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(American Journal of Pathology. 2003;163:711-721.)
© 2003 American Society for Investigative Pathology

An Angiogenic Switch in Macrophages Involving Synergy between Toll-Like Receptors 2, 4, 7, and 9 and Adenosine A_2A Receptors

Grace Pinhal-Enfield^*, Madhuri Ramanathan^*, Gyorgy Hasko, Stefanie N. Vogel, Andrew L. Salzman, Geert-Jan Boons^¶ and S. Joseph Leibovich^*

From the Department of Cell Biology and Molecular Medicine,^* and the Department of Surgery, New Jersey Medical School, UMDNJ, Newark, New Jersey; the Department of Microbiology and Immunology, University of Maryland Baltimore, Baltimore, Maryland; Inotek Pharmaceuticals Corporation, Beverly, Massachusetts; and The Complex Carbohydrate Research Center,^¶ University of Georgia, Athens, Georgia

Adenosine A_2A receptor (A_2AR) 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_2AR agonists and adenosine to up-regulate VEGF, while simultaneously strongly down-regulating TNF expression. In the absence of adenosine or A_2AR agonists, Porphyromonas gingivalis (P. gingivalis) LPS and PAM₃CAG (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_2AR agonists, but not A₁R 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_2AR agonists, TLR2, 7, and 9, but not TLR4 agonists, also synergistically up-regulate VEGF, while down-regulating 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_2AR 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_2AR agonists. While adenosine and A_2AR agonists strongly down-regulate TNF protein expression induced by TLR2, 3, 4, 7, and 9 agonists, TNF mRNA and NF-B activation are not reduced. We propose a novel signaling pathway in murine macrophages involving synergy between TLRs 2, 4, 7, and 9 and A_2ARs, that up-regulates VEGF and down-regulates TNF expression, thus acting as an angiogenic switch. This angiogenic switch may play an important role in ischemia when TLR agonists are present, providing an interface between innate immunity and wound healing.

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