Adenosine A2a receptors induce heterologous desensitization of chemokine receptors - PubMed (original) (raw)

Adenosine A2a receptors induce heterologous desensitization of chemokine receptors

Ning Zhang et al. Blood. 2006.

Abstract

Adenosine, released by cells in an injurious or hypoxic environment, possesses potent anti-inflammatory effects by inhibiting the production of proinflammatory cytokines and superoxide anions (O2-). We hypothesized that adenosine compounds also induced heterologous desensitization of chemokine receptors, which played a critical role in leukocyte trafficking. Our studies using adenosine receptor subtype-specific agonists revealed that pretreatment with adenosine compounds suppressed RANTES-induced chemotaxis and Ca2+ flux through activation of A2a adenosine receptor. Adenosine compounds also desensitized IL-8- and MCP-1-induced chemotaxis, but not that induced by fMLP. Activation of protein kinase A (PKA), a component of the signaling pathway induced by the A2a receptor, was sufficient to desensitize RANTES-induced chemotaxis. Inhibition of PKA reversed the desensitization effects of adenosine compounds, suggesting that PKA was necessary for A2a receptor-mediated heterologous desensitization. In a mouse model, prior activation of A2a receptors blocked RANTES-induced recruitment of leukocytes in an air pouch. Moreover, the A2a receptor-induced cross-desensitization also reduced the susceptibility of monocytes to infection by an R5 strain of HIV-1. Our results suggest that activation of A2a adenosine receptors suppresses chemokine receptor function, and such receptor cross-talk was based on the simple mechanism of PKA-mediated heterologous desensitization, thus contributing to the antiinflammatory activity of adenosine.

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Figures

Figure 1.

Figure 1.

Chemotactic effects of adenosine. (A) Semiquantitative RT-PCR revealed the expression of A1, A2a, A2b, and A3 receptors on human neutrophils, monocytes, and lymphocytes. (B) Adenosine (from left, 0, 0.1, 1, 10, 100, 1000 μM) and CPA (from left, 0, 0.01, 0.1, 1, 10, 100 μM), but not CGS21680 (from left, 0, 0.1, 1, 10, 100, 1000 μM) induced chemotaxis by human monocytes. (*P < .05). (C) Adenosine and its analogs did not induce a transient Ca2+ influx. ATP and RANTES/CCL5 induced robust Ca2+ influx.

Figure 2.

Figure 2.

Role of A2a receptor in adenosine-induced heterologous desensitization of RANTES receptors. (A) Effects of adenosine (0.1 mM), NECA (10 μM), CGS21680 (2 μM), ZM241385 (2 μM)/NECA (10 μM), and CPA (0.3 μM) on RANTES-induced chemotaxis of human monocytes. (B) Effects of CGS21680 at 2 μM and 0.2 μM, CGS21680 (0.2 μM)/ZM241385 (0.1 μM) on RANTES-induced chemotaxis of human monocytes. (C) Effects of CGS21680 (2 μM) on RANTES (RAN), MCP-1 (MCP), and fMLF-induced chemotaxis of human monocytes. (D) Effects of CGS21680 (2 μM) on CXCL8/IL-8-induced chemotaxis of human neutrophils. (E) Effects of adenosine (Ade, 0.1 mM), NECA (10 μM), CGS21680 (2 μM), ZM241385 (2 μM)/NECA (10 μM), and CPA (0.3 μM) on RANTES-induced (50 ng/mL) Ca2+influx in human monocytes. (F) Effects of CGS21680 (2 μM) on MCP-1- and fMLF-induced Ca2+influx (*P < .001, by 2-way ANOVA analysis; **P < .02, Student t analysis).

Figure 3.

Figure 3.

Role of PKA in A2a receptor-mediated heterologous desensitization of RANTES receptors. (A) Effect of CGS21680 on cAMP accumulation in human monocytes. (B) Effect of a potent PKA inhibitor, cAMPs, Rp-isomer, TEA salt, at 100 μM (PKAI), on the inhibitory effects of CGS21680 (2 μM) on RANTES-induced chemotaxis. Effect of the potent PKA activator, _N_6-benzoyl-cAMP, at 1 and 10 μM (PKAA), on RANTES-induced chemotaxis in the absence of CGS21680. (C) Effects of PKAA at 1 μM on RANTES-induced Ca2+ influx. (D) Effects of a PKA inhibitor, cAMPs, Rp-isomer (PKAI), at 100 μM on CGS21680-induced inhibitory effects on RANTES-mediated Ca2+ influx. (E) Effects of a PKA inhibitor, cAMPs, Rp-isomer (PKAI), at 1 μM on CGS21680-induced inhibitory effects on MCP-1-mediated Ca2+-influx. *P < .001, by 2-way ANOVA analysis; **P < .03, by Student t analysis.

Figure 4.

Figure 4.

In vivo assay of RANTES-induced leukocyte recruitment into murine air pouches. The air pouches were initially treated with PBS, 20 μM CGS21680, or 30 μM ZM241385 together with 20 μM CGS21680 for 30 minutes, then stimulated with either 1 μg/mL RANTES, 20 μM CGS21680, 3 μM CPA, or the combination of CGS21680 and 30 μM ZM241385, for 4 hours. The cells were then harvested in PBS supplemented with EDTA and heparin, stained, and counted. (This graph represents data from one of 3 in vivo experiments, each bar represents 4 mice; *P < .01.)

Figure 5.

Figure 5.

Effect of activation of A2a receptors on CCR5-mediated HIV-1 viral infection. (A) FACS analysis revealed a decrease in the CCR5 level on activated human monocytes after pretreatment with RANTES for 30 minutes at 37°C. (B) FACS analysis revealed a decrease in the CCR5 level on activated human monocytes after pretreatment with CGS21680 (2 μM) for 30 minutes at 37°C. Pretreatment with cAMPs, Rp-isomer (PKAI) at 100 μM reversed the inhibitory effects by CGS21680. (C) Homologous competition binding analysis, using 125 I-labeled RANTES, on the binding affinity (_K_d) and binding sites (Bmax) on human monocytes. (D) Treatment with CGS21680 (2 μM) decreased RANTES binding affinity by 5-fold and RANTES binding sites by 39% (2-way ANOVA analysis of panel C versus panel D yield P < .001). (E) Effect of pretreatment with CGS21680 on the susceptibility of monocytes to R5 HIV-1. Monocytes were pretreated with either CGS21680 (CGS), the adenosine receptor antagonist ZM241385 (ZM), or both (ZM/CGS) at concentrations from 1 to 100 μM, and after 1 hour, the monocytes were exposed to HIV-1 strain JRFL. Cells were washed after 1 hour and returned to culture for 48 hours, and the supernatants were collected. The HIV p24 level in the supernatants was determined by ELISA. Data are representative of 6 experiments.

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