Regulation of eotaxin-3/CCL26 expression in human monocytic cells

Abstract

Eotaxin-3/CCL26 is an agonist for chemokine receptor 3 (CCR3) and a natural antagonist for CCR1, CCR2 and CCR5. CCL26 expression by non-haematopoietic cells has been well documented; however, no studies to date have demonstrated CCL26 expression by leucocytes. In this study, we investigated the ability of human monocytic cells to produce CCL26 in response to cytokines. We found that interleukin-4 (IL-4) increased the expression of CCL26 messenger RNA (mRNA) and protein in U937 cells, in human monocytes and in human monocyte-derived macrophages. Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) alone did not induce CCL26 expression, yet these pro-inflammatory cytokines synergized with IL-4 to increase CCL26 protein expression. Signal transducer and activator of transcription 6 (STAT6) was not affected by costimulation with TNF-alpha, suggesting that the synergy between IL-4 and TNF-alpha occurs at a step downstream of STAT6 activation. Co-incubation of interferon-gamma (IFN-gamma) with IL-4 had no effect on CCL26 protein release. By contrast, pretreatment with IFN-gamma decreased total STAT6 protein, blocked IL-4-mediated STAT6 phosphorylation and decreased IL-4-mediated CCL26 mRNA expression and protein release. These data show that IL-4 and pro-inflammatory cytokines such as TNF-alpha, IL-1beta and IFN-gamma regulate CCL26 synthesis in human monocytic cells, which may be important in regulating monocyte inflammatory responses.

Figure 1

CCL26 messenger RNA (mRNA) is expressed in monocytic cells following stimulation with interleukin (IL)-4. (a and d) U937 cells, (b and e) monocyte-derived macrophages (MDMs), or (c and f) monocytes were cultured for 24 hr in medium alone (Con) or in mediim containing 10 ng/ml of IL-4, tumour necrosis factor-α (TNF-α), IL-1β or interferon-γ (IFN-γ). RNA was isolated from the cells using TRIzol. (a–c) The expression of mRNA for CCL26 and β-actin was evaluated using the reverse transcription–polymerase chain reaction (RT-PCR), and the PCR products were visualized by gel electrophoresis in the presence of ethidium bromide. Data are representative of results obtained from at least three independent experiments. (d–f) The expression of mRNA for CCL26 was evaluated using real-time PCR and data were normalized using 18S ribosomal RNA (rRNA) expression. Values for cytokine-stimulated cells were compared with values 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. **P<0·01 indicates statistical significance compared with the control.

Figure 2

Interleukin (IL)-4-induced CCL26 messenger RNA (mRNA) expression in monocytic cells is concentration-dependent and time-dependent. (a) U937 cells and (c) monocyte-derived macrophages (MDMs) were cultured for 24 hr in medium alone or in medium containing increasing amounts of IL-4 (0·1–50 ng/ml). (b) U937 cells and (d) MDMs were cultured in medium alone, or in medium containing 10 ng/ml of IL-4, for 1–72 hr. RNA was extracted from the cells using TRIzol. The expression of mRNA for CCL26 was evaluated by real-time polymerase chain reaction (PCR) and the data were normalized using 18S ribosomal RNA (rRNA) expression. Values for cytokine-stimulated cells were compared with those of control cells and data were then expressed as the mean ± standard error of the mean (SEM) of −delta delta Ct (−ddCt) from at least four independent experiments. **P<0·01 indicates statistical significance compared with the control; and ns is not significant.

Figure 3

Interleukin (IL)-4 induces CCL26 protein release from U937 cells and monocyte-derived macrophages (MDMs). U937 cells were cultured (a) for 24 or 48 hr in the absence or presence of 0·1–50 ng/ml of IL-4 or (b) for 24–72 hr in the presence of 10 ng/ml of IL-4. (c) MDMs were cultured for 24 or 48 hr in the absence or presence of 10 ng/ml of IL-4. Supernatants were collected and the levels of CCL26 protein were measured using enzyme-linked immunosorbent assay (ELISA). (a and b) Data are expressed as mean ± standard error of the mean (SEM) of at least three independent experiments. (c) Data are expressed as the mean of eight independent experiments. **P<0·01 and ***P < 0·001 indicates statistical significance compared with the control; † indicates statistical significance as compared to cells stimulated with the same concentration of IL-4 for 24 hr; and ns is not significant. Con, control.

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.

Figure 5

Treatment with interferon-γ (IFN-γ) prevents subsequent interleukin (IL)-4-induced CCL26 messenger RNA (mRNA) and protein expression in U937 cells. U937 cells were cultured for 48 hr in medium alone (0) or in medium containing 0·1–100 ng/ml of IFN-γ. The U937 cells were then washed and incubated with fresh serum-free medium, either alone or containing IL-4 (1 or 10 ng/ml), for 24 hr. (a) RNA was isolated from the cells using TRIzol. The expression of mRNAs for CCL26 and β-actin was evaluated using the reverse transcription–polymerase chain reaction (RT-PCR). PCR products were visualized by gel electrophoresis in the presence of ethidium bromide. Data are representative of the results obtained 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 < 0·001 compared to the Control (0) with no IL-4; ns, not significant.

Figure 6

Treatment with interferon-γ (IFN-γ) decreases total signal transducer and activator of transcription 6 (STAT6) protein and subsequent interleukin (IL)-4-induced STAT6 phosphorylation in U937 cells. (a) U937 cells were cultured for 24 or 48 hr in medium alone (none) or in medium containing 100 ng/ml of IFN-γ. U937 cells were then washed and incubated with fresh serum-free medium alone or with medium containing IL-4 (10 ng/ml) or IL-4 and IFN-γ for 10 min (co-inc). Cell lysates were harvested in 2 × Laemelli sample buffer and analysed by Western blotting for phosphorylated STAT6 (P-STAT6), total STAT6 (T-STAT6) and β-actin. (a) The blots shown are representative of the results obtained in three independent experiments. (b) Densitometry was used to measure the levels of total STAT6 and β-actin, and the values were expressed as a percentage of the density levels of each respective protein present in U937 cells cultured for 48 hr in the presence of medium alone (none) ± standard error of the mean (SEM) from four independent experiments. **P < 0·01 compared to cells cultured for 24 hr in the presence of medium alone.