Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brasil.
PLoS One. 2012;7(2):e31205. doi: 10.1371/journal.pone.0031205. Epub 2012 Feb 13.
Macrophages are key elements in the inflammatory process, whereas depending on the micro-environmental stimulation they exhibit a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype. Extracellular ATP can act as a danger signal whereas adenosine generally serves as a negative feedback mechanism to limit inflammation. The local increase in nucleotides communication is controlled by ectonucleotidases, such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto-5'-nucleotidase/CD73 (ecto-5'-NT). In the present work we evaluated the presence of these enzymes in resident mice M1 (macrophages stimulated with LPS), and M2 (macrophages stimulated with IL-4) macrophages. Macrophages were collected by a lavage of the mice (6-8 weeks) peritoneal cavity and treated for 24 h with IL-4 (10 ng/mL) or LPS (10 ng/mL). Nitrite concentrations were measured using the Greiss reaction. Supernatants were harvested to determine cytokines and the ATPase, ADPase and AMPase activities were determined by the malachite green method and HPLC analysis. The expression of selected surface proteins was evaluated by flow cytometry. The results reveal that M1 macrophages presented a decreased ATP and AMP hydrolysis in agreement with a decrease in NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M2. In contrast, M2 macrophages showed a higher ATP and AMP hydrolysis and increased NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M1 macrophages. Therefore, macrophages of the M1 phenotype lead to an accumulation of ATP while macrophages of the M2 phenotype may rapidly convert ATP to adenosine. The results also showed that P1 and P2 purinoreceptors present the same mRNA profile in both phenotypes. In addition, M2 macrophages, which have a higher ATPase activity, were less sensitive to cell death. In conclusion, these changes in ectoenzyme activities might allow macrophages to adjust the outcome of the extracellular purinergic cascade in order to fine-tune their functions during the inflammatory set.
巨噬细胞是炎症过程中的关键因素，根据微环境的刺激，它们表现出促炎（经典/M1）或抗炎/修复（替代/M2）表型。细胞外 ATP 可以作为危险信号，而腺苷通常作为限制炎症的负反馈机制。核苷酸通讯的局部增加受细胞外核苷酸酶控制，如细胞外三磷酸二核苷酸磷酸水解酶（E-NTPDase）家族和外核苷酸酶/CD73（ecto-5'-NT）的成员。在本工作中，我们评估了这些酶在驻留小鼠 M1（用 LPS 刺激的巨噬细胞）和 M2（用 IL-4 刺激的巨噬细胞）巨噬细胞中的存在。用 LPS（10ng/ml）或 IL-4（10ng/ml）处理从小鼠腹腔灌洗中收集的巨噬细胞 24 小时。用 Greiss 反应测定亚硝酸盐浓度。收集上清液以测定细胞因子，并通过孔雀绿法和 HPLC 分析测定 ATPase、ADPase 和 AMPase 活性。通过流式细胞术评估选定表面蛋白的表达。结果表明，与 M2 相比，M1 巨噬细胞的 ATP 和 AMP 水解减少，NTPDase1、-3 和外核苷酸酶表达降低。相比之下，M2 巨噬细胞显示出更高的 ATP 和 AMP 水解以及增加的 NTPDase1、-3 和外核苷酸酶表达与 M1 相比。因此，M1 表型的巨噬细胞导致 ATP 的积累，而 M2 表型的巨噬细胞可能迅速将 ATP 转化为腺苷。结果还表明，P1 和 P2 嘌呤能受体在两种表型中具有相同的 mRNA 谱。此外，具有更高 ATPase 活性的 M2 巨噬细胞对细胞死亡的敏感性较低。总之，这些细胞外酶活性的变化可能使巨噬细胞能够调整细胞外嘌呤能级联的结果，以微调它们在炎症过程中的功能。
