Th1 Cytokines Regulate Adenosine Receptors and Their Downstream Signaling Elements in Human Microvascular Endothelial Cells

Abstract

We and others have shown that adenosine, acting at its receptors, is a potent modulator of inflammation and angiogenesis. To better understand the regulation of adenosine receptors during these processes we studied the effects of IL-1, TNF-α, and IFN-γ on expression and function of adenosine receptors and select members of their coupling G proteins in human dermal microvascular endothelial cells (HMVEC). HMVEC expressed message and protein for A2A and A2B, but not A1 or A3 receptors. IL-1 and TNF-α treatment increased message and protein expression of A2A and A2B receptor. IFN-γ treatment also increased the expression of A2B receptors, but decreased expression of A2A receptors. Resting HMVEC and IFN-γ-treated cells showed minimal cAMP response to the selective A2A receptor agonist 2-[2-(4-chlorophenyl)ethoxy]adenosine (MRE0094). In contrast, MRE0094 stimulated a dose-dependent increase in cAMP levels in TNF-α-treated cells that was almost completely blocked by the A2A receptor antagonist ZM-241385 (4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol). The nonselective adenosine receptor agonist 5′-(N-ethylcarboxamido)adenosine increased cAMP levels in both TNF-α- and IFN-γ-treated cells, but not control cells, and its effect was only partially reversed by ZM-241385 in TNF-α-treated cells and not affected in IFN-γ-treated cells. HMVEC expressed a higher level of G protein β1 isoform than β4 isoform. Although none of the cytokines tested affected Gβ1 expression, both IL-1 and TNF-α significantly up-regulated Gβ4 expression. These findings indicate that inflammatory cytokines modulate adenosine receptor expression and function on HMVECs and suggest that the interaction between proinflammatory cytokines and adenosine receptors may affect therapeutic responses to anti-inflammatory drugs that act via adenosine-dependent mechanisms.

Tumor Necrosis Factor-α Prevents Desensitization of Gαs-Coupled Receptors by Regulating GRK2 Association with the Plasma Membrane

“…We found that TNF-␣ inhibits agonist-dependent desensitization of A 2A Rs in human monocytoid THP-1 cells, an observation that further elucidates the TNF-␣-stimulated change in receptor function. These findings provide a more solid explanation for the enhanced function and activity of A 2A Rs observed after TNF-␣ and IL-1 treatment in our previous studies (Khoa et al, 2001(Khoa et al, , 2003. Together, these data suggest that TNF-␣ and probably some other inflammatory cytokines promote A 2A R signaling and function through multiple effects, not only enhancing the receptor number but also regulating mediators of receptor signaling and desensitization.…”

“…We and others have demonstrated that the inflammatory cytokines IL-1 and TNF-␣ increase the function and expression of adenosine receptors, especially A 2A Rs, in what seems to be feedback regulation of inflammation (Khoa et al, 2001(Khoa et al, , 2003Bshesh et al, 2002;Trincavelli et al, 2002). However, in our prior studies on several human cell types, cytokine-induced changes in A 2A R number are probably not sufficient to account for the increase in the receptor's activities (Khoa et al, 2001(Khoa et al, , 2003, leading us to speculate that TNF-␣ and other inflammatory mediators may also increase receptor function by modulating the desensitization mechanisms of adenosine receptors.…”

Purinergic Signaling and Blood Vessels in Health and Disease

“…It has also been suggested that cytokines modulate the expression of A 2A and A 2B receptors in human microvascular endothelial cells (Khoa et al, 2003). Regulation of Ang-II-induced reactive oxygen species (ROS) production and redox-signaling by A 2A receptors on endothelial cells has been reported; inhibition of A 2A receptors protects endothelial cells from acute Ang-IIinduced oxidative stress and endothelium dysfunction (Thakur et al, 2010).…”

Adenosine A2A receptor agonist ameliorates EAE and correlates with Th1 cytokine‐induced blood brain barrier dysfunction via suppression of MLCK signaling pathway

“…Using reverse transcription‐polymerase chain reaction, A2A receptor has been detected in all brain regions 26. Trincavelli reported that IL‐1 and TNF‐α up‐regulate A2A receptors in rat PC12 cells 17; more recently, Nguyen found that Th1 cytokines (IL‐1, TNF‐α, and IFN‐γ) significantly enhance A2A receptor expression in human microvascular endothelial cells 6. We found strong immunopositive A2A receptor signals on the vasculature of MS and EAE lesions.…”

“…Endothelial cells express several or all four known adenosine receptors, including A1, A2a, A2b, and A3 6. Adenosine limits the secretion of the cytokines IL‐6 and IL‐8 by acting on A2A receptors 7.…”