Adenosine Receptors and Mammalian Toll-Like Receptors: Synergism in Macrophages
- Mark E. Olah1 and
- Charles C. Caldwell2
- 1Department of Pharmacology and Cell Biophysics,
- 2Department of Surgery, College of Medicine, University of Cincinnati, Cincinnati, OH 45267
For many years, adenosine has had the reputation of being a “retaliatory metabolite” in that several actions of adenosine
are protective during periods of cellular stress (1). This designation was originally based in large part on the ability of adenosine to equalize energy supply to metabolic demand.
More recently, the role of adenosine as a protective signaling molecule has been explored extensively at both the physiological
and signal transduction levels in regard to its cardioprotective effects observed in cardiac preconditioning (2, 3), during ischemia–reperfusion injury (4), and in cerebral (5) and hepatic (6, 7) ischemia. During ischemic events, hypoxia promotes increased accumulation of adenosine to levels substantially greater than
those observed under normoxic conditions. Adenosine is able to produce its protective effects through activation of cell surface
G protein–coupled adenosine receptors (ARs) of which four subtypes are recognized, the A1 AR, A2A AR, A2B AR, and A3 AR (8).
The immune system represents another context in which the protective effects of adenosine are observed (9). Specifically, activation of the A2A AR inhibits several processes in polymorphonuclear leukocytes including degranulation (10), production of oxygen free radicals (11), and tumor necrosis factor–α (TNFα) (12), and adhesion to and migration across vascular endothelium (13, 14). Similarly, in macrophages, activation of the A2A AR decreases expression of TNFα and interleukin- (IL)-12 while increasing expression of the anti-inflammatory cytokine IL-10
(15). Adenosine may also modulate immune responses downstream of TNFα release, because adenosine suppresses TNFα-induced the activation
of the transcription factor nuclear factor–κ B (NF-κ B) in leukemic, lymphoid, and epithelial cells (16). Thus, in the immune system, adenosine may again act as a physiological “brake” in that, an unremitting inflammatory response
may result in organ damage or possibly lethal systemic inflammation. Indeed, the beneficial response to A2A AR agonists in ischemia–reperfusion injury may in large part reflect the anti-inflammatory effects of AR activation (4).
Two recent publications (17, 18) have demonstrated that activation of the A2A AR in murine macrophages produces, in addition to events typically deemed as anti-inflammatory such as downregulation of
TNFα, a regulation of vascular endothelial growth factor (VEGF). Treatment of macrophages with A2A AR agonists alone produces a relatively modest increase in VEGF secretion. However, in the presence of activators of mammalian
toll-like receptors (TLRs) that, by themselves, have negligible effects on VEGF expression, stimulation of the A2A AR results in increased VEGF secretion to a level similar to that produced under hypoxia, the best characterized and perhaps
most potent inducer of VEGF expression.
TLRs, of which at least ten family members exist, are critical components of the innate immune response. TLRs recognize and
are activated by foreign ligands such as lipopolysaccharides, a component of the outer membrane of Gram-negative bacteria,
bacterial DNA that contains unmethylated CpG dinucleotides, and flagellins (19, 20). Upon activation, TLRs convey a transmembrane signal(s) that ultimately results in the induction of pro-inflammatory cytokines,
and thus the immune response (19, 20). The role of the A2A AR in the observed synergy with TLRs to increase greatly VEGF expression was convincingly demonstrated through characterization
with AR subtype–selective agonists and antagonists, as well as through the examination of macrophages isolated from A2A AR-deficient mice (17). The dramatic effects observed upon coactivation of these two very different classes of receptors prompts speculation as
to the physiological importance of these interactions in the immune system and angiogenesis.
The ability of A2A AR agonists to inhibit TLR-induced TNFα production and yet, to act synergistically with TLR agonists to increase VEGF expression
led Pinhal-Enfield and coworkers (18) to suggest that these actions allow macrophages to switch from an inflammatory to an angiogenic phenotype. The term “angiogenic
switch” was introduced by Hanahan and Folkman (21) to describe of the events that promote vascular development in tumors. In this paradigm, either an increase in pro-angiogenic
factors or a decrease in anti-angiogenic factors, or both, creates an environment that promotes tumor growth through the development
of new vessels from the pre-existing vasculature (Figure 1⇓). A post-inflammation, angiogenic phenotype of macrophages would be of significance in wound healing, and indeed, activation
of the A2A AR is associated with enhanced wound healing in vivo (22).
Over the last decade, the most extensively examined pro-angiogenic molecule has been VEGF, a secreted protein that, through
activation of tyrosine kinase receptors, promotes key events in angiogenesis that include increases in vascular permeability,
and stimulation of endothelial cell proliferation and migration (23, 24). Interestingly, adenosine acting primarily through A2A AR or A2B AR also regulates endothelial cell function and promotes angiogenesis (25 –30). As with VEGF, adenosine- promoted angiogenesis may be considered beneficial in contexts such as wound healing (22) and myocardial ischemia (2), or detrimental in disease states such as cancer (31) or in retinopathy of prematurity (29). AR activation modulates VEGF expression; however, the direction of this regulation varies in an AR subtype–specific and
cell type–dependent fashion. For instance, activation of the A2A AR in macrophages stimulates VEGF expression (17,18), whereas this receptor subtype promotes VEGF downregulation in pheochromocytoma PC12 cells (32, 33). Stimulation of the A2B AR leads to increased VEGF expression in retinal endothelial cells (34), human microvascular endothelial cells (35), and human mast cells (30). Though the AR subtype was not precisely defined, an A2 AR has been implicated in increased VEGF expression in vascular smooth muscle cells (36). Future studies should further define the dependence of adenosine-induced endothelial cell responses (and subsequent angiogenesis)
on VEGF production. Additional AR subtypes may also be involved in regulation of angiogenesis through direct or indirect effects:
the activation of A3 AR results in increased expression of angiopoietin-2 in mast cells (30).
From the standpoint of the activation of receptors and their dowmsream effectors, the synergistic effect of A2A AR and TLR activation on VEGF production is provocative. Activation of TLRs does not increase A2A AR expression (17), thus the observed synergism is apparently not a simple amplification of the mechanism responsible for the relatively modest
increase of VEGF expression observed with A2A AR agonists alone. The A2A AR is classically associated with activation of the Gsα –adenylyl cyclase–cAMP-dependent protein kinase (PKA) cascade; however, the increase of VEGF in response to A2A AR and TLR activation is not sensitive to inhibition of PKA (17). The A2A AR may signal through G protein–coupled mechanisms that are not dependent on PKA activation (27, 33, 37). Thus, a possible role for these mechanisms (and even an absolute requirement for G protein–coupling) require future study.
The A2A AR–TLR synergism is not dependent on nitric oxide, which is required for the weak induction of VEGF by “TLR-only” agonists
(17). Based on the current data, it is tempting to speculate that simultaneous activation of TLRs and the A2A AR engages a signaling mechanism that is not stimulated by either receptor alone. TLRs, upon activation by ligand, act as
scaffolds with multiple signaling molecules including the MyD88 adaptor protein, the IL-1 receptor-associated kinase (IRAK),
and TNF receptor–associated kinase (TRAF6) recruited to the receptor (19, 20). Historically, G protein–coupled receptors were thought to be almost exclusively restricted to signaling by direct association
with G protein α subunits. More recently, however, G protein–coupled receptors are known to associate with additional proteins
involved in varied, but not necessarily mutually exclusive, processes including signal transduction, scaffolding, and cellular
localization (38, 39). Indeed, recent reports describe the physical association of the A2A AR with the glutamate mGlu5 receptor (40) and α-actinin (41). With strategies directed at detecting protein–protein interactions, such as yeast two-hybrid screening and resonance energy
transfer approaches, it may be possible to identify agonist-regulated association of the A2A AR and members of the TLR family. Because concomitant activation of A2A AR and TLRs result in increased VEGF mRNA expression (17), it may be hypothesized that the presently unidentified signaling cascade engaged by receptor coactivation ultimately regulates
VEGF gene transcription. However, regulation of VEGF expression may also occur through a modulation of transcript stability
(42).
In conclusion, apparently another facet of the protective effects of adenosine has been identified through the effects of
A2A AR activation on VEGF expression in macrophages with presumably important consequences in wound healing. Future studies should
reveal further information regarding the physiological significance of this response as well as reveal new signal transduction
mechanisms for both the A2A AR and TLRs.
Figure 1.
Adenosine facilitates the angiogenic switch by macrophages. Upon bacterial infection, macrophages respond by the secretion of leukocyte recruiting mediators. Macrophages also secrete
cytokines that will initiate a Type 1 response by the adaptive immune system. This inflammatory response leads to increased
tissue damage and hypoxia as the bacterial infection is resolved (green). Extracellular adenosine concentrations increase
as a result of cellular damage and hypoxia (grey). Adenosine facilitates the angiogenic switch by inhibiting IL-12 and enhancing
VEGF and IL-10 production by macrophages (dark pink).
- © American Society for Pharmacology and Experimental Theraputics 2010
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Mark E. Olah, PhD, (left) is Assistant Professor of Pharmacology and Cell Biophysics at the University of Cincinnati College of Medicine. His
research interest is signal transduction by adenosine receptors with particular focus on vascular endothelial growth factor
regulation and endothelial cell responses. Address correspondence to MEO. Email: Mark.Olah{at}UC.edu; fax 513-558-1169. Charles C. Caldwell, PhD, (right) is an Assistant Professor of Surgery in the Trauma, Sepsis and Inflammation Research Group at the University of
Cincinnati College of Medicine. He is currently studying the involvement of purinergic receptors and hypoxia inducible factors
on lymphocytes during trauma injuries and sepsis.
