Bharadwaj Uddalak, Zhang Rongxin, Yang Hui, Li Min, Doan Linh X, Chen Changyi, Yao Qizhi
Molecular Surgeon Research Center, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas 77030, USA.
J Surg Res. 2005 Jul 1;127(1):29-38. doi: 10.1016/j.jss.2005.02.020. Epub 2005 Apr 21.
Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein as well as a secreted protein and has recently been reported to be an immunomodulatory molecule. The objective of this study was to determine the effect of CypA on dendritic cell (DC) differentiation, activation, and functional maturation. The role of p38 MAP kinase in DC functions was also investigated.
KG1 cells (CD34+ human myeloblastic cell line) were treated with cytokines (GM-CSF+IL-4) and/or CypA and expression of cell surface markers was analyzed by FACS analysis. The antigen-uptake capacity of different DCs was determined by FITC-dextran uptake assay. Antigen-presentation capacity of DCs was determined by allogeneic mixed lymphocyte reaction (MLR) by [3H] thymidine incorporation assay. To assess the T cell polarization stimulated by KG1 derived DCs, various Th1 and Th2 cytokines secreted by allostimulated CD4+ and CD8+ T cells were determined by Bioplex cytokine assay. Total and phosphorylated p38 MAPK activity in CypA treated DCs was detected by Bioplex p38 total and phosphoprotein assay.
During the differentiation of KG1 cells to immature DCs, cell surface expression of CD11b was increased by 30.6% for CypA alone, 55% for CypA plus cytokines, and 44% for cytokines alone. Similarly, CypA alone increased the cell surface expression of CD11c by 59% as compared to CypA plus cytokines (68%) and cytokines alone (50%). CypA up-regulated the antigen uptake capacity of the immature DCs to a greater extent (5 times) as compared to cytokines alone (2.5 times). Moreover, CypA augmented the capacity of DCs to present antigens to allogenic CD8+ T cells, and also increased the secretion of Th1 type cytokines TNF-alpha and IFN-gamma from the allogenic CD4+ T cells. Furthermore, CypA induced the phosphorylation and hence activation of MAP kinase p38. Pre-treatment with SB-203580, a p38 inhibitor, significantly reduced MLR stimulatory capacity of CypA-induced DCs in both CD8+ and CD4+ T cells (P < 0.05).
CypA enhances DC differentiation and maturation by up-regulating CD11b and CD11c expression. CypA can also augment DC antigen uptake and antigen presentation, which may be mediated by the p38 signaling pathway.
亲环素A（CypA）是一种广泛分布的细胞内蛋白，也是一种分泌蛋白，最近有报道称其为一种免疫调节分子。本研究的目的是确定CypA对树突状细胞（DC）分化、激活和功能成熟的影响。同时也研究了p38丝裂原活化蛋白激酶在DC功能中的作用。
用细胞因子（GM-CSF+IL-4）和/或CypA处理KG1细胞（CD34+人髓母细胞系），通过流式细胞术分析细胞表面标志物的表达。通过FITC-葡聚糖摄取试验测定不同DC的抗原摄取能力。通过[3H]胸苷掺入试验的同种异体混合淋巴细胞反应（MLR）测定DC的抗原呈递能力。为了评估KG1来源的DC刺激的T细胞极化，通过Bioplex细胞因子测定法测定同种异体刺激的CD4+和CD8+T细胞分泌的各种Th1和Th2细胞因子。通过Bioplex p38总蛋白和磷酸化蛋白测定法检测CypA处理的DC中总p38 MAPK和磷酸化p38 MAPK的活性。
在KG1细胞分化为未成熟DC的过程中，单独使用CypA时CD11b的细胞表面表达增加了30.6%，CypA加细胞因子时增加了55%，单独使用细胞因子时增加了44%。同样，与CypA加细胞因子（68%）和单独使用细胞因子（50%）相比，单独使用CypA时CD11c的细胞表面表达增加了59%。与单独使用细胞因子（2.5倍）相比，CypA在更大程度上（5倍）上调了未成熟DC的抗原摄取能力。此外，CypA增强了DC向同种异体CD8+T细胞呈递抗原的能力，还增加了同种异体CD4+T细胞分泌Th1型细胞因子TNF-α和IFN-γ。此外，CypA诱导了丝裂原活化蛋白激酶p38的磷酸化并因此激活。用p38抑制剂SB-203580预处理显著降低了CypA诱导的DC在CD8+和CD4+T细胞中的MLR刺激能力（P<0.05）。
CypA通过上调CD11b和CD11c的表达增强DC的分化和成熟。CypA还可以增强DC的抗原摄取和抗原呈递，这可能由p38信号通路介导。
