Interleukin-27 induces a STAT1/3- and NF-kappaB-dependent proinflammatory cytokine profile in human monocytes

Abstract

IL-27 is a heterodimeric cytokine bridging innate and adaptive immunity by playing a role in the activation of naive T cells and in development of Th1 cells. Additionally, recent evidence supports a role for IL-27 in the activation of monocytic cells. Both pro-inflammatory and anti-inflammatory activities have been attributed to IL-27; however, the role played by IL-27 in the activation of human monocytic cells in terms of cytokine production has not been well described. Our results show that IL-27 is a strong inducer of proinflammatory cytokine and chemokine expression, including enhancement of IL-6, IP-10, MIP-1alpha, MIP-1beta, and TNF-alpha expression in human primary monocytes. Furthermore, we observed that IL-27-induced cytokine and chemokine production was mediated by STAT1, STAT3, and NF-kappaB activation. Understanding how IL-27 exerts its effects on monocytic cells will identify important molecular mechanisms in the regulation of immune responses, particularly in the modulation of monocyte activation.

FIGURE 1.

IL-27 induces expression pro-inflammatory cytokines and chemokines in primary human monocytes. Primary human monocytes were isolated by negative selection (StemCell Technologies). Monocytes were cultured in either media alone or in the presence of IL-27 for 24 h. A, cell supernatants were harvested and subjected to cytokine array analysis of 36 different cytokines. Cytokines were classified into 6 subsets with similar functions such as: (i) chemokines, (ii) Th1 cytokines, (iii) anti-inflammatory cytokines, (iv) cytokines involved in inflammation and cell differentiation, (v) IL-12 and IL-17 family cytokines, and (vi) Th2 cytokines. Densitometry analysis of the cytokine arrays showed IL-27 significantly induced IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α by a greater than 2-fold increase in chemiluminescence detection. * indicates a greater than 2-fold increase in expression levels. B, same cell culture supernatants used for the cytokine array in panel A were analyzed for IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α expression by ELISA. ND, not detected.

FIGURE 2.

IL-27 induced expression of IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α in primary human monocytes. Primary human monocytes were isolated by negative selection and were cultured in either media alone or IL-27 for the following conditions: (A) cells were treated with IL-27 for 0 to 48 h, and cell supernatants were analyzed for IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α expression by ELISA. B, cells were treated with IL-27 for 24 h and IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α expression were detected by intracellular cytokine staining by flow cytometry. Representative histograms are shown with cells cultured in medium alone (shaded histogram) overlaid with the histogram for IL-27-treated cells (dark line). C, cells were treated with IL-27 for times ranging from 2–8 h. Levels of mRNA for IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α expression were measured by RT-PCR. 18 S rRNA was used as a loading control. Results shown are from one donor and representative of five different donors.

FIGURE 3.

IL-27 induces expression of IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α in THP-1 cells. THP-1 cells were cultured in either media alone or IL-27 for the following conditions: (A) cells were treated with IL-27 for 0 to 48 h and cell culture supernatants were analyzed for IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α expression by ELISA. B, cells were treated with IL-27 for 24 h and IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α expression were detected by intracellular cytokine staining by flow cytometry. Representative histograms are shown with cells cultured in medium alone (shaded histogram) overlaid with the histogram for IL-27-treated cells (dark line). C, cells were treated with IL-27 for times ranging from 2 to 16 h. Levels of mRNA for IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α expression were measured by RT-PCR. 18 S rRNA was used as a loading control. Results shown in this figure are representative of five separate experiments.

FIGURE 4.

IL-27-induced cytokine production is mediated by STAT1 and STAT3 activation. THP-1 cells were either cultured in media or IL-27 under the following conditions: (A) cells were treated with IL-27 for times ranging from 5 to 30 min. Cell pellets were harvested and whole cell lysates were separated by PAGE. Membranes were probed with anti-phospho-STAT1 (p-STAT1) or anti-phospho-STAT3 (p-STAT3) antibodies. Membranes were stripped and re-probed with pan STAT1 or pan STAT3 antibodies as indicated. B, cells were treated with IL-27 for times ranging from 5 to 30 min. Cell pellets were harvested and nuclear protein extracts were subjected to EMSA to measure DNA binding activity of STAT1 and STAT3. Cold competitor (CC) lanes contained 200X excess of unlabeled probes compared with biotin-labeled probes. Supershift (SS) antibodies specific for STAT1 or STAT3 were able to interfere with complex formation. Results shown are representative of five separate experiments. C, THP-1 cells cultured in medium alone were transfected with control, STAT1, or STAT3 siRNA, followed by RT-PCR analysis with primers specific for either STAT1 or STAT3 expression. 18 S rRNA primers were used as a control. D, THP-1 cells were transfected with control, STAT1, or STAT3 siRNA and were either cultured in medium alone or in the presence of IL-27 for 4 h. RT-PCR analysis for IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α expression was performed using cytokine-specific primers. Detection of 18 S rRNA was used as a loading control. Results shown in this figure are representative of five separate experiments.

FIGURE 5.

IL-27-induced cytokine production is dependent on NF-κB activation. THP-1 cells were either cultured in media or IL-27 under the following conditions: (A) cells were treated with IL-27 for times ranging from 5 to 120 min. Cell pellets were harvested and whole cell lysates were separated by PAGE. Membranes were probed with anti-phospho-NF-κB p50 (p-NF-κB p50) antibody. Membranes were stripped and re-probed with anti- pan-NF-κB p50 (NF-κB p50) and anti-β-actin antibodies as indicated. B, cells were treated with IL-27 for times ranging from 5 to 60 min. Cell pellets were harvested and nuclear protein extracts were subjected to EMSA to measure DNA binding activity of NF-κB. Cold competitor (CC) lanes contained 200× excess of unlabeled probes compared with biotin-labeled probes. Supershift antibodies specific for NF-κB p50 (SSp50) or NF-κB p60 (SSp65) were able to interfere with complex formation. Results shown are representative of five separate experiments. C, THP-1 cells cultured in medium alone were pretreated with the NF-κB inhibitor CAPE for the concentrations indicated. Nuclear extracts were subjected to EMSA analysis for NF-κB DNA binding. D, THP-1 cells were pretreated with NF-κB inhibitor, CAPE, and were either cultured in medium alone or in the presence of IL-27 for 4 h. RT-PCR analysis for IL-6, IP-10, MIP-1α, MIP-1β, TNF-α, and RANTES expression was performed using cytokine-specific primers. Detection of 18SrRNA was used as a loading control. E, primary monocytes were pretreated with CAPE followed by culture in either medium alone or in the presence of IL-27 for 4 h. RT-PCR analysis for IL-6, IP-10, MIP-1α, MIP-1β, TNF-α, and RANTES expression was performed using cytokine-specific primers. Detection of 18 S rRNA was used as a loading control. Results shown in this figure are representative of five separate experiments.