A role for reactive oxygen species in endotoxin-induced elevation of MOR expression in the nervous and immune systems☆
Introduction
Lipopolysaccharide (LPS), an outer-membrane component of gram-negative bacteria, is a well characterized endotoxin that activates the immune system, and, in particular, induces inflammation (Parrillo et al., 1990, Dunn, 1991, Raetz and Whitfield, 2002, Lopez-Bojorquez et al., 2004, Rumpa et al., 2010). In some cases, endotoxemia progresses to severe sepsis, resulting in multiple organ dysfunction, septic shock, and death (Lopez-Bojorquez et al., 2004, Qu et al., 2009). Morbidity associated with severe sepsis is high. There are one million deaths from sepsis worldwide, and approximately 25–30% of the cases are due to gram-negative bacterial infection (Rumpa et al., 2010).
The host-mediated response to endotoxemia involves the secretion of inflammatory cytokines and mediators as well as the activation of the coagulation and complement cascades (Dunn, 1991, Lopez-Bojorquez et al., 2004, Andreasen et al., 2008). The increased levels of circulating inflammatory cytokines resulting from LPS endotoxemia exacerbate systemic inflammation. Our previous studies showed that the levels of the pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6, are elevated in both the serum and brain of rats treated systemically with LPS (Ocasio et al., 2004, Chen et al., 2005). Other studies have reported an increase in the secretion of IL-1β and TNF-α from macrophages following LPS treatment (Evans et al., 1991, Hsu and Wen, 2002). Our previous findings also indicated that LPS couples the immune and nervous systems via actions mediated by pro-inflammatory cytokines on the hypothalamic–pituitary–adrenal (HPA) axis and that such cross-talk is necessary in order to maintain homeostasis in response to infection (Chang et al., 1998).
Inflammatory cytokines can modulate the expression of the mu-opioid receptor (MOR) in both neuronal and immune cells. In 1998, we reported that co-treatment with IL-1α and IL-1β increases MOR expression in microvascular endothelial cells (Vidal et al., 1998). IL-6 increases MOR expression and MOR binding in SH-SY5Y neuroblastoma cells (Borner et al., 2004), and TNF-α increases MOR expression in human T lymphocytes, Raji B cells, U937 monocytes, primary human polymorphonuclear leukocytes, and mature dendritic cells (Kraus et al., 2003).
The activation of the opioidergic pathway via the MOR leads to suppression of the immune response (Gaveriaux-Ruff et al., 1998, Wang et al., 2002). Chronic administration of morphine, a MOR agonist, desensitizes the pro-inflammatory cytokine-mediated effects on the HPA axis and deregulates the immune response in rats (Chang et al., 1995, Chang et al., 1996, Chang et al., 2001, Chen et al., 2005). In addition, deregulation of immune responses by exogenous opioids leads to many of the complications associated with LPS-induced endotoxic shock (Chang et al., 1998, Chang et al., 2001, Chen et al., 2005).
Reactive oxygen species (ROS) are highly reactive molecules produced during cellular respiration (McCord and Fridovich, 1978, Bast and Goris, 1989, Bayir, 2005). Both intracellular and extracellular ROS are maintained at non-lethal levels by superoxide dismutases, catalases, and a thiol-reducing buffer consisting of glutathione and thioredoxion (Nakamura et al., 1997, Gamaley and Klyubin, 1999). However, disease and stress can alter a cell's ability to effectively regulate ROS. Elevated levels of ROS can damage proteins, DNA, RNA, and cell membranes by hydroxyl radical attack, and induce apoptosis (McCord and Fridovich, 1978, Machlin and Bendich, 1987, Bast and Goris, 1989). Exposure to LPS increases the production of ROS in murine macrophages (Hsu and Wen, 2002, Kim et al., 2004), and the accumulation of ROS is a promoting factor in the development of sepsis in rats (Bayir, 2005). Thus, ROS appears to play a key role in the LPS-induced inflammatory response and the subsequent incidence of sepsis.
In this study, an in vitro conditioned medium (CM) model was used to investigate the mechanism by which LPS exposure alters MOR expression in immune and neuronal cells. Specifically, we examined the effects of LPS-induced ROS accumulation on MOR expression in TPA-differentiated HL-60 (TPA-HL-60) macrophage-like cells (Rovera et al., 1979, Kowalski and Denhardt, 1989). We also assayed the conditioned medium from the LPS-treated TPA-HL-60 cells for TNF-α, GM-CSF, IL-1β, IL-8, IL-10, IL-12p70, IL-2, IL-6, and INFγ to determine if LPS-induced ROS had a modulating effect on the cytokine secretion profile. We then evaluated MOR expression in SH-SY5Y neuroblastoma cells (Ciccarone et al., 1989) cultured in CM from the LPS-treated TPA-HL-60 cells. We have previously used undifferentiated SH-SY5Y cells to study MOR expression (Zadina et al., 1993, Zadina et al., 1994, Yu et al., 2003), and others have reported the use of SH-SY5Y as a neuronal cell model since it presents several neuronal markers, including tyrosine and dopamine-β-hydroxylase activity, uptake of norepinephrine, and core Mr 68,000 and Mr 150,000 neurofilament proteins (Ciccarone et al., 1989, Gao et al., 2001, Ruffels et al., 2004, Wu et al., 2007). Our findings indicate that ROS plays a key role in LPS-induced modulation of MOR expression in both neuronal and immune cells.
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Cell culture and treatments
Human HL-60 promyelocytic leukemic cells (ATCC, Manassas, VA) were grown in RPMI 1640 medium supplemented with 20% FBS, 100 U penicillin, and 100 μg/mL streptomycin (Gibco, Invitrogen Corp., Grand Island, NY). Experimental 12-well plates (BD Biosciences, VWR, West Chester, PA) were seeded with HL-60 cells at 5 × 105 cells/mL in 1 mL/well. In this study, HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA) into macrophage-like cells (TPA-HL-60) over a period of 48 h
Effect of LPS on ROS accumulation in TPA-HL-60 cells
HL-60 cells, differentiated for 48 h with 16 nM TPA (TPA-HL-60 cells), were treated with 0.125, 0.250, or 0.500 LPS for 24 h. Increased ROS production and intracellular accumulation of ROS have been reported in rat cardiomyocytes (Yuan et al., 2009) and rat microglia (Wang et al., 2004) using these concentrations of LPS. After LPS treatment, 20 μM of the ROS indicator, DHR123, was added for 30 min. DHR123 passively diffuses into cells where it undergoes oxidation to form the fluorophore, rhodamine
Discussion
In this study, we found that LPS stimulated the intracellular accumulation of ROS and the expression of the MOR in TPA-HL-60 cells, a macrophage cell model, and that CM from those cultures significantly increased MOR expression in SH-SY5Y cells, a neuronal cell model. These findings suggest that an indirect ROS signaling mechanism could be responsible, at least in part, for the modulation of LPS-stimulated MOR expression in SH-SY5Y cells.
Cytokines have been shown to regulate MOR expression (
Acknowledgments
The authors would like to express their thanks to Dr. Louaine Spriggs for her editorial assistance on this manuscript.
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This study was supported, in part, by the National Institutes of Health/National Institute on Drug Abuse (R01 DA007058 and K02 DA016149 to SLC).