2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine inhibit TNF-α and CXCL10 production from activated primary murine microglia via A2A receptors

Abstract

Background: Some cells, tissues and organs release 2',3'-cAMP (a positional isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'-AMP plus 3'-AMP and convert these AMPs to adenosine (called the extracellular 2',3'-cAMP-adenosine pathway). Recent studies show that microglia have an extracellular 2',3'-cAMP-adenosine pathway. The goal of the present study was to investigate whether the extracellular 2',3'-cAMP-adenosine pathway could have functional consequences on the production of cytokines/chemokines by activated microglia.

Methods: Experiments were conducted in cultures of primary murine microglia. In the first experiment, the effect of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production was determined. In the next experiment, the first protocol was replicated but with the addition of 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) (0.1 μM; antagonist of adenosine receptors). The last experiment compared the ability of 2-chloro-N(6)-cyclopentyladenosine (CCPA) (10 μM; selective A1 agonist), 5'-N-ethylcarboxamide adenosine (NECA) (10 μM; agonist for all adenosine receptor subtypes) and CGS21680 (10 μM; selective A2A agonist) to inhibit LPS-induced TNF-α and CXCL10 production.

Results: (1) 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine similarly inhibited LPS-induced TNF-α and CXCL10 production; (2) DPSPX nearly eliminated the inhibitory effects of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production; (3) CCPA did not affect LPS-induced TNF-α and CXCL10; (4) NECA and CGS21680 similarly inhibited LPS-induced TNF-α and CXCL10 production.

Conclusions: 2',3'-cAMP and its metabolites (3'-AMP, 2'-AMP and adenosine) inhibit LPS-induced TNF-α and CXCL10 production via A2A-receptor activation. Adenosine and its precursors, via A2A receptors, likely suppress TNF-α and CXCL10 production by activated microglia in brain diseases.

Keywords: 2′,3′-cAMP; 2′-AMP; 3′-AMP; A(2A) receptors; Adenosine; Adenosine receptors; CXCL10; Primary microglia; TNF.

Figure 1. Profile of the effects of LPS on release of cytokines and chemokines in primary murine microglia with LPS

Primary microglia were treated with LPS (100 ng/mL) or phosphate-buffered saline (PBS) for 24 hours. Released cytokines and chemokines were measured using multianalyte profiling (LabMAP™ system; Luminex Corporation, Austin, TX) with a Milliplex^® kit (EMD Millipore, Billerica, MA). Production of cytokines and chemokines are expressed as percentage of basal levels (PBS-treated controls). <DL, NS and S indicate less than assay detection limit, non-significant change and significant change, respectively.

Figure 2. Adenosine, 2’,3’-cAMP, 3’-AMP and 2’-AMP inhibit lipopolysaccharide (LPS) induced TNF-α and CXCL10 production in primary murine microglia

Primary microglia were treated without and with LPS (100 ng/mL) and without and with adenosine (30 µM), 2’,3’-cAMP (30 µM), 3’-AMP (30 µM), or 2’-AMP (30 µM) for 24 hours. TNF-α and CXCL10 production were measured by ELISA. Data represent means and SEMs and are expressed as % of TNF-α and CXCL10 levels in LPS-treated cells in the absence of purines. LPS treatment alone resulted in TNF-α levels of 2692±371 pg/ml and CXCL10 levels of 9828±1033 pg/ml. “a” indicates significantly different from corresponding group without LPS, “b” indicates significantly different from “No Purine” LPS-treated group, and “c” indicates significantly different from “Adenosine” LPS-treated group.

Figure 3. 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) blocks the effects of adenosine, 2’,3’-cAMP, 3’-AMP and 2’-AMP on lipopolysaccharide (LPS) induced TNF-α and CXCL10 production in primary murine microglia

Primary microglia were treated with DPSPX (0.1µM) and without and with LPS (100 ng/mL) and without and with adenosine (30 µM), 2’,3’-cAMP (30 µM), 3’-AMP (30 µM), or 2’-AMP (30 µM) for 24 hours. TNF-α and CXCL10 production were measured by ELISA. Data represent means and SEMs and are expressed as % of LPS treated control. LPS treatment in the presence of DPSPX resulted in TNF-α levels of 512±22 pg/ml and CXCL10 levels of 11,246±533 pg/ml. “a” indicates significantly different from corresponding group without LPS and “b” indicates significantly different from “No Purine” LPS-treated group.

Figure 4. Activation of A_2A, but not A₁, adenosine receptors results in inhibition of lipopolysaccharide (LPS) induced TNF-α and CXCL10 production

Primary microglia were co-treated with LPS (100 ng/mL) and NECA (10 µM), CGS21680 (10 µM), or CCPA (10 µM). TNF-α and CXCL10 production were measured by ELISA. Data represent means and SEMs and are expressed as % of LPS treated control. LPS treatment in the presence of ADA resulted in TNF-α levels of 1291±67 pg/ml and CXCL10 levels of 2238±158 pg/ml. “a” indicates significantly different from corresponding group without LPS and “b” indicates significantly different from “No Purine” LPS-treated group.