CONTRIBUTION OF CELL MARKERS TO THE STUDY OF RMS PATHOGENESIS

Red mark syndrome (RMS) is a chronic non-lethal skin disease affecting farmed rainbow trout (O. mykiss) in U.K., Austria, Germany, Italy (Galeotti et al., 2011), Serbia and U.S.A. Histology shows a lymphocyte/macrophage infiltration in scale pockets, dermis and ipodermis. Aiming at the full comprehension of RMS aetio-pathogenesis, we focused on the mechanisms of cell recruitment/activation in the skin lesions, in order to elucidate if and how an hypothetical microbial agent might trigger the host inflammatory response. Samples of skin from infected fish were evaluated by histology, immunohistochemistry and electron microscopy. The following markers were used: rabbit to human CD3 (A-0452, Dako); rabbit to rainbow trout IgT/IgM (Prof. Sunyer); rabbit to salmonid HSP70 (AS05061A, Agrisera); rabbit to human GM-CSFRα (sc-690, Santa Cruz Biotech.); mouse to PCNA (2586, Cell Signaling Technology); mouse to AE1/AE3 Cytokeratin (M3515, Dako); mouse to E Cadherin (M3612, Dako). Anti trout IgT and IgM marked a limited number of scattered cells in the dermis and ipodermis. Anti CD3 marked a relevant number of cells composing the skin infiltrate. HSP70 marked monocyte/macrophages, dendritic like-cells and endothelial cells, within the scale pockets involved by inflammation. GM-CSFRα positive monocyte-macrophage were scattered in the derma, surrounding the scales. Anti-Cytokeratin and E Cadherin marked the epithelial cells. PCNA positive cells have been detected in epidermis, dermis and hypodermis, as well among infiltrating lymphocytes, stromal fibroblasts and vascular endothelial cells. HSP70 are considered not only as acute phase proteins, but also as molecules able to mediate immunity and inflammation (Pockley, 2003); they can be released by several cell populations in response to various stimuli. Briefly HSP70 could act as an “antigen” inducing a severe T lymphocyte response, leading to an auto-immune like reaction. Dendritic cells and APCs are stimulated by HSP70 to release TNF-α, IL1-and GM-csf. We might speculate that a microbial agent promotes HSP70 expression by macrophages/endothelial cells, within scale pockets. HSP70 might be also internalized by skin APCs. The pro-inflammatory cytokines released could then trigger the local inflammatory process. The GM-csf stimulates the development of osteoclasts. Skin APCs could express HSP70 and therefore stimulate T cell proliferation. These findings might justify the severe cell infiltration detectable in the skin of RMS affected fish.