LIPOPOLYSACCHARIDE-STIMULATED MACROPHAGE MCP-1 PRODUCTION DEPENDS UPON SPLEEN TYROSINE KINASE

X X CHIA1, D NIKOLIC-PATERSON1,2, G TESCH1,2

1Department of Nephrology, Monash Health, Clayton, Australia, 2Centre for Inflammatory Diseases, Monash University, Clayton, Australia

Aim: To investigate the role of spleen tyrosine kinase (SYK) in inflammatory cell signalling pathways in macrophages.

Background: SYK is expressed in B-lymphocytes and myeloid cells. Inhibition of SYK has been shown to prevent or reduce disease severity in animal models of inflammatory kidney disease, including LPS-induced acute kidney injury. This protection is associated with reduced expression of monocyte chemoattractant protein-1 (MCP-1), an important chemokine recruiting macrophages to sites of inflammation.

Methods: LPS stimulation assay was performed in cultured primary mouse bone marrow-derived macrophages (BMMac) and RAW264.7 murine macrophage cells. SYK inhibition was achieved using an SYK inhibitor (SYKi), GS-492429, and Sykf/f Csf1rCre mice with selective Syk gene knockout in myeloid cells. RNA expression of inflammatory markers was examined by RT-PCR and protein secretion over 24 hours into culture media was examined by ELISA.

Results: MCP-1 mRNA expression was significantly upregulated 6 hours after LPS stimulation, and this was reduced by 33% by SYKi in RAW264.7 cells (p<0.01). SYKi reduced LPS-induced MCP-1 secretion by 74% (p<0.01) in RAW264.7 cells over 24 hours. This finding was confirmed using BMMacs in which Syk knockout reduced LPS-induced MCP-1 secretion by 48% (p=0.01). Interestingly, SYKi treatment did not affect NFκB activation (IκBα degradation by Western blot) or up-regulation of TNF mRNA levels at 1 hr post-LPS. Finally, delaying SYKi addition until 1hr after LPS stimulation still inhibited MCP-1 secretion by 60% (p<0.01) in RAW264.7cells.

Conclusion: SYK inhibition reduces MCP-1 expression and release from macrophages after LPS stimulation. This appears to be independent of NFkB activation. These new finding highlight the potential role of SYK in inflammatory kidney disease.


Biography:

Dr Xiu Xian Chia is a Nephrologist currently working towards a PhD at the Department of Nephrology, Monash Health. Dr Chia has a special interest in chronic kidney disease and is investigating the role of spleen tyrosine kinase signalling in models of acute kidney injury and chronic kidney disease.

Recent Comments
    Categories