The Anti-Inflammatory Effects of Low Concentrations of Carbon Monoxide in Human Airway Smooth Muscle Cells
Carbon monoxide (CO) has emerged as a potential therapy exerting cytoprotective effects in models of inflammatory diseases including asthma. Similar studies implicate human airway smooth muscle cells (HASMC) as contributing to CO’s anti-inflammatory effects by inhibiting the release of pro-inflammatory cytokines including granulocyte macrophage colony stimulating factor (GM-CSF). However, the mechanism related to the decrease in GM-CSF release requires further exploration. Evidence demonstrates similarities in effect on oxidative stress and metabolism and subsequent effects on inflammation between hypoxic adaptation and low concentrations of CO. Hypoxic adaptation is associated with increased levels of hypoxia inducible factor-1 (HIF-1), a stress factor limited by oxygen. Therefore, if CO limits oxygen binding, as suggested by its increased affinity to and inhibition of cytochrome c oxidase, CO’s anti-inflammatory effect could be a result of HIF-1α induction. We hypothesize that CO inhibits GM-CSF release by HASMC via its effects on oxidative stress, metabolism and cellular adaptation similar to hypoxia. Immunoblotting, cytochemical staining and cell-based, immuno-based and biochemical assays were used to compare the effects of CO (250-500 ppm) and hypoxia (10-0% O2) over time as they relate to mitochondrial metabolism, pro- and anti-oxidant levels, GM-CSF release and HIF-1α expression. The major findings were that dose response patterns for CO and hypoxia suggest an inverse association between both the level of CO and hypoxic severity and GM-CSF release. A biphasic effect for cellular ROS, mitochondrial metabolic activity, and ATP levels occurs in response to both CO and hypoxia, producing an early significant increase followed by a significant decrease as compared to control conditions. This indicates that metabolism is initially up-regulated with CO and hypoxia, possibly inducing a change in signaling downstream, ultimately resulting in decreased GM-CSF release. Chemically inhibiting cytochrome c oxidase and ROS levels resulted in decreased GM-CSF release similarly to CO and hypoxia. However, HIF-1α induction does not occur in HASMC exposed to CO or 10% O2, as is seen with 0% O2. Overall, these data suggest that CO’s mechanism is associated with changes in cellular ROS levels and mitochondrial activity, partially mirroring a hypoxic mechanism in terms of its anti-inflammatory effect within HASMC.