Biochem. J. (2005) 391
(575–580) (Printed in Great Britain)
Lipopolysaccharide rapidly modifies adenosine receptor transcripts in murine and human macrophages: role of NF-kB in A2A adenosine receptor induction
Lauren J. MURPHREE*, Gail W. SULLIVAN†, Melissa A. MARSHALL† and Joel LINDEN*†1
*Department of Pharmacology, University of Virginia Health System, PO Box 801394, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, U.S.A., and †Department of Medicine, University of Virginia Health System, PO Box 801394, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, U.S.A.
The A2A adenosine receptor (A2AAR) mediates anti-inflammatory actions of adenosine in a variety of cell types. LPS (lipopolysaccharide) was reported to induce a small (<2-fold) increase in the expression of A2AAR mRNA in human monocytes and monocytic cell lines. We investigated the effects of LPS on the expression of adenosine receptor mRNAs in primary mouse IPMF (intraperitoneal macrophages), human macrophages and Wehi-3 cells. Treatment with 10 ng/ml LPS for 4 h produced a >100-fold increase in A2AAR mRNA. LPS-induced increases in mRNA for A2AAR and TNFa (tumour necrosis factor a) are reduced by 90% in IPMF pretreated with the NF-kB (nuclear factor kB) inhibitor, BAY 11-7082 {(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; 10 µM}. In Wehi-3 cells exposed to LPS, A2AAR and A2BAR transcripts are elevated by 290- and 10-fold respectively, the A1AR transcript is unchanged and the A3AR transcript is decreased by 67%. The induction of A2AAR mRNA by LPS is detectable after 1 h, reaches a peak at 6 h at 600 times control and remains elevated beyond 24 h. The ED50 (effective dose) of LPS is 2.3 ng/ml. A2AAR receptor number, measured by 125I-ZM241385 binding to whole cells, is undetectable in naïve cells and increases linearly at a rate of 23 receptors·cell-1·min-1 to a Bmax of 348 fmol/mg (28000 receptors/cell) in 20 h. The increase in receptor number is correlated with an increase in the potency of an A2A agonist (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; referred to as ATL146e) to stimulate cAMP in these cells. After LPS pretreatment, the potency of the A2A agonist, ATL146e, to reduce TNFa release from IPMF was increased by 200-fold. The results support the hypothesis that regulation of adenosine receptor expression, especially up-regulation of the A2AAR, is part of a delayed feedback mechanism initiated through NF-kB to terminate the activation of human and mouse macrophages.
Key words: adenosine receptor, ATL146e, inflammation, lipopolysaccharide, macrophage, tumour necrosis factor a (TNFa).
Abbreviations used: AR, adenosine receptor; ADA, adenosine deaminase; ATL146e, 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; BAY 11-7082, (E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; EIA, enzyme immunoassay; FBS, foetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 125I-ZM241385, 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]-triazin-5-yl amino]ethyl)-3-[125I]iodophenol; IPMF, intraperitoneal macrophages; LPS, lipopolysaccharide; NECA, 5´-N-ethylcarboxamidoadenosine; NF-kB, nuclear factor kB; TLR, Toll-like receptor; TNFa, tumour necrosis factor a.
1To whom correspondence should be addressed, at Cardiovascular Research Center, University of Virginia Health System, Charlottesville, VA 22908, U.S.A. (email jlinden@virginia.edu).
Received 2 June 2005; accepted 15 July 2005
Published as BJ Immediate Publication 15 July 2005, doi:10.1042/BJ20050888
The Biochemical Society, London ©2005
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