Expression of CX3CL1/fractalkine by mesangial cells in vitro and in acute anti-Thy1 glomerulonephritis in rats.
BACKGROUND
Mesangial cells (MCs) can promote glomerular macrophage accumulation in glomerulonephritis through production of a variety of chemokines. This study investigated the potential of MCs to synthesize CX3CL1/fractalkine, a CX3C chemokine, both in vitro and in acute anti-Thy1 glomerulonephritis in rats.


METHODS
Anti-Thy1 glomerulonephritis was induced in Wistar rats by a single injection of mouse anti-rat Thy1.1 antibody intravenously. Glomerular mRNAs for CX3CL1/fractalkine, CCL2/monocyte chemoattractant protein (MCP)-1, and their cognate receptors, CX3CR1 and CCR2, were determined by northern blot analysis or reverse-transcription polymerase chain reaction. CX3CL1/fractalkine mRNA and protein expression in vivo was localized by in situ hybridization and immunohistochemistry. Monocytes/macrophages and activated MCs were detected by immunohistochemistry. Regulation of CX3CL1/fractalkine expression in cultured MCs was determined by northern and western blot analysis.


RESULTS
After induction of anti-Thy1 disease, glomerular CX3CL1/fractalkine mRNA was significantly up-regulated, peaking at 2 h and sustaining into day 5 of the nephritis. A corresponding increase in urinary CX3CL1/fractalkine protein was evident after day 1 of the nephritis, but became more prominent during the MC proliferative phase (days 3-5). Meanwhile, induction of glomerular CCL2/MCP-1 mRNA and urinary CCL2/MCP-1 protein occurred within 24 h, and was barely detectable after day 3 of the nephritis. Urinary CCL2/MCP-1, but not CX3CL1/fractalkine, correlated with glomerular macrophage accumulation (r = 0.936, P<0.01) and glomerular CCR2 mRNA expression (r = 0.965, P<0.01). In contrast, only urinary CX3CL1/fractalkine coincided temporally to glomerular mRNA for CX3CR1 (r = 0.809, P < 0.01). Combined in situ hybridization and immunohistochemistry revealed that activated MCs were a major source for CX3CL1/fractalkine mRNA and protein during days 3-5 of the nephritis. Incubation of cultured MCs with tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, platelet-derived growth factor (PDGF)-AB or basic fibroblast growth factor (bFGF) significantly up-regulated CX3CL1/fractalkine mRNA and protein expression. This cytokine- and growth factor-stimulated CX3CL1/fractalkine expression could be abolished by the nuclear factor-kappaB inhibitors, curcumin and MG132.


CONCLUSIONS
Our data demonstrate that activated MCs are a source for the augmented glomerular CX3CL1/fractalkine expression during the proliferative phase of acute anti-Thy1 glomerulonephritis. Up-regulation of MC CX3CL1/fractalkine by TNF-alpha, IL-1beta, PDGF-AB and bFGF is mediated, at least in part, via the nuclear factor-kappaB signalling pathway. The differential expression of CCL2/MCP-1 and CX3CL1/fractalkine may sequentially recruit distinct subsets of monocytes to the glomerulus during acute anti-Thy1 glomerulonephritis.
Chen
Yung-Ming
YM
Department of Internal Medicine, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei 10016, Taiwan.
Hu-Tsai
Mi-I
MI
Lin
Shuei-Liong
SL
Tsai
Tun-Jun
TJ
Hsieh
Bor-Shen
BS
D000818:Animals; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C / Q000096:biosynthesis* / Q000276:immunology; D005920:Glomerular Mesangium / Q000276:immunology / Q000378:metabolism*; D005921:Glomerulonephritis / Q000139:chemically induced / Q000276:immunology / Q000378:metabolism*; D007518:Isoantibodies / Q000096:biosynthesis* / Q000276:immunology; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000276:immunology; D051381:Rats; D017208:Rats, Wistar
D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C; C515539:Cx3cl1 protein, mouse; C515540:Cx3cl1 protein, rat; D007518:Isoantibodies; D008565:Membrane Proteins; C035678:anti-Thy antibody