| Objective:To investigate the quantity and function of dendritic cells (DCs),explorethe effect of DCs on immune imbalance and regulations on T helper lymphocytes,and explain the immunopathogenesis of acquired severe aplastic anemia (SAA).Methods:Twenty-six untreated patients,9 recovered patients with SAA and 16 asnormal controls were studied.The quantities of mDCs (HLA-DR~+Lin~-CD11c~+ cells),pDCs (HLA-DR~+Lin~-CD123~+ cells) in peripheral blood mononuclear cells (PBMNCs)were measured with flow cytometry and the expressions of CD80 mRNA and CD86mRNA in PBMNC with semiquantitive RT-PCR.The mDCs were induced frommonocytes in bone marrow with rhIL-4 and rhGM-CSF in vitro,and then co-culturedwith allogenic lymphocytes (mixture lymphocyte reaction,MLR).The growth rate oflymphocyte was measured by MTT colorimetry and the concentrations of IL-12 andIFNγin MLR supernatant were measured by ELISA.The correlation between thegrowth rate and the concentration of IL-12 was analyzed,as well as the correlationbetween the growth rate and the concentration of IFNγ.The expressions of T-betmRNA and GATA3 mRNA in PBMNC were also measured by semiquantitiveRT-PCR and the levels of IFNγand IL-4 in plasma were measured by ELISA.Thecorrelation between the expression of T-bet mRNA and the ratio of mDC/pDC,aswell as the IL-4 level were analyzed.Results:1.The percentages of mDCs in PBMNCs were (0.44±0.25) % and (0.68±0.31) % inuntreated and recovered SAA patients respectively,either of which was higher thanthat of controls [(0.29±0.10)%] (P＜0.05,P＜0.01).The percentages of pDCs were(0.20±0.20)%,(0.26±0.20)% and (0.29±0.13)%,and there was no statisticsignificance among three groups (P＞0.05).The mDCs/pDCs ratios were 3.25±2.70and 2.95±0.86 in untreated and recovered groups respectively,either of which washigher than that of controls (1.15±0.56) (P＜0.01,P＜0.05).No statistic significanceof mDCs/pDCs was found between untreated and recovered patients in spite of higherpercentage of mDCs of recovered patients compared with untreated patients(P＞0.05).2.The expression rate of CD80 and CD86 mRNA was 88.4% in untreated cases,100% in recovered cases and 68.8% in controls.The relative mRNA expressions ofCD86 were 1.42±0.16,1.06±0.12 and 1.10±0.07 in 3 groups,respectively.The expression in untreated cases was higher than that of recovered and controls (P＜0.05).The relative mRNA expressions of CD80 in 3 groups were 1.10±0.14,1.07±0.12 and1.01±0.15 respectively,which had no statistic significance (P＞0.05).3.When mDCs and lymphocytes were co-cultured at the ratio of 1:50,the growth rateof lymphocyte in untreated cases was (322.13±171.07)%,which was higher than thatof recovered and controls [(180.90±79.12)%,(192.25±91.93)%] (P＜0.05).Therewas no statistic significance between the recovered and controls (P＞0.05).The mDCsof 8 recovered cases and 4 controls were co-cultured with allogenic lymphocytes atthe ratio of 1:100,1:50,1:20 and 1:10,but no statistic significance was foundbetween two groups at each ratio (P＞0.05).Cross MLR showed only untreated casesversus normal or normal versus normal group had statistic significance (P＜0.05).Theconcentrations of IL-12 and IFNγin supernatant in untreated cases were higher thanthose of the recovered or controls (P＜0.05),but there were no statistic significancesbetween the recovered and controls (P＞0.05).The concentration of IL-12 in MLRsupernatant correlated positively with the growth rate of lymphocyte (r=0.529,P=0.001),which was also had positive correlation with concentration of IFNγ(r=0.381,P=0.024).4.The relative mRNA expressions of T-bet in PBMNC were 0.37±0.07,0.20±0.07and 0.17±0.05 in untreated patients,recovered patients and controls respectively.The expression of T-bet in untreated patients was higher than that of recoveredpatients or controls (P＜0.05).There was no statistic significance of GATA3expression among 3 groups (P＞0.05).T-bet/GATA3 ratio is 0.72±0.13 in untreatedpatients,which was higher than that of recovered patients (0.33±0.08) and controls(0.35±0.11) (P＜0.05).The plasma level of IFNγin untreated cases was (50.9±1.1)pg/ml,which was higher than that of recovered [(49.7±0.9) pg/ml] andcontrols[(49.7±0.7) pg/ml].There was significantpositive correlation between T-betmRNA expression and the ratio of mDCs/pDCs (r=0.445,P＜0.01),as well as T-betand IFNγ(r=0.402,P＜0.01).Conclusions:1.There was higher percentage of mDCs in peripheral blood and imbalance ofmDCs/pDCs in SAA patients.The percentage of mDCs and mDCs/pDCs ratio mightnot reach to normal range in SAA patients despite their normal blood regular test,indicating that immunosuppressive therapy should not be withdrawn too earlier.The mDCs/pDCs ratio could be an index for discontinuation.2.The CD86 mRNA in PBMNC was up-expressed in untreated SAA patients.The monocyte-derived mDCs was more potential to stimulate lymphocyteproliferation and the growth rate of lymphocyte had positive correlation with IL-12and IFNγin MLR supernatant,indicating enhanced function of mDCs in SAApatients.3.The PBMNC T-bet mRNA,which was a transcriptive factor related with Thlpolarization,was up-expressed.Its relative expression had positive correlation withmDCs/pDCs ratio and the level of IFNγin plasma.These results confirmed that SAAwas really a Thl autoimmune disease.The higher level of type 1 lymphokine inplasma in those patients was related with Th0 polarizing to Thl,which might beregulated by mDCs upstream.
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