The Underlying Mechanism For Immune Tolerance Through Intrabone Infusion Of Endosteal Bone Marrow Cells

BackgroundAllogeneic composite tissue transplantation is one of possible approaches that areavailable to cope with extensive defects caused by trauma, this process has always beenhalted by side effects caused by intensive immune tolerance inducing protocol.Hematopoietic stem cell transplantation combined with solid organ transplantation such askidney, heart, liver transplantation has provided a new perspective into the possible role ofmixed chimerism and hematopoietic stem cells. All these studies could help pave the roadfor composite tissue allotransplantation. But the optimal channel for bone marrow cellstransplantation and possible roles of regulatory cells in both donor and recipient bonemarrow have not been fully depicted. Through direct injection of allogeneic stem cells intorecipient bone marrow cavity or donor vascularized bone marrow transplantation, it issuggested that the recipient and donor bone marrow microenvironments which are intactin above two models, may contribute to the immune tolerance privilege of bone marrow.Through intravital cell tracking technics, in the early stage after allogeneic stem cell transplantation the hematopoietic stem cells are bound for the endosteal regions which islining the inner cavity of bone rather than ‘cavernous’ central bone cavity; To furtherprobe the specific roles of endosteal region, following flushing out the central bonemarrow cells by PBS, the endosteal region bone marrow cells are harvested throughenzymatic digestion. After comparison for their competence to maintain the hematopoieticstem cells and reconstituting the irradiated recipient hematopoietic system, it wasconfirmed that endosteal bone marrow cells have increased ability in both the two abilitiesinvolved. Consequently the endosteal region and the microenvironment within may take acritical part in the immune tolerance induction.ObjectiveIn our series of experiments, we intend to compare EBMCs and CBMCs in terms oftheir abilities to induce the long-term survival of allogeneic skin graft, and characterize theprofile of immune tolerance related gene expression both before and after preconditioningand intrabone bone marrow transplantation. The early trafficking ability of CBMCs andEBMCs to different regions of bone marrow is also compared by trafficking CFSE labeledbone marrow cells.Methods1. Preconditioning the recipient mice with rapamycin and low dosage (4.8Gy) of totalbody irradiation, different components(CBMCs or EBMCs) of bone marrow cellswere harvested and injected into recipient mice in intrabone fashion depicted byKushida in2000, both intravenous and intrabone approaches were used in thisexperiment. After4weeks, when the hematopoietic stem cells were supposed to haverecovered, allogeneic full-thickness skin grafting was done, the textures and colorswere closely observed for any rejection signs. After about6weeks after skin grafting,the chimerism levels in bone marrow and spleen was measured.2. CXCL-12and regulatory T cells related genes such as Foxp3, interleukin-10, TGF-βect. were assessedthrough real-time PCR both before and after transplantation, acomparison was drawn between CBMCs and EBMCs, and between pre transplantationand post transplantation in CBMCs and EBMCs components respectively. 3. Central bone marrow cells labeled with CFSE were infused into unilateral femur bonemarrow cavity.12hours later, the intensity of CFSE was measured in different bonesand cell components (CBMCs or EBMCs) through flow cytometry. Comparsionbetween them was made to give us an insight into where the intabone-injected bonemarrow cells prefer tomigrating.ResultsThe injection of endosteal bone marrow cells (average21.5days of survival) couldinduce longer the survival of skin allograftsthan the injection of CMBCs (Intrabonetransplantation: average survival period,,18days; Intravenous transplantation,averagesurvival period,,15days). IL-10, Foxp3, N-cadherin not TGF-βwere highly expressed inEBMCs before transplantation;4weeks after transplantation, the expression of IL-10increased in both CBMCs and EBMCs cell populations, while the expression of CXCL-12,Foxp3, N-cadherin decreased in the EBMCs. CFSE-labeled EBMCs was more likely tomigrate into contralateral non-injected femur bone marrow than CBMCs do, while forinfusion of both EBMCs and CBMCs, at an early stage (12hours after transplantation),more cells were bound for the endosteal region.ConclusionCompared with injection of CBMCs through either intrabone or intravenous bonemarrow transplantation, the intrabone infusion of endosteal bone marrow cells couldinduce longer allograftssurvival. And the IL-10may be one of the critical cytokine thatmediate the immune tolerance induction through intrabone marrow transplantation.Through the intrabone bone marrow cells approach, more cells are bound for the endostealregion at an early stage.