Generation of primary monocyte-like cultures from rainbow trout head kidney leukocytes

Abstract

Trout primary kidney monocyte-like cultures (T-PKM) were generated by incubating head kidney leukocytes in the presence of cell-conditioned medium (CCM). This technique was adapted from procedures that were previously used to cultivate in vitro-derived kidney macrophages (IVDKM) from the goldfish. Flow cytometric analysis of the initial T-PKM cultures, identified three cell sub-populations, but only one of these sub-populations survived extensive cultivation periods (i.e. >8 days) in the presence of CCM. Functionally, reactive oxygen intermediate (ROI) production was detected following stimulation of T-PKM with PMA. However, these cells failed to produce reactive nitrogen intermediates (RNI) in response to immunological stimuli. In contrast, goldfish IVDKM were capable of producing both ROI and RNI. Using the dihydrorhodamine (DHR) assay and flow cytometry, we identified two ROI-producing sub-populations in goldfish IVDKM but only a single ROI-producing sub-population was present after extended cultivation of T-PKM. This T-PKM sub-population was subsequently sorted using the flow cytometer and shown to possess monocyte-like morphology by microscopic and cytometric analysis. Thus, acquisition of antimicrobial functions following cultivation of kidney leukocytes of rainbow trout and goldfish is markedly different, and may be due to the failure of trout monocyte-like cells to undergo a final differentiation step in vitro.