Endotoxin recognition in fish results in inflammatory cytokine secretion not gene expression

Macrophages are phagocytes that have a central role in the organization of the immune system after an infection. These cells can recognize specific molecular components of micro-organisms (pathogen-associated molecular patterns, PAMPs) via specific receptors (PRRs) and elicit specific cellular responses. In the past, the expression of immune genes in response to different PAMPs has been characterized in different fish species. However, little is known about actual cytokine release. We characterized the secretion of tumour necrosis factor (TNF)-alpha in primary macrophage cultures of rainbow trout (Oncorhynchus mykiss) in response to several PAMPs by Western blot and compared this to the induction of TNF-alpha gene expression as well as other pro-inflammatory cytokines such as interleukin (IL)-1 beta and IL-6 and anti-viral molecules such as INF-alpha and Mx protein (Mx). We show that lipopolysaccharide (LPS) and zymosan are major inducers of TNF-alpha secretion, which is not initially linked to the induction of TNF-alpha mRNA expression. The secretion of TNF-alpha, but intriguingly not the expression, is also stimulated by ultrapure LPS meaning that, in fish, contaminants of commercial LPS preparations are better inducers of the inflammatory response. Moreover, we have characterized the signaling pathways that are activated by different PAMPs and the link between those pathways and the final step of TNF-alpha secretion: TNF-alpha shedding by TNF-alpha converting enzyme (TACE/ADAM17). For the first time, we show that, in fish macrophages, TNF-alpha is processed by TACE-like activity and this cleavage is dependent upon the activation of ERK, p38MAPK and JNK signaling pathways by LPS.