| In this study, a series of experiments were conducted on the basis of the primary macrophage culture, in order to investigate the specific functions of highly unsaturated fatty acids (HUFAs) on the physiological and immune responses of macrophages. The present studies include (1) Isolation, characterization and primary culture of head kidney macrophages of large yellow croaker. (2) In vitro effects of arachidonic acid (ARA, 20:4n-6) on nutritional physiology and immunity of large yellow croaker head kidney macrophages. (3) In vitro effects of eicospentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3) on nutritional physiology and immunity of large yellow croaker head kidney macrophages. The results are as follows:(1) Head kidney macrophages from large yellow croaker were isolated by density gradient sedimentation and characterized under light microscope and electronic microscope. Compared to 31%/45% Percoll gradient, 31%/45% Percoll gradient contributed to higher isolation efficiency of macrophages. After over-night incubation and two washes to remove non-adherent cells, macrophage purity exceeded over 90%. Cells were maintained in Leibowitz L-15 medium supplemented with 5% fetal bovine serum (FBS). Best cell viability assessed by MTT assay was observed at 22°C treatment compared to18°C and 26°C. The cells,like typical macrophages, exhibited strong adhesion and plasticity as well as high phagocytic capacity for engulfing yeasts. Macrophages were incubated with lipopolysaccharide (LPS) from Escherichia coli 055:B5 for 3h and 24h. LPS modulated superoxide oxide anion (O2-) production in time and concentration dependent way, only 3h incubation with LPS could significantly enhance O2- production of PMA-triggered macrophages, while after 24h incubation, O2- production was significantly decreased by each LPS incubation level. Macrophages did not show increased nitric oxide (NO) production level when exposed to LPS alone and LPS combined with PMA, human tumor necrosis factorα(hrTNF-α) or calcium ionophore A23187, respectively.(2) Cells were incubated in medium supplemented with graded levels of ARA (5μM,25μM,50μM,100μM,200μM and 1000μM) in the form of fatty acid bovine serum albumin (FA-BSA) complex for various duration of time, respectively. Following stimulation, results showed that with increasing priming time and ARA concentration, cell viability decreased significantly. Especially after 36h, 200μM and 1000μM ARA significantly decreased cell viability (P <0.01). In the meanwhile, higher levels of ARA (200μM and 1000μM) led to significant increase in production of lipid peroxide (P<0.05). Significantly increased phagocytosis percentage (PP) value was found on treatments with 25μM and 100μM ARA(P<0.05), while 1000μM ARA significantly suppressed PP value (P<0.01). Enhanced intracellular O2? synthesis was found on treatments with 100μM and 200μM ARA. The fact that ARA significantly promoted the production of secretory phospholipase A2 (sPLA2) seemed to be irrelevant to increased ARA concentration, while prostaglandin E2 (PGE2) production was significantly increased in ARA dose dependent way, and the highest PGE2 level was found in 1000μM ARA treatment. Noteworthy, no pronounced effect was observed on Interleukin 1β(IL-1β) production among graded levels of ARA, and levels of IL-1βin cell culture supernatant were fairly low (only 6pg/ml or so) .(3) Cells were incubated in medium supplemented with graded levels of EPA or DHA (5μM,25μM,50μM,100μM,200μM and 1000μM) in the form of FA-BSA complex for various duration of time, respectively. Following stimulation, results showed that with increasing priming time and EPA or DHA concentration, cell viability decreased significantly. Especially after 24h and 36h, high levels (200μM and 1000μM) of EPA or DHA significantly decreased cell viability (P <0.01). In the meanwhile, higher levels (200μM and 1000μM) of DHA led to significant increase in production of lipid peroxide (P <0.05) while no significant effect was observed by EPA treatments. PP value on treatments with 5μM DHA was significantly enhanced (P <0.05), while PP value was significantly suppressed by high levels (200μM and 1000μM) of EPA or DHA(P<0.01). Significantly enhanced intracellular O2? synthesis was found on treatments with EPA but significantly decreased O2? production on DHA treatments (P <0.05). The production of sPLA2 was significantly inhibited by each EPA or DHA supplemental level (P<0.01). Decreased PGE2 production was found on treatments with EPA or DHA with the exception of 1000μM EPA or DHA treatments, where sudden significantly increased PGE2 level was found (P <0.01). No pronounced effect was observed on IL-1βproduction by graded levels of EPA or DHA level, and IL-1βlevel in cell culture supernatant was fairly low (only 6pg/ml or so).In conclusion, it is the first time to investigate the effects of ARA, EPA or DHA on immunity of large yellow croaker macrophages based on an in-vitro model. Results showed that regulatory effects exerted by HUFAs on isolated macrophages were closely related to HUFAs doses, incubation time and their respective biochemical properties. Fish cell culture techniques is also proved to be a promising tool in the investigation between HUFAs and immunity, which could help to unveil the mechanisms by which fatty acids modulate fish immunity. |
PDF Full Text Request