Differential Macrophage Activation Alters the Expression Profile of NTPDase and Ecto-5′-Nucleotidase

Rafael Fernandes Zanin,Letícia Scussel Bergamin,Ana Maria Oliveira Battastini,Alfeu Zanotto Filho,José Cláudio Fonseca Moreira,Maria Rosa Chitolina Schetinger,Angela Terezinha De Souza Wyse,Fernanda Bueno Morrone,Elizandra Braganhol,Jean Sévigny,Luís Felipe Ingrassia Campesato,Tobias Eckle

doi:10.1371/journal.pone.0031205

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.

Highlights

Macrophages play key functions in the inflammatory process and are characterized by a marked phenotypic heterogeneity depending on their micro-environmental stimulation [1,2]
Characterization of activated macrophages Primary macrophages were stimulated with LPS or IL-4 in order to obtain differentiated macrophages with distinct phenotypes, which were characterized by evaluating the arginase/iNOS activities, Ym1 and FIZZ1 mRNA expression and cytokine production. iNOS is up-regulated in response to inflammatory stimuli as macrophages shift towards the classical/M1 phenotype and become involved in the initiation of the immune response, while the expression of arginase is induced by Th2-type cytokines characterizing the alternative/M2 phenotype [2,3]
These results confirm that macrophages used in these experiments were differentiated into two phenotypes: macrophages classically activated by LPS (M1) and alternatively activated by IL-4 (M2), and this protocol was applied to further experiments as described below

Summary

Introduction

Macrophages play key functions in the inflammatory process and are characterized by a marked phenotypic heterogeneity depending on their micro-environmental stimulation [1,2]. These cells exhibit diverse biochemical properties that influence pathobiology with classical/M1 and alternative/M2 polarization representing phenotypic extremes [3]. Classical activation is induced by microbial agents and/or T helper cell type 1 (Th1) cytokines and interferon-c (IFN-c), being associated with the production of large amounts of nitric oxide (NO) and proinflammatory cytokines (IL-1b, IL-6, IL-12 and TNF-a), which are involved in cytotoxicity and microbial killing [4,5]. Alternative activation is induced by Th2 cytokines (IL-4 and/or IL13), and is characterized by anti-inflammatory and tissue repair properties [3]. Alternative activated macrophages are characterized by an increase in the extracellular matrix remodeling associated with the expression of matrix proteins, such as fibronectin, bIGH3, fibrogenesis, and a high expression of arginase, which is related to repair properties [9,10,11,12]

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Results

Conclusion