| Objective: aGVHD was a common complication of allogeneic hematopoietic stem cell transplantation(allo-HSCT), which induced by T cells. rhG-CSF was the standard mobilizing agent for peripheral blood stem cell(PBSCs) transplantation, rencently a lot of clinical studies have demonstrated that rhG-CSF mobilized PBSC products can induced the immune tolerance . rhGM-CSF as another standard mobilizing agent has several dedaults, for example greater toxicity and poor mobilization. A study showed mobilization might proceed well with a combination of rhG-CSF and rhGM-CSF after a mobilization failure with rhG-CSF alone in some individuals. SM Devine etal[2] suggested that compared to rhG-CSF allografts collected following mobilization with rhGM-CSF alone may be associated with a diminished risk of moderate to severe acute GVHD; Vij etal[3] suggested that combined use of rhG-CSF and rhGM-CSF for mobilization could reduce the number of T cells infused into the recipients which might confer a decreased risk of the development of GVHD compared with the single use of rhG-CSF. The purpose of this study is to investigate the effect of in vivo administration of rhG-CSF and ————————————————————————————————————————/or rhGM-CSF on mice immune system function.Methods:Male BALB/C mice were randomly assigned to receive the mobilizing regimens as follows: rhG-CSF alone at 250 ug·kg-1·d-1 subcutaneously once daily on days 1-5; rhGM-CSF alone at 250 ug·kg-1·d-1 subcutaneously once daily on days 1-5; the combination of rhG-CSF(250 ug·kg-1·d-1) and rhGM-CSF(250 ug·kg-1·d-1) subcutaneously once daily on days 1-5; PBS was used for control injections. Mice were sacrificed 24 hours after the last injection and the spleens harvested, single-cell suspension of spleen cells obtained as previously described[29]. T cell subgroups, suppressor T cells (CD8 + CD28 - , CD4 +CD25 +) , type 1 and type 2 dendritic cells (DCs), expression of CD28 on T cells were determined by multicolor flow cytometry. MTT was used to determine the T cell proliferation capacity. Semi-quantitative RT-PCR was used to determine spleen T cells intracellur IL-4 mRNA and IFN-γmRNA expression.Results:1. The capacities of splenic T cells proliferation: Cytokines treatments significantly decreased the capacities of splenic T cells proliferation compared with the PBS treatment (P<0. 05) , the rhGM-CSF group being more significantly decreased (P< 0.01).2. Differential expression of Lymphocytes and T cells subgroups: In vivo administration of cytokines decreased the percentage of lymphocytes (P<0.05) ; rhGM-CSF signifycantly decreased the CD3+ cells and CD4+ cells(P<0.05); rhGM-CSF alone and the combination group significantly decreased the CD8+ cells(P<0.05) , but there was no significantly difference between rhGM-CSF alone and the combination group(P>0.05) ; rhGM-CSF alone significantly decreased the CD4 + /CD8 + ratio (P<0.05) .3. Effect of rhG-CSF and/or rhGM-CSF administration on the expression of CD28 on T cells: Cytokines treatments signifycantly decreased CD3+CD28+ cells(P< 0.05), rhGM-CSF alone most significantly decreased (P<0.05); there were no significantly difference on the expression of CD4+CD28+cells or CD8+CD28+cells among all groups(P>0.05).4. The percentage of suppressor T cells following rhG-CSF and/or rhGM-CSF administration: Cytokines treatments signifycantly increased the percentage of CD8 + CD28 - suppressor T cells compared to the PBS treatment (P<0.05) , rhGM-CSF alone most significantly increased (P<0.05); Cytokines treatments significantly increased the percentage of CD4 + CD25 + suppressor T cells(P<0.05),rhGM-CSF alone most significantly increased(P<0.05).5. Differential expression of type 1 and type 2 dendritic cells(DCs): Cytokines treatments significantly increased the percentage of DC2( P<0.05), but there was no significant difference of DC1(P>0.05), the DC1/DC2 ratios were lower (P <0.05).6. The level of IL-4mRNA and INF-γmRNA following rhG-CSF and/or rhGM-CSF administration: Cytokines treatment significantly increased the expression of IL-4mRNA compared with the PBS treatment (P<0.05), rhG-CSF significantly decreased the expression of INF-γmRNA compared with the PBS treatment( P<0.05), rhGM-CSF significantly increased the expression of INF-γmRNA compared with the PBS treatment( P <0.05) ,the ratio of IFN-γmRNA/IL-4mRNA were significantly decreased after cytokines treatment compared with the PBS treatment( P<0.05).Conclusion:1. rhG-CSF mobilized PBSC products can induced the immune tolerance by their effects on the proliferation capacity and function of T lymphocytes, and decreased the risk of aGVHD after allo-HSCT.2. rhGM-CSF can induced the immune tolerance and decreased the risk of aGVHD by the function CD4+CD25+ T-regulatory cells and DC2 type dendritic cells.3. The combination rhGM-CSF and rhG-CSF is poorly synergistic in the induction of immune tolerance, rhGM-CSF was associated with lower rates of aGVHD.
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