LETTERS Phagocyte-derived catecholamines enhance acute inflammatory injury Michael A. Flierl 1 , Daniel Rittirsch 1 , Brian A. Nadeau 1 , Anthony J. Chen 1 , J. Vidya Sarma 1 , Firas S. Zetoune 1 , Stephanie R. McGuire 1 , Rachel P. List 1 , Danielle E. Day 1 , L. Marco Hoesel 1 , Hongwei Gao 1 , Nico Van Rooijen 3 , Markus S. Huber-Lang 4 , Richard R. Neubig 2 & Peter A. Ward 1 It is becoming increasingly clear that the autonomic nervous system and the immune system demonstrate cross-talk during inflammation by means of sympathetic and parasympathetic path- ways 1,2 . We investigated whether phagocytes are capable of de novo production of catecholamines, suggesting an autocrine/paracrine self-regulatory mechanism by catecholamines during inflam- mation, as has been described for lymphocytes 3 . Here we show that exposure of phagocytes to lipopolysaccharide led to a release of catecholamines and an induction of catecholamine-generating and degrading enzymes, indicating the presence of the complete intracellular machinery for the generation, release and inactiva- tion of catecholamines. To assess the importance of these findings in vivo, we chose two models of acute lung injury. Blockade of a 2 -adrenoreceptors or catecholamine-generating enzymes greatly suppressed lung inflammation, whereas the opposite was the case either for an a 2 -adrenoreceptor agonist or for inhibition of cate- cholamine-degrading enzymes. We were able to exclude T cells or sympathetic nerve endings as sources of the injury-modulating catecholamines. Our studies identify phagocytes as a new source of catecholamines, which enhance the inflammatory response. Although tumor, rubor, dolor and calor (the Latin terms for swell- ing, redness, pain and heat) are classical features of acute inflam- mation, the proximal trigger of these responses is neuronal in origin. Similarly, the systemic acute-phase response to infection involves neurone-dependent responses (fever and activation of the hormonal stress response), which are regulated mainly by the hypo- thalamus 2 . Innate immunity and inflammation are the first lines of defence 4 , with initial responses involving non-specific cellular and humoral pathways, resulting in mediator release 1 . Immune and pro- inflammatory mediators that are subsequently released rapidly activ- ate neuronal responses that amplify local immune and inflammatory responses designed to contain pathogens and trigger systemic neuro- endocrine and regional neural responses that seek to return the sys- tem to a homeostatic state 2 . One of the key pathways involved in the neuroendocrine–immune modulating network is the autonomic nervous system. Recently, vagal parasympathetic signalling has been shown to have an important regulatory function in inflammation through cholinergic receptors on phagocytic cells 1,5,6 . Functional interplay of the adrenergic nervous system with the immune/inflam- matory system may counterbalance effects of the parasympathetic nervous system. The presence and synthesis of catecholamines in lymphocytes were first described more than a decade ago 3,7 . Catecholamines can thereby modulate the proliferation, differenti- ation and apoptosis of lymphocytes and the production of cytokines through adrenoreceptors expressed on T and B cells 8–12 . To invest- igate whether macrophages and neutrophils (polymorphonuclear cells; PMNs) might be able to generate and release catecholamines, as has been suggested 13,14 , cells were incubated with bacterial lipopo- lysaccharide (LPS) and levels of noradrenaline and adrenaline in cell supernatants were determined. As shown in Fig. 1a–d, noradrenaline and adrenaline levels increased significantly in cell supernatants 15 min after exposure of macrophages and PMNs to LPS, decreasing at 1 h and 2 h, and then increasing by 4 h. Experiments were repeated with human PMNs exposed to the potent complement anaphyla- toxin C5a (10 nM), with virtually identical results (data not shown). On the basis of these findings, we conducted experiments to evaluate the presence of two key enzymes involved in catecholamine synthesis in macrophages and PMNs after stimulation with LPS. Tyrosine hydroxylase (TH) is known to be the rate-limiting step in catecho- lamine synthesis, whereas dopamine b-hydroxylase (DBH) accounts for the final step of noradrenaline synthesis, converting dopamine to noradrenaline 15 . Macrophages or blood PMNs were incubated for 15 min and 4 h with 30 ng ml 21 LPS. Thereafter, messenger RNA expression for catecholamine-generating enzymes was analysed by real-time polymerase chain reaction (PCR). Low baseline mRNA levels for both key enzymes (TH and DBH) were detected in unstimulated macrophages. At 4 h after cell exposure to LPS, mRNAs for both enzymes were significantly upregulated (Fig. 1e, f). PMNs expressed low constitutive levels of mRNA for TH and DBH, but both were clearly upregulated 4 h after the addition of LPS (Fig. 1g, h). The actions of catecholamines can be terminated in three ways: by reuptake into nerve terminals, by diffusion into extracellular fluids or by metabolic transformation. Two enzymes are essential in the metabolic inactivation of catecholamines: monoa- mine oxidase (MAO) and catechol O-methyltransferase (COMT). Unstimulated macrophages contained mRNA for both COMT and MAO-A (Fig. 2a, b). After exposure of cells to LPS, the quantity of mRNA for COMT decreased whereas that for MAO-A increased. In accordance with this, when proteins were analysed by western blot- ting, exposure to LPS caused a decrease in macrophage COMT (Fig. 2c), whereas the same intervention brought about higher levels of MAO-A protein (Fig. 2d). Similar patterns were found in PMNs. Incubation with LPS decreased the level of mRNA for COMT (Fig. 2e) and increased that of mRNA for MAO-A (Fig. 2f). COMT protein amounts decreased when PMNs were exposed to LPS (Fig. 2g); the opposite was true for MAO-A (Fig. 2h). During COMT-mediated O-methylation of catecholamines, S-adenosylmethionine (SAM) serves as the methyl donor and is subsequently converted to S-adenosyl-L-homocysteine (SAH) after donation of the methyl group to the substrate. It has been known for many years that elevated SAH levels can serve as a strong inhibitor of the COMT-mediated O-methylation metabolism of catechols 16,17 . Increased phagocyte 1 Department of Pathology, 2 Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. 3 Department of Cell Biology and Immunology, Vrije Universiteit, 1081BT Amsterdam, The Netherlands. 4 Department of Trauma-, Hand- and Reconstructive Surgery, University of Ulm Medical School, 89075 Ulm, Germany. Vol 449 | 11 October 2007 | doi:10.1038/nature06185 721 Nature ©2007 Publishing Group