Development and Screening of a Marker to Detect Activated Rainbow Trout Leukocytes

Abstract

Monoclonal antibodies (mAbs) have been essential tools in the elucidation of the immune system of mammals, and their application to identify surface molecules on leukocytes have allowed important functions of these cell to be identified (such as receptors that bind antigens, ligands involved in cell to cell signaling or in initiating immune response activity). Not only have mAbs been used to discriminate cells during different stages of cell development, but have also assisted in understanding the dynamics of molecules expressed during functional processes. Such molecules detected on human leukocytes are called human leukocyte differentiation antigens or HLDA. In order to group the antibodies that detect similar molecules and have similar patterns of reaction, immunologists have organised the mAbs that bind to these antigens into Clusters of Differentiation (CD). So far, there are about 350 leukocyte surface molecules detected by mAbs with a CD nomenclature for human leukocytes (www.hcdm.org). In fish immunology there is a great need to produce mAbs that are able to differentiate the various components of the fish immune system to assist in the elucidation of the fish immune system. The present study was an endeavour to develop and characterise mAbs that could be accredited to such scheme. A better understanding of the fish immune system is urgently required so that effective strategies of control can be developed for significant diseases during fish farming. Monoclonal antibodies were prepared by immunizing mice with thymic leukocytes from rainbow trout. The leukocytes were activated with the lectin Concanavalin A to promote the activation and proliferation of the target T-cell population. The selection of clones producing antibodies during screening was performed on the basis of the response of the supernatant from hybridomas using three consecutive assays. First, selection was determined by the positive staining of cells from the thymus in a Dot blot assay. Secondary screening was performed by means of flow cytometry (FCM) and the criterion for selection was the preferential detection of leukocytes gated in the lymphocyte region. Finally, the positive supernatants from hybridomes were evaluated to determine their effectiveness in the detection of modifications in the labelled cells during a multiple way activation by detection of foreign histocompatibility complex enhanced with mitogens. Monoclonal antibody TcOm15 was selected from 564 hybridomas produced and then used to stain cells from various Rainbow Trout tissues. It was clear from FCM, microscopy and Western blot analysis that mAb TcOm15 not only reacted with thymic cells but also with cells from other tissues. Differential staining of cells with mAb TcOm15 was observed with 27.1 ±1.4 % of leukocytes from peripheral blood leukocytes (PBL) stained in comparison to 2.0 ±0.2 % from the thymus, 13.8 ±0.4 % from the spleen, and 5.6 ±0.6 % cells stained from head kidney. The labeled cells showed characteristics of lymphocytes and monocytes, presenting a distinctive staining in immunohistochemistry and confocal microscopy. Western blot analysis, using electrophoresed proteins under denaturing conditions with leukocytes from several different tissues, showed that mAb TcOm15 did not detect a single protein. At least three proteins appeared to be identified by the mAb at 105, 160 and 200 kDa. The proteins were identified as α Actinin-4, non-erythroid Spectrin αII chain or Ig-like protein and non-muscle Myosin (MYH10) by MALDI-TOF analysis. Three of these identities are for compositional molecules for the cytoskeleton of different types of cells, and one it is associated to immunoglobulin superfamily. The identification of these proteins by mAb TcOm15 suggests an ability of this mAb to detect a specific function, possibly related with the synchronicity of expression or interaction of cytoskeleton-membrane proteins forming a multiprotein complex. Another possibility is as a carrier role for a protein during interactions. Colocalization of the mAb with F actin from the cytoskeleton was also observed suggesting the possibility that mAb TcOm15 detects a specific site in a multi-protein complex from the cytoskeleton. The molecule detected showed down-regulation in a dose dependant way with Concanavalin A and the expression was almost lost following stimulation of cells with phorbol 12-myristate 13-acetate stimulation. Leukocytes from the PBL and thymus up-regulated the expression of the TcOm15 molecule under mitogenic conditions in vitro, and results from in vivo experiments suggested the possibility of up-regulation on thymic cells. In conclusion, the results obtained in the present study provide information on a potentially useful marker (mAb TcOm15) for a cytoskeleton-membrane antigen that is modulated during stimulation of teleost lymphocytes. Additionally, this may enable insights into the relationship between cytoskeletal proteins and membrane associated immunoglobulin. Future research is necessary in order to explain this relationship and to determine the functional participation of the TcOm15 molecule during the activation of rainbow trout cells.