Porcine IgG Fc Receptors And Its Role In Antibody-dependent Enhancement Of PRRSV Infection

Fc receptors (FcR) are a group of crucial molecules expressed on the surface of immune cells, which bind the Fc region of immunoglobulins with specific affinities and have a number of important biological functions. FcRs play a critical role in immune regulation by providing a link between the humoral and cellular immune responses. Three classes of FcγR (FcγRI, II, III), present in human and mouse, are the best characterized. In this study, three classes of porcine FcγRs was cloned and characterized. The relationship between immune suppression and dynamic expression of porcine activation and inhibitory FcγRs after infection with PRRSV isolates was investigated. Porccine FcγRII was been proved playing a crucial role in mediation the antibody-dependent enhancement (ADE) of PRRSV infection by construction the Marc-145 cell line with stable poFcγRII expression.By screening a translated EST database (which is composed of sequences from specieces other than human and mouse) with the protein sequence of the human CD64,we identified six putative overlapping porcine homologues. With this sequence information,a pair of primers was designed and the full-length cDNA encoding porcine FcγRI we isolated (poFcγRI) from peripheral blood leucocyte RNA using RT-PCR. Porcine FcγRI contains an open-reading frame (ORF) of 1038 bp length, encoding a 346 amino acids glycoprotein. Extracellular domain of poFcγRI consists of three Ig-like domains and four N-glycosylation sites. The predicted amino acid sequence of whole poFcγRI was found to be 79%, 69% and 57% identitical with human, bovine and mouse counterpart; however, a comparison of the three extracellular domains alone between the four species shows a higher identity of 77%, 74% and 70% respectively. This is in agreement with the generally highly conserved nature of the individual FcR binding domains. In order to examine the cell distribution of poFcγRI, RT-PCR was performed on mRNA from selective cellular subsets. Porcine FcγRI is expressed in PAM, monocytes, and PMN. A higher abundance of transcripts was found in PAM and monocytes than in PMN, where only a trace transcript was found. Then, a cDNA for the complete coding region of poFcγRI was subcloned into the expression vector pcDNA3, and transfected into COS-7 cells. The COS-7 cells transfected with the poFcγRI cDNA were able to bind chicken erythrocytes sensitized with porcine IgG.Three classes of porcine FcγR, named FcγRI, FcγRII and FcγIII, was further characterized. Of which, poFcγRII belongs to inhibitory Fc receptor, as an ITIM motif was found in intracellular domain. Whereas, porcine FcγRII and FcγIII, devoid of know signal motif, seem to be activatory Fc receptors and that they may form an activation receptor complex withγchain on the surface of immune cells. Two isoforms of poFcγRII, FcγRIIB1 and FcγRIIB2 were further identified. FcγRIIB1 has an in-frame insertion, which increases the cytoplasmic region from 47 to 99 amino acids. The two isoforms have ITIM motif in the intracellular domain, so they both belong to the inhibitory receptors.The paired expression of activating and inhibitory molecules on the same cell is the key for the generation of a balanced immune response. After binding with the activating Fc receptor, immune cells can be activated and a variety of immunological effects were induced by antigen-antibody complex. The role of inhibitory receptors is exactly the opposite. Thus, it is important to study the activating and inhibitory Fc receptor transcription dynamicas after PRRSV infection for further invesitgaiton the immune suppression by PRRSV infection. Using real-time PCR, the activating and inhibitory Fc receptor transcription dynamic in porcine peripheral blood leucytes was investigated after infection with PRRSV isolates. The expression of activation receptor, FcγRI and FcγRIII, was found to be rapidly reduced, and the inhibitory Fc receptor, poFcγRII, had a slight increase after infection. PRRSV infection rapidly induced high levels of PRRSV-specific antibodies, but the the immune effector cells were not to be activated by antibody and antigen-antibody complex due to the decreased expression of activating FcγRs. This may be the molecular basis of immunosuppression after PRRSV infection.Antibody-dependent enhancement (ADE) is one of the major problems in prevention and control PRRS. In this study, poFcγRII gene was transfected to Marc-145 cells. After G418 selection and continuous cloning, construction of Marc-145 cell line with stable expression of poFcγRII was achieved. Then, Marc-145 cell lines with stable poFcγRII expression was infected with PRRSV BJ-4 strain and 1:320 dilution of anti-PRRSV IgG together or PRRSV alone. The harvested virus of Marc-145 cell lines infection with PRRSV and antibody together was significantly higher than that of PRRSV absence of antibody. Thus, poFcγRII mediated the ADE of PRRSV infection. The existing evaluation criteria for the effect of PRRSV vaccine is determined by detection of the PRRSV vaccine specific antibodies. As ADE of PRRSV infection, PRRSV-specific antibodies could not protect pigs from PRRSV infection and contribute to infection in some cases. In this study, Marc-145 cell lines with stable poFcγRII expression can be used for evaluation the ADE effect of antibodies, so it will provide technical support for develop ping efficient PRRSV vaccines.