Activation of the Adenosine A3 Receptor in RAW 264.7 Cells Inhibits Lipopolysaccharide-Stimulated Tumor Necrosis Factor- Release by Reducing Calcium-Dependent Activation of Nuclear Factor- B and Extracellular Signal-Regulated Kinase 1/2

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)and other inflammatory cytokines. Previous studies have shown that the nucleoside adenosine suppresses LPS-stimulated TNFrelease 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 TNFrelease in a mouse (RAW 264.7) cell line. Treatment of RAW 264.7 cells with LPS (3 g/ml) increased TNFrelease, which was reduced in a dose-dependent manner by adenosine analogs N-(3-iodobenzyl)-adenosine-5 -N-methyluronamide (IB-MECA) and R-phenylisopropyladenosine and reversed by selective A3AR blockade. The increase in TNFrelease was preceded by an increase in intracellular Ca levels. Inhibition of intracellular Ca release by IB-MECA, a selective agonist of the A3AR, or with BAPTA-AM, an intracellular Ca chelator, reduced LPSstimulated TNFrelease. Activation of the A3AR or inhibition of intracellular Ca release also reduced LPS-stimulated nuclear factorB (NFB) 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 TNFand NO via the A3AR, involves suppression of intracellular Ca signaling, leading to suppression of NFB and ERK1/2 pathways. Gram-negative bacteria represent a major group of pathogens responsible for causing serious infections. The lethal effect of such infections is linked to the biological effects of a glycolipid, lipopolysaccharide (LPS), or endotoxin (Morrison and Ryan, 1987). LPS activates cell signaling through the Toll-like receptor (TLR) 4 (Janeway and Medzhitov, 2002). All TLR activate a common signaling pathway, which culminates in the activation of nuclear factorB (NFB) transcription factors, as well as the mitogen-activated protein kinases (MAPK) (Aderem and Ulevitch, 2000). This process leads to the transcriptional activation of genes involved in the inflammatory response, such as inducible nitric-oxide synthase (iNOS), tumor necrosis factor (TNF), and interleukin (IL)-1 (Barnes and Karin, 1997). In the unstimulated cells, NFB is localized to the cytoplasm, where stimulus-induced activation promotes its translocation to the nucleus to transactivate NFB target genes. The translocation process is initiated by stimulusmediated phosphorylation and degradation of I B, an inhibitory binding protein present in the cytosol, releasing the 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. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.091868. ABBREVIATIONS: LPS, lipopolysaccharide; NFB, nuclear factorB; AR, adenosine receptor; IB-MECA, N-(3-iodobenzyl)-adenosine-5 -Nmethyluronamide; 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]quinazolin5-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, enzymelinked immunosorbent assay; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; NECA, 5 -N-ethylcarboxamidoadenosine. 0022-3565/06/3161-71–78$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 316, No. 1 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 91868/3067856 JPET 316:71–78, 2006 Printed in U.S.A. 71 at A PE T Jornals on A ril 2, 2017 jpet.asjournals.org D ow nladed from active NFB homodimer or heterodimers, which is free to enter the nucleus and regulate gene expression (Karin and Ben-Neriah, 2000). MAPK are a group of related serine/threonine protein kinases, which also participate in LPS-mediated immune cell activation (Han et al., 1994; Dong et al., 2002). The MAPK family includes the extracellular signal-related kinase (ERK)1/2, p38 MAPK, and c-Jun NH2-terminal kinase. These MAPK modulate cellular responses through phosphorylation of transcription factors and activation of downstream kinases. Inhibition of individual MAPK pathways suppresses downstream cytokine production. For example, inhibition of p38 MAPK reduces TNFand IL-8 (Foey et al., 1998; Manthey et al., 1998) and IL-10 levels (Foey et al., 1998) in LPS-stimulated monocytes. In contrast, inhibition of ERK produced only partial inhibition of TNFand IL-10 but no change in IL-1 production (Foey et al., 1998). The nucleoside adenosine is a potent endogenous regulator of the inflammatory response by interacting with the P1 purinergic receptors. These receptors include the A1 adenosine receptor (AR), the A2A and A2BAR, and the A3AR subtypes (Olah and Stiles, 2000). Three of these, the A1, A2A, and A3AR subtypes, are expressed on monocytes and macrophage cell lines (Sajjadi et al., 1996). Adenosine suppresses LPSstimulated TNFproduction by macrophage lineage cells (Bouma et al., 1994; Haskó et al., 1996; Sajjadi et al., 1996), presumably through activation of the A2A (Bouma et al., 1994; Haskó et al., 1996) or the A3AR subtype (Haskó et al., 1996; Sajjadi et al., 1996). However, the mechanism mediating this anti-inflammatory action is not known. This study was undertaken to better understand the mechanism by which adenosine mediates its anti-inflammatory action in a murine macrophage (RAW 264.7) line. This cell line is a widely used model for studying the effect of LPS on signal transduction pathways. Our data indicate that activation of the A3AR suppresses LPS-stimulated intracellular Ca accumulation, resulting from inhibition of intracellular Ca release and plasma membrane Ca entry. We also show that a rise in intracellular Ca promotes NFB and ERK1/2 activation, signaling pathways linked to TNFand iNOS production. Materials and Methods Materials. LPS (Escherichia coli 0111:B4), R-phenylisopropyladenosine (R-PIA), IB-MECA, EGTA, pyrrolidine dithiocarbamate (PDTC), and PD98059 were from Sigma-Aldrich (St. Louis, MO). MRS1220, DPCPX, and ZM241385 were from Tocris Cookson Inc. (Ellisville, MO). Antibodies for pERK1/2 and I Bwere obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and antibodies for p42 ERK were from Calbiochem (San Diego, CA). ECL kits for Western blotting were purchased from ECL Western Blotting System (GE Healthcare, Little Chalfont, Buckinghamshire, UK). BAPTA-AM was from Calbiochem. Cell Culture. The mouse macrophage cell line RAW 264.7 was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 5% CO2, and 100% humidity. Cells at 3 10/ml were added to tissue culture plates, allowed to adhere overnight, and then stimulated with 3 g/ml LPS. Adenosine receptor agonists, antagonists, and other drugs were added at the times