Figure 5. STAT3-dependent regulation of IP-10 by Mϕs and B cells. (A) IP-10 production in the B cell–Mϕ co-cultures in the presence of neutralizing Abs against IL-6 and IL-6R. Data are from five independent experiments using cells from different donors. *, P = 0.0168, paired Student’s t test. (B) IP-10 production of Mϕs stimulated with 50 ng/ml recombinant IL-6 for 18 h. Data are from four independent experiments using cells from different donors. *, P = 0.0289, paired Student’s t test. (C) Mϕs cultured with IL-6 or in combination with 50 µM AG490 for 15 min and stained with phosphoflow Abs against pSTAT3 (Y705). The dashed line indicates the isotype control. (D) Production of IP-10 and IL-8 by Mϕs stimulated with recombinant IL-6 in the presence of AG490. (E) Mϕs were transfected with a STAT3 siRNA or control siRNA. Total STAT3 expression was measured at 72 h after transfection by flow cytometry. (F) Mϕs transfected with a STAT3 siRNA or control siRNA were stimulated with IL-6 for 48 h, and IP-10 was measured. Data are from four independent experiments using cells from different donors. *, P = 0.04, paired Student’s t test. (G) Total B cells were cultured with recombinant IL-6 or IP-10 for from 2 to 30 min and stained with phosphoflow Abs against pSTAT3 (Y705). Data are representative of at least three experiments. (H) Total B cells were cultured with or without 100 ng/ml IP-10 for 15 min, and total RNA was extracted for the measurement of IL-6 mRNA by qPCR. Data represent experiments using cells from five different donors. *, P = 0.04, paired Student’s t test. (I) Supernatants of B cells cultured with or without IP-10 were collected at various time points and measured for IL-6 by Luminex assay. Data are mean ± SEM from experiments using cells from three donors. **, P < 0.01, paired Student’s t test. (J) Total B cells were cultured with recombinant IP-10 or in combination with 10 µg/ml of an mAb against IL-6 for 15 min. Cells were stained with phosphoflow Abs against pSTAT3 (Y705). (K) Mϕs were generated in vitro from patients with hyper-IgE syndrome (STAT3+/−) or healthy donors (STAT3+/+). Sorted naive B cells (from patients or healthy donors) were preactivated with anti-BCR before being co-cultured with Mϕs in the presence of CpG for 6 d and analyzed for PC development. Data show the phenotype for CD38+CD138+ PCs. (L) Supernatant from a day 4 co-culture was collected for the measurement of IP-10. Data are representative of two independent experiments using cells from three patients. (D and L) Mean ± SD of duplicate (L) or triplicate (D) cultures is shown.
Figure 5.

STAT3-dependent regulation of IP-10 by Mϕs and B cells. (A) IP-10 production in the B cell–Mϕ co-cultures in the presence of neutralizing Abs against IL-6 and IL-6R. Data are from five independent experiments using cells from different donors. *, P = 0.0168, paired Student’s t test. (B) IP-10 production of Mϕs stimulated with 50 ng/ml recombinant IL-6 for 18 h. Data are from four independent experiments using cells from different donors. *, P = 0.0289, paired Student’s t test. (C) Mϕs cultured with IL-6 or in combination with 50 µM AG490 for 15 min and stained with phosphoflow Abs against pSTAT3 (Y705). The dashed line indicates the isotype control. (D) Production of IP-10 and IL-8 by Mϕs stimulated with recombinant IL-6 in the presence of AG490. (E) Mϕs were transfected with a STAT3 siRNA or control siRNA. Total STAT3 expression was measured at 72 h after transfection by flow cytometry. (F) Mϕs transfected with a STAT3 siRNA or control siRNA were stimulated with IL-6 for 48 h, and IP-10 was measured. Data are from four independent experiments using cells from different donors. *, P = 0.04, paired Student’s t test. (G) Total B cells were cultured with recombinant IL-6 or IP-10 for from 2 to 30 min and stained with phosphoflow Abs against pSTAT3 (Y705). Data are representative of at least three experiments. (H) Total B cells were cultured with or without 100 ng/ml IP-10 for 15 min, and total RNA was extracted for the measurement of IL-6 mRNA by qPCR. Data represent experiments using cells from five different donors. *, P = 0.04, paired Student’s t test. (I) Supernatants of B cells cultured with or without IP-10 were collected at various time points and measured for IL-6 by Luminex assay. Data are mean ± SEM from experiments using cells from three donors. **, P < 0.01, paired Student’s t test. (J) Total B cells were cultured with recombinant IP-10 or in combination with 10 µg/ml of an mAb against IL-6 for 15 min. Cells were stained with phosphoflow Abs against pSTAT3 (Y705). (K) Mϕs were generated in vitro from patients with hyper-IgE syndrome (STAT3+/−) or healthy donors (STAT3+/+). Sorted naive B cells (from patients or healthy donors) were preactivated with anti-BCR before being co-cultured with Mϕs in the presence of CpG for 6 d and analyzed for PC development. Data show the phenotype for CD38+CD138+ PCs. (L) Supernatant from a day 4 co-culture was collected for the measurement of IP-10. Data are representative of two independent experiments using cells from three patients. (D and L) Mean ± SD of duplicate (L) or triplicate (D) cultures is shown.

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