Isolation of fish macrophage and its physiology

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Neutrophils of Tilapia Oreochromis niloticus were collected from the swim bladder 24 hrs after infection with 1 mg/fish of formalin-killed Eschericia coli in Hanks' balanced solution. The neutrophils were sonicated using an ultrasonic processor for 60 sec. To collect inflammatory macrophages, the fish were injected with 0.2 mL of the neutrophil cytolysates into the swim bladders, and cells that exuded into the organ were harvested at 4 days after injection. The cells were smeared and stained with Giemsa, non-specific esterase, and peroxidase stains to identify the cell types. Macrophages occupied 40% of the exudate cells. To purify the inflammatory macrophages, the exudate cells were incubated in L-15 medium 25℃ for 48 hrs or subjected to a gradient-centrifuge procedure with Ficoll (1.065 in gradient, at 1,500 rpm for 30 min). Both the methods enabled to purify the macrophages more than 90%.In order to examine the macrophage physiology, the phagocytic activities and various cytokines levels (TNF-α, IL-1β, IL-10, and IL-12) with a ELISA method using rabbit IgG against them were surveyed on the macrophages. Besides, immunostimulants like glucan raised their physiological parameters of macrophages. Tilapia were kept under self-feeding for 1 week to make lower stressful condition. An analysis on the blood cortisol confirmed the lower cortisol level in the fish kept under the self-feeding than the ones kept under the usual feeding, meaning the self-feeding provides the low stress condition to the fish. The macrophages collected from the fish kept under the self-feeding showed the higher phagocytotic activities and cytokine productivities of macrophages. Thus, it appears that the stressful condition influences the macrophage physiology.