Abstract
Bacterial lipopolysaccharide (LPS) activates the immune system and promotes inflammation via Toll-like receptor (TLR) 4, which regulates the synthesis and release of tumor necrosis factor (TNF)-α and other inflammatory cytokines. Previous studies have shown that the nucleoside adenosine suppresses LPS-stimulated TNF-α release in human UB939 macrophages by activating an adenosine A3 receptor (A3AR) subtype on these cells. In this study, we examined the mechanism(s) underlying A3AR-dependent inhibition of TNF-α release in a mouse (RAW 264.7) cell line. Treatment of RAW 264.7 cells with LPS (3 μg/ml) increased TNF-α release, which was reduced in a dose-dependent manner by adenosine analogs N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) and R-phenylisopropyladenosine and reversed by selective A3AR blockade. The increase in TNF-α release was preceded by an increase in intracellular Ca2+ levels. Inhibition of intracellular Ca2+ release by IB-MECA, a selective agonist of the A3AR, or with BAPTA-AM, an intracellular Ca2+ chelator, reduced LPS-stimulated TNF-α release. Activation of the A3AR or inhibition of intracellular Ca2+ release also reduced LPS-stimulated nuclear factor-κB (NF-κB) activation and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Similar inhibition by A3AR was observed for LPS-stimulated inducible nitric-oxide synthase. These data support the contention that inhibition of LPS-stimulated release of inflammatory molecules, such as TNF-α and NO via the A3AR, involves suppression of intracellular Ca2+signaling, leading to suppression of NF-κB and ERK1/2 pathways.
Footnotes
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This work was supported by a grant from the Central Research Committee of Southern Illinois University School of Medicine and by National Institutes of Health Grant RO1-DC02396.
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doi:10.1124/jpet.105.091868.
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ABBREVIATIONS: LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; AR, adenosine receptor; IB-MECA, N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide; R-PIA, R-phenylisopropyladenosine; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase(s); PDTC, pyrrolidine dithiocarbamate; PD98059, 2′-amino-3′methoxyflavone; MRS1220, N-(9-chloro-2-(2-furanyl)[1,2,4]-triazolol[1,5-c]quinazolin-5-benzeneacetamide; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol; iNOS, inducible nitric-oxide synthase; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidtetra(acetoxymethyl)ester; EMSA, electrophoretic mobility shift assay(s); TLR, Toll-like receptor(s); IL, interleukin; TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; NECA, 5′-N-ethylcarboxamidoadenosine.
- Received July 1, 2005.
- Accepted September 26, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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