of live cells using trypan blue exclusion assay (C). Results are presented as mean ± S.E. (n = 5). ^ap < 0.05 difference with control group; ^bp < 0.05, difference with 0.25 μg/ml hsBAFF group; ^cp < 0.05 S6K1/Erk1/2 shRNA group vs GFP shRNA group.