Pronephric leucocytes of Cyprinus carpio: Isolation, separation and characterization

Abstract

Since the teleost pronephros is an important source of diverse immunocytes, suspensions of pronephric cells from young adult carp have been characterized. In freshly prepared suspensions, adherent, spreading cells (macrophages?) constituted <3% of the total population. Granulocytes and lymphocytes were co-dominant (<80%) leucocyte types. Continuous Percoll density gradient centrifugation yielded discrete subpopulations with these ϱ values and cytological characteristics: Fraction I & II ϱ = 1.055 − 1.070 thrombocytes, monocytes, macrophages, and lymphocytes. Fraction III ϱ = 1.080 − 1.090 granulocytes, type 1. Fraction IV ϱ = 1.105 − 1.110 erythrocytes and granulocytes, type 2. Fraction V ϱ = 1.118 − 1.125 granulocytes, type 3. Fraction VI ϱ = 1.140 − 1.150 granulocytes, type 4. Granulocyte motility increased markedly over the first 24 hr in vitro, and was enhanced by components washed from intact yeast. The subtypes of granulocytes were distinguishable by not only the ϱ values, but also on the basis of cell size, ultra-structure of the granules, and their histochemical and phagocytic characteristics. After simultaneous in vivo injection of Bacillus megaterium (Gram + ve), Aeromonas hydrophila(Gram − ve) and Saccharomyces cerevisiae (yeast), individual pronephric leucocytes were found capable of phagocytosing all three types of particle. Granulocytes which had phagocytosed B. megaterium were slower than macrophages in their ability to kill the bacteria. Encounter with B. megaterium or S. cerevisiae in vitro elicited a clumping reaction which involved mostly the larger leucocytes [granulocytes]. Both adherent cells and non-adherent cells were phagocytic in vitro.