Granulocyte-macrophage colony-stimulating factor negatively regulates early IL-10-mediated responses

Abstract

Aim: Treatment of inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. IL-10 is a cytokine with therapeutic potential, but until now has not been as successful as previously anticipated. A reason for this may be that IL-10 responsiveness depends on the environment of the inflamed tissue. In this study we investigated whether GM-CSF is able to influence IL-10-mediated responses.

Methodology: Dendritic cells and macrophages were differentiated from mouse bone marrow and treated or depleted from GM-CSF prior to analyze their response to IL-10. Activity was assessed by measuring cytokine expression upon lipopolysaccharide stimulation, IL-10-induced signaling and down-stream gene expression.

Conclusion: This study describes that GM-CSF negatively regulates IL-10-mediated responses.

Keywords: dendritic cells; granulocyte-macrophage colony-stimulating factor (GM-CSF); interleukin-10 (IL-10); macrophages; signal transduction.

Figure 1.. IL-10-mediated signaling in dendritic cells and macrophages.

(A) Macrophages and dendritic cells were pretreated with IL-10 and were stimulated with 100 ng/ml lipopolysaccharide and the inhibition of tumor necrosis factor-α expression was determined at 2 and 24 h (n = 3, error bars represent standard error). (B) Phosphorylation of tyrosine 705 (Y705) and serine 727 (S727) of STAT3 by IL-10 (0, 1, 10 and 100 ng/ml) were analyzed in macrophages and dendritic cells using western blot. (C & D) Relative induction of STAT3 phosphorylation was quantified for Y705 (C) and S727 (D) upon western-blot analysis (n = 3 or 4 for Y705 and S727, respectively, error bars represent standard error). (E) Macrophages and dendritic cells were treated with 100 ng/ml IL-10 and the activation of intracellular signaling pathways was analyzed with a PathScan Array. A representative figure is given for two independent experiments. Asterisk(s) indicate significant differences as determined by a Welch's t-test (*p < 0.05; **p < 0.01).

Figure 2.. GM-CSF suppresses early IL-10-mediated responses.

Macrophages (A–C) and dendritic cells (D–F) were cultured for the last 24 h in the presence or absence (±) of GM-CSF. Cells were pretreated for 20 min with IL-10 followed by stimulation with 100 ng/ml lipopolysaccharide. The inhibition of TNF-α expression was determined after 24 h of stimulation (A & D). Alternatively, cells were pre-treated for 20 min with 10 ng/ml IL-10 and then stimulated with 100 ng/ml lipopolysaccharide. The inhibition of TNF-α expression was determined after 2 h of stimulation (B & E). Absolute expression levels of TNF-α for the abovementioned experiments are also given (C & F). All bars represent the average of 3–5 biological replicates (n = 3–5, error bars indicate standard error) and asterisk(s) indicate significant differences as determined by a Welch's t-test (*p < 0.05; **p < 0.01).

Figure 3.. GM-CSF negatively influences IL-10 activity without affecting STAT3 activation.

(A) Macrophages were treated for 24 h with GM-CSF or left untreated. Cells were then activated with IL-10 (0, 1, 10 and 100 ng/ml) and phosphorylation of tyrosine 705 (Y705) and serine 727 (S727) of STAT3 were analyzed by western blot. (B) Relative socs3 transcript levels were analyzed by quantitative PCR in both macrophages and dendritic cells that were treated overnight by GM-CSF or were left untreated (n = 3, error bars represent standard error). Fold induction of socs3 expression upon IL-10 treatment (100 ng/ml) was calculated using the 2^ΔCt method using HPRT as a reference gene and is depicted above the bars in the graph (macrophages/dendritic cells). (C) Macrophages and dendritic cells were treated overnight with GM-CSF or left untreated. Cells were then stimulated with 100 ng/ml IL-10 and the activation of intracellular signaling pathways was analyzed by PathScan Array. A representative figure is given for two independent experiments. Asterisk(s) indicate significant differences as determined by a Welch's t-test (*p < 0.05; **p < 0.01).