title: Endotoxin recognition in fish results in inflammatory cytokine secretion not gene expression.
abstract: 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)-α 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-α gene expression as well as other pro-inflammatory cytokines such as interleukin (IL)-1β and IL-6 and anti-viral molecules such as INF-α and Mx protein (Mx). We show that lipopolysaccharide (LPS) and zymosan are major inducers of TNF-α secretion, which is not initially linked to the induction of TNF-α mRNA expression. The secretion of TNF-α, 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-α secretion: TNF-α shedding by TNF-α converting enzyme (TACE/ ADAM17). For the first time, we show that, in fish macrophages, TNF-α is processed by TACE-like activity and this cleavage is dependent upon the activation of ERK, p38MAPK and JNK signaling pathways by LPS.
authors.affiliation: Departament Biologia Cellular, Immunologia i Fisiologia Animal Universitat Autónoma de Barcelona, Barcelona, Spain.
authors.forename: Nerea
authors.initials: N
authors.lastname: Roher
authors.forename: Agnes
authors.initials: A
authors.lastname: Callol
authors.forename: Josep V
authors.initials: JV
authors.lastname: Planas
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Goetz
authors.forename: Simon A
authors.initials: SA
authors.lastname: MacKenzie
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000072198:ADAM17 Protein; D000818:Animals; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004731:Endotoxins / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics*; D053583:Interleukin-1beta / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D017686:Oncorhynchus mykiss; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism*; D015054:Zymosan / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D004731:Endotoxins; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D047428:Protein Kinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; D015054:Zymosan; D003513:Cycloheximide; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D051722:ADAM Proteins; D000072198:ADAM17 Protein