Table 1.
Demographic data for subjects providing primary human airway epithelial cells.
Fig 1.
Effect of IFNγ in the presence and absence of TNFα on the release of CXCL9, CXCL10 and CXCL11 from BEAS-2B and primary human airway epithelial cells.
BEAS-2B cells, n = 11 (open bars, panels a, c and e) or human primary airway epithelial cells from non-smokers (n = 4) (closed bars), smokers (n = 5) (horizontal lines) or patients with COPD (n = 7) (hatched bars) (panels b, d and f) were incubated for 20h in the absence or presence of IFNγ (10ng/ml) or IFNγ + TNFα (10ng/ml). Media was harvested and the concentrations of CXCL9 (panels a and b), CXCL10 (panels c and d) and CXCL11 (panels e and f) were measured by ELISA. Data are presented as mean ± SEM, where * represents p<0.05, **p<0.01 and ***p<0.001.
Fig 2.
Effect of JAK inhibitors, PF956980 and PF1367550 and dexamethasone on IFNγ and IFNγ + TNFα-stimulated release of CXCL9, CXCL10 and CXCL11 from BEAS-2B cells.
BEAS-2B cells were pre-treated and incubated for 1h with either PF956980 (○), PF1367550 (●), or dexamethasone (▲) followed by stimulation for 20h with IFNγ (10ng/ml) (panels a-c) or IFNγ + TNFα (10ng/ml) (panels d-f). Media was harvested and the concentrations of CXCL9 (panels a and d), CXCL10 (panels b and e) and CXCL11 (panels c and f) were measured by ELISA. Data are presented as mean±SEM, n = 7–8.
Table 2.
Selectivity of PF1367550.
Table 3.
IC50 values for PF956980 and PF1367550 on IFNγ and IFNγ+TNFα-stimulated release of CXCL9, CXCL10 and CXCL11 from BEAS-2B cells.
Fig 3.
Effect of JAK inhibitors, PF956980 and PF1367550 and dexamethasone on IFNγ and IFNγ+TNFα-stimulated release of CXCL9, CXCL10 and CXCL11 from human primary airway epithelial cells.
Human primary airway epithelial cells were pre-treated for 1h with either PF956980 (○), PF1367550 (●), or dexamethasone (▲) followed by stimulation for 20h with IFNγ (10 ng/ml) (panels a-c) or IFNγ + TNFα (10 ng/ml) (panels d-f). Media was harvested and the concentrations of CXCL9 (panels a and d), CXCL10 (panels b and e) and CXCL11 (panels c and f) were measured by ELISA. Data are presented as mean ± SEM, n = 10–13.
Table 4.
IC50 values for PF956980 and PF1367550 on IFNγ and IFNγ+TNFα-stimulated release of CXCL9, CXCL10 and CXCL11 from human primary airway epithelial cells from non-smokers, smokers and COPD patients.
Table 5.
IC50 values for PF956980 and PF1367550 on IFNγ and IFNγ+TNFα-stimulated release of CXCR3 chemokinesCXCL9, CXCL10 and CXCL11 from human primary airway epithelial cells.
Fig 4.
Effect of JAK inhibitors, PF956980 and PF1367550 on IFNγ and IFNγ + TNFα-stimulated phosphorylation of STAT-1 in BEAS-2B cells.
BEAS-2B cells were pre-treated for 1h with either PF956980 or PF1367550 followed by stimulation for 10 min with IFNγ (10ng/ml) (○) or IFNγ + TNFα (●). Cells were harvested and western blots performed for phosphorylated STAT-1 and total STAT-1 and quantified by densitometry. Data are representative of n = 4 independent experiments (panels a and c) and presented as mean±SEM, n = 4 (panels b and d).
Fig 5.
Effect of JAK inhibitors, PF956980 and PF1367550 on IFNγ and IFNγ + TNFα-stimulated phosphorylation of STAT-1 in human primary airway epithelial cells.
Human primary airway epithelial cells were pre-treated for 1h with either 1μM PF956980 (PF956) or 1μM PF1367550 (PF136) followed by stimulation for 10 min with either IFNγ (10ng/ml) or IFNγ + TNFα. Cells were harvested and western blots performed for phosphorylated STAT-1 and total STAT-1 and quantified by densitometry. Data are representative of n = 4 independent experiments (panel a) and presented as mean ± SEM, n = 4 (panel b).
Fig 6.
Effect of JAK inhibitors, PF956980 and PF1367550 on IFNγ and IFNγ + TNFα-stimulated DNA binding of STAT-1 and STAT-3 in BEAS-2B cells.
BEAS-2B cells were pre-treated with either PF956980 (PF956) or PF1367550 (PF136) for 1h followed by stimulation with IFNγ (10ng/ml) or IFNγ + TNFα. After 1 h, nuclear extracts of the cells were prepared and TransAM assays for STAT-1 (panel a) and STAT-3 (panel b) were performed. Data are presented as fold change from baseline ± SEM; n = 4.
Fig 7.
Effect of JAK inhibitors, PF956980 and PF1367550 on IFNγ and IFNγ + TNFα-stimulated expression of CXCL9, CXCL10, and CXCL11.
BEAS-2B cells were pre-treated with either PF956980 (checked bars), PF1367550 (hashed bars) or dexamethasone (10-6M white bar) for 1h followed by stimulation with IFNγ (10ng/ml) or IFNγ + TNFα for 6 h. Gene expression of CXCL9 (panels a and b), CXCL10 (panels c and d) and CXCL11 (panels e and f) were quantified by Taqman RT-PCR. Data are presented as fold change form stimulus ± SEM; n = 3 where 1 indicates the level of mRNA induced by IFNγ or IFNγ + TNFα in the absence of inhibitors.