Molecular Mechanism Of Antigen Processing By Dendritc Cell For Inactivate Foot-and-mouth Disease Virus

Foot-and-mouth disease(FMD)is caused by the FMDV, which is a highly contagiousvesicular disease of cloven-hoofed animals, such as pigs, cattle, and sheep.The mechanismthat animals mount an immune response against FMDV antigens has not yet been profiled.It has been well-documented that initiation of adaptive immunity requires presentation ofantigens by dendritic cells (DCs).DCs can cross present antigens. And this leads to theconclusion that states DCs are the most powerful antigen-presenting cells in the body.However, it is unclear that if DC is capable of processing and presenting FMDV antigen.FMDV infects animals usually by way of digestive tract, respiratory tract, and damagedskin. In any infected animal, FMDVs were replicated in great deal at pharynx. So, pharynxis regarded as the most important organ in host defence. However, the distribution of DCsin the bovine pharynx has not been completely defined. To document the morphology ofDCs in both pharynx and the draining lymph node, retropharyngeal lymph node, pharynxesand lymph nodes were collected from in ten healthy cattle. Immunohistochemistry wasconducted with usage of S-100 antibody. The results showed that DCs were scatteringlydistributed in the mucosal epithelium of the pharynx. In the draining lymph nodes, bothfollicular dendritic cells (FDCs) and interdigitating dendritic cells(IDC)were intensivelystained in the cortex. Surprisingly, densely populated DCs in the medulla were clearlyshown with S-100 antibody. There is a growing body of evidence that peripheral DCsmigrate into the draining lymph nodes immediately after encountering the invadingpathogens. However, no datum is available that states the dynamics of FMDV antigen-bearing DCs in vivo. To address the issue of whether pharyngeal DCs take active part inthe antigen processing and presentation of FMDV, BALB/c mice were inoculated orallywith the inactivated FMDV vaccine at different time points. The draining lymph nodeswere harvested as usual. The dynamics of pharyngeal DCs after orally challenging withinactivated FMDV was immunohistochemically revealed. In contrast to the control group,many DCs were present in cortex at 24h post-vaccination. The number of DCs wasdramatically increased at 48h post-vaccination and the accumulation was predominantlydetected in paracortical area. The migration of DCs reached their peaking stage inparacortical area at 72h post- vaccination. At 96h post-vaccination, the quantity of DCsdeclined remarkably and scattered either in the cortex or the medulla. DCs were found toemerge again primarily in cortex at 120h post-vaccination. Accordingly, the serum levelsof IFN-γwere determined with ELISA at different timepoints when BALB/c mice wereinoculated orally with the inactivated FMDV vaccine. The results showed that the serumlevels of IFN-γin vaccinated mice the serum slightly raised (P＞0.05) at 24hpost-vaccination. At 48h post-vaccination, in contrast to the control group, the content of IFN-γin the serum was increased considerably(P＜0.01). The content of IFN-γin theserum reached its peaking stage and have a significant difference to the control group(P＜0.01). The content of IFN-γin the serum declined at 96h post-vaccination, having nosignifican difference at 120h post-vaccination(P＞0.05).These results indicated that pharyngeal DCs were the antigen presenting cells whichpresent FMDV antigen to naive T lymphocytes, leading to the release of IFN-γ. Toelucidate the mechanism of processing of FMDV antigen in DC, monocyte-deriveddendritic cells(MoDC) of BALB/c mouse were generated, and the resulting MoDCs wereidentified by immunohistochemistry and FACS analysis.Subsequently, MoDCs werepulsed with inactivate FMDV vaccine. Ubiquitinated FMDV antigens were detectd byWestern blotting with FK1 antibody or FK2 antibodies. The results showed thatubiquitinated FMDV antigens were detected after DCs were pulsed 0.5h. The molecularweight of ubiquinated FMDV antigen was approximately 75KD. At 3h of antigen pulse,the content of ubiquitinated FMDV antigens increased. However, the content ofubiquitinated proteins did not show any recognizable fluctuation from then on. The resultsshowed that the content of FK2-positive proteins were much more than the FK1-positiveproteins. It can be deduced that inactivated FMDV either were degraded in the proteasomewith the resultant of CD8+T cell epitopes, or were degraded in the lysosome withgenerating some CD4+T cell epitopes. Taking together, antigens of inactivated FMDV canbe degraded both in proteasome and lysosome. It is notebale that producing epitopesgenerated in lysosomes predominantly initiate the differentiation of CD4+T lymphocytesinto Thl subste which is indicative of high level of serum IFN-γas determined with ELISAprocedure.In summary, DCs were the antigen presenting cells of killed FMDV. FMDV antigenswere degraded both in the ubiquitin-proteasome pathway and endosome-lysosomepathway.