Real-time gene expression analysis in carp (Cyprinus carpio L.) skin: Inflammatory responses to injury mimicking infection with ectoparasites

We studied a predictive model of gene expression induced by mechanical injury of fish skin, to resolve the confounding effects on the immune system induced by injury and skin parasite-specific molecules. We applied real time quantitative PCR (RQ-PCR) to measure the expression of the pro-inflammatory cytokines CXCa, CXCb, interleukin (IL)1-beta, tumor necrosis factor alpha (TNFalpha), and the receptors IL1R1, CXCR1 and CXCR2 in skin of Cyprinus carpio after mechanical injury. We also studied the expression of the anti-inflammatory cytokine IL-10. Most obvious, specific up-regulation of the chemokine CXCa, the chemokine receptor CXCR1 and the pro-inflammatory cytokine IL-beta was detected at 2-3h after injury. In order to correlate gene expression patterns after injury with cell migration, we studied chemotaxis of head kidney leukocytes towards lysates of epithelioma papulosum cyprini (EPC) cells. Neutrophilic granulocytes were shown to migrate towards epithelial lysates. Using immunohistochemistry we observed that the early inflammatory response after injury involved an influx of cells, most probably neutrophilic granulocytes, into the injured area. This suggests that the increased expression of pro-inflammatory genes is related to a rapid influx of neutrophilic granulocytes