Lipopolysaccharide rapidly modifies adenosine receptor transcripts in murine and human macrophages: role of NF-kappaB in A(2A) adenosine receptor induction

Abstract

The A(2A) adenosine receptor (A(2A)AR) 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 A(2A)AR mRNA in human monocytes and monocytic cell lines. We investigated the effects of LPS on the expression of adenosine receptor mRNAs in primary mouse IPMPhi (intraperitoneal macrophages), human macrophages and Wehi-3 cells. Treatment with 10 ng/ml LPS for 4 h produced a >100-fold increase in A(2A)AR mRNA. LPS-induced increases in mRNA for A(2A)AR and TNFalpha (tumour necrosis factor alpha) are reduced by 90% in IPMPhi pretreated with the NF-kappaB (nuclear factor kappaB) inhibitor, BAY 11-7082 {(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; 10 microM}. In Wehi-3 cells exposed to LPS, A(2A)AR and A(2B)AR transcripts are elevated by 290- and 10-fold respectively, the A(1)AR transcript is unchanged and the A(3)AR transcript is decreased by 67%. The induction of A(2A)AR 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. A(2A)AR receptor number, measured by 125I-ZM241385 binding to whole cells, is undetectable in naïve cells and increases linearly at a rate of 23 receptors x cell(-1) x min(-1) to a B(max) 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 A(2A) 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 A(2A) agonist, ATL146e, to reduce TNFalpha release from IPMPhi was increased by 200-fold. The results support the hypothesis that regulation of adenosine receptor expression, especially up-regulation of the A(2A)AR, is part of a delayed feedback mechanism initiated through NF-kappaB to terminate the activation of human and mouse macrophages.

Figure 1. Effect of LPS treatment on expression of A_2AAR mRNA in Wehi-3 cells as determined by real-time, quantitative PCR

(A) Time course of A_2AAR expression upon stimulation with 100 ng/ml LPS. (B) Dose–response of A_2AAR expression as measured after 4 h of LPS treatment. Transcript levels were normalized to GAPDH levels and all data points are the means±S.E.M. for at least three independent experiments performed in duplicate and quantitative PCR performed in triplicate.

Figure 2. Effects of LPS and BAY 11-7082 on mRNA and protein expression in macrophages

(A) Fold changes in transcripts following treatment of various cells with 10 ng/ml LPS for 4 h. (B) Effect of pretreating IPMΦ with 10 μM BAY 11-7082 (BAY 11) on the transcription of A_2AR, TNFα mRNAs and TNFα protein. Control TNFα levels are 3.6±1.2 pmol/ml. Data are pooled for three to seven experiments.

Figure 3. Effect of LPS treatment on A_2AAR receptor expression and function in Wehi-3 cells

(A) Saturation binding isotherms for control cells and cells treated for 4–20 h with LPS (n=3; where n is the number of replicates). (B) Scatchard transformations of the data in (A) fitted with linear regressions demonstrating binding to a single site. (C) Change over time in the number of A_2AARs (¹²⁵I-ZM241385 binding sites) after treatment with LPS. The data are fitted to a straight line (coefficient of determination r²=0.998). (D) Change in cAMP (TNFα-treated – basal) in response to A_2AAR agonist stimulation. After stimulation with or without 10 nM ATL146e and 50 nM ZM241385 (¹²⁵I-ZM) (in four combinations shown in D), cells were incubated for 20 min at 37 °C in the presence of 50 μM rolipram. The cAMP concentrations in the supernatant were measured by EIA. The baseline level of cAMP was 0.2 pmol/ml.

Figure 4. Effect of LPS treatment on whole-cell radioligand binding and functional potency of ATL146e in IPMΦ

Macrophages were harvested from Balb/c mice as described in the Experimental section. (A) Cells treated with or without 10 ng/ml LPS for 20 h were removed from the tissue-culture plates with PBS +10 mM EDTA and gentle scraping. After washing in PBS, cells were resuspended and radioligand binding was performed with approx. 0.3 nM ¹²⁵I-ZM241385 for 3 h at 4 °C. Non-specific binding was measured in the presence of 100 nM ZM241385. Data represent the means±S.E.M., n=3. (B) Cells were pretreated with or without 10 ng/ml LPS for 18 h, rinsed twice with PBS, and then restimulated with 10 ng/ml LPS±ATL146e in various concentrations. Cell supernatants were harvested after 4 h and assayed for TNFα. The IC₅₀ of ATL146e for control and LPS-pretreated cells was 20 and 0.1 nM respectively. Each point represents the mean±S.E.M. for three independent experiments.