Fig.5.

rhSlit2 attenuates bTSH-induced IL-23p19 while enhancing IL-12p35 induction in fibrocytes. Knocking-down Slit2 in GD-OF enhances IL-23p19 induction but attenuates the IL-12p35 response. (A and D) Fibrocytes and GD-OF were incubated with rhSlit2 (50 ng/ml) for 7–9 d, then received diluent or bTSH (5 mIU/mL) for 6 h. Cell layers were harvested, RNA extracted, reverse-transcribed and subjected to RT-PCR for the targets indicated. Values were normalized to their respective GAPDH signals. (B) Fibrocytes were treated as in A, media were harvested and subjected to IL-23 and IL-12 specific ELISAs. Values were normalized to respective cell layer protein content. (C) Fibrocytes were covered with GD-OF-conditioned medium that had been incubated for 48 h with uncoated beads or those coated with anti-Slit2 (100 μg) or isotype IgG (100μg), as described in “Methods”. Fibrocyte monolayers were processed as in (A). (D) Confluent GD-OF were transfected with scrambled (control) (3μg) siRNA or Slit2-targeting siRNA (3μg), were incubated for 3 d, and treated with diluent or bTSH for 6 h. They were then processed as in (A). (E) GD-OF were treated as in (D), media were collected and subjected to ELISAs for IL-23 and IL-12 as in (B). Data are expressed as the mean ± SD, *, p<0.05; **, p<0.01, ***, p<0.001 of three independent determinations.