LPS induces IL-10 production by human alveolar macrophages via MAPKinases- and Sp1-dependent mechanisms

Abstract

Background: IL-10 is a cytokine mainly produced by macrophages that plays key roles in tolerance to inhaled antigens and in lung homeostasis. Its regulation in alveolar macrophages (HAM), the resident lung phagocytes, remains however unknown.

Methods: The present study investigated the role of intracellular signalling and transcription factors controlling the production of IL-10 in LPS-activated HAM from normal nonsmoking volunteers.

Results: LPS (1-1000 pg/ml) induced in vitro IL-10 production by HAM, both at mRNA and protein levels. LPS also activated the phosphorylation of ERK, p38 and JNK MAPkinases (immunoblots) and Sp-1 nuclear activity (EMSA). Selective inhibitors of MAPKinases (respectively PD98059, SB203580 and SP600125) and of Sp-1 signaling (mithramycin) decreased IL-10 expression in HAM. In addition, whilst not affecting IL-10 mRNA degradation, the three MAPKinase inhibitors completely abolished Sp-1 activation by LPS in HAM.

Conclusion: These results demonstrate for the first time that expression of IL-10 in lung macrophages stimulated by LPS depends on the concomitant activation of ERK, p38 and JNK MAPKinases, which control downstream signalling to Sp-1 transcription factor. This study further points to Sp-1 as a key signalling pathway for IL-10 expression in the lung.

Figure 1

Time and dose dependency of IL-10 production in HAM stimulated by LPS. HAM were stimulated with LPS (1 μg/ml) and supernatant were collected after different incubation time and assayed for IL-10 content by ELISA for Panel A and by real time PCR for Panel C. Panel B: HAM were stimulated for 24 hours with increasing concentration of LPS and supernatant were assayed for IL-10 by ELISA.

Figure 2

Activation of MAP kinases in HAM stimulated by LPS. Panel A: HAM were stimulated 30 min with increasing doses of LPS and phosphorylated ERK, p38 and JNK were detected by Western-blotting as described in Materials and Methods. Panel B: HAM were incubated with LPS (1 μg/ml) during different time and phosphorylated ERK, p38 and JNK were detected by Western-blotting. Below the blots, the graph represents the phosphorylated MAP kinases to β-actin ratio obtained by densitometric analysis of each bands using Quantity One Sotware.

Figure 3

Inhibition of MAPkinases activation by PD98059, SB203580, SP600125. HAM were preincubated during 1 h with inhibitors (50 μM) and then stimulated 30 min with LPS (1 μg/ml). Levels of phosphorylated ERK, p38 and JNK MAP kinases were evaluated by Western-blot analysis. Each bands was normalised by performing phospho MAP kinase to β-actin ratio as by the graph representing the densitometric analysis.

Figure 4

Effect of specific MAP kinase inhibitors on the production of IL-10 in HAM. HAM were preincubated (1 h) with increasing concentrations of inhibitors and then were stimulated with LPS (1 μg/ml) during 24 h. Supernatants were assayed to determine the production of IL-10 by ELISA.

Figure 5

Effect of mithramycin on IL-10 production in HAM. HAM were preincubated (1 h) with increasing concentration of mithramycin and were stimulated with LPS (1 μg/ml) during 24 h. Supernantants were assayed by ELISA to determine IL-10 production.

Figure 6

EMSA analysis of Sp1 binding activity in nuclear proteins of HAM. Panel A : HAM were treated or not with LPS (1 μg/ml) during 2 h. Nuclear proteins were extracted following procedure described in Materials and Methods. Lane 1: free labelled probe only (without nuclear proteins) – Lane 2: Nuclear proteins from control HAM incubated with labelled probe specific for Sp1 – Lane 3: idem lane2 except that HAM were treated 2 h with LPS – Lane 4: idem lane 3 except that an excess of unlabelled (cold) probe was added in the binding reaction – Lane 5: idem lane 3 except that a mutant labelled probe (mutation in consensus binding site) is used instead of the specific labelled probe for Sp1 – Lane 6: idem lane 3 except that nuclear proteins were preincubated with a specific anti-Sp1 antibody before addition of the specific labelled probe. Panel B represents a quantitative analysis of a EMSA gel from HAM following stimulation by LPS. Data are mean ± SD from 6 experiments; *p value = 0.0411 (Mann-Whitney U test).

Figure 7

Sp1 binding activity in nuclear proteins of HAM. HAM were preincubated 1 h or not with inhibitors (50 μM for PD98059, SB203580 and SP600125 or 500 nM for mithramycin) and then stimulated with LPS (1 μg/ml) during 2 hours. Control HAM were HAM cultured with medium alone. Nuclear proteins were extracted as previously described and were then incubated in the binding buffer with specific labelled probe and then subjected to electrophoresis for separation of the complexes.

Figure 8

IL-10 mRNA in HAM stimulated with LPS. Panel A : HAM were preincubated 1 h with inhibitors (25 μM for PD98059, SB203580 and SP600125 or 100 nM for mithramycin) and then stimulated for 24 h with LPS. RNA was extracted as described in details in Materials and Methods. After reverse transcription, specific primers were used to amplify fragment of IL-10 mRNA and the relative abundance of IL-10 compared to β-actin is represented in the graph. Data are mean ± SD from 3 experiments; *p value < 0.05 versus (One sample t test). Panel B: HAM were incubated with LPS (1 μg/ml) for 16 h, translation was stopped using Actinomycin D 2.5 μg/ml and inhibitors (PD98059 or SB203580, 25 μM) were added. IL-10 mRNA was assayed at different time points by real time PCR and corrected for β-actin mRNA.