Limits...
An anti-inflammatory role for C/EBPδ in human brain pericytes.

Rustenhoven J, Scotter EL, Jansson D, Kho DT, Oldfield RL, Bergin PS, Mee EW, Faull RL, Curtis MA, Graham SE, Park TI, Dragunow M - Sci Rep (2015)

Bottom Line: Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1β (IL-1β).C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression.Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology and Clinical Pharmacology [2] Centre for Brain Research.

ABSTRACT
Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes. Cellular inflammatory responses are orchestrated by transcription factors but information on transcriptional control in pericytes is lacking. Because the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) is induced in a number of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses. Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1β (IL-1β). To investigate the function of the induced C/EBPδ in pericytes we used siRNA to knockdown IL-1β-induced C/EBPδ expression. C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression. Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry. Our results show that knock-down of C/EBPδ expression in pericytes following immune stimulation increased chemokine and adhesion molecule expression, thus modifying the human brain pericyte inflammatory response. The induction of C/EBPδ following immune stimulation may act to limit infiltration of peripheral immune cells, thereby preventing further inflammatory responses in the brain.

No MeSH data available.


Related in: MedlinePlus

Characterisation of adult human brain pericyte cultures.Primary human brain cell cultures at passage five were stained for Hoechst (blue) and cell specific markers αSMA (a), PDGFRβ (b), NG2 (c), P4H (d), Fibronectin (e), CD45 and GFAP (f). Positive controls of astrocytes (GFAP; g) and microglia (CD45; h) at passage two are included. Scale bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4499812&req=5

f1: Characterisation of adult human brain pericyte cultures.Primary human brain cell cultures at passage five were stained for Hoechst (blue) and cell specific markers αSMA (a), PDGFRβ (b), NG2 (c), P4H (d), Fibronectin (e), CD45 and GFAP (f). Positive controls of astrocytes (GFAP; g) and microglia (CD45; h) at passage two are included. Scale bar = 50 μm.

Mentions: Immunocytochemical analysis of early passage cell cultures obtained from human middle temporal gyrus tissue reveals a mixed population of astrocytes, microglia and pericytes as described previously10. Under our in vitro conditions microglia and astrocytes do not proliferate and are diluted out in subsequent passages. To ensure no contamination from brain glia in pericyte cultures these were grown until passage five before use. Late passage cultures showed positive immunocytochemical staining for the pericyte markers alpha smooth muscle actin (αSMA; Fig. 1a), platelet derived growth factor receptor beta (PDGFRβ; Fig. 1b) and neural/glial antigen 2(NG2; Fig. 1c) as well as the fibroblast markers prolyl-4-hydroxylase (P4H; Fig. 1d) and fibronectin (Fig. 1e). There were no cells positive for the microglia marker CD45 or the astrocyte marker glial fibrillary acidic protein (GFAP; Fig. 1f) at passage five onwards. Positive controls of GFAP (Fig. 1g) and CD45 (Fig. 1h) staining at passage two are shown.


An anti-inflammatory role for C/EBPδ in human brain pericytes.

Rustenhoven J, Scotter EL, Jansson D, Kho DT, Oldfield RL, Bergin PS, Mee EW, Faull RL, Curtis MA, Graham SE, Park TI, Dragunow M - Sci Rep (2015)

Characterisation of adult human brain pericyte cultures.Primary human brain cell cultures at passage five were stained for Hoechst (blue) and cell specific markers αSMA (a), PDGFRβ (b), NG2 (c), P4H (d), Fibronectin (e), CD45 and GFAP (f). Positive controls of astrocytes (GFAP; g) and microglia (CD45; h) at passage two are included. Scale bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499812&req=5

f1: Characterisation of adult human brain pericyte cultures.Primary human brain cell cultures at passage five were stained for Hoechst (blue) and cell specific markers αSMA (a), PDGFRβ (b), NG2 (c), P4H (d), Fibronectin (e), CD45 and GFAP (f). Positive controls of astrocytes (GFAP; g) and microglia (CD45; h) at passage two are included. Scale bar = 50 μm.
Mentions: Immunocytochemical analysis of early passage cell cultures obtained from human middle temporal gyrus tissue reveals a mixed population of astrocytes, microglia and pericytes as described previously10. Under our in vitro conditions microglia and astrocytes do not proliferate and are diluted out in subsequent passages. To ensure no contamination from brain glia in pericyte cultures these were grown until passage five before use. Late passage cultures showed positive immunocytochemical staining for the pericyte markers alpha smooth muscle actin (αSMA; Fig. 1a), platelet derived growth factor receptor beta (PDGFRβ; Fig. 1b) and neural/glial antigen 2(NG2; Fig. 1c) as well as the fibroblast markers prolyl-4-hydroxylase (P4H; Fig. 1d) and fibronectin (Fig. 1e). There were no cells positive for the microglia marker CD45 or the astrocyte marker glial fibrillary acidic protein (GFAP; Fig. 1f) at passage five onwards. Positive controls of GFAP (Fig. 1g) and CD45 (Fig. 1h) staining at passage two are shown.

Bottom Line: Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1β (IL-1β).C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression.Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology and Clinical Pharmacology [2] Centre for Brain Research.

ABSTRACT
Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes. Cellular inflammatory responses are orchestrated by transcription factors but information on transcriptional control in pericytes is lacking. Because the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) is induced in a number of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses. Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1β (IL-1β). To investigate the function of the induced C/EBPδ in pericytes we used siRNA to knockdown IL-1β-induced C/EBPδ expression. C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression. Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry. Our results show that knock-down of C/EBPδ expression in pericytes following immune stimulation increased chemokine and adhesion molecule expression, thus modifying the human brain pericyte inflammatory response. The induction of C/EBPδ following immune stimulation may act to limit infiltration of peripheral immune cells, thereby preventing further inflammatory responses in the brain.

No MeSH data available.


Related in: MedlinePlus