The Connection of Monocytes and Reactive Oxygen Species in Pain

Citation: Hackel D, Pflu cke D, Neumann A, Viebahn J, Mousa S, et al. ( The Connection of Monocytes and Reactive Oxygen Species in Pain Dagmar Hackel 0 Diana Pflu cke 0 Annick Neumann 0 Johannes Viebahn 0 Shaaban Mousa 0 Erhard Wischmeyer 0 Norbert Roewer 0 Alexander Brack 0 Heike Lydia Rittner 0 Louis S. Premkumar, Southern Illinois University School of Medicine, United States of America 0 1 Klinik und Poliklinik f u r Anaesthesiologie, Universita tsklinikum , W u rzburg, Germany, 2 Klinik f u r Anesthaesiologie m. S. operative Intensivmedizin, Campus Benjamin Franklin , Charite - Universita tsmedizin Berlin, Germany, 3 Institut f u r Physiologie, Universita t Wu rzburg , Germany, 4 Klinik f u r Anaesthesiology, Campus Virchow , Charite - Universita tsmedizin Berlin , Germany The interplay of specific leukocyte subpopulations, resident cells and proalgesic mediators results in pain in inflammation. Proalgesic mediators like reactive oxygen species (ROS) and downstream products elicit pain by stimulation of transient receptor potential (TRP) channels. The contribution of leukocyte subpopulations however is less clear. Local injection of neutrophilic chemokines elicits neutrophil recruitment but no hyperalgesia in rats. In meta-analyses the monocytic chemoattractant, CCL2 (monocyte chemoattractant protein-1; MCP-1), was identified as an important factor in the pathophysiology of human and animal pain. In this study, intraplantar injection of CCL2 elicited thermal and mechanical pain in Wistar but not in Dark Agouti (DA) rats, which lack p47phox, a part of the NADPH oxidase complex. Inflammatory hyperalgesia after complete Freund's adjuvant (CFA) as well as capsaicin-induced hyperalgesia and capsaicin-induced current flow in dorsal root ganglion neurons in DA were comparable to Wistar rats. Macrophages from DA expressed lower levels of CCR2 and thereby migrated less towards CCL2 and formed limited amounts of ROS in vitro and 4-hydroxynonenal (4-HNE) in the tissue in response to CCL2 compared to Wistar rats. Local adoptive transfer of peritoneal macrophages from Wistar but not from DA rats reconstituted CCL2-triggered hyperalgesia in leukocyte-depleted DA and Wistar rats. A pharmacological stimulator of ROS production (phytol) restored CCL2-induced hyperalgesia in vivo in DA rats. In Wistar rats, CCL2-induced hyperalgesia was completely blocked by superoxide dismutase (SOD), catalase or tempol. Likewise, inhibition of NADPH oxidase by apocynin reduced CCL2-elicited hyperalgesia but not CFA-induced inflammatory hyperalgesia. In summary, we provide a link between CCL2, CCR2 expression on macrophages, NADPH oxidase, ROS and the development CCL2-triggered hyperalgesia, which is different from CFA-induced hyperalgesia. The study further supports the impact of CCL2 and ROS as potential targets in pain therapy. - Funding: This study was supported by the Interdisciplinary Center of Clinical Studies University of Wuerzburg N-113. This publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding programme Open Access Publishing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. In inflammation leukocyte subpopulations may play different roles in the generation of hyperalgesia. Intraplantar injection of the neutrophilic chemokine CXCL2/3 (macrophage inflammatory protein, MIP-2) leads to a selective accumulation of neutrophils. However, in contrast to complete Freunds adjuvant induced (CFA) inflammation with similar numbers of neutrophils in the tissue, CXCL2/3 induces no mechanical or thermal hyperalgesia [1]. In the early phase of CFA inflammation, neutrophils tonically release opioid peptides resulting in basal analgesia, which could counterbalance proalgesic effects [2]. Therefore, other cell populations appear to be responsible for inflammatory hyperalgesia. Monocytes and macrophages are major contributors to inflammatory infiltrate in later phases of inflammation [3]. CCL2 is an important and well-characterized monocytic chemokine [4,5,6]. CCL2 is a critical player in neuropathic pain and might be important to inflammatory pain [6,7]. Injection of CCL2 in the paw elicits thermal and mechanical hyperalgesia [8]. Furthermore, exposure of macrophages to CCL2 results in the release of reactive oxygen species (ROS), proinflammatory cytokines (e.g. IL-1, TNF-a, MCP-1) and profibrotic growth factors (e.g. PDGF, TGF-b) [9,10]. The phagocyte NADPH oxidase complex generates ROS. ROS play a role in the pathogenesis of acute and chronic pain and have been postulated as mediators of inflammatory [11] and neuropathic pain e.g. chemotherapy-induced neuropathic pain [12]. ROS-induced oxidative stress during inflammation results in highly reactive lipid peroxidation products like 4-hydroxynonenal (4-HNE) protein adducts [13]. Potential targets of ROS are transient receptor potential vannilloid 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1) expressed on nociceptors. ROS production by the phagocyte NADPH oxidase complex is achieved by two catalytic domains including gp91phox and p22phox a regulatory domain containing p40phox, p67phox and p47phox, coded by ncf1. NADPH oxidase can be inhibited by apocynin [14]. The NOX2 complex (the phagocyte NADPH oxidase) yields superoxide anion, which is further transformed into hydrogen peroxide and hydroxyl radicals [15]. Superoxide dismutase (SOD) catalyses the transformation of superoxide into oxygen and hydrogen peroxide. Catalase catalyses the decomposition of hydrogen peroxide to water and oxygen [16]. Thus, they are an important antioxidant defense in nearly all cells exposed to ROS. Dark Agouti (DA) rats develop an increased susceptibility to arthritis due to a genetic polymorphism for ncf1 encoding p47phox [17]. This variant of ncf1 results in a reduced release of ROS from all leukocyte populations including peritoneal macrophages [18]. As a consequence macrophages are not able to suppress the T-cell response, which in turn leads to an increased arthritis [17]. In comparison, Wistar rats are less susceptible to adjuvant-induced arthritis [19]. The present study examines the question whether the formation of ROS from monocytes is important for the development of CCL2-induced hyperalgesia. We compared DA rats with reduced activity of NADPH oxidase to Wistar rats with normal NADPH oxidase. Specifically we investigated 1) the contribution of macrophages and ROS to CCL2-induced hyperalgesia using cross over adoptive transfer experiments in DA and Wistar rats 2) inflammatory pain and TRPV1 responsiveness in both strains 3) CCR2 expression, leukocyte migration and ROS and HNE generation in response to CCL2 in both strains and 4) specific role of ROS and NADPH oxidase in CCL2-induced hyperalgesia in Wistar rats. Materials an (...truncated)