Figure 4.

Tumour necrosis factor-α (TNF-α) and interleukin (IL)-1β synergize with IL-4 to induce enhanced CCL26 expression from U937 cells. U937 cells were cultured for 48 hr in medium alone (Control) or in medium containing 10 ng/ml of IL-4, TNF-α, IL-1β or IFN-γ, alone, or in combination with IL-4. (a) RNA was extracted from the cells using TRIzol. The expression of CCL26 mRNA was evaluated by real-time polymerase chain reaction (PCR) and data were normalized using 18S ribosomal RNA (rRNA) expression. Values for cytokine-stimulated cells were compared with those for control cells and data were then expressed as the mean ± standard error of the mean (SEM) of −delta delta Ct (−ddCt) from at least three independent experiments. (b) Supernatants were collected and CCL26 protein was measured using enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± standard error of the mean (SEM) of at least three independent experiments. *P < 0·05; **P < 0·01 and ***P < 0·001 compared to stimulation with IL-4 alone. U937 cells were stimulated with (c) IL-4 or (d) IL-4 and TNF-α for 1–360 min. Cell lysates were harvested in 2 × Laemelli sample buffer and analysed by Western blotting for phosphorylated signal transducer and activator of transcription 6 (p-STAT6) and Total STAT6. The blots shown are representative of the results obtained in more than three independent experiments. Densitometry was performed, the ratio of the signal in P-STAT6 to total STAT6 was determined and the value is shown.