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

Trout primary kidney monocyte-like cultures (T-PKM) were generated by incub ating head kidney leukocytes in the presence of cell-conditioned medium (CC M). This technique was adapted from procedures that were previously used to cultivate in vitro-derived kidney macrophages (IVDKM) from the goldfish. F low cytometric analysis of the initial T-PKM cultures, identified three cel l sub-populations, but only one of these sub-populations survived extensive cultivation periods (i.e. >8 days) in the presence of CCM. Functionally, r eactive oxygen intermediate (ROI) production was detected following stimula tion of T-PKM with PMA. However. these cells failed to produce reactive nit rogen intermediates (RNI) in response to immuno logical stimuli. In contras t, goldfish IVDKM were capable of producing both ROI and RNI. Using the dih ydrorhodamine (DHR) assay and Row cytometry, we identified two ROI-producin g sub-populations in goldfish IVDKM but only a single ROI-producing sub-pop ulation was present after extended cultivation of T-PKM. This T-PKM sub-pop ulation was subsequently sorted using the how cytometer and shown to posses s monocyte-like morphology by microscopic and cytometric analysis. Thus, ac quisition of antimicrobial functions following cultivation of kidney leukoc ytes of rainbow trout and goldfish is markedly different. and may be due to the failure of trout monocyte-like cells to undergo a final differentiatio n step in vitro. (C) 2001 Elsevier Science Ltd. All rights reserved.