Purpose/objective. IL-6 is a pleiotropic cytokine that provokes a broad range of cellular and physiological responses including inflammation and oncogenesis. While the expression of IL-6 by human blood monocytes is well established, the potential for inducing IL-6 by human neutrophils remains controversial. Accordingly, the aim of this study was to identify whether human neutrophils produce IL-6 in response to LPS that is the major inducer of this cytokine in human monocytes through TLR4 activation. Material and methods. Granulocytes (neutrophils >96,5%) and Percoll-purified human blood peripheral monocytes (>85%) were stimulated with ultra pure LPS for 1-6 hours for gene expression studies by RT-qPCR and for overnight for protein detection by ELISA. Polymerase II (Pol II) recruitment to the transcription start site (TSS) within IL-6 gene promoter and the level of histone 4 acetylation (H4Ac) at IL-6 promoter in human neutrophils and monocytes was assessed by chromatin immunoprecipitation (ChIP). Results. The secretion level of IL-6 protein by neutrophils upon LPS stimulation remained near the detection limit. In accordance, neither significant induction of IL-6 mRNA nor its primary transcript could be detected in neutrophils activated with LPS in 6 hour time frame while IL-6 mRNA expression by human monocytes was at the maximum level. Furthermore, the lack of transcriptional activity at IL-6 locus in human neutrophils activated by LPS was confirmed by absence of Pol II recruitment to its promoter. In contrast, significant level of Pol II binding to the IL-6 promoter could be detected in human monocytes already after 1h of activation by LPS and the recruitment was further increased more than 4-fold after 5h of activation. Importantly, differences in the level of H4Ac, an epigenetic marker of transcriptionally active chromatin, were found at the IL-6 promoter in human neutrophils and monocytes. Readily detectable signal of H4Ac at the IL-6 promoter of resting monocytes increased 30-fold upon 3h LPS stimulation. In contrast, neither significant level of H4Ac nor its induction upon LPS stimulation could be observed in human neutrophils. Conclusions. These results suggest differential capacity of human neutrophils and monocytes to express IL-6 upon LPS activation that is determined in epigenetic level.
Studies on the molecular mechanisms controlling the differential capacity of human neutrophils and monocytes to express IL-6 in response to LPS
Zimmermann, Maili;TAMASSIA, Nicola;CASTELLUCCI, Monica;ROSSATO, Marzia;BAZZONI, Flavia;CASSATELLA, Marco Antonio
2012-01-01
Abstract
Purpose/objective. IL-6 is a pleiotropic cytokine that provokes a broad range of cellular and physiological responses including inflammation and oncogenesis. While the expression of IL-6 by human blood monocytes is well established, the potential for inducing IL-6 by human neutrophils remains controversial. Accordingly, the aim of this study was to identify whether human neutrophils produce IL-6 in response to LPS that is the major inducer of this cytokine in human monocytes through TLR4 activation. Material and methods. Granulocytes (neutrophils >96,5%) and Percoll-purified human blood peripheral monocytes (>85%) were stimulated with ultra pure LPS for 1-6 hours for gene expression studies by RT-qPCR and for overnight for protein detection by ELISA. Polymerase II (Pol II) recruitment to the transcription start site (TSS) within IL-6 gene promoter and the level of histone 4 acetylation (H4Ac) at IL-6 promoter in human neutrophils and monocytes was assessed by chromatin immunoprecipitation (ChIP). Results. The secretion level of IL-6 protein by neutrophils upon LPS stimulation remained near the detection limit. In accordance, neither significant induction of IL-6 mRNA nor its primary transcript could be detected in neutrophils activated with LPS in 6 hour time frame while IL-6 mRNA expression by human monocytes was at the maximum level. Furthermore, the lack of transcriptional activity at IL-6 locus in human neutrophils activated by LPS was confirmed by absence of Pol II recruitment to its promoter. In contrast, significant level of Pol II binding to the IL-6 promoter could be detected in human monocytes already after 1h of activation by LPS and the recruitment was further increased more than 4-fold after 5h of activation. Importantly, differences in the level of H4Ac, an epigenetic marker of transcriptionally active chromatin, were found at the IL-6 promoter in human neutrophils and monocytes. Readily detectable signal of H4Ac at the IL-6 promoter of resting monocytes increased 30-fold upon 3h LPS stimulation. In contrast, neither significant level of H4Ac nor its induction upon LPS stimulation could be observed in human neutrophils. Conclusions. These results suggest differential capacity of human neutrophils and monocytes to express IL-6 upon LPS activation that is determined in epigenetic level.
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
