RESUMO
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.
Assuntos
Galectinas , Neutrófilos , Espécies Reativas de Oxigênio , Galectinas/genética , Galectinas/metabolismo , Galectinas/farmacologia , Humanos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Reactive oxygen species (ROS) can be generated in mammalian cells via both enzymatic and non-enzymatic mechanisms. In sperm cells, while ROS may function as signalling molecules for some physiological pathways, the oxidative stress arising from the ubiquitous production of these compounds has been implicated in the pathogenesis of male infertility. In vitro studies have undoubtedly shown that spermatozoa are indeed susceptible to free radicals. However, many reports correlating ROS with sperm function impairment are based on an oxidative stress scenario created in vitro, lacking a more concrete observation of the real capacity of sperm in the production of ROS. Furthermore, sample contamination by leukocytes and the drawbacks of many dyes and techniques used to measure ROS also greatly impact the reliability of most studies in this field. Therefore, in addition to a careful scrutiny of the data already available, many aspects of the relationship between ROS and sperm physiopathology are still in need of further controlled and solid experiments before any definitive conclusions are drawn.
RESUMO
El dimetil sulfóxido es un secuestrador inactivante del radical hidroxilo (úOH), e inhibe, de manera proporcional a la dosis, la quimioluminiscencia (QL) de luminol (QLU) y de lucigenina (QLC) en leucocitos polimorfonucleares neutrófilos (PMN) activados con estimulantes solubles y partículas de zimosán opsonizado (ZO). Los resultados indican la inhibición de la QLU en respuesta al ionóforo de calcio A23187 puede deberse al secuestro de úOH por DMSO, mientras que la inhibición de QLC sugiere que el DMSO afecta negativamente a la oxidasa de membrana de PMN. Ello se confirmó al observar el DMSO inhibió el consumo de O2 en PMN activados con FMLP y ZO. Cuando el DMSO se añadió luego de estimulación con FMLP y ZO, no hubo inhibición de la QLU, pero sí de la inducida por A23187. El labado de PMN expuestos a DMSO causó un incremento en la QLU en respuesta a la estimulació con FMLP y ZO. Ello es congruente con la hipótesis de que el DMSO interfiere con la activación de las subunidades de membrana de la oxidasa por las unidades reguladoras citoplasmáticas. Estos resultados implican que el DMSO puede inhibir la QL en fagocitos tanto mediante secuestro de úOH como por interferencia con la producción de superóxido por la oxidasa de membrana.
Dimethylsulfoxide (DMSO), a hydroxyl radical scavenger, exerted a dose dependent inhibition on the luminol and lucigenin-enhanced chemiluminiscent responses of human neutrophils activated with soluble and particulate stimulants. DMSO inhibition of the luminol chemiluminescence induced by calcium ionophore A23187 was probably due to .OH scavenging, whereas inhibition of the lucigenin chemiluminiscence suggested DMSO negatively affects the NADPH-dependent membrane oxidase of neutrophils. In agreement with this, DMSO moderately inhibited O2 consumption in PMN suspensions stimulated with chemotactic peptide and opsonized zymosan. DMSO inhibition of chemotactic peptide and opsonized zymosan-induced luminol chemiluminescence was observed only when added before or in conjunction with stimulants, whereas A23187-induced chemiluminescence was inhibited by DMSO regardless of time of addition. Washing of DMSO-treated PMN resulted in increased luminol enhanced chemiluminescence in response to chemotactic peptide and opsonized zymosan. This is consistent with the idea that DMSO may be interfering with activation of the membrane subunits of the oxidase by translocation and docking of the cytoplasmic, regulatory subunits...