The Effect Of Mast Cell In Donor Liver’s Protection And Recipient’s Acute Rejection

Recently, more and more researchers found that mast cell (MC) plays importantrole in tranplantation immunology. But there are still lots of problems that need to besolved, such as the effect of MC degranulation in liver I/R injury, the effect onrecipient survival and its mechanism of the status of preconditioning donor liver withMC modulator and the effect of MC degranulation on recipient’s acute rejection.In this research, we used rat I/R injury model, orthotopic liver transplantationmodel and heterotopic heart transplantation model to explore the there problemsabove respectively. In the end, we hope that we can find the function of MC moreextensively and provide some theory to improve transplantation prognosis.Part I The study of the effect of mast cell degranulation inischemia and reperfusion injury in liverBackground: The degranulation of MCs plays a role in I/R injury in many organs.However, a recent study found that MCs are not involved in I/R injury. We doubtedthe result.Materials and methods: A warm hepatic I/R injury model of1h ischemia followedby24h of reperfusion was used. MC modulation was induced via cromolyn injectionor a method called MC depletion using compound48/80. The effects of MCmodulation were evaluated by toluidine blue staining and assessment of mast celltryptase in sera. The result of modulation was evaluated by hematoxylin and eosinstaining graded by Suzuki criteria, alanine transaminase (ALT) and aspartatetransaminase (AST) levels in sera, and malondialdehyde (MDA) levels in liverhomogenates. Results: First, MC degranulation peaked after2h of reperfusion and liver damagepeaked after approximately6h of reperfusion. Second, a method called MC depletionpreviously used in the skin worked similarly in the hepatic setting. Third, stabilizationof MCs with cromolyn or depletion of MCs with compound48/80each decreasedhepatic I/R injury as measured by Suzuki’s score, ALT, AST and MDA.Conclusions: MC degranulation promotes hepatic I/R injury in rats. Part II The effect and its mechanism of prolonging recipientsurvival after orthotopic liver transplantation bypreconditioning donor liver with mast cell degranulationmodulatorsBackground: Immunosupressive therapy can cause opportunistic infection and tumorformation after transplantation. In part I, we have verified that stabilization of MCs inliver with cromolyn (CRM) or depletion MCs with compound48/80(CMP48/80) canalleviate liver ischemia and reperfusion injury. We wanted to veryfy whetherpreconditioning with cromolyn or compound48/80can alleviate AR and prolongsurvival as well as its mechanism.Materials and Methods: We used the male-DA-to-female-Lewis-rat orthotopic livertransplantation (OLT) model. Donors were preconditioned with CRM in a MCstabilizing way (CRM group) or CMP48/80in a MC depleting way (CMP48/80group). Rats preconditioned with phosphate buffered saline were used as controls(PBS group). After preconditioning, OLT surgeries were carried out. OLT Male-Lewis-to-female-Lewis-rats were used as the syngeneic group (Syn group).Results: Rats in the PBS group died at7.40±1.14days. Rats in the CRM and CMP48/80groups died at days17.40±1.67and14.20±2.28, respectively. Rats in CRM and CMP48/80groups prolonged the survival as compared with PBS group (p <0.05). Rats in the syngeneic group survived more than60days. Rejection activityindexes (RAIs) and liver functions were all alleviated through CRM or CMP48/80preconditioning. Interferon-γ (IFN-γ) mRNA expressions were reduced andinterleukin-10(IL-10) mRNA levels were higher in allografts in the CRM and CMP48/80groups, compared with the PBS group. These were confirmed by testing serumIFN-γ and IL-10.Conclusion: Preconditioning donor livers with CRM in a MC stabilizing way or CMP48/80with a MC depleting way can reduce AR and prolong survival of recipientsafter OLT. The mechanism of such effect may result from shifting the balance ofTh1/Th2to Th2pole. Part III The study of the effect of mudulating recipient’smast cell on acute rejection after transplantation and itsmechnismBackground: The effect of recipient’s mast cell (MC) in acute rejection is still notvery clear. We wanted to find whether MC degranulate in recipient aftertransplantation and whether the degranulation could promote acute rejection and thepossible mechanismMaterials and Methods: In vitro, we isolated primary rat skin mast cell (RSMC) andrat peritoneal mast cell (RPMC), examined the expression of C5a receptor in thesurface of these cells and detected the degranulation rate of these cells after irritatingwith C5a. In vivo, we used a heterotopic heart transplantation models from DA rats toLewis rats. We divided the recipients according to drugs which were injected to themas follows：(1) rats injected with PBS (PBS group),(2) rats injected with cromolyn(CRM group),(3) rats injected with3mg/kg cyclosporine A (CsA3group),(4) rats injected with3mg/kg cyclosporine A and cromolyn (CsA3+CRM group),(5) ratsinjected with5mg/kg cyclosporine A (CsA5group),(6) rats injected with5mg/kgcyclosporine A and cromolyn (CsA5+CRM group) and (7) syngeneic transplantationgroup (Syn group). The survival period was observed in every group. C5a, IL-10andIFN-γ in recipient’s sera were examined at the5th,10th and15th day aftertransplantation. Mast cell degranulation was examined with mast cell degranulationkit. The degree of rejection under hematoxylin and eosin staining of transplantedhearts were analyzed. The transcriptional levelsof IL-10mRNA and IFN-γ mRNA intransplanted hearts were tested.Results: In vitro,58.69%–67.64%RSMCs were C5a receptor positive cells.1.23%–1.89%RPMCs expressed C5a receptors. But after stimulating with lipopolysaccharide(LPS), the expressing rate of C5a receptor of RPMCs could arrive at73.52%–41.14%.C5a could cause degranulation of RSMC and irritated RPMC, but not RPMC. And thedegranulation of these MCs could be blocked with cromolyn. In vivo, the C5a andMC degranulation levels elevated as contrasted with Syn group after transplantation.Cromolyn could decrease the level of MC degranulation. But there were nosignificant differences of the survival periods between CRM group and PBS group,CsA3+CRM group and CsA3group, CsA5+CRM group and CsA5group. Therejection degrees of transplanted hearts, the level of IL-10and IFN-γ, the IFN-γmRNAs and IL-10mRNA were not different among these groups.Conlusion: The levels of C5a and MC degranulation are elevated in the recipientsafter heterotopic transplantation of hearts. C5a can promote the degranulation of MCand cromolyn can alleviate the degree of MC degranulation. But the survival oftransplanted hearts and the degree of rejection were not alleviated after treating withcromolyn.