Figure 7

LPS-induced nuclear NF-IL6-IR is linked to ACTH and increased TNFα in the rat pituitary. (A, a) Representative microphotographs show that nuclear (DAPI, blue) NF-IL6-IR (red) co-localized with some ACTH-positive cells (green) 6 h after LPS (100 μg/mL, (a) but not PBS treatment (A) in primary cell cultures of the anterior pituitary lobe. (B, b) Moreover, LPS (100 μg/mL) -induced TNFα-IR (green) in S100 protein-positive folliculostellate cells (red) 6 h after stimulation (b), compared to PBS controls (B). (C, c and D, d) In a different set of experiments, pretreatment (30 min, 100 μM) with the JAK-STAT inhibitor AG490 reduced LPS (100 μg/mL) -induced NF-IL6-IR partly in ACTH-IR cells (green) of the anterior pituitary lobe 6 h after stimulation (d), compared to solvent (cremophor, Cre) -treated controls (c). Brightness, contrast, and color balance were adjusted for better representation of the actual data. Scale bars in A (applies to A, a, B and b) and C (applies to C, c, D and d) represent 10 μm. (E) Relative TNFα concentration in cell culture supernatants, in PBS- or LPS-stimulated and cremophor (Cre) or AG490 preincubated primary cell cultures are presented as percentage of LPS- and Cre-stimulated wells (100%) as calculated for each experiment. Means of two to three wells of the means (of each independent experiment, n = 3) were calculated and revealed a dramatic LPS- and Cre-induced TNFα-increase that was abolished by AG490 preincubation. (F) Counting of ACTH-IR cells as percentage of all cells in anterior pituitary cell cultures showed a significant increase of ACTH-positive cells after AG490 pretreatment in LPS-stimulated cultures (means of three independent experiments). AG490 did not show any significant effect on the amount of ACTH-positive cells in PBS-treated controls. * LPS & Cre vs. LPS & AG490; *** P <0.001, ** P <0.01.