Scuticociliate cysteine proteinases modulate turbot leucocyte functions

The effects exerted by cysteine proteinases isolated from the histiophagous ciliate Philasterides dicentrarchi on the phagocytic functions of turbot pronephric leucocytes (PL) were investigated. The enzymes were tested at concentrations of 125, 250 and 500 μg ml−1, and it was found that the viability of the leucocytes was not affected after treatment for 24 h. Leucocyte migration was inhibited by the cysteine proteinases in a dose-dependent manner, whereas the ascitic fluid obtained from turbot experimentally infected with P. dicentrarchi induced high chemotactic activity in the turbot PL. The proteinases did not affect yeast cell phagocytosis but increased intracellular production of the superoxide anion (O2−). Stimulation with the proteinases did not alter the PGE2 levels in supernatants from 24-h cultures of PL, however, β-glucans (100 μg ml−1) provoked a large increase in PGE2 levels, which were inhibited after addition of 10 μg ml−1 of indomethacin, a non-selective inhibitor of COX2 enzymatic activity. The mean PGE2 level in ascitic fluid from turbot, experimentally infected with P. dicentrarchi, was 500 pg ml−1, and the addition of low levels of PGE2 (62.5 pg ml−1) to PL cultures stimulated O2− production, although addition of PGE2 at concentrations higher than 250 pg ml−1 blocked the increase in stimulation. Addition of cysteine proteinases to 24-h cultures of PL also increased mRNA levels in the pro-inflammatory cytokine interleukin-1β. The results revealed the capacity of cysteine proteinases isolated from P. dicentrarchi to modulate the innate immune response of turbot, which together with the inflammation mediators produced during infection, may play an important role in pathogenesis of the disease and in the survival of the parasite.