Comparative analysis of the immune responses of CcIgZ3 in mucosal tissues and the co-expression of CcIgZ3 and PCNA in the gills of common carp (Cyprinus carpio L.) in response to TNP-LPS

Abstract

Fish live in an aquatic environment rich in various microorganisms and pathogens. Fish mucosal-associated lymphoid tissue (MALT) plays a very important role in immune defence. This study was conducted to characterize the immune response mediated by CcIgZ3 in common carp (Cyprinus carpio.) and investigate the proliferating CcIgZ3+ B lymphocytes in gill. We determined the expression of CcIgZ3 in many different tissues of common carp following stimulation by intraperitoneal injection of TNP-LPS (2,4,6-Trinitrophenyl hapten conjugated to lipopolysaccharide) or TNP-KLH (2,4,6-Trinitrophenyl hapten conjugated to Keyhole Limpet Hemocyanin). Compared with TNP-KLH, TNP-LPS can induce greater CcIgZ3 expression in the head kidney, gill and hindgut, especially in the gill. The results indicate that the gill is one of the main sites involved in the immune response mediated by CcIgZ3. To examine the distribution of CcIgZ3+ B lymphocytes, immunohistochemistry (IHC) experiments were performed using a polyclonal antibody against CcIgZ3. The results indicated that CcIgZ3 was detected in the head kidney, hindgut and gill. To further examine whether CcIgZ3+ B lymphocytes proliferate in the gills, proliferating CcIgZ3+ B cells were analysed by immunofluorescence staining using an anti-CcIgZ3 polyclonal antibody and an anti-PCNA monoclonal antibody. CcIgZ3 and PCNA (Proliferating Cell Nuclear Antigen) double-labelled cells in the gills were located within the epithelial cells of the gill filaments of common carp stimulated with TNP-LPS at 3 dps and 7 dps, and relatively more proliferating CcIgZ3+ B cells appeared in the gills of common carp at 7 dps. These data imply that CcIgZ3+ B cells in the gills might be produced by local proliferation following TNP-LPS stimulation. In summary, compared with those in TNP-KLH, CcIgZ3 preferentially affects the gills of common carp following challenge with TNP-LPS. CcIgZ3+ B cells proliferate in the gills to quickly produce the CcIgZ3 antibody. In addition, CcIgZ3+ B cells can be activated to induce a strong immune response very early locally in the gill and produce the antibody CcIgZ3, which helps exert an immune-protective effect. These results suggest that an effective vaccine can be designed to promote production of the mucosal antibody CcIgZ3.