Adenosine A 2A receptor (A 2A R) agonists synergize with Escherichia coli (E. coli) LPS [toll-like receptor (TLR)4 agonist] to up-regulate vascular endothelial growth factor (VEGF) expression in murine macrophages. Here, we demonstrate that TLR2, TLR7, and TLR9, but not TLR3 and TLR5 agonists, also synergize with A 2A R agonists and adenosine to up-regulate VEGF, while simultaneously strongly down-regulating TNF␣ expression. In the absence of adenosine or A 2A R agonists, Porphyromonas gingivalis (P. gingivalis) LPS and PAM 3 CAG (TLR2 agonists), resiquimod (R848) (TLR7 agonist), and non-methylated CpG DNA (TLR9 agonist) strongly up-regulate TNF␣ expression, with no effect on VEGF. In the presence of adenosine or A 2A R agonists, but not A 1 R agonists, TLR2, 4, 7, and 9 agonists strongly up-regulate VEGF expression, while simultaneously down-regulating TNF␣. C57BL/ 10ScN (TLR4 deletion mutant) macrophages produce TNF␣ in response to TLR2, 3, 7, and 9 agonists, but not the TLR4 agonist E. coli LPS. With adenosine or A 2A R agonists, TLR2, 7, and 9, but not TLR4 agonists, also synergistically up-regulate VEGF, while downregulating TNF␣ expression. Polyinosinic-polycytidilic acid (poly(I:C)) (TLR3 agonist) stimulates TNF␣ expression in macrophages from both C57BL/10ScSn and C57BL/10ScN mice, but has little effect on VEGF expression in the presence of adenosine or A 2A R agonists. R-flagellins from Serratia marcescens (S. marcescens) and Salmonella muenchen (S. muenchen) do not stimulate TNF␣ expression in either C57BL/ 10ScSn or C57BL10/ScN mice, and have no effect on VEGF production in the presence of adenosine or A We have shown previously that VEGF expression by murine macrophages is synergistically up-regulated by Escherichia coli (E. coli) lipopolysaccharide (LPS) acting through TLR4 receptors, and adenosine A 2A agonists acting through A 2A Rs. 3 Treatment of macrophages with 2-[p-(2-carboxylethyl)-phenylethyl amino]-5Ј-N-ethyl-carboxamido-adenosine (CGS21680) (a specific adenosine A 2A R agonist), or 5Ј-N-ethyl-carboxamido-adenosine (NECA) (a non-specific adenosine A 2 R agonist), together with E. coli LPS, strongly up-regulates VEGF expression above the level induced by CGS21680 or NECA alone, while LPS alone does not induce VEGF expression. This synergistic up-regulation is independent of hypoxia, NO, and protein kinase-A and is stronger than that induced by hypoxia alone. 3 Mammalian toll-like receptors (TLRs) are members of a family of proteins that resemble the Drosophila toll protein. 4,5 Toll plays a role in dorsal-ventral patterning in the developing fly embryo, and also plays a key role in regulating the innate immune response of adult flies to fungi. 4,5 In mammals, TLR receptors also play a key role in the innate immune response. TLR receptors respond to bacteria and bacterial products by transmitting a ligandinduced trans-membrane signal that induces the expression of cytokines such as TNF␣, IL-1,

Murine macrophages are activated by interferon-γ (IFNγ) and/or TLR agonists such as bacterial endotoxin (LPS) to express an inflammatory (M1) phenotype characterized by expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as TNF-α and IL-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα) dependent manner. Macrophages activated in this way are designated as “alternatively activated” (M2a) macrophages. We have shown previously that adenosine A2A receptor (A2AR) agonists act synergistically with TLR2, 4, 7 and 9 agonists to switch macrophages into an “M2-like” phenotype that we have termed “M2d”. Adenosine signaling suppresses TLR-dependent expression of TNF-α, IL-12, IFN-γ and several other inflammatory cytokines by macrophages, and induces expression of VEGF and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα−/− mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10 and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α) or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. Use of these markers in vivo to identify “M2” macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.

Under normoxic conditions, macrophages from C57BL mice produce low levels of vascular endothelial growth factor (VEGF). Hypoxia stimulates VEGF expression by approximately 500%; interferon-gamma (IFN-gamma) with endotoxin [lipopolysaccharide (LPS)] also stimulates VEGF expression by approximately 50 to 150% in an inducible nitric oxide synthase (iNOS)-dependent manner. Treatment of normoxic macrophages with 5'-N-ethyl-carboxamido-adenosine (NECA), a nonselective adenosine A(2) receptor agonist, or with 2-[p-(2-carboxyethyl)-phenylethyl amino]-5'-N-ethyl-carboxamido-adenosine (CGS21680), a specific adenosine A(2A) receptor agonist, modestly increases VEGF expression, whereas 2-chloro-N(6)-cyclopentyl adenosine (CCPA), an adenosine A(1) agonist, does not. Treatment with LPS (0 to 1000 ng/ml), or with IFN-gamma (0 to 300 U/ml), does not affect VEGF expression. In the presence of LPS (EC(50) < 10 ng/ml), but not of IFN-gamma, both NECA and CGS21680 synergistically up-regulate VEGF expression by as much as 10-fold. This VEGF is biologically active in vivo in the rat corneal bioassay of angiogenesis. Inhibitors of iNOS do not affect this synergistic induction of VEGF, and macrophages from iNOS-/- mice produce similar levels of VEGF as wild-type mice, indicating that NO does not play a role in this induction. Under hypoxic conditions, VEGF expression is slightly increased by adenosine receptor agonists but adenosine A(2) or A(1) receptor antagonists 3,7-dimethyl-1-propargyl xanthine (DMPX), ZM241385, and 8-cyclopentyl-1,3-dipropylxanthine (DCPCX) do not modulate VEGF expression. VEGF expression is also not reduced in hypoxic macrophages from A(3)-/- and A(2A)-/- mice. Thus, VEGF expression by hypoxic macrophages does not seem to depend on endogenously released or exogenous adenosine. VEGF expression is strongly up-regulated by LPS/NECA in macrophages from A(3)-/- but not A(2A)-/- mice, confirming the role of adenosine A(2A) receptors in this pathway. LPS with NECA strongly up-regulates VEGF expression by macrophages from C(3)H/HeN mice (with intact Tlr4 receptors), but not by macrophages from C(3)H/HeJ mice (with mutated, functionally inactive Tlr4 receptors), implicating signaling through the Tlr4 pathway in this synergistic up-regulation. Finally, Western blot analysis of adenosine A(2A) receptor expression indicated that the synergistic interaction of LPS with A(2A) receptor agonists does not involve up-regulation of A(2A) receptors by LPS. These results indicate that in murine macrophages there is a novel pathway regulating VEGF production, that involves the synergistic interaction of adenosine A(2A) receptor agonists through A(2A) receptors with LPS through the Tlr4 pathway, resulting in the strong up-regulation of VEGF expression by macrophages in a hypoxia- and NO-independent manner.

Macrophages are an important source of vascular endothelial growth factor (VEGF). Adenosine A 2A receptor (A 2A R) agonists with Toll-like receptor (TLR) 2, 4, 7, and 9 agonists synergistically induce macrophage VEGF expression. We show here using VEGF promoter-luciferase reporter constructs that the TLR4 agonist Escherichia coli lipopolysaccharide (LPS) and the A 2A R agonists NECA and CGS21680 synergistically augment VEGF transcription in macrophages and that the HRE in the VEGF promoter is essential for this transcription. We examined whether LPS and/or NECA induce HIF-1␣ expression. HIF-1␣ mRNA levels were increased in LPS-treated macrophages in an NF-B-dependent manner; NECA strongly increased these levels in an A 2A R-dependent manner. LPS induced luciferase expression from a HIF-1␣ promoter-luciferase construct in an A 2A R-independent manner. Further stimulation with NECA did not increase HIF-1␣ promoter activity, indicating that the A 2A R-dependent increase in HIF-1␣ mRNA is post-transcriptional. LPS/NECA treatment also increased HIF-1␣ protein and DNA binding levels. Deletion of putative NF-B-binding sites from the VEGF promoter did not affect LPS/NECA-induced VEGF promoter activity, suggesting that NF-B is not directly involved in VEGF transcription. Taken together, these data indicate that LPS/NECA-induced VEGF expression involves transcriptional regulation of the VEGF promoter by HIF-1␣ through the HRE. HIF-1␣ is transcriptionally induced by LPS and post-transcriptionally up-regulated in an A 2A R-dependent manner. INTRODUCTIONMacrophages play a key role in induction of angiogenesis, which is crucial for wound healing, fibroproliferative responses, and solid tumor development (Crowther et al., 2001). When stimulated, macrophages secrete an array of cytokines and growth factors, including the potent angiogenic factor vascular endothelial growth factor (VEGF). VEGF is an endothelial cell-specific mitogen and plays an important role in vascular development and angiogenesis during embryogenesis, wound healing, solid tumor growth, and certain chronic fibroproliferative inflammatory diseases (Ferrara and Davis-Smyth, 1997). Macrophages are exquisitely sensitive to their microenvironment and produce VEGF in a tightly regulated manner (Crowther et al., 2001).Macrophages produce elevated levels of VEGF in response to a variety of stimuli, including hypoxia and endotoxin (lipopolysaccharide [LPS]) together with interferon-␥ (IFN-␥), and growth factors, and cytokines such as TGF-␣, TGF-␤, IL-1␤, and IL-6 (Goldman et al., 1993;Pertovaara et al., 1994;Levy et al., 1995;Cohen et al., 1996;Gille et al., 1997;Xiong et al., 1998). Other stimuli such as hydrogen peroxide and nitric oxide (NO) have also been implicated in VEGF up-regulation (Kimura et al., 2000;Cho et al., 2001). We have shown previously that VEGF expression by murine macrophages is synergistically up-regulated by Escherichia coli LPS acting through Toll-like receptor, TLR4, receptors with adenosine acting through A 2A receptors (A 2A Rs; Leibovich e...

Synergy between Toll-like receptor (TLR) and adenosine A 2A receptor (A 2A R) signaling switches macrophages from production of inflammatory cytokines such as tumor necrosis factor-␣ to production of the angiogenic growth factor vascular endothelial growth factor (VEGF). We show in this study that this switch critically requires signaling through MyD88, IRAK4, and TRAF6. Macrophages from mice lacking MyD88 (MyD88 ؉/؉ wounds and stimulated angiogenesis but had no significant effect on healing of MyD88 ؊/؊ wounds. These results suggest that the synergistic interaction between TLR and A 2A R signaling observed in vitro that switches macrophages from an inflammatory to an angiogenic phenotype also plays a role in wound healing in vivo.