Experimental Study On Prolonging Skin Allografts Survival By Recombinant CD200 Gene

Background: With the use of immunosuppressive drugs that efficientlycontrol acute rejection, organ transplantation has achieved impressivedevelopment.Nevertheless,clinical transplantation still faces some importantproblems:lifelong immunosuppression is associated with toxicity,opportunistic infections and a high incidence of cancer.Therefore, it is key toinduce immune tolerance in transplant.With the development inMolecular-Biology and the grown up of gene transfer vectors, gene therapyhas been an novel means of tolerance induction in transplantation.CD200, formerly known as OX-2, is a highly conserved type Itransmembrane glycoprotein that is expressed on a variety of celltypes,inclufing thymocytes, B cells, active T cells, follicular dendriticcells,neuronal cells and endothelium.Expression of the receptor forCD200(CD200R) is restricted to myeloid-derived APCs and certainpopulations of T cells.Several studies have shown that CD200 imparts animmunoregulatory signal through CD200R, leading to the suppression ofT-cell-mediated immune responses.Increased allograft survival followingportal vein immunization with alloantigen correlates with an increase inCD200 expression on both hepatic and splenic DCs.Tolerance in this setting isreversed with a monoclonal antibody to CD200.CD200+DCs or solubleCD200 decrease type-1 cytokine production( IL-2, IFN-γ) and promoteproduction of type-2 cytokines( IL-4, IL-10 and TGF-β) in vitro and prolongallograft and xenograft survival in vivo. CD200-deficient mice have acompromised capacity to down-regulate APC activation in the steadystate,resulting in chronic central nervous system inflammation,an exaggeratedinflammatory response to trauma and an increased susceptibility to developboth experimental autoimmune encephalitis and collagen-induced arthritis.Taken together, these and other studies provide evidence that CD200-CD200Rsignaling results in the down-regulation of immune responses and that in theabsence of CD200 tolerance to peripheral self-antigens is easily broken.Objective: We tested the ability of suppressed rejection of skin allograftsin mice by electrotransfer of CD200 gene recombinant plasmid into mousemuscle, and explored the potential mechanisms of action for CD200.Methods: Different inbred strain male C57BL/6(H-2b)and male BALB/c(H-2d) mice were used as skin transplantation donors and recipients.On thesame day of skin transplant, 100μg CD200 gene recombinant plasmid intomouse muscle by electrotransfer. Survival time of skin grafts were recorded.Muscle tissue of injected and electrotransfered plasmid that were retrieved onday 7 ,15 ,30. mRNA transcription and protein expression of CD200 gene inmuscle tissue were measured by RT-PCR and immunofluorescence technique.Histologic evaluation of skin grafts that were retrieved on day 7 post transplantthat were undertake using hematoxylin and eosin-stained paraffin sections.Mixed lymphocyte reaction(MLR) against donor and thirdpart alloantigenswere measured by MTT.Results: Both RT-PCR and immunofluorescence analysis could detect inmuscle tissue transcription and expression of CD200 gene on day 7. Thetranscription and expression of CD200 gene could be detected in much higherlevel on day 15. The in vivo transcription and expression of CD200 gene couldlast more than 30 days. In comparision with blank control group( 8.67±1.75days),treatment of allograft recipients with pcDNA3 group( 8.00±1.55 days)and pcDNA3-CD200 group( 11.78±1.86 days) resulted in significantprolongation of skin allografts survival. In allografts from recipients treatedwith pcDNA-CD200, inflammation cells were significantly reduced comparedwith blank control groups.The stimulation index(SI) of MLR for C57BL/6 inpcDNA3-CD200 group were lower than those in blank control group( on day15 0.11±0.02 vs 0.19±0.03 p<0.05).For the thirdpart also have similar result.Conclusion: The transcription and expression of CD200 gene could bedetected in much higher level in vivo after by electrotransfer of CD200 generecombinant plasmid into mouse. CD200 are effective agents in counteringtransplant rejection and can significantly prolong survival time of skinallografts.It can reduce infiltrating inflammation cells in the skin grafts andinhibit the MLR. These data strongly suggest that CD200 play important rolesin allograft rejection, and it will have a positive effect on preventingtransplantation rejection.