The effects of two protocols (density gradient versus hypotonic lysis) used for leukocyte isolation from three major lymphoid tissue of fish (head-kidney, spleen and blood) were examined on some cell functional activities (tissue leucocytes distributions, phagocytosis, basal and burst oxidative activities) classically used to estimate the fish immune status. Experiments were conducted on roach (Rutilus rutilus), a cyprinid fish model often studied in different eco-physiological contexts (aquaculture, ecotoxicology …). All of immune endpoints were assessed either immediately after cell isolation or after a 12 h of incubation in order to observe if a post-isolation incubation may influence the leukocytes activities. Compared to the density gradient, hypotonic lysis is associated with granulocytes enrichments of cell suspensions. This is particularly true for leukocyte suspensions isolated from head kidney where granulocytes are naturally abundant. However, important variabilities in leukocyte distributions were observed in head kidney and spleen cells samples obtained by the use of hypotonic lysis for two incubation conditions used (no incubation or 12 h of incubation at 4 °C). The density gradient protocol leads to a transitory increase in basal ROS production in spleen lymphocytes and macrophages The blood leukocytes isolated by this same method exhibit high basal oxidative activities after 12 h of incubation at 4 °C and for the three leukocyte types (lymphocytes, monocytes and granulocytes). The hypotonic lysis is associated with an increase in PMA-induced ROS production especially in head kidney leukocytes. The increases in cell oxidative activities are consistent with increases in granulocyte proportions observed in leukocyte suspensions obtained by hypotonic lysis. Finally, the two protocols have no effect on leukocyte mortality and phagocytic activity. Within limits of our experimental conditions, the spleen is the organ whose leukocyte oxidative activities (stimulated or not) are only slightly influenced by the methods used for leukocyte isolation. This is also the case for the anterior kidney, but for this tissue, it is necessary to incubate the isolated cells for 12 h at 4 °C before functional analyses. Each of the two methodologies used has advantages and disadvantages. The hypotonic lysis allows to isolate a greater variety of leukocytes types whereas the density gradient used ensures a better stability of cells distributions over time. However, for the same fish species and for the same tissue, the method used to isolate leukocytes influences results and must be taken into consideration during acquired data analysis for evaluation of fish immune status.
In aquatic ecosystems, detected endotoxins or lipopolysaccharide (LPS) contamination can be linked to cyanobacteria blooms or enterobacterial contaminations. Water contaminations by endotoxins is of particular interest as these microbiological molecules may be released in freshwater often in combination with other chemicals (pharmaceutics…). Their immunomodulatory properties rise interest towards their proinflammatory action on mammalians and aquatic vertebrates. Despite the fact that some E.coli LPS serotypes such as O55:B5 are often used as immunostimulant in experimentations, no data are available concerning some other serotypes as O157:H7 even if they may significantly contaminate freshwater ecosystems, particularly in agricultural field contexts.We aimed to compare here the immunomodulation patterns potentially induced by two E.coli LPS serotypes (O55:B5 and O157:H7) alone or in combination with diclofenac, an anti-inflammatory drugs which chronically contaminate European freshwaters. Using an ex vivo approach, we studied immune parameters (Oxidative activity, Phagocytosis and cytotoxicity) of roach (Rutilus rutilus) leucocytes populations (lymphocytes, monocytes/macrophages and granulocytes) isolated from blood, head-kidney and spleen in response to endotoxins exposure. Leucocytes were exposed from 6 to 12 hours to sub-lethal concentrations of LPS and/or diclofenac (1µg/mL LPS, 0,1µM diclofenac) determined from preliminary results.Like diclofenac, endotoxins induced significant decrease in intracellular ROS production by leucocytes at time 0. After 12 hours of exposure to the E.coli O55:B5 LPS / diclofenac combination, the oxidative activity was stimulated in spleen leucocytes and, simultaneously, they showed a significant decrease in phagocytosis. Moreover, while the E.coli O157:H7 LPS alone showed tendencies to decrease phagocytic activity, the two endotoxin / diclofenac combinations significantly decreased it in head-kidney leucocytes after 6 hours.All these results will be set versus an ongoing in vivo experiment which would allow us to study more inflammation aspects like cytokines and immune-related factors production.Whereas environmental monitoring and studies of endotoxins presently concern only human health like in wastewater units, our results rise the questioning on environmental reality of endotoxins and their potential immunomodulations in fish.
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