title: Differential macrophage activation alters the expression profile of NTPDase and ecto-5'-nucleotidase.
abstract: 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.
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brasil. rafaelzaninn@gmail.com
authors.forename: Rafael Fernandes
authors.initials: RF
authors.lastname: Zanin
authors.forename: Elizandra
authors.initials: E
authors.lastname: Braganhol
authors.forename: Letícia Scussel
authors.initials: LS
authors.lastname: Bergamin
authors.forename: Luís Felipe Ingrassia
authors.initials: LF
authors.lastname: Campesato
authors.forename: Alfeu Zanotto
authors.initials: AZ
authors.lastname: Filho
authors.forename: José Cláudio Fonseca
authors.initials: JC
authors.lastname: Moreira
authors.forename: Fernanda Bueno
authors.initials: FB
authors.lastname: Morrone
authors.forename: Jean
authors.initials: J
authors.lastname: Sévigny
authors.forename: Maria Rosa Chitolina
authors.initials: MR
authors.lastname: Schetinger
authors.forename: Angela Terezinha
authors.initials: AT
authors.lastname: de Souza Wyse
authors.forename: Ana Maria Oliveira
authors.initials: AM
authors.lastname: Battastini
mesh_terms: D015720:5'-Nucleotidase / Q000032:analysis* / Q000235:genetics; D000241:Adenosine / Q000096:biosynthesis / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020869:Gene Expression Profiling; D008262:Macrophage Activation / Q000235:genetics*; D051379:Mice; D011755:Pyrophosphatases / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D018047:Receptors, Purinergic P1 / Q000235:genetics; D018048:Receptors, Purinergic P2 / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D018047:Receptors, Purinergic P1; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D015720:5'-Nucleotidase; D011755:Pyrophosphatases; D000241:Adenosine