| Background The recruitment and activation and entry to graft of recipient leukocytes in response to a transplanted organ consititute the initial steps of a complex immune reaction with oxygen radicals and lytic enzymes released, which ultimately culminates in rejection of the graft. Chemokines play a central role in this process.Chemokines consist of a large family of cytokines with four major subfamilies of CXC ( ), CC ( ), CX3C ( ) and C. Chemokine bind to G protein coupled member receptors. Chemokines were considered as potential targets for immunomodulation and inhibiting immune rejection,if their bindings to receptors were blocked. However,about 50 human chemokines and nearly 20 receptors have been identified and characterized at present.Multiple chemokines are usually induced at inflammatory sites and many different chemokine receptors can be expressed by a single type of cell. It is difficult to inhibit inflammatory reponses by antagonizing a single chemokine or its receptor.Molluscum contagiosum virus(MCV) ,a member of the poxvirus family, the MCI48 ORF of which encoding a chemokine homolog MC148P consisting of 104 amino acids. MC148P can bind to chemokine receptors CCR1 , CCR2, CCR5, CCR8, CXCR1, CXCR2 and CXCR4 and inhibit the attraction of multiple leukocyte subjects to a b chemokines. MC148P antagonize chemokines the most broadly to this day.In the present study, adenovirus-mediated gene transfer of MC148 was performed both in vivo and in vitro to antagonize chemokines,which washypothesized to inhibit the recruitment of leukocyte to graft and to prevent,to some extent, immune rejection. This study might provide a novel way to control the immune response. Methods:1. MC148 was amplified from DNA of MCV isolates and sequenced .The product was excised by BamH I and EcoR I and then cloned into the cloning vector pUC19. The cloned MC148 was sequenced again.2. The cloned insert was excised from pUC19-MC148 and blunted, then ligated to cosmid vector pAxCAwt. The shuttle plasmid pAxCAwt-MC148 was packaged and co-transfected into 293 cells with DNA-TPC through homologous recombination. The recombinant Ad-MC148 was confirmed and the titer was determined.3.MC148P protein was obtained from the supernatant of 293 cells transfected with recombinant Ad-MC148 and served as antagonist against MCP-1 and RANTES.4.The bioactivity of MC148 gene was tested in a model of skin transplantation (from Lewis to Wistar rats).Briefly, four groups were set up:(1)Donor skin samples were transfected with Ad-MC148 before transplantation (n=6) (2)Ad-MC148 was injected peritoneally into recipients followed by skin transplantation (n=6).(3) Ad-MC148 was injected peritoneally into recipients initially and seven days after skin transplantation respectively(n=6).(4) Control group.Results:1.MC148 gene was successfully cloned and ligated into pUC19 vector. The sequence of the cloned gene was identical to that reported in GenBank.2. Recombinant Ad-MC148 was constructed,which only propagate in 293 cells with El gene. The titer of the recombinant was as high as 3.56 109 PFU/ml.3.The transcription of MC148 gene inserted into Ad-MC148 was confirmed by RT-PCR and protein SDS-PAGE .The expression was found as early as day 1, reached the peak on either day 2 or 3, and could sustain for about 10 days.4.The chemotaxis of monocyte and lymphocyte induced by MCP-1 and RANTES was inhibited by MC148P protein as shown by the phenominen of the decrease of intraflux of calcium.5.The survival time of transplanted skin was prolonged in Ad-MC148 transfected group, i.e., group 1 15.0 2.0days vs control group 8.5 3.4days (p<0.01), group 2 21.2 4.1days and group 3 26.2 4.5 days vs control group respectively(p<0.01 and p<0.001).Conclusion: MC148 could antagonize b chemokines and inhibit chemota- xis and recruitment of lymphocytes,which might be responsible for long-term survival of skin grafts under the condition of MC148 gene delivery. This study might provide a novel way to control immune respons...
|
PDF Full Text Request