Effect Of PRRSV Infection On Processing And Presentation Function Of Porcine Pulmonary Alveolar Macrophages For Exogenous Antigen

Porcine reproductive and respiratory syndrome virus (PRRSV) can cause reproductive failure in sows and gilts, respiratory diseases in piglets and immunosupression in pigs. Porcine pulmonary alveolar macrophages (PAM) are important target cells of PRRSV. In order to explore the effect of PRRSV infection on processing and presentation function of PAM for exogenous antigen at cellular and molecular level, PAM in vitro and in vivo infected with PRRSV HB-2(sh)/2002 were collected at different time to analyze the expression of surface molecules, functional molecules associated with exogenous antigen processing and presentation, antigen presentation ability and PRRSV nucleic acid kinetics. Meanwhile, apoptosis in PAM associated with PRRSV infection was observed.SLA-DR, invariant (Ii) chain, CD40 and PRRSV ORF7 were amplified by RT-PCR from PAM and cloned according to their sequences available from GenBank respectively. After sequencing, competitive depletion clones for the cDNA molecules 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 percentages of SLA-DR cells of PAM were analyzed by flow cytometry (FCM). According to their respective standard competitive curves, mRNA expressions of SLA-DR, SLA-DM, Ii chain and costimulatory molecules CD40, CD80 and CD86 in PAM were investigated by qcPCR.Results showed that, in vitro the lowest percentage of SLA-DR cells of PAM, the lowest mRNA transcription levels of SLA-DR and SLA-DM occured at 12h post infection(PI) ; the lowest mRNA transcription levels of Ii chain was observed at 24h PI; The mRNA transcription levels of CD40, CD80 and CD86 were high in late stage of infection. \n vivo, the percentage of SLA-DR cells of PAM increased transiently at day 3 PI, declined quickly at day 7 PI and remained low level thereafter. The mRNA transcription level of SLA-DR in PRRSV-infected PAM was lower than that of control PAM until day 28 PI, but no significant difference. The mRNA expression of SLA-DM decreased at day 3 PI (p<0.01), ascended at day 42 PI (p<0.05). The mRNA expression of Ii chain declined to neap at day 3 PI (p<0.01), increased at day 28 PI and day 42 PI (p<0.01). CD40 mRNA expression fell to neap at day 3 PI (p<0.01), ascended quickly at day 7 PI (p<0.01), then decreased and resumed to normal level thereafter. CD80 mRNA expression decreased sharply at day 3 PI (pO.Ol), then rose and peaked at day 14 PI (p<0.01), went down to the normal level thereafter. CD86 mRNA expression decreased sharply at day 28 PI (p<0.05), then increased and recovered. The results showed that PRRSV affected markly processing and presentation of PAM for exogenous antigen in early inoculated stage, resulting in a down regulation antigen presentation function of PAM for exogenous antigen and onset of impairment for humoral immune responses of swine.Antigen presentation ability of PAM was detected by autologous mixed lymphocyte reaction (AMLR). Results showed that antigen presentation ability of PRRSV-inoculated PAM tended to decrease at day 3, 7, 28 and 42 PI. A reduction of antigen presenting capacity is indicative of a persistent suppression on antigen presentation function in porcine alveolar macrophages, which mightbe the mechanism of immunosupressio by PRRSV.Apoptosis of PAM and PRRSV nucleic acid kinetics were observed by flow cytometry (FCM) with FITC-Annexin V/PI kit and qcPCR, respectively.In vitro two peaks of apoptosis were detected, the first at 4h PI and the second at 36h PI. In vivo the peak of apoptosis was observed at day 28 PI (p<0.01). In vitro the highest level of viral RNA was present at 4h PI, decreased sharply thereafter. In vivo the highest level of viral RNA appeared at day 3 PI, decreased sharply thereafter and was dectected until day 42 PI. Results showed that PRRSV could induce apoptosis in PAM, PRRSV replicated in PAM for a fairly long period, the peak of apoptosis occured when the copy number of PRRSV RNA was very small, high leve...