Detection of Reactive Oxygen Species in Human Neutrophils Under Various Conditions of Exposure to Galectin

Reactive oxygen species (ROS) have been extensively studied in biology in the past years. This class of molecules can be derived from endogenous sources (e.g., phagocytic cells as neutrophils, eosinophils, monocytes, macrophages, and organelles as mitochondria and peroxisomes) and participate in physiological and pathological conditions. The beneficial and harmful effects of ROS depend on redox regulation, which establishes the balance between their production and the activity of antioxidant systems to prevent oxidative stress in vivo. Neutrophils are the immune effectors most well depicted with an intense oxidative burst in response to tissue inflammation. Several proteins and members of the galectin family are involved in this fine modulation of ROS production by neutrophils. Interestingly, studies have indicated that Galectin-1 (Gal-1) can up- or downregulate ROS production by neutrophils even when exposed to N-formyl-Met-Leu-Phe (fMLP) or Phorbol Myristate Acetate (PMA), both of which are potent neutrophil stimulants that trigger high levels of ROS production. Similarly, Galectin-3 (Gal-3) induces ROS in neutrophils from a sterile or nonsterile inflammatory environment, possibly creating a negative loop that could control ROS production. Besides, superoxide production is also induced by Galectin-8 (Gal-8) and Galectin-9 (Gal-9) in neutrophils but in a different manner. We describe herein the luminol and lucigenin-dependent chemiluminescence technique by using a luminometer as a method of assessment to measure ROS production by human neutrophils isolated and exposed to purified human recombinant Gal-1. The protocol described herein could be applied for the investigation of the role of other galectins in the modulation of ROS production by neutrophils.