Mechanisms involved in apoptosis of carp leukocytes upon in vitro and in vivo immunostimulation

• Apoptosis is an important regulatory process during in vitro and in vivo immunostimulation.
• During peritonitis, carp inflammatory neutrophils and monocytes/macrophages died by apoptosis.
• Zymosan and PMA-induced apoptosis of neutrophils was associated with loss MMP.
• Zymosan-induced apoptosis of carp neutrophils is NADPH oxidase dependent.
• PMA-induced apoptosis of carp neutrophils is protein kinase, but not NADPH oxidase dependent.

During inflammation leukocyte activity must be carefully regulated, as high concentrations and/or prolonged action of pro-inflammatory mediators e.g. reactive oxygen species (ROS) can be detrimental not only for pathogens but also for host tissues. Programmed cell death – apoptosis is a most effective regulatory mechanism for down regulation of leukocyte activity, but little is known about this process in fish.We aimed to reveal the mechanisms of initiation and regulation of apoptosis in carp neutrophilic granulocytes and macrophages.During zymosan-induced peritonitis in carp, activated inflammatory neutrophilic granulocytes and monocytes/macrophages died by apoptosis. This correlated with a strong production of ROS, but pretreatment of the fish with NADPH oxidase inhibitor only slightly decreased late apoptosis. Interestingly in vitro incubation with zymosan or phorbol ester, but not lipopolisaccharide and poli I:C induced apoptosis of head kidney neutrophilic granulocytes. This coincided with loss of mitochondrial membrane potential. Moreover, in zymosan-stimulated neutrophilic granulocytes NADPH oxidase inhibitor not only reduced the production of ROS but also apoptosis. A similar effect was not observed in cells stimulated with phorbol ester, where DPI reduced ROS production, but not apoptosis. In PMA-stimulated neutrophilic granulocytes both the respiratory burst and apoptosis were reduced by protein kinase inhibitor. Furthermore, a short neutrophil stimulation either with PMA or with zymosan did induce caspase-independent apoptosis.These results show that in carp, apoptosis is an important regulatory process during in vitro and in vivo immunostimulation. In neutrophils, protein kinase, but not NADPH oxidase, is involved in PMA-induced apoptosis while apoptosis induced by zymosan is ROS-dependent.