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.

Figure 1. Characterization of resident, LPS- and IL-4-stimulated macrophages.

(A) iNOS and (B) Arginase activities: iNOS activity was estimated by the NO²⁻ (nitrite) accumulation in the supernatant of cultured cells and Arginase activity was evaluated by measuring the formation of urea from arginine (3.10⁵ cells). *Significantly different from the two other groups (p<0.001). (C) FIZZ1 and Ym1 expression in stimulated macrophages was evaluated by qPCR. Expression was normalized to β-actin signals as described in Material and Methods. *Significantly different from resident and LPS-stimulated macrophages (p<0.001). (D) IL-10 and (E) TNF-α cytokines were measured from supernatants of macrophage cultures. *Significantly different from stimulated and resident macrophages (p<0.001). Data show mean ± SD of at least three independent experiments. Significant difference between groups was determined by ANOVA, followed by Tukey's test.

Figure 2. NTPDase activity on mouse macrophages after phenotype differentiation.

(A) Resident, M1 (stimulated with LPS) and M2 (stimulated with IL-4) macrophages were incubated in 48-well plates with ATP, ADP or AMP as described in Materials and Methods (section 2.5). Specific activity values were expressed as nmol Pi/min/mg protein. The data represent the mean ± S.D. (n = 5) with pooled macrophages from 6 to 8 mice per experiment carried out separately. Data were analyzed by ANOVA, followed by Tukey's test. (*) Significantly different from resident macrophages; (^#) significantly different from LPS-stimulated macrophages (p<0.05). (B–D) Metabolism of extracellular ATP by HPLC; resident (B), M1 (stimulated with LPS) (C) and M2 (stimulated with IL-4) (D) macrophages were incubated in 48-well plates with 100 µM ATP in 200 µl of incubation medium as described in Material and Methods. An aliquot of the supernatant was withdrawn at 0, 20, 40, 60 and 120 min and the presence of ATP, ADP, AMP were determined. Data are mean ± SD values from three experiments in triplicates. (E–G) The same procedure utilized to evaluate the metabolism extracellular of ATP was used to AMP metabolism - adenosine (ADO) and inosine (INO) - Resident (E), M1 (stimulated with LPS) (F) and M2 (stimulated with IL-4) macrophages (G). Data are mean ± SD values from three experiments in triplicates. (H, I) Amount of nucleotides/nucleosides at different time of incubation. Data are mean ± SD values from three experiments in triplicates.

Figure 3. PCR quantification of NTPDase1, -2, -3 and ecto-5′-nucleotidase mRNAs.

The total mRNA amount were normalized to β-actin signals and expressed as 2^−Δ/ΔCT. Data show mean±SD for real time PCR experiments performed in triplicate with RNA purified from three independent experiments with pooled macrophages from 8 to 10 mice per experiment. M1 (stimulated with LPS) and M2 (stimulated with IL-4) macrophages were compared to resident macrophages (*) p<0.001, and (#) p<0.01 M2 (stimulated with IL-4) compared to M1 (stimulated with LPS) macrophages, two-way ANOVA with Tukey's post-hoc test.

Figure 4. Activated macrophages express different protein levels of NTPDases and Ecto-5′-nucleotidase.

The cells were stained 24 h after stimulation with antibodies to CD11b and to NTPDase1, -2 and -3, or Ecto-5′NT, and when necessary, with secondary FITC- or Alexa 488-conjugated antibodies. Macrophages were primed either with LPS to generate M1 macrophages, with IL-4 to generate M2 macrophages, or left unstimulated (resident). FL-1 represent the intensity of staining with the indicated Abs. (A) The controls were performed using guinea pig serum (Sigma) and rabbit serum (Invitrogen) as primary antibodies as detailed in Materials and Methods section. The results presented were subtracted from the data presented in the following panels according to the Abs used. (B) Dot plot with percentage of double positive cells for CD11b and NTPDase1, -2, -3 or Ecto-5′NT. (C) Mean fluorescence intensity (MFI) from panel B is shown for Ecto-5′NT and (D) depicts NTPDase1, -2, -3 MFIs. Data are representative of at least three independent experiments with pooled macrophages from 7 to 9 mice per experiment. (*) p<0.05 indicates changes in expression when compared against resident macrophages and (#) p<0.05 indicate significant difference between M1 and M2 macrophages, two-way ANOVA with Tukey's post-hoc test.

Figure 5. PCR quantification of P2X7 mRNA.

The total mRNA amount were normalized to β-actin signals and expressed as 2^−Δ/ΔCT. Data show mean±SD for real time PCR experiments performed in triplicate with RNA purified from three independent experiments with pooled macrophages from 5 mice per experiment. There were no significant differences in the P2X7 mRNA among the groups.

Figure 6. The NTPDase activity on macrophages after phenotype differentiation alters the susceptibility to ATP induced cell death.

(A) After macrophages differentiation these cells were treated for 3 h with 2 mM ATP in the presence or absence of potato apyrase (apy; 2 U) or P2X7 receptor antagonists (3 µM KN-62, 10 µM A438079). The total number of the cells was counted in five random fields in visible filter and the cells positive for PI were counted in same fields but with ultra-violet filter. Data show mean±SD of three experiments. Significantly different from resident macrophages for ***p<0.001 or *p<0.05, two-way ANOVA with Tukey's post-hoc test. (B, C, D) UV and (E, F, G) visible representative images (magnification: 40×). M1 (stimulated with LPS) and M2 (stimulated with IL-4) macrophages were compared to resident macrophages.