Functional characterization of mast cells and histamine in the immune system of gilthead seabream (Sparus aurata L.)

Abstract

The immune system of teleost fish has been studied for decades but, despite the important advances made in our related knowledge, much is still unknown, especially about the presence and role of mast cells (MCs) and histamine. The gilthead seabream (Sparus aurata L.), a marine teleost fish of great commercial value in the Mediterranean aquaculture, has been widely used for better understanding the fish immune response. Moreover, and due to the fact that the gilthead seabream is a hermaphrodite specie, many studies has focus on the effects that the synthetic estrogen 17?-ethinylestradiol (EE2), widespread in the aquatic environment, exerts in this specie. However, no studies have been focused on peritoneal leukocytes. Within the above framework, the aim of this thesis has been to advance our knowledge of the role of MCs and histamine in the immune response of the gilthead seabream. The first chapter ended up with the development of a protocol to isolate peritoneal MCs of gilthead seabream. Using discontinuous density gradient centrifugations, cell culture, magnetic-activated cell sorting, and specific monoclonal antibodies (mAb), 95% pure MC fractions were obtained. Isolated peritoneal MCs possess histamine which is released by the compound 48/80 (Co 48/80), the classical mast cell activator. Moreover, IL-1? and IL-8 gene expression was induced in these cells by stimulation with bacterial DNA. This MC isolation protocol will be useful in the knowledge advance of the vertebrate inflammatory mechanisms evolution. In the second chapter, we study the effect of histamine and Co 48/80 on the innate and adaptive immune response of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with the pathogen, Vibrio anguillarum, and their effects on peritoneal exudate and head kidney were analyzed. Histamine and Co 48/80 alter the percentage of peritoneal exudate and head kidney immune cells, increase the ROS production by peritoneal leukocytes and impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Moreover, both, histamine and Co 48/80, reduce the expression of the gene encoding histamine receptor 2 in peritoneal leukocytes. These results show that although systemic administration of histamine and Co 48/80 is safe, neither histamine nor Co48/80 can be regarded as efficient adjuvants for gilthead seabream intraperitoneal vaccination. In the third chapter, we report the production and characterization of a mAb, GB10, which specifically recognizes gilthead seabream MCs. GB10 reacted with 20-30% of peritoneal leukocytes. GB10 rapidly induce death of gilthead seabream MCs, as demonstrated by flow cytometry. In vivo assays, in which GB10 will be intraperitoneally injected in gilthead seabream, will help to understand the evolutionary aspects of both MCs and histamine. Finally, in the fourth chapter, we analyze the effects of EE2 in the peritoneal immune response of gilthead seabream. Juvenile fish were fed a pellet diet supplemented with EE2 for 76 days and intraperitoneally injected with hemocyanin plus imject alum adjuvant at the end of EE2 treatment and 92 days later. The dietary intake of EE2 induces the expression of genes encoding for the nuclear estrogen receptor ? and the G protein-coupled estrogen receptor 1 in the peritoneal leukocytes. Interestingly, the dietary intake of EE2 induced an inflammatory response in the peritoneal exudate, which was largely maintained for several months after the cessation of the treatment and, besides, modulates histamine receptor gene expression. Taken together, the study provides fresh information about endocrine immune disruption, focusing on peritoneal leukocytes.