Apoptotic Pathways Of Piglet’s Lymphocytes Induced By PCV2

Porcine circovirus type2(PCV2) was demonstrated to be a causative agent of porcine circovirus disease (PCVD) in pigs. It is all over the world since first emerged in western Canada in1991. Because the virus related with immunosuppression in pigs, it has been caused enormous loss to pig industry of whole world. At the20th international pig’s disease conference in2008, people have already classified PCVD as the No.1disease endangering pig industry’s development at present. Typical pathohistological findings are lymphocyte depletion of follicular and interfollicular areas together with macrophage infiltration of lymphoid tissues. The mechanism of lymphocyte depletion is in disputing, some research discovered that apoptosis is the reason of lymphocyte depletion, but others have opposite view. In an attempt to understand the mechanism of lymphocyte depletion induced by PCV2, we design vitro experiment and vivo experiment.In vitro:Five conventional piglets with free of antibodies against PCV2and porcine reproductive and respiratory syndrome virus (PRRSV) were used for splenocytes collection. Lymphocytes and macrophages (Mφ) isolated from spleen were distributed into four groups: lymphocytes control, co-culture with Mφ with or without PCV2inoculation (PCV2+Mφ or Mφ group), and PCV2inoculation only. The apoptosis of lymphocytes was measured with flow cytometry (FCM) at0,6,12and24h post-inoculation (HPI), and Fas and Fas ligand (FasL) mRNA in the lymphocytes were evaluated by semiquantitative RT-PCR and the cAMP/cGMP concentration by radioimmunoassay at the same time. The apoptotic rate in PCV2+Mcp group was significantly higher than other groups at24h HPI (p<0.05). The expression of Fas mRNA in PCV2+Mcp group was significantly elevated at6h (p<0.05, compared with lymphocytes control) and was the highest among four groups at24h HPI. The expression of FasL in lymphocytes was the same as that of Fas. The relationship between apoptotic rate and expression of Fas and FasL mRNA was significantly positive correlation (apoptotic rate and Fas:p=0.000, r=0.606; apoptotic rate and FasL:p=0.000, r=0.579). There is no difference between the PCV2/PCV2+Mφ and control/Mφ group in any time (p>0.05). When compared with control and PCV2group, down-regulation of the cAMP/cGMP concentration and cAMP/cGMP ratio was seen in6HPI in Mφ/PCV2+Mcp group (p<0.05),and the same change of cGMP concentration in12HPI and cAMP/cGMP ratio in24HPI (p<0.05),the reverse changes of cGMP concentration in24HPI and cAMP/cGMP ratio in12HPI was seen compared with control and PCV2group (p<0.05).The results demonstrated that macrophages could mediate the surface expression of Fas/FasL on lymphocytes and increase lymphocytes apoptosis induced by PCV2in vitro.To understand the mechanism of the apoptosis of lymphocytes in vivo, we producted PCVD by PCV2infection. Method:nineteen piglets without antibody to PCV2and PCV2nucleic acid were allotted into four groups:a control group of four piglets, and three experimental groups of five piglets per group and sacrificed at0,14,21, or35DPI with PCV2. The clinical signs of the pigs were observed every day.The level of andibody to PCV2, cytochrome C concentration and PCV2nucleic acid in serum was detected by ELISA and PCR. Samples of the following tissues were collected:liver, kidney, heart, lung and lymphoid tissue inclouding spleen, mesenteric lymph node, inguinal lymph nodes and bronchial lymph nodes. Tissues were fixed in10%neutral buffered formalin, embedded in paraffin wax, and stained with haematoxylin and eosin (HE) for microscopic study. Results: significant up-regulation of the andibody to PCV2and cytochrome C concentration was tested when compared with the control group, and the positive correlations between the level of andibody against PCV2and cytochrome C concentration in sera. All the piglets from the three experimental groups were sufferd from viremia. The most severity pathological change was lymphoid depletion of follicular and interfollicular areas together with macrophages and eosinophilic granulocytes infiltration of lymphoid tissues and the granulomatous inflammatory reaction in lung and liver especially at21and35DPI. Conclusion:PCVD was reproducted by PCV2infection (21DPI and35DPI). Cytochrome C could be an indicator of PCVD in vivo.When successful reproduced of PCVD, we researched the mechanism of the apoptosis induced by PCV2in the most significance periphery immune organ:inguinal lymph nodes and spleen in vivo. Method:The apoptotic rates of lymphocytes were detected with flow cytometry, the transcriptions of Fas/FasL, bax/bcl-2and CaMK II mRNA were tested by semiquantitative RT-PCR, caspase-3,-8and-9activities were measured with spectrophotometry, the cytosolic Ca2+concentrations were evaluated with spectrofluorometry, activities of Ca2+-ATPase were determined by malachite green colorimetric assay and concentrations of the cAMP/cGMP were evaluated by radioimmunity in inguinal lymph nodes and spleens, as well as the levels of sFas/sFasL were also detected in serum.Results in inguinal lymph nodes:(1) PCV2infection was found to be associated with apoptosis of porcine inguinal lymph nodes, and the apoptosis of inguinal lymph nodes in three experimental groups (14,21, and35DPI) was significantly higher than in the control group (0DPI)(p<0.01).(2) Transcription of the FasL mRNA was upregulated by PCV2infection, particularly at21and35DPI; The transcription of Fas mRNA were down-regulated in35DPI (p<0.05).(3) The transcription of bax mRNA and bax/bcl-2ratio were up-regulated in14DPI (p<0.05).(4) The activities of caspase-3and caspase-8were obviously increased at21DPI and no significant changes at14,35DPI.(5) Significant up-regulation of cytosolic Ca2+concentration was seen when compared with the control group (p<0.01) and was highest at21d. The activities of Ca2+-ATPase were seen down-regulation in21DPI when compared with the control group (p<0.01). No statistical significant difference was observed in the transcription of CaMKⅡmRNA (p>0.05).(6) The concentrations of cAMP and cGMP were no statistical significant difference between the three experimental groups and the control group(p>0.05), but up-regulation for the cAMP/cGMP ratio was seen in all the experimental groups(p<0.05).Results in spleen:(1) The apoptotic rates of spleen in three experimental groups were significantly higher than in the control group (0DPI)(p<0.01).(2) Expressions of the FasL mRNA were upregulated by PCV2infection, particularly at21and35DPI; but expressions of the Fas mRNA were down-regulated at35DPI.(3) The transcriptions of bax mRNA were downregulated at14DPI (p<0.05), but they were increased at35DPI (p<0.05). Bax/bcl-2ratiowas detected obvious decreasing (p<0.05) at14and21DPI.(4) The activities of caspase-3and caspase-8were gradually increased after PCV2infection and were significant up-regulation than control group (p<0.01). The activities of Caspase-9were obviously upregulated at21DPI, then were slightly depressed at35DPI, but were higher than control group (p<0.05).(5) The concentration of cytosolic Ca2+were significantly up-regulated in three experiment groups at p<0.01, and were gradually heighteded after PCV2infection. The activities of Ca2+-ATPase at21and35DPI were lower than the control group (p<0.01).(6) The relative levels of CaMKⅡmRNA were significantly up-regulated at35DPI (p<0.05).(7) The concentrations of cAMP in the three experiment groups were lower than control group(p<0.05), and cAMP/cGMP ratio were also decreased especially at21and35DPI. The data presented above indicate that:(1) PCV2could induce apoptosis of splenic lymphocytes and macrophages could increase the apoptosis in vitro, and the apoptosis is related to Fas/FasL pathway.(2) Cytochrome C in serum could be an indicator of the development PCVD in vivo.(3) Fas/FasL pathways are very important to the apoptosis of lymphcytes induced by PCV2both in vitro and in vivo.(4) The apoptosis of lymphcytes in inguinal lymph nodes and spleen induced by PCV2are related to the cytosolic Ca2+concentration.