Purinergic Signaling on Leukocytes Infiltrating the LPS-Injured Lung

Extracellular nucleotides and nucleosides have been implicated as important signaling molecules in the pathogenesis of acute lung injury (ALI). While adenosine is known to inhibit T cell activation, little information is available as to ATP and NAD degrading enzymes, the expression of ATP and adenosine receptors/transporters in different T cell subsets. ALI was induced by challenging mice with intra-tracheal instillation of 60 µl (3 µg/g) LPS. After 3 d and 7 d blood, lung tissue and bronchoalveolar lavage was collected and immune cells were analyzed using flow cytometry. The transcriptional phenotype of T helper cells, cytotoxic and regulatory T cells sorted by FACS was assessed by measuring the expression profile of 28 genes related to purinergic signaling using TaqMan Array Micro Fluidic Cards. Catabolism of ATP, NAD and cAMP by activated CD4+ T cells was evaluated by HPLC. CD73 was found to be highly abundant on lymphoid cells with little abundance on myeloid cells, while the opposite was true for CD39. After ALI, the abundance of CD39 and CD73 significantly increased on all T cell subsets derived from lung tissue and bronchoalveolar space. Expression analysis in T cell subsets of the lung revealed ATP (Cd39, Cd73) and NAD (Cd38, Cd157, Cd296, Pc-1) degrading enzymes. However, only transcription of Cd38, Cd39, Cd73, Ent1 and A2a receptor was significantly upregulated after ALI in T helper cells. CD4+ T cells from injured lung rapidly metabolized extracellular ATP to AMP and adenosine but not NAD or cAMP. These findings show that lung T cells – the dominant cell fraction in the later phase of ALI – exhibit a unique expression pattern of purinergic signaling molecules. Adenosine is formed by T cells at an enhanced rate from ATP but not from NAD and together with upregulated A2a receptor is likely to modulate the healing process after acute lung injury.细胞外核苷酸与核苷已被证实为急性肺损伤（acute lung injury, ALI）发病机制中的重要信号分子。尽管腺苷抑制T细胞活化的功能已被阐明，但针对ATP与NAD降解酶、以及不同T细胞亚群中ATP及腺苷受体/转运蛋白的表达特征，目前仍缺乏足够的研究数据。本研究通过向小鼠气管内滴注60 μl（3 μg/g）脂多糖（lipopolysaccharide, LPS）诱导急性肺损伤模型。于造模后第3天及第7天采集小鼠血液、肺组织与支气管肺泡灌洗液，通过流式细胞术（flow cytometry）分析其中的免疫细胞组分；采用荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分离辅助性T细胞、细胞毒性T细胞与调节性T细胞后，利用TaqMan微流体阵列卡（TaqMan Array Micro Fluidic Cards）检测28个嘌呤能信号通路相关基因的表达谱，以评估上述T细胞亚群的转录表型。通过高效液相色谱法（high performance liquid chromatography, HPLC）评估活化CD4+ T细胞对ATP、NAD及cAMP的代谢分解情况。研究发现，CD73在淋巴样细胞中高表达，而在髓系细胞中表达量极低；CD39的表达模式则恰好相反。急性肺损伤造模后，肺组织与支气管肺泡腔来源的所有T细胞亚群表面CD39与CD73的表达量均显著升高。对肺组织内T细胞亚群的表达分析显示，其可表达ATP（Cd39、Cd73）与NAD（Cd38、Cd157、Cd296、Pc-1）降解酶类。但在急性肺损伤造模后的辅助性T细胞中，仅Cd38、Cd39、Cd73、Ent1及A2a受体的转录水平发生显著上调。来自损伤肺组织的CD4+ T细胞可快速将细胞外ATP代谢为AMP与腺苷，但无法代谢NAD或cAMP。上述研究结果表明，作为急性肺损伤后期主要细胞组分的肺T细胞，其嘌呤能信号通路分子的表达模式具有独特性。T细胞可通过增强的速率将ATP代谢为腺苷（而非NAD），结合上调表达的A2a受体，该过程或可调控急性肺损伤后的修复进程。