No abstract

Macrophages are important in the induction of new blood vessel growth during wound repair, inflammation and tumour growth. We show here that tumour necrosis factor-alpha (TNF-alpha), a secretory product of activated macrophages that is believed to mediate tumour cytotoxicity, is a potent inducer of new blood vessel growth (angiogenesis). In vivo, TNF-alpha induces capillary blood vessel formation in the rat cornea and the developing chick chorioallantoic membrane at very low doses. In vitro, TNF-alpha stimulates chemotaxis of bovine adrenal capillary endothelial cells and induces cultures of these cells grown on type-1 collagen gels to form capillary-tube-like structures. The angiogenic activity produced by activated murine peritoneal macrophages is completely neutralized by a polyclonal antibody to TNF-alpha, suggesting immunological features are common to TNF-alpha and the protein responsible for macrophage-derived angiogenic activity. In inflammation and wound repair, TNF-alpha could augment repair by stimulating new blood vessel growth; in tumours, TNF-alpha might both stimulate tumour development by promoting vessel growth and participate in tumour destruction by direct cytotoxicity.

No abstract

Adenosine has been implicated in suppressing the proinflammatory responses of classically activated macrophages induced by Th1 cytokines. Alternative macrophage activation is induced by the Th2 cytokines interleukin (IL)-4 and IL-13; however, the role of adenosine in governing alternative macrophage activation is unknown. We show here that adenosine treatment of IL-4- or IL-13-activated macrophages augments the expression of alternative macrophage markers arginase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and macrophage galactose-type C-type lectin-1. The stimulatory effect of adenosine required primarily A(2B) receptors because the nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased both arginase activity (EC(50)=261.8 nM) and TIMP-1 production (EC(50)=80.67 nM), and both pharmacologic and genetic blockade of A(2B) receptors prevented the effect of NECA. A(2A) receptors also contributed to the adenosine augmentation of IL-4-induced TIMP-1 release, as both adenosine and NECA were less efficacious in augmenting TIMP-1 release by A(2A) receptor-deficient than control macrophages. Of the transcription factors known to drive alternative macrophage activation, CCAAT-enhancer-binding protein β was required, while cAMP response element-binding protein and signal transducer and activator of transcription 6 were dispensable in mediating the effect of adenosine. We propose that adenosine receptor activation suppresses inflammation and promotes tissue restitution, in part, by promoting alternative macrophage activation.

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,