Role of adenosine A_2B receptors in inflammation

Abstract

Recent progress in our understanding of the unique role of A_2B receptors in the regulation of inflammation, immunity and tissue repair was considerably facilitated with the introduction of new pharmacological and genetic tools. However, it also led to seemingly conflicting conclusions on the role of A_2B adenosine receptors in inflammation with some publications indicating pro-inflammatory effects and others suggesting the opposite. This chapter reviews the functions of A_2B receptors in various cell types related to inflammation and integrated effects of A_2B receptor modulation in several animal models of inflammation. It is argued that translation of current findings into novel therapies would require a better understanding of A_2B receptors functions in diverse types of inflammatory responses in various tissues and at different points of their progression.

I. Introduction

The extracellular accumulation of adenosine contributes to the regulation of inflammation, immunity and tissue repair. Adenosine exerts its action by interacting with four adenosine receptor subtypes, A₁, A_2A, A_2B and A₃ that belong to the family of seven transmembrane G protein-coupled receptors (Fredholm et al., 2001a).

Among adenosine receptor subtypes the A_2B receptor has the lowest affinity to adenosine requiring micromolar concentrations to become functional, whereas the affinities of other adenosine receptor subtypes are significantly higher, rendering them active well below micromolar concentrations of adenosine (Fredholm et al., 2001b). Therefore, it is likely that A_2B receptors remain silent under the resting conditions when extracellular adenosine concentrations are low, estimated between ten and few hundred nM range (Fredholm, 2007), but their role becomes more important in pathophysiological conditions when adenosine concentrations are the highest.

Because both A_2A and A_2B receptor subtypes stimulate adenylate cyclase, A_2B receptors were often viewed as being a redundant low affinity version of A_2A receptors. However, there is also strong evidence that A_2B receptors play a non-redundant role distinct from, and often opposite to that of A_2A receptors.

II. Tools to study A_2B adenosine receptor function

Recent progress in our understanding of the unique role of A_2B receptors was considerably facilitated with the introduction of new pharmacological and genetic tools. Results from using these complementary approaches, however, have not always agreed, with some publications indicating pro-inflammatory effects and others suggesting the opposite. We will address these issues later in this chapter, but first will discuss potential pitfalls in using genetic and pharmacological tools that might have contributed to inadvertent misinterpretation of results.

Generation of mice deficient in A_2B adenosine receptor provided a powerful tool for investigating its function in vivo and in vitro. Several studies have found significant differences between A_2BKO and WT mouse phenotypes at rest. These differences include increases in basal TNF-α secretion, leukocyte adhesion and vascular permeability as well as changes in expression of E-selectin, P-selectin, ICAM-1, IκB and P2Y1 receptors (Eckle et al., 2008a;Yang et al., 2010;Yang et al., 2006). This is surprising because basal levels of adenosine are thought to be too low to activate A_2B receptors. As with many gene knockouts there is always concern that potential compensatory changes in the expression of other proteins may confound the specific role of A_2B receptors in any given process. Therefore, it is important to keep in mind limitations of gene knockouts so that appropriate controls can be included and correct conclusions can be made.

A number of potent and selective A_2B receptor antagonists have been synthesized over the last decade (for review see (Kalla and Zablocki, 2009)). They were used together with antagonists of other adenosine receptor subtypes to elucidate the role of A_2B receptors in inflammatory models. When interpreting these studies, however, attention should be paid to the use of antagonists at concentrations that remain selective for the receptor to be targeted. For example, DPCPX and ZM241385 are often used as selective A₁ and A_2A antagonists, respectively, but the fact that they can also bind to human and mouse A_2B receptors at low nanomolar concentrations (Kreckler et al., 2006;Linden et al., 1999) is often overlooked. Table in this chapter summarizes binding affinities of adenosine antagonists at human and mouse adenosine receptor subtypes commonly used to study the role of A_2B receptors.

Table.

Affinity of commonly used antagonists to human (h) and mouse (m) adenosine receptor subtypes determined in radioligand binding assays (inhibition constants in μM).

Compounds	Subtypes
	A1	A2A	A2B	A3
ALT-801	h 5.0a	h 0.7a	h 0.02a	h 6.3a
	m 5.2a	m 3.5a	m 0.2a	m >10a
CVT 6883	h 1.9b	h 3.3b	h 0.02b	h 1.0b
MRS 1706	h 0.2c	h 0.1c	h 0.001c	h 0.2c
MRS 1754	h 0.4c	h 0.5c	h 0.002c	h 0.6c
	m 0.009d	m × 10d	m 0.003d	m > 10d
PSB1115	h > 10b	h > 10b	h 0.05b	h > 10b
DPCPX	h 0.004e	h 0.1e	h 0.05f	h 4.0e
	m 0.002d	m 0.6d	m 0.09d	m > 10d
ZM 241385	h 0.3g	h 0.0008g	h 0.03f	h > 10g
	m 0.2d	m 0.0007d	m 0.03d	m > 100d

^a(Kolachala et al., 2008a),

^b(Kalla and Zablocki, 2009),

^c(Kim et al., 2000)

^d(Kreckler et al., 2006),

^e(Klotz et al., 1998),

^f(Linden et al., 1999),

^g(Ongini et al., 1999)

The goal of attaining selectivity for A_2B agonists has been more elusive than for antagonists but several potent A_2B agonists have been developed recently (for review see (Baraldi et al., 2009)). BAY 60-6583 has been described as a high-affinity (EC₅₀ of 3 nM) and specific A_2B agonist based on its effects on the activity of reporters co-expressed together with human adenosine receptors (Eckle et al., 2007). However, the reported affinity of BAY 60-6583 to A_2B receptors determined by radiolgand binding were approximately two order magnitude lower (Ki of 330 – 750 nM) (Auchampach et al., 2009). It is likely, therefore, that the affinity of BAY 60-6583 to A_2B receptors was overestimated in a functional reporter assay, possibly due to the significant receptor reserve in cells overexpressing A_2B receptors (Linden et al., 1999). Comparative binding studies at all four adenosine receptor subtypes would be necessary to validate the selectivity of this compound toward A_2B receptors.

In summary, the combination of novel genetic and pharmacological approaches provides powerful tools to dissect the role of adenosine signaling through A_2B receptors in physiological and pathological processes. As important as these advances are, we wanted to alert the reader to the importance of taking into account the pitfalls and limitations of these approaches for the correct interpretation of results of studies on the role of A_2B receptors in adenosine-dependent regulation of inflammatory responses.

III. A_2B receptors on immune cells

A. Neutrophils, lymphocytes, platelets

A_2B receptors are ubiquitously expressed and, therefore, it is not surprising that they are present on various cells of hematopoietic origin. In most cases, A_2B receptors are co-expressed with A_2A receptors. A_2B receptor transcripts are found in neutrophils (Fredholm et al., 1996) lymphocytes (Gessi et al., 2005) and even platelets (Amisten et al., 2008). Little is known about their specific functions in these cells and A_2A receptors appear to predominate (Csoka et al., 2008;Fredholm et al., 1996;Yang et al., 2010).

Recently, the neuronal guidance molecule netrin-1 was proposed to inhibit neutrophil migration through activation of A_2B receptors located on these cells (Rosenberger et al., 2009). Although intriguing, the issue of interactions between netrin-1 and A_2B receptors remains controversial. It has been previously hypothesized that A_2B receptors can also serve as receptors for netrin-1 (Corset et al., 2000). However, there is evidence against a direct effect of netrin-1 on A_2B receptors. First, binding assays in COS cells overexpressing A_2B receptors failed to demonstrate netrin-1 binding to these receptors (McKenna et al., 2008). Second, the lack of effects of 100 μM DPCPX and ZM241385, known to inhibit human A_2B receptors (see Table), on the netrin-1-dependent inhibition of neutrophil migration (Rosenberger et al., 2009) argues against this hypothesis.

B. Mast cells

1. Role of A_2B receptors in mast cell degranulation

Adenosine has distinct effects on mast cell degranulation of pre-formed mediators and on release of newly generated cytokine/growth factors. Adenosine is known to potentiate antigen-induced degranulation of mast cells. This effect of adenosine is mediated by A₃ adenosine receptors in rodent (Ramkumar et al., 1993;Salvatore et al., 2000), but perhaps not in human (Walker et al., 1997) or canine (Auchampach et al., 1997) mast cells.

The role of A_2B receptors in mast cell degranulation is less clear. A_2B, but not A₃ receptors both stimulated directly and potentiated the effects of the calcium ionophore A23187 on degranulation of canine BR mastocytoma cells (Auchampach et al., 1997). Conversely, A_2B receptors were proposed to play an inhibitory role in degranulation of mouse bone marrow derived mast cells (BMMCs), based on the finding that A_2BKO mice show an exaggerated antigen-induced mast cell degranulation (Hua et al., 2007). Acknowledging the implausibility of explaining this phenomenon by tonic stimulation of the low-affinity A_2B receptor by endogenous adenosine, Hua et al proposed the alternative explanation that A_2B receptors may be constitutively active in wild type BMMCs even in the absence of an agonist. However, we determined that A_2B receptors expressed in wild type BMMCs display no constitutive activity. Furthermore, our work demonstrated that A_2B receptors do not inhibit the A₃ receptor-mediated potentiation of antigen-induced degranulation of BMMCs (Ryzhov et al., 2008d). It is likely therefore that exaggerated antigen-induced degranulation in BMMCs deficient of A_2B receptors is unrelated to the loss of adenosine signaling function of A_2B receptors. Compensatory developmental changes in mice or rearrangement of proteins normally coupled to the A_2B receptor as a result of the A_2B knockout may have contributed in this phenomenon.

In contrast, pharmacological studies suggested that A_2A but not A_2B receptors can attenuate antigen-induced degranulation in human cord blood-derived mast cells (Suzuki et al., 1998) and primary human lung mast cells (Duffy et al., 2007). This effect was attributed to the ability of A_2A receptors to close K+ channel KCa3.1 by a cAMP-independent mechanism (Duffy et al., 2007).

2. Role of A_2B receptors in cytokine/growth factor secretion from mast cells

The role of A_2B receptors in regulation of cytokine/growth factor secretion from mast cells is better understood than its effects on degranulation. Studies in HMC-1 cells showed that only A_2B, but not A_2A or A₃ receptors stimulate secretion of angiogenic factors IL-8 and VEGF, and the Th2 cytokines IL-13 and IL-4 (Feoktistov and Biaggioni, 1995;Feoktistov et al., 2003;Ryzhov et al., 2006;Ryzhov et al., 2004). Conditioned media from A_2B receptor-activated mast cells stimulated human umbilical vein endothelial cell (HUVEC) proliferation and migration, and induced capillary tube formation. These pro-angiogenic effects of A_2B receptor-stimulated mast cells were attributed primarily to VEGF release, because they were blocked by anti-VEGF antibody (Feoktistov et al., 2003). Co-culturing B lymphocytes with A_2B receptor-stimulated mast cells induced IgE production by B lymphocytes, an effect that appeared to be secondary to increased secretion of Th2 cytokines IL-4 and IL-13 by the mast cells (Ryzhov et al., 2004).

Like human HMC-1 cells, mouse BMMCs express A₃, A_2A and A_2B, but not A₁ receptors (Feoktistov et al., 2003;Meade et al., 2002;Ryzhov et al., 2008d). Activation of adenosine receptors stimulated IL-13 and VEGF secretion only in wild type (WT) but not in A_2BKO BMMCs. Contrary to adenosine action on degranulation, which is only apparent in antigen-stimulated BMMCs, these effects do not require activation of FcεRI receptors, since they are evident even in the absence of antigen (Ryzhov et al., 2008d).

The notion that only A_2B, but not A_2A receptors co-expressed in the same cells, were able to stimulate cytokine/growth factor secretion could seemed paradoxical because both receptor subtypes were thought to act by stimulation of adenylate cyclase through Gs proteins. However, studies in HMC-1 revealed that in contrast to A_2A receptors, A_2B receptors are also coupled to phospholipase C (PLC), as evidenced by increase in inositol phosphate production with consequent mobilization of intracellular calcium. These A_2B receptor-dependent pathways are stimulated through a cholera toxin- and pertussis toxin-insensitive G-protein, presumably of the Gq family (Feoktistov and Biaggioni, 1995). In addition, stimulation of A_2B receptors activates the small GTP-binding protein p21ras. This event triggers ERK signaling pathway with sequential stimulation of Raf, MEK1/2 and ERK1/2 protein kinase activities (Feoktistov et al., 1999). We have also demonstrated the coupling of adenosine receptors to JNK and p38 MAPK signaling pathways (Feoktistov et al., 1999). The fact that A_2B receptors are coupled to multiple intracellular signaling pathways in mast cells, explains their ability to regulate the generation and secretion of diverse cytokines and growth factors.

A_2B receptors stimulate release of VEGF, IL-13 and IL-8 from mast cells by mechanisms that involve activation of ERK and p38 MAPK (Ryzhov et al., 2006;Ryzhov et al., 2008d). Stimulation of the receptor tyrosine kinase c-kit with stem cell factor (Meade et al., 2002) or the receptor complex ST2/IL1RAP with IL-33 (Silver et al., 2010) synergized with A_2B receptors in the upregulation of IL-8 from HMC-1 cells. Functional analysis of cells transfected with full-length and truncated receptor constructs revealed that the A_2B receptor C-terminus is important for coupling to Gs and Gq proteins. However, the A_2B receptor C-terminus is not essential for upregulation of IL-8. Instead, integrity of the third intracellular loop of the A_2B receptor was crucial for IL-8 stimulation (Ryzhov et al., 2009).

Whereas A_2B receptor-mediated stimulation of IL-8 and IL-13 is cAMP-independent, stimulation of adenylate cyclase was required (but not sufficient) for upregulation of VEGF and IL-4 (Ryzhov et al., 2006;Ryzhov et al., 2008d). The dual coupling of A_2B receptors to Gs/Gq proteins with concurrent stimulation of diverse intracellular pathways is necessary for adenosine-dependent regulation of IL-4 production in HMC-1. A_2B adenosine receptors induce IL-4 generation via Gq-mediated stimulation of PLC_β, inositol trisphosphate-mediated mobilization of intracellular Ca2+ and activation of NFAT by calcineurin. This process is potentiated via Gs-mediated stimulation of adenylate cyclase and activation of protein kinase A (PKA), and may involve the increase in protein levels of NFATc1. Thus, the existence of cross-talk between Gq- PLC_β and Gs-adenylate cyclase signaling pathways in regulation of IL-4 secretion enables A_2B receptors, coupled to both Gq and Gs, to effectively stimulate IL-4 production in mast cells (Ryzhov et al., 2006).

C. Dendritic cells

1. Dendritic cell functions

Dendritic cells play an important role in bridging innate and adaptive immunity. It is generally accepted that conventional dendritic cells arise from bone-marrow hematopoietic progenitors or peripheral blood monocytes that migrate into peripheral tissues and differentiate into immature dendritic cells. Immature dendritic cells in tissues are constantly sampling their microenvironment for the presence of antigens. Upon activation by pathogens and other inflammatory stimuli, dendritic cells undergo phenotypical maturation and migrate toward the secondary lymphoid organs. On reaching these organs, dendritic cells develop into mature cells capable to present antigens to naïve T lymphocytes, thus initiating the development of adaptive immune responses (Dominguez and Ardavin, 2010). The presence of A_2B receptors on monocytes and dendritic cells (Novitskiy et al., 2008) suggests that their activation may influence both differentiation and maturation of dendritic cells.

2. Role of A_2B receptors in dendritic cell differentiation

Using a combination of genetic and pharmacological approaches, we have recently shown that stimulation of A_2B receptors in vitro and in vivo induces generation of a phenotypically and functionally distinct subset of dendritic cells. These “adenosine-differentiated” cells are impaired in their ability to induce T-cell proliferation and IFN-γ production. These cells also produce high levels of immunomodulatory cytokines IL-6, IL-10 and TGF-β. It is possible that by up-regulating IL-10 and TGF-β, adenosine-differentiated dentritic cells could affect Th1-mediated immune reactions, induce the generation of regulatory T cells, and polarize the immune response toward a Th2 type. Because these cells also express high levels of the tolerance-inducing enzymes indoleamine 2,3-dioxygenase (IDO) and arginase, they can impair T-cell signal transduction and function. Adenosine-differentiated dendritic cells also secrete high levels of angiogenic factors VEGF and IL-8. Both immunosuppressive and pro-angiogenic properties of these cells could be beneficial for tumor growth. Indeed, our studies in vivo demonstrated that the presence of “adenosine-differentiated” dendritic cells significantly promoted tumor growth in a mouse Lewis lung carcinoma model (Novitskiy et al., 2008).

3. Role of A_2B receptors in dendritic cell maturation

In addition to modulation of cell differentiation, A_2B receptors were shown also to affect maturation of dendritic cells. A_2B receptors inhibited Th1 immune response-promoting cytokines IL-12 p70 and IL-23 but enhanced IL-10 secretion by TLR-activated bone-marrow-derived dendritic cells. Moreover, stimulation of A_2B receptors during dendritic cell maturation increased expression and enzymatic activity of IDO and arginase in LPS-activated dendritic cells (Ben et al., 2008). A_2B receptors were shown to be responsible for formation of a dendritic cell fraction expressing low levels of MHC-II and co-stimulatory molecule CD86. These cells were characterized by an increased expression of A_2B receptor transcripts compared to the rest of dendritic cell population. Only this subset of “adenosine-matured” dendritic cells expressed lower levels of IL-12p40 but higher levels of IL-10 and had poor capacity to stimulate CD4+ T cells, compared to cells matured in the absence of adenosine stimulation (Wilson et al., 2009). Thus, stimulation of A_2B receptors, not only during dendritic cell differentiation but also during maturation, may lead to the formation of a cell population capable of impairing Th1 differentiation of CD4+ T cells and promoting immune tolerance. The role of A_2B receptors in adenosine actions on both differentiation and maturation of dendritic cells appears to be non-redundant because specific stimulation of other adenosine receptor subtypes did not produce similar effects. Furthermore, these effects were observed only in WT but not A_2BKO cells and were inhibited by selective A_2B antagonists (Ben et al., 2008;Novitskiy et al., 2008;Wilson et al., 2009).

D. Monocytes/Macropages

Adenosine has been recognized as an important regulator of monocyte/macrophage functions. Some responses to adenosine are mediated by cAMP-dependent mechanisms. These responses include the inhibition of LPS-induced TNF-α production (Kreckler et al., 2009), the potentiation of IL-10 production (Nemeth et al., 2005), and the inhibition of proliferation induced by monocyte colony stimulating factor (M-CSF) (Xaus et al., 1999b). The role of A_2B receptors in adenosine-dependent regulation of these monocyte/macrophage functions is often masked by A_2A receptors co-expressed in the same cells. A_2B receptors can control cAMP-dependent functions only in those cell models where the expression of dominant A_2A receptors is negligible or absent. For example, macrophages generated from mouse bone marrow in vitro expressed predominantly A_2B adenosine receptors and only negligible levels of A_2A receptors. Stimulation of A_2B, but not A_2A receptors increased cAMP levels in this cell preparation and inhibited M-CSF-induced macrophage proliferation by upregulating the cyclin-dependent kinase inhibitor p27Kip-1 via activation of cAMP-PKA pathway (Xaus et al., 1999b). IFN-γ further upregulated the expression of A_2B receptors on bone marrow-derived macrophages resulting in an increased cAMP production in response to stimulation with NECA, which in turn down-regulated both MHC-II and iNOS expression (Xaus et al., 1999a). Due to negligible expression of A_2A receptors on RAW264.7 cells, A_2B receptors were capable of inhibiting TNF-α production and augmenting IL-10 production in this macrophage-like cell line following activation with LPS (Nemeth et al., 2005).

Similarly, A_2B receptors inhibited LPS-induced TNF-α release in peritoneal macrophages isolated from mice lacking A_2A receptor. However, selective inhibition of A_2B receptors had no effect on the adenosine-dependent inhibition of LPS-induced TNF-α secretion from WT mouse peritoneal macrophages due to the dominant role of A_2A receptors in regulation of this cAMP-dependent event (Kreckler et al., 2006). Indeed, NECA-induced cAMP accumulation was similar in peritoneal macrophages obtained from WT and A_2BKO animals, indicating the dominant role of A_2A receptors in this process. Furthermore, the absence of A_2B adenosine receptors did not affect adenosine receptor-dependent suppression of LPS-activated TNF-α release from peritoneal macrophages (Ryzhov et al., 2008c). Comprehensive pharmacological analysis of adenosine-dependent inhibition of LPS-induced TNF-α release from human primary monocytes (Zhang et al., 2005) and alveolar macrophages (Buenestado et al., 2010) revealed that this effect was exclusively mediated by A_2A receptors, corroborating the findings in mouse peritoneal macrophages.

However, A_2B receptors may have distinctive functions in macrophages even in the presence of otherwise dominant A_2A receptors. Pre-treatment of mouse primary alveolar macrophages with the selective A_2B antagonist MRS 1706 or genetic ablation of A_2B receptors resulted in a loss of NECA-stimulated increases in osteopontin expression (Schneider et al., 2010). Likewise, pharmacological inhibition with selective A_2B antagonists or genetic ablation of A_2B receptors completely abrogated NECA-induced increase in IL-6 release from peritoneal macrophages (Ryzhov et al., 2008c). Of interest, elevation of cAMP in murine macrophages attenuates LPS-induced TNF-α secretion (Kreckler et al., 2009), but has no effect on basal IL-6 release (Tang et al., 1998). It is possible that the differential regulation of TNF-α and IL-6 secretion by A_2A and A_2B receptors in mouse peritoneal macrophages can be explained by coupling of these receptors to distinct intracellular pathways. Further studies are needed to delineate the signaling pathways linking activation of A_2B receptors to cytokine production in macrophages.

IV. A_2B receptors on endothelial cells

A. Endothelial cells in inflammation

Vascular endothelium lines all vessels in the body and serves as a dynamic and selective barrier regulating the flow of nutrients, biologically active molecules and cells across blood vessel walls. Endothelial cells, in close cooperation with other cell types, play an important role in inflammation and subsequent tissue remodeling. Activation of endothelial cells by inflammatory stimuli increases vascular permeability and the expression of adhesion molecules on the endothelial surface, which in turn promote edema, and leukocyte attachment and extravasation leading to initiation of an inflammatory cascade. Later in inflammation, endothelial cells play a central role in the expansion, regression and remodeling of pre-existing blood vessels, a process commonly known as angiogenesis. Adenosine has been implicated in modulation of all these events (Sands and Palmer, 2005).

Differential expression of cell surface adenosine receptors is part of the phenotypic heterogeneity of endothelial cells, and endothelial responses to adenosine can differ depending on the relative expression of adenosine receptor subtypes. For example, HUVECs express predominantly A_2A adenosine receptors, whereas the human microvascular endothelial cells HMEC-1 express predominantly A_2B receptors (Feoktistov et al., 2002). Predominant expression of A_2B receptors has been demonstrated also in cardiac microvascular endothelial cells (Ryzhov et al., 2008b) and endothelial cells of high endothelial venules (Takedachi et al., 2008). Furthermore, there are multiple factors that can modify an endothelial phenotype, including mechanical forces, biologically active compounds, the composition of extracellular matrix, and contact with circulating and tissue-based cells. Conditions present in inflammatory processes provide powerful stimuli for such phenotypic changes. The Th1 cytokines IL-1 and TNF-α increase expression of both A_2A and A_2B adenosine receptors in human dermal microvascular endothelial cells. IFN-γ treatment increases the expression of A_2B receptors, but decreases the expression of A_2A receptors (Khoa et al., 2003). Hypoxia, a condition often present in inflamed tissues, also selectively increases A_2B expression in endothelial cells (Eltzschig et al., 2003;Feoktistov et al., 2004) by a mechanism that involves the oxygen-sensitive HIF-1α-dependent transactivation of the A_2B receptor promoter (Kong et al., 2006). An increase in the A_2B receptor expression may not only increase the effects of adenosine but also affect their outcomes. For example, HUVECs express predominantly A_2A adenosine receptors, and do not produce VEGF in response to adenosine (Feoktistov et al., 2002). Hypoxia decreased A_2A and increased A_2B receptor expression in these cells. Consistent with these changes in receptor expression, adenosine stimulated VEGF release under hypoxic but not normoxic conditions, indicating that hypoxia increased the expression of A_2B receptors that were functionally coupled to upregulation of VEGF (Feoktistov et al., 2004).

B. Role of A_2B receptors in the expression of endothelial adhesion molecules

Adenosine has been shown to inhibit the expression of adhesion molecules and leukocyte recruitment by activated endothelial cells. Whereas A_2A receptors have been implicated in these effects both in vitro and in vivo (Palmer and Trevethick, 2008), the role of A_2B receptors is less understood.

Genetic ablation of A_2B receptor in mice produced a phenotype that was interpreted as supporting an inhibitory role of A_2B receptors in the expression of endothelial adhesion molecules and leukocyte recruitment. Intravital examination of mesenteric venules revealed an increased number of leukocytes rolling or adhered to the vascular wall of A_2BKO mice as compared to WT mice. Based on these findings and also on the increased levels of the adhesion molecules ICAM-1, P-selectin and E-selectin in protein extracts isolated from mesenteric arteries of A_2BKO mice, it has been suggested that A_2B receptors tonically downregulate the expression of endothelial adhesion molecules and leukocyte recruitment (Yang et al., 2006). However, these effects of A_2B receptor ablation in vivo are likely to be secondary to the increased basal plasma levels of TNF-α, a potent activator of endothelial adhesion molecules (Mackay et al., 1993). In contrast, our studies showed that stimulation of A_2B receptors in cardiac microvascular endothelial cells induced rapid cell surface expression of P-selectin by a mechanism likely involving exocytosis of the content of Weibel-Palade bodies (Ryzhov et al., 2008b). Further studies are needed to elucidate the role of endothelial A_2B receptor activation on the expression of adhesion molecules involved in the recruitment of inflammatory cells by activated endothelium.

C. Role of A_2B receptors in regulation of endothelial barrier function

Numerous studies in vitro have shown that adenosine acting on A_2A or/and A_2B receptors decreases endothelial permeability (for review see (Biaggioni and Feoktistov, 2005)). In vivo, mice lacking either apyrase (CD39) or ecto-5′-nucleotidase (CD73), enzymes involved in the generation of extracellular adenosine, had a higher leakage of albumin through endothelium in various tissues, as measured by the Evans Blue technique (Eltzschig et al., 2003;Thompson et al., 2004). Remarkably, a similar phenotype was found in mice lacking A_2B receptors but not other adenosine receptor subtypes (Eckle et al., 2008a). The increased basal vascular permeability was even further increased in A_2BKO mice subjected to ambient hypoxia (Eckle et al., 2008a) supporting the previous evidence obtained in vitro on the role of A_2B receptors in regulation of endothelial barrier function. Studies in bone marrow chimeric mice suggested a predominant role of vascular A_2B receptors but not those located on bone marrow-derived cells in this response. Surprisingly, A_2AKO mice did not demonstrate loss of barrier function in vivo (Eckle et al., 2008a) as it would be expected from in vitro studies that implicated A_2A receptors in adenosine-dependent regulation of permeability of several endothelial cell types (Umapathy et al., 2010;Wang and Huxley, 2006). Although some of the differences in vascular permeability between A_2BKO and A_2AKO mice used in these studies may be attributed to their different genetic background (C57Bl6 and CD1, respectively), these results can be explained by the predominant role of A_2B receptors in hypoxic endothelial cells due to hypoxia-inducible factor-1α (HIF-1α)-dependent increase in A_2B receptor expression (Kong et al., 2006). HIF-1α was also shown to transactivate the promoter of CD73 (Synnestvedt et al., 2002) and repress the promoter of the equilibrative nucleoside transporter (Eltzschig et al., 2005). Taken together, these effects of hypoxia would upregulate A_2B receptors and produce high levels of adenosine at the endothelial surface, thus promoting A_2B receptor signaling. However the reason for the augmented vascular permeability in A_2BKO mice at rest, when extracellular adenosine levels are low, is less clear. Explanation of this phenomenon will require further investigation.

Reduction of endothelial permeability by adenosine seems to be mediated by the cAMP-PKA-dependent pathway, because this effect was mimicked by reagents elevating cAMP or stimulating PKA. These effects were associated with a rearrangement of the F-actin component of the cytoskeleton, enhanced cell-surface expression of cell–cell junctional protein VE-cadherin and an involvement of myosin-light-chain phosphatase (Umapathy et al., 2010). However, the exact mechanism of adenosine-induced endothelial barrier enhancement remains largely unknown. It has been proposed that it can be explained in part by relaxation of actin cytoskeletal tension, as a result of phosphorylation by PKA of actin-associated, vasodilator-stimulated phosphoprotein (Comerford et al., 2002). Inhibition of RhoA-dependent pathway has been implicated in adenosine- and cAMP-dependent regulation of endothelial barrier function (Harrington et al., 2004;Waschke et al., 2004). In addition, stimulation of ERK via A_2B receptors was also proposed to promote barrier function through dephosphorylation of the myosin II regulatory light chains (Srinivas et al., 2004).

D. Role of A_2B receptors in regulation of endothelial cell proliferation

Depending on the endothelial cell studied, either A_2A or A_2B receptors have been implicated in stimulation of endothelial cell proliferation. Adenosine A_2B receptors have been shown to mediate the proliferative actions of adenosine in human retinal microvascular endothelial cells (Afzal et al., 2003;Grant et al., 2001;Grant et al., 1999;Mino et al., 2001), in porcine coronary artery and rat aortic endothelial cells (Dubey et al., 2002). The proliferative effects of adenosine on endothelial cells are mediated at least partly by stimulating the production of growth factors that facilitate new blood vessel formation. Adenosine increased VEGF production in pig cerebral microvascular endothelial cells (Fischer et al., 1995), but the adenosine receptor subtype involved in VEGF upregulation in these cells remains uncertain because of non-specific concentrations of antagonists used in that study. Takagi and associates showed that adenosine upregulates VEGF mRNA in bovine retinal microvascular cells via A_2A receptors (Takagi et al., 1996).

However, adenosine upregulated VEGF mRNA expression and protein secretion via A_2B receptors in human retinal endothelial cells (Grant et al., 1999). VEGF is not the only angiogenic factor modulated by adenosine in endothelial cells. In human retinal microvascular endothelial cells, A_2B receptor activation upregulated also basic fibroblast growth factor (bFGF) and insulin-like factor-1 (Grant et al., 1999). In immortalized human dermal microvascular endothelial cells HMEC-1, stimulation of A_2B adenosine receptors upregulated bFGF and IL-8 in addition to VEGF (Feoktistov et al., 2002). The intracellular mechanisms mediating these effects are not clear. Although some data suggest that cAMP may play a role in the effects of adenosine on VEGF secretion (Takagi et al., 1996), other studies found that cAMP-PKA-independent stimulation of MAPK pathways was primarily responsible for pro-angiogenic effects of A_2B receptors on microvascular endothelial cells (Feoktistov et al., 2002;Grant et al., 2001;Grant et al., 1999).

It is commonly accepted that VEGF production is regulated by HIF-1α. However, A_2B receptors can upregulate VEGF production in microvascular endothelial cells by a HIF-1α-independent mechanism (Feoktistov et al., 2004). Because adenosine levels are increased in hypoxia, stimulation of A_2B receptors on endothelial cells can complement HIF-1α-dependent actions of hypoxia in the regulation of angiogenesis. Stimulation of A_2B receptors results in secretion of additional angiogenic factors (IL-8) not induced by hypoxia per se (when adenosine is scavenged by adenosine deaminase) and in greater VEGF production from endothelial cells (Ryzhov et al., 2007). Thus, A_2B receptor-dependent release of angiogenic factors can contribute to the overall effect of hypoxia and provide an autocrine pathway regulating endothelial cell growth during chronic inflammation or in the resolution phase of acute inflammation.

V. A_2B receptors on epithelial cells

A. Intestinal epithelial cells

Epithelial cells participate in inflammatory processes by maintaining mucosal integrity, producing biologically active mediators and modulating local immune responses. Functional adenosine A_2B receptors are expressed on intestinal epithelial cells. Epithelial A_2B receptors were shown to upregulate chloride secretion through the activation of apical cystic fibrosis conductance regulator (CFTR) (Strohmeier et al., 1995). The effect of A_2B receptors on epithelial secretion has received particular attention because of its potential relevance to intestinal inflammation. As part of the pathophysiology of these disorders, neutrophils are recruited into intestinal crypts, where they release AMP, which is then converted to adenosine at the epithelial cell surface by CD73. It is adenosine that then acts on epithelial A_2B receptors to stimulate chloride secretion (Madara et al., 1993). This pathway normally serves to hydrate the mucosal surface, thereby protecting the intestine by preventing the translocation of bacteria, bacterial products and antigens to lamina propria. When stimulated during inflammation, chloride secretion facilitates fluid movement to lumen in order to flush pathogens from mucosal surface, thus contributing to inflammation-associated diarrhea (Kolachala et al., 2008b).

Epithelial A_2B receptors were also shown to modulate the release of proteins involved in intestinal inflammation. Stimulation of A_2B receptors on colonic epithelial cells induced an increase in IL-6 secretion into the luminal compartment at levels sufficient to activate neutrophils (Sitaraman et al., 2001). Stimulation of A_2B receptors also induced fibronectin synthesis and secretion from the apical surface of intestinal epithelial cells (Walia et al., 2004). Apical fibronectin significantly enhanced the adherence and invasion of Salmonella typhimurium to cultured epithelial cells as well as consequent IL-8 secretion (Walia et al., 2004). Intracellular mechanisms mediating these effects were suggested to involve stimulation of IL-6 and fibronectin transcription via cAMP/PKA-mediated activation of nuclear cAMP-responsive element-binding protein (CREB) (Sitaraman et al., 2001;Walia et al., 2004).

A_2B receptor functions on intestinal epithelium can be modulated by INF-γ and TNF-α, factors elevated at various stages of inflammation. TNF-α reportedly increase A_2B receptor expression in colonic epithelial cells by a post-transcriptional mechanism possibly involving downregulation of A_2B receptor-specific micro RNA miR27b and miR128a (Kolachala et al., 2010), whereas INF-γ affects A_2B receptor signaling through the direct inhibition of adenylate cyclase expression (Kolachala et al., 2005).

B. Pulmonary epithelial cells

A_2B receptors have been suggested to play an important role in regulation of ion and water transport in airway epithelium. Airborne particles, including pathogens, are absorbed by the mucus layer lining the airways, where they are inactivated by the innate mucosal defense system and removed via mucociliary and cough clearance. This process is largely depends on active transepithelial salt transport involving the CFTR and amiloride-sensitive epithelial Na+ channel (ENaC). The role of A_2B receptors in the regulation of CFTR was demonstrated in the human airway epithelial Calu-3 cell line and primary bronchial epithelial cells (Huang et al., 2001;Lazarowski et al., 2004). Like in intestinal epithelium, A_2B receptors stimulate CFTR by a mechanism involving cAMP/PKA-dependent pathway (Lazarowski et al., 2004). In bronchial ciliated epithelium, CFTR can inhibit ENaC, and activation of CFTR in the presence of ENaC inhibition generates Cl^- secretion and liquid transport to maintain airway surface liquid volume (Donaldson and Boucher, 2007). In distal airspaces of the lung, sodium movement through ENaC and chloride movement through CFTR may be coupled (Reddy et al., 1999), and activation of CFTR-mediated chloride transport can result in fluid absorption (Fang et al., 2006). Therefore, adenosine-dependent, cAMP-mediated pathways could promote alveolar ion absorption and fluid clearance. Indeed, adenosine receptors were shown to regulate alveolar liquid clearance in rat type II pneumocytes (Factor et al., 2007), and high levels of A_2B receptor expression were described in murine type II alveolar epithelial cells (Cagnina et al., 2009).

In addition to stimulation of cAMP-dependent pathway, apical adenosine A_2B receptors in Calu-3 cells can regulate anion secretion through stimulation of basolateral K_Ca channels via PLC/Ca2⁺ signaling. This pathway synergizes with cAMP-dependent modulation of apical CFTR channels for transepithelial anion secretion and a mixed secretion of chloride and bicarbonate (Wang et al., 2008). The mechanism of A_2B receptor-mediated tranepithelial liquid transport appears even more complicated in view of the recent report that A_2B receptor signaling regulates gap junctional intercellular communication between epithelial cells by the release of PGE₂ and subsequent activation of basolateral EP₄ receptors. It has been suggested that this mechanism may contribute to the spread of ions, second messenger and/or co-factor exchange between cells to fully activate CFTR and ensure efficient Cl^- secretion (Scheckenbach et al., 2010).

Physical stimulation of airway surfaces was suggested to evoke liquid secretion by producing ATP that then locally converted to adenosine, and sensed by A_2B adenosine receptors (Huang et al., 2001). In nasal epithelial cells A_2B receptor agonists elicited sustained responses in ciliary beat frequences (Morse et al., 2001). Thus, A_2B receptors were proposed to stimulate mucociliary clearance in response to injurious stimuli to remove them from airway surfaces. Whether tonic stimulation of A_2B receptors is required at rest in order to maintain the mucus clearance is less clear given the low affinity of this receptor subtype. A recent report by Rollins et al appeared to support this hypothesis by demonstrating that the selective A_2B antagonist ATL801 inhibited autoregulation of airway surface liquid height in human bronchial epithelial cells (Rollins et al., 2008). However, the lack of effects of micromolar concentrations of DPCPX and ZM241385 (Rollins et al., 2008) known to inhibit human A_2B receptors (see Table) adds uncertainty to the authors’ conclusion.

A_2B receptors can also regulate cytokine secretion from pulmonary epithelium. Stimulation of A_2B receptors on human primary bronchial epithelial cells (HBECs) upregulated the expression of several cytokines including CXCL2, CXCL3 and IL-19 (Zhong et al., 2005). A_2B receptor-stimulated IL-19 released from HBECs was able to activate the monocytic cell line THP-1 and induce TNF-α secretion. In turn, TNF-α released from THP-1 upregulated the expression of A_2B receptors in HBECs, thus providing a positive feedback loop to facilitate the effects of adenosine (Zhong et al., 2005).

VI. Role of A_2B receptors on fibroblasts

The human fibroblasts cell line VA13 was the first cell type where a low affinity adenosine receptor was originally described back in 1980 (Bruns, 1980), and later designated as A_2B (Bruns et al., 1986). Fibroblasts represent a heterogeneous population of cells, which may differ in phenotype and function not only between anatomical sites but even within a single tissue where immature cells (often called mesenchymal fibroblasts) exist with fibroblasts of various degree of differentiation. Fibroblasts play an important role in the progression of inflammation by secreting various factors that define the tissue microenvironment and modulate immune cell functions. They also contribute to tissue remodeling by increased proliferation, differentiation and generation of various components of the extracellular matrix (for review see (Flavell et al., 2008)).

Stimulation of A_2B receptors on mouse cardiac fibroblasts was shown to promote IL-6 release. This effect was independent of the Gs–cAMP–PKA pathway, but required protein kinase C δ and p38 MAPK activation (Feng et al., 2010). In contrast, A_2B receptor-mediated increase of IL-6 release from human gingival fibroblasts was attributed, at least in part, to the activation of Gs–cAMP–PKA pathway (Murakami et al., 2000). Stimulation of these cells with high concentrations (10–50 μM) of adenosine agonists potentiated IL-6 and IL-8 release induced by IL-1β and upregulated the expression of hyaluronate synthase mRNA (Murakami et al., 2001). In contrast, stimulation of A_2B receptors on synovial fibroblasts counteracted the effects of IL-1β by decreasing MMP1 mRNA expression (Boyle et al., 1996).

The role of A_2B receptors in regulation of IL-6 secretion under normoxic and hypoxic conditions was studied in human pulmonary fibroblasts. Stimulation of A_2B receptors under normoxic conditions increased the release of IL-6 and promoted the differentiation of human lung fibroblasts to myofibroblasts. Hypoxia amplified these effects by upregulating the expression of A_2B adenosine receptors. The findings that adenosine increases the release of IL-6, and this cytokine in turn induces differentiation of fibroblasts into myofibroblasts suggest a mechanism whereby adenosine could participate in the remodeling process of chronic inflammatory diseases (Zhong et al., 2005).

Studies in corpus cavernosal fibroblast cells isolated from WT and A_2BKO mice suggested that A_2B receptors upregulate the expression of TGF-β1 and promote fibrosis. Pharmacological analysis in WT cells showed that NECA increased TGF-β1 mRNA expression and procollagen I mRNA levels, which was completely abolished by the A_2B receptor specific antagonist, MRS1754. Furthermore, this effect was not seen in the A_2BKO cells (Wen et al., 2010).

In contrast, A_2B receptors diminished collagen production and proliferation of rat cardiac fibroblasts (Dubey et al., 2001;Epperson et al., 2009). The diverse effects of A_2B receptors on fibroblasts of different origins can be attributed to the known phenotypical and functional heterogeneity of these cells.

VII. Role of A_2B receptors in animal models of inflammation

The recent generation of A_2BKO mice and the development of selective A_2B antagonists have made it possible for the testing of an integrated role of A_2B receptors in complex animal models of acute and chronic inflammation. Results have been interpreted as evidence for either “pro-inflammatory” or “anti-inflammatory” role of A_2B receptors, and results from genetic and pharmacological approach have not always been in agreement.

A_2BKO mice appear to have a pro-inflammatory phenotype compared to WT controls, characterized by elevated TNF-α plasma concentrations (Yang et al., 2006) and increased vascular permeability for albumin in the colon, kidney and lung (Eckle et al., 2008a). This is surprising because this is seen even at rest, when the low extracellular adenosine levels are not expected to stimulate A_2B receptors. Short-term exposure of mice to ambient hypoxia induced significantly higher vascular leak in all organs of A_2BKO mice compared to WT control (Eckle et al., 2008a). A_2BKO mice had also increased pulmonary albumin leakage after acute lung injury produced by either mechanical ventilation (Eckle et al., 2008b) or LPS inhalation (Eckle et al., 2008b;Schingnitz et al., 2010), compared to WT controls. In parallel to pulmonary edema, an increase in neutrophil infiltration and tissue levels of IL-1β, IL-6 and TNF-α was also higher in A_2BKO mice. Studies using bone marrow chimeric mice showed that A_2B receptors located on stromal cells rather than those on bone marrow-derived cells contribute to these differences in lung injury between A_2BKO and WT animals (Eckle et al., 2008b;Schingnitz et al., 2010).

Furthermore, it has been reported that genetic deficiency of A_2B receptors increased the mortality of mice suffering from cecal ligation and puncture-induced sepsis. The increased mortality of A_2B knockout mice was associated with increased inflammatory indices measured in the spleen, heart, and plasma in comparison with WT animals. Again, experiments using bone-marrow chimeras revealed that it is the lack of A_2B receptors on nonhematopoietic cells that is primarily responsible for the increased inflammation of septic A_2B receptor-deficient mice (Csoka et al., 2010). Another study employing bone-marrow chimeras suggested that vascular A_2B receptors also play an important role in protective effects of ischemic preconditioning in a mouse model of acute renal failure from ischemia (Grenz et al., 2008). Thus, it is possible that an increase in vascular permeability in mice lacking A_2B receptors may explain, at least in part, the observed exacerbation of acute tissue injury seen in these different disease models.

This evidence obtained in models of acute tissue injury seems to support an “anti-inflammatory” role of A_2B receptors. However, even similar models of acute injury may produce opposite results in different tissues. For example, A_2B receptors were proposed to play a protective role in gastro-intestinal model of ischemia-reperfusion injury based on enhanced intestinal injury observed in A_2BKO mice (Hart et al., 2009). In contrast, the lungs of A_2BKO mice were significantly protected in a pulmonary ischemia–reperfusion model, as evidenced by reduced pulmonary artery pressure, increased lung compliance, decreased myeloperoxidase, and reduced levels TNF-α, IL-6, CXCL-1, CCL2 and CCL5 (Anvari et al., 2010). These results would suggest a “pro-inflammatory” role of A_2B receptors in the lung, in contrast to their “anti-inflammatory” actions in the intestine. Experiments using bone-marrow chimeras also suggested that these effects were due to A_2B receptor activation primarily on resident pulmonary cells and not bone marrow-derived cells (Anvari et al., 2010).

It becomes increasingly clear that the final outcome of genetic ablation of A_2B receptors may be dependent not only on the tissue but also on the model of inflammation studied. Although genetic ablation of adenosine A_2B receptors in mice has been shown to facilitate acute inflammatory responses to antigen challenges in passively sensitized mice (Hua et al., 2007), this is not the case in chronic inflammation, a process dependent on the complex interplay between multiple cells and inflammatory factors. We studied the effects of A_2B receptor gene ablation in the context of ovalbumin-induced chronic pulmonary inflammation. We found that repetitive airway allergen challenge induced a significant increase in adenosine levels in fluid recovered by bronchoalveolar lavage (BAL). Genetic ablation of A_2B receptors significantly attenuated allergen-induced chronic pulmonary inflammation as evidenced by a reduction in the number of BAL eosinophils and in peribronchial eosinophilic infiltration. The most striking difference in the pulmonary inflammation induced in A_2BKO and WT mice was the lack of allergen-induced IL-4 release in the airways of A_2BKO animals, in line with a significant reduction in IL-4 protein and mRNA levels in lung tissue. In addition, attenuation of TGF-β1 release in airways of A_2BKO mice correlated with reduced airway smooth muscle and goblet cell hyperplasia/hypertrophy. It was concluded, therefore, that genetic removal of A_2B adenosine receptors in mice leads to inhibition of allergen-induced chronic pulmonary inflammation and airway remodeling (Zaynagetdinov et al., 2010). This conclusion was also supported by pharmacological evidence; antagonism of adenosine A_2B receptors by the selective A_2B antagonist CVT-6883 resulted in inhibition of airway inflammation induced by chronic exposure to allergen (Mustafa et al., 2007).

Inhibition of adenosine A_2B receptors by the selective A_2B antagonist CVT-6883 also reduced pulmonary inflammation and airway remodeling in adenosine deaminase (ADA)-deficient mice (Sun et al., 2006). These mice are characterized by elevated lung tissue levels of adenosine and exhibit a lung phenotype with features of lung inflammation, bronchial hyperresponsiveness, enhanced mucus secretion, increased IgE synthesis, and elevated levels of pro-inflammatory cytokines and angiogenic factors that could be reversed by lowering adenosine levels with exogenous ADA (Blackburn et al., 2000). Paradoxically, genetic removal of the A_2B receptors from ADA-deficient mice led to enhanced pulmonary inflammation and airway destruction. The authors suggested that loss of pulmonary barrier function in A_2BKO mice and excessive airway neutrophilia contributed to the enhanced tissue damage observed in this model (Zhou et al., 2009). In addition, TNF-α levels, known to be elevated in A_2BKO mice at rest (Ryzhov et al., 2008c;Yang et al., 2006), were also markedly increased in the lungs of ADA/A_2B double-knockout mice, which could activate pathways that influence the trafficking of neutrophils in the lung (Zhou et al., 2009). To explain the opposite effects of A_2B receptor antagonism and genetic ablation, the authors emphasized that pharmacological inhibition of A_2B receptors was introduced later in the disease process, 10 days after triggering pulmonary disease by withdrawing mice from ADA replacement therapy (Zhou et al., 2009). Thus, the differential effects of pharmacological antagonism and genetic deletion of A_2B receptors in ADA-deficient mice may indicate the importance of timing when elimination of A_2B receptor signaling could either promote or attenuate the development of pulmonary disease in ADA-deficient mouse model. The tonic increase in TNFα in A_2BKO but not in WT mice may also contribute to this phenomenon.

The use of A_2BKO mice has also resulted in different outcomes of bleomycin-induced pulmonary fibrosis depending on the research model used. Bleomycin instilled directly into the respiratory tract produced the acute damage resulting in extensive apoptosis of airway epithelial cells, followed by infiltration of granulocytes early after the challenge. These mice developed severe fibrosis due to failed wound healing. In contrast, in the intraperitoneal bleomycin model, lung injury and inflammation are not induced acutely, but mice do develop chronic pulmonary fibrosis. The fibrotic process was significantly reduced in A_2BKO mice in the intraperitoneal bleomycin model, but not in the intratracheal model, compared to corresponding WT controls (Zhou, 2010). In contrast, pharmacological antagonism of A_2B receptors with CVT-6883 attenuated pulmonary inflammation and fibrosis in WT mice subjected to lung injury induced by intratracheal instillation of bleomycin (Sun et al., 2006). These differences in the results obtained from genetic and pharmacological targeting of A_2B receptors could be explained by the loss of pulmonary vascular barrier seen in A_2BKO mice even at rest (Eckle et al., 2008a). This phenotype would be expected to amplify any model of acute pulmonary injury that involves an acute phase of inflammation with extensive edema and neutrophil infiltration.

The role of A_2B receptors in promoting intestinal inflammation was demonstrated in both acute and chronic mouse models of colitis. Genetic removal or pharmacological antagonism of A_2B receptors with ATL-801 attenuated clinical and histological features of intestinal inflammation in parallel with reduction of IL-6 and CXCL-1 secretion in several mouse models of experimental colitis (Kolachala et al., 2008a;Kolachala et al., 2008c). In contrast, both genetic ablation and pharmacological antagonism of A_2B with PSB1115 promoted intestinal inflammation in a mouse model of acute colitis as reflected by increased weight loss, colonic shortening, and disease activity indices (Frick et al., 2009). The reason for disparate results of these studies is unclear but may reflect the complexity of events orchestrated in these models when small differences in protocols or in composition of intestinal flora present in animals may greatly affect outcomes.

Adding more to controversy on the role of A_2B receptors in inflammatory responses, A_2B receptors were proposed to play a role in protective effects of ischemic preconditioning in models of myocardial ischemia-reperfusion injury (Eckle et al., 2007;Kuno et al., 2007). Interestingly, in situ ischemic preconditioning conferred cardioprotection in A₁KO, A_2AKO, or A₃KO mice but not in A_2BKO mice (Eckle et al., 2007). On the other hand, a recent comprehensive study also employing A_2BKO mice, and using the selective A_2B antagonist ATL-801 both in mouse and rat models of myocardial ischemia-reperfusion injury argued against contribution of A_2B receptors at least in the acute phase of ischemic preconditioning (Maas et al., 2010).

Stimulation of angiogenesis and modulation of immune response by A_2B receptors may play an important role in promotion of cancer growth, which can be considered in broad terms to share characteristics with chronic inflammatory processes. Indeed, Lewis lung carcinoma tumors grown in host animals lacking A_2B adenosine receptors contained significantly lower levels of VEGF and displayed lower intra-tumor vascular density compared to tumors grown in WT animals. This difference in neovascularization and tumor tissue VEGF levels was due to A_2B receptor-dependent VEGF production by host tumor-associated cells. Furthermore, analysis of host immune cells in tumors suggested that A_2B receptor signaling may favor the expansion of myeloid-derived suppressor cells. These observations raise the interesting possibility that host A_2B receptors on immune cells not only stimulate tumor angiogenesis, but also suppress immune surveillance, thus engaging two distinct mechanisms to promote tumor survival and growth (Ryzhov et al., 2008a). It is also possible that not only tumors, but also infectious agents may exploit host A_2B receptors for their advantage. Stimulation of host A_2B receptors was implicated in persistence of chlamidal infection (Pettengill et al., 2009) and establishing of leishmania infection (de Almeida Marques-da-Silva et al., 2008). However, the role of other adenosine receptor subtypes in these infections is not known and the mechanisms responsible for these effects need to be defined in detail.

VIII. Conclusion

To be beneficial, the inflammatory reaction must be acute, destroying an injurious agent within a short period of time and in a localized area, while inducing an immune response. This is achieved through a complex series of events involving multiple cell types and secreted factors. Given the actions of adenosine to return the host to a homeostatic state, it is not surprising that A_2B receptors may operate to control the extent of acute inflammatory responses. On the other hand, premature suppression of acute inflammation by adenosine may promote its progression to a chronic status to the detriment of the host.

We believe that labeling A_2B receptors as “anti-inflammatory” or “pro-inflammatory” may be overly simplistic and misleading because it fails to appreciate just how complex each of the different types of inflammation is and conveys little about precise mechanisms. For example, the inflammation in a model of ventilation-induced lung injury is clearly different from the inflammation in a model of allergen-induced chronic lung disease. Therefore, it is not surprising that inhibition of A_2B receptors produced opposite effects in these models. As we illustrated in this chapter, A_2B receptors may play different roles even in similar types of inflammation but occurring in different tissues. Furthermore, A_2B receptors can play different roles at different points in the progression of inflammation. In this respect, A_2B receptor functions may be reflective of pleiotropic effects of the secreted factors. As an example, IL-6 was shown to promote inflammation in models of chronic inflammatory diseases, whereas in models of acute inflammation it can suppress local and systemic acute inflammatory responses (Gabay, 2006). Moreover, various combinations of cytokines released from A_2B receptor-activated cells can produce different outcomes. TGF-β1 produced by “adenosine-differentiated” dendritic cells (Novitskiy et al., 2008) favors the emergence of adaptive Tregs, but in combination with IL-4 or IL-6, it may stimulate Th9 and Th17 cells, respectively (Ernst et al., 2010).

Targeting the low affinity A_2B receptor, as opposed to other adenosine receptor subtypes, in the development of novel therapeutic approaches for treatment of inflammatory diseases is especially appealing because these receptors are likely activated only in the pathophysiological environment, while remaining silent in normal tissues. This characteristic could provide specificity in therapy of certain immune-related disorders, while decreasing likelihood of side effects. However, translation of current findings into novel therapies would require a better understanding of A_2B receptors functions in diverse types of inflammatory responses in various tissues and at different points of their progression.

Abbreviations

ADA

adenosine deaminase

BAL

bronchoalveolar lavage

bFGF

basic fibroblast growth factor

BMMC

bone marrow-derived mast cell

CFTR

cystic fibrosis transmembrane conductance regulator

ENaC

epithelial sodium channel

ERK

extracellular signal-regulated kinase

HIF-1α

hypoxia-inducible factor 1-alpha

HBEC

human bronchial epithelial cell

HMC-1

human mast cell-1

HMEC-1

human microvascular endothelial cell-1

HUVEC

human umbilical vein endothelial cell

ICAM

inter-cellular adhesion molecule

IDO

indoleamine 2,3-dioxygenase

IL1RAP

interleukin-1 receptor accessory protein

INF-γ

interferon-gamma

JNK

c-Jun N-terminal kinase

KO

knockout

LPS

lipopolysaccharide

MAPK

mitogen-activated protein kinase

M-CSF

macrophage colony-stimulating factor

MHC-II

major histocompatibility complex class II

MMP-1

matrix metalloproteinase-1

NFAT

nuclear factor of activated T-cells, PKA, protein kinase A

PLC

phospholipase C

TGF-β1

transforming growth factor beta one

TLR

toll-like receptors

TNF-α

tumor necrosis factor alpha

VEGF

vascular endothelial growth factor

WT

wild type