Figure 6.

Cytokine mediated effects on LPS-induced epithelial production. (a) NO production in response to stimulation with LPS (100 ng/ml) or TNF (5 ng/ml) and IFN-γ (20 ng/ml) for 24 hr. (b) Reduction of the LPS/IFN-γ induced NO synthesis by preincubation with IL-4 and IL-13 but not IL-11 or TGF-β for 12 hr. The lower total NO production as compared to Fig. 2(e) is explained by the extended culture time.¹⁹ (c) Quantitative iNOS mRNA analysis of m-IC_cl2 cells preincubated in the absence or presence of IL13 or IL-4 for 12 hr and subsequently stimulated with LPS (100 ng/ml) and IFN-γ (20 ng/ml) for 24 hr. Values are presented as RQ ± RQ maximum and minimum, respectively, and indicate epithelial iNOS mRNA expression normalized to HPRT1. (d) Immunoblot of m-IC_cl2 cells pretreated for 12 hr (left) or 24 hr (right) with medium alone, IL-4, IL-13, or IL-11 as indicated and subsequently stimulated with LPS/IFN-γ for 24 hr; 120 µg total cell lysate were loaded in each lane. The results are representative for three independent experiments. The numbers indicate NO concentrations in the cell supernatant from the experiment. Note that because of a different medium volume to cell ratio, reduced NO quantities are found as compared to (b) and (g). (e) Quantitative arginase 1 mRNA analysis of m-IC_cl2 cells pretreated for 12 hr with medium, IL-13, IL-11. (f) Quantitative arginase 1 mRNA analysis of m-IC_cl2 cells after incubation with medium, IL-13, IL-4, or IL-11 for 24 hr and subsequent stimulation with LPS/IFN-γ for 24 hr. (g) Both addition of 4 U/ml purified arginase or pretreatment with IL-4 or IL-13 for 24 hr to the culture of m-IC_cl2 cells stimulated with IFN-γ/LPS for 24 hr led to a reduction of NO production. The results from stimulation assays and Western blot are representative for three independent experiments. *P < 0·05, **P < 0·01.