Developmental and Stem Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
J Allergy Clin Immunol. 2011 Oct;128(4):854-863.e1. doi: 10.1016/j.jaci.2011.07.039. Epub 2011 Aug 25.
Inherited or acquired defects in purine nucleoside phosphorylase (PNP) impair purine metabolism, as well as the survival and function of T lymphocytes. However, the effects of PNP deficiency on thymocyte development are not well known.
We sought to study thymocyte development in PNP-deficient (PNP-KO) mice.
Maturation, proliferation, and apoptosis were determined in thymocytes from PNP-KO mice and hematopoietic stem cells from these mice grown ex vivo into thymocyte-like cells.
Reduced percentages of CD4(+)CD8(+) double-positive (DP) thymocytes with normal percentages of CD4(-)CD8(+) and CD4(+)CD8(-) single-positive thymocytes were found in the thymi of PNP-KO mice. Similarly, reduced DP-like thymocytes grew ex vivo from hematopoietic stem cells of PNP-KO mice. Thymi of PNP-KO mice contained increased apoptotic DP thymocytes. Increased apoptosis of PNP-deficient DP thymocytes occurred after exposure to deoxyguanosine (dGuo), although not after Fas ligation, and could be prevented by restoring PNP activity within the cells. In DP thymocytes from PNP-KO mice, dGuo caused mitochondrial membrane potential dissipation and induced release of cytochrome c from the mitochondria followed by nuclear DNA fragmentation. Inhibition of the caspase pathway prevented dGuo-induced nuclear DNA fragmentation but not mitochondrial membrane potential dissipation, indicating that PNP deficiency induces apoptosis that is initiated in the mitochondria of DP thymocytes. 5-Bromo-2-deoxyuridine incorporation demonstrated that PNP deficiency does not interfere with DP or single-positive thymocyte proliferation.
PNP is important for the survival of DP thymocytes. Accumulation of dGuo in cases of PNP deficiency leads to mitochondria-initiated apoptosis of DP thymocytes, which can be prevented by restoring PNP activity in the cells.
嘌呤核苷磷酸化酶（PNP）的遗传或获得性缺陷会损害嘌呤代谢以及 T 淋巴细胞的存活和功能。然而，PNP 缺乏对胸腺细胞发育的影响尚不清楚。
我们试图研究 PNP 缺陷（PNP-KO）小鼠中的胸腺细胞发育。
通过测定 PNP-KO 小鼠的胸腺细胞以及这些小鼠的造血干细胞在体外生长为类胸腺细胞的成熟、增殖和凋亡来确定。
在 PNP-KO 小鼠的胸腺中，发现 CD4(+)CD8(+)双阳性（DP）胸腺细胞的百分比降低，而 CD4(-)CD8(+)和 CD4(+)CD8(-)单阳性胸腺细胞的百分比正常。同样，从 PNP-KO 小鼠的造血干细胞中生长的 DP 样胸腺细胞也减少。PNP-KO 小鼠的胸腺中含有凋亡增加的 DP 胸腺细胞。PNP 缺乏的 DP 胸腺细胞在暴露于脱氧鸟苷（dGuo）后会发生凋亡，尽管 Fas 连接后不会发生凋亡，并且可以通过在细胞内恢复 PNP 活性来预防。在 PNP-KO 小鼠的 DP 胸腺细胞中，dGuo 导致线粒体膜电位耗散，并诱导细胞色素 c 从线粒体释放，随后细胞核 DNA 片段化。抑制半胱天冬酶途径可防止 dGuo 诱导的细胞核 DNA 片段化，但不能防止线粒体膜电位耗散，表明 PNP 缺乏诱导 DP 胸腺细胞中线粒体起始的凋亡。5-溴-2-脱氧尿苷掺入表明 PNP 缺乏不会干扰 DP 或单阳性胸腺细胞的增殖。
PNP 对 DP 胸腺细胞的存活很重要。在 PNP 缺乏的情况下，dGuo 的积累会导致 DP 胸腺细胞的线粒体起始凋亡，通过在细胞中恢复 PNP 活性可以预防这种凋亡。
