| Porcine circovirus type 2 (PCV2) is the primary aetiological agent of postweaning multisystemic wasting syndrome (PMWS) that is an important emerging disease in swine. Porcine alveolar macrophage (PAM) is both an important target cell of PCV2 and an important immune cell. In order to explore the effects of PCV2 infection on PAM functions at the cellular, molecular, and genie level, PAMs from forty-day-old healthy piglets inoculated oro-nasally with PCV2 BF strain were collected at different days post inoculation (DPI) to detect the expression of surface molecules, function molecules associated with antigen presentation, and cytokines. Meanwhile, PAMs apoptosis associated with PCV2 infection was observed.Swine function molecules associated with antigen presentation, including SLA-DM, CD80, CD86, and cytokines interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-a ), IL-8, monocyte chemotactic protein-1 (MCP-1), IL-18, and granulocyte-macrophage colony stimulating factor (GM-CSF) were amplified by RT-PCR from PAMs and cloned according to their sequences available in GenBank respectively. After sequencing, competitive depletion clones for the cDNA molecules mentioned above were generated by reverse PCR. Then, the linear regression equations were obtained after construction of the standard competitive curves respectively by quantitative competitive PCR (qcPCR) assay.The apoptosis of PAMs was observed by flow cytometry (FCM) with FTTC-Annexin V/PI kit and agrose gel electrophoresis. The phagocytosis ability of PAMs was detected by phagocytosing chicken erythrocytes, and Fc receptor expressed on PAMs was determined by EA assay. Results showed no PAMs apoptosis was observed throughout the experiment from the above two assays, indicating that PCV2 could induce no apoptosis on PAMs. As compared to the control, Fc receptor expression decreased apparently in 7 DPI, then increased and recovered. PAM phagocytosis shared the same changes. These results demonstrated that the ability of PAMs to phagocytose and eliminate pathogens weakened transiently after infection with PCV2.The number of cells expressing major histocompatibility complex (MHC) antigen was detected by FCM. The mRNA expression of SLA-DM, CD80, and CD86 was analyzed by qcPCR according to their respective standard competitive curve constructed. Results revealed that there were no difference in SLA Class I antigen between PCV2-infected macrophages and the control throughout the experiment, while a decrease in the number of the infected cells expressing SLA-DR was observed at 7 DPI, then no changes were detected. There were a similar profile of mRNA expression among SLA-DM, CD80, and CD86, they declined to neap at 3 DPI, ascended quickly at 7 DPI, resumed to normal thereafter. The decreases in those important function molecules associated with antigen presentation, is suggestive of a downregulation of PAM antigen presentation.The PAMs from PCV2 infected and control piglets were investigated for mRNA expression of the cylokines IL-1β , TNF-a , IL-8, MCP-1, IL-10, IL-12p40, IL-18, and GM-CSF by qcPCR accordingto their respective standard competitive curve constructed. Altered cytokine mRNA expression patterns were observed. IL-10 mRNA expression increased in 7 DPI, then fell to normal at 14 DPI. After a sharp decrease in the mRNA expression of several cytokines as IL-12p40, IL-18, GM-CSF, and MCP-1 at 3 DPI, these cytokines rose and peaked at 14 or 21 DPI, went down to the normal thereafter. IL-8 mRNA expression was less at 3 and 7 DPI, but more at 14 DPI. The expression of IL-1β mRNA was higher in 14 DPI, then declined, whereas TNF-a mRNA expression was not affected. In conclusion, those cytokine mRNA imbalance, which were characterized by overexpression of IL-10 mRNA, and by decreases in the expression of several cytokines as IL-12p40, IL-18, GM-CSF, is indicative of a macrophage immunoiegulation impairment.Taken together, the present study demonstrated that PCV2 infection resulted in phagocytosis decrease, reduction of antigen presentin... |
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