Human monocytes respond to extracellular cAMP through A2A and A2B adenosine receptors

Abstract

In this study, we test the hypothesis that cAMP, acting as an extracellular mediator, affects the physiology and function of human myeloid cells. The cAMP is a second messenger recognized as a universal regulator of several cellular functions in different organisms. Many studies have shown that extracellular cAMP exerts regulatory functions, acting as first mediator in multiple tissues. However, the impact of extracellular cAMP on cells of the immune system has not been fully investigated. We found that human monocytes exposed to extracellular cAMP exhibit higher expression of CD14 and lower amount of MHC class I and class II molecules. When cAMP-treated monocytes are exposed to proinflammatory stimuli, they exhibit an increased production of IL-6 and IL-10 and a lower amount of TNF-α and IL-12 compared with control cells, resembling the features of the alternative-activated macrophages or M2 macrophages. In addition, we show that extracellular cAMP affects monocyte differentiation into DCs, promoting the induction of cells displaying an activated, macrophage-like phenotype with reduced capacity of polarized, naive CD4(+) T cells into IFN-γ-producing lymphocytes compared with control cells. The effects of extracellular cAMP on monocytes are mediated by CD73 ecto-5'-nucleotidase and A2A and A2B adenosine receptors, as selective antagonists could reverse its effects. Of note, the expression of CD73 molecules has been found on the membrane of a small population of CD14(+)CD16(+) monocytes. These findings suggest that an extracellular cAMP-adenosine pathway is active in cells of the immune systems.

Keywords: dendritic cells; ecto-5′-nucleotidases; macrophages.

Figure 1.. Exogenous cAMP up-regulates CD14 and prevents the expression of MHC class I and class II molecules on human monocytes.

Histogram plots show the phenotype of human CD14 monocytes, isolated from the PBMC of healthy donors, cultured for 6 days with medium alone (A) or cAMP (0.5 mM; B). Cells were stained using anti-CD14, -HLA-ABC, and -HLA-DR mAb and analyzed by flow cytometry. Histogram plots are representative of five experiments performed. (C) The histogram bars show the means (+se) of the relative mean fluorescence intensities (mfi) of the different markers analyzed from five independent experiments performed. *P < 0.05, significant differences between cells cultured in the presence or absence of cAMP.

Figure 2.. Upon LPS stimulation, monocytes exposed to exogenous cAMP produce proinflammatory and regulatory cytokines.

Human CD14 monocytes, isolated from the PBMC of healthy donors, were cultured with medium alone or cAMP (0.5 mM). After 6 days, cells were incubated with LPS (200 ng/ml) for 48 h. The accumulation of IL-6 (A), TNF-α (B), IL-10 (C), and IL-12 (D) cytokines in culture supernatants was evaluated by ELISA assay. The data shown represent the mean (+sd) of three independent experiments.

Figure 3.. Exogenous cAMP impairs differentiation of human monocytes into DCs.

Dot and histogram plots show the phenotype of human CD14 monocytes, isolated from the PBMC of healthy donors, cultured for 6 days with GM-CSF (50 ng/ml) and IL-4 (35 ng/ml) in the presence of medium alone (A) or cAMP (0.5 mM; B). Cells were double-stained using anti-CD14-FITC, anti-CD1a-PE mAb and analyzed by flow cytometry. Dot and histogram plots are representative of one experiment out of 12 performed. The histogram bars show the means (+se) of the relative mfi of CD14 (C) and CD1a (D) expression from 12 independent experiments performed.

Figure 4.. Monocytes exposed to exogenous cAMP produce proinflammatory and regulatory cytokines and exhibit a reduced capacity to skew the immune response toward a Th1 profile.

Human CD14 monocytes, isolated from the PBMC of healthy donors, were cultured with medium alone or cAMP (0.5 mM). After 6 days, cells were incubated with LPS (200 ng/ml) for 48 h. The accumulation of IL-6 (A), TNF-α (B), IL-10 (C), and IL-12 (D) cytokines in culture supernatants was evaluated by ELISA. The data shown represent the mean (+sd) of three independent experiments. Alternatively, after 6 days, cells cultured in medium alone (E) or exogenous cAMP (F) were washed, starved for 8 h, and cocultured with purified CD4⁺CD45RA⁺ T cells at a 1:5 ratio. After 11 days of culture, the production of IL-4 and IFN-γ by CD4⁺ T lymphocytes was analyzed by flow cytometry after intracellular staining, using anti-CD4-PerCP, -IFN-γ-FITC, and -IL-4-PE mAb. Dot plots show the percentage of cytokine-producing cells on CD4⁺-gated populations. The data shown are from one representative experiment of three performed. (G) The histogram bars (+sd) show the means of the percentages of IFN-γ-positive CD4⁺ T cell from six independent experiments performed.

Figure 5.. Rapid metabolism of cAMP in the supernatants of monocytes induced to differentiate into DCs.

Monocytes were cultured in the presence of cAMP (0.5 mM), and its concentration was evaluated at different time-points (at 0, 10 and 30 min, and 1, 3, 6, 12, 18, and 24 h) by ELISA assay. The results are expressed as percentage, considering 100% as the concentration of cAMP measured at the beginning of culture (To). Means ± sem of five analyzed donors run in duplicate are presented.

Figure 6.. Dose-dependent effects of NECA on DC differentiation.

Human CD14 monocytes, isolated from the PBMC of healthy donors, were cultured for 6 days with GM-CSF (50 ng/ml) and IL-4 (35 ng/ml) in the presence of medium alone, scalar doses of NECA (0.001–10 μM), or cAMP (0.5 mM). Cells were double-stained using anti-CD14-FITC and anti-CD1a-PE mAb and analyzed by flow cytometry. The histograms show the mean fluorescence intensity of CD14 (A) and CD1a (B) expression.

Figure 7.. Nonselective adenosine receptor antagonist prevents the effects of cAMP and NECA on DC differentiation from monocytes.

Dot plots show the phenotype of human CD14 monocytes, isolated from the PBMC of healthy donors and cultured for 6 days with GM-CSF (50 ng/ml) and IL-4 (35 ng/ml) in the presence of medium alone (A), cAMP (B), cAMP plus DPSPX (C), NECA (D), NECA plus DPSPX (E), FSK (F), or FSK plus DPSPX (G). Cells were double-stained using anti-CD14-FITC, anti-CD1a-PE mAb and analyzed by flow cytometry. Dot plots are representative of one experiment out of three performed.

Figure 8.. Selective A2A and A2B adenosine receptor antagonists prevent the effects of cAMP on DC differentiation from monocytes.

Human CD14 monocytes, isolated from the PBMC of healthy donors, were cultured for 6 days with GM-CSF (50 ng/ml) and IL-4 (35 ng/ml) in the presence of medium alone, cAMP (0.5 mM), NECA (5 μM), or scalar doses of A2A antagonist CSC plus cAMP or plus NECA or scalar doses of A2B antagonist MRS1754 plus cAMP or plus NECA. Cells were stained using anti-CD14-FITC, or anti-CD32 mAb and analyzed by flow cytometry. The histograms show the mean fluoresce intensity of CD14 (A, B, E, and F) and CD32 (C, D, G, and H) expression.

Figure 9.. Selective CD73 inhibitor partially prevents the effects of cAMP on DC differentiation from monocytes.

Human CD14 monocytes, isolated from the PBMC of healthy donors, were cultured for 6 days with GM-CSF (50 ng/ml) and IL-4 (35 ng/ml) in the presence of medium alone, cAMP (0.5 mM), or scalar doses of AOPCP (1 and 10 μM) plus cAMP. Cells were stained using anti-CD14-FITC and anti-CD1a-PE mAb and analyzed by flow cytometry. The histograms show the percentages of CD1a (E) and CD14 (F) expression.

Figure 10.. Expression of CD73 on CD14⁺CD16⁻ and CD14⁺CD16⁺ macrophage/monocyte subsets.

Human CD14 monocytes, isolated from the PBMC of healthy donors, were purified by positive selection, using anti-CD14-conjugated magnetic microbeads. Cells were triple-stained with anti-CD14, -CD16, and -CD73 mAb, and the expression of CD73 was evaluated on CD14⁺CD16⁻ (A) and on CD14⁺CD16⁺ (B) subpopulations. The percentages of CD14⁺CD16⁺ in different healthy donors is reported in panel C. The histograms show the percentages of CD73 expression on CD14⁺CD16⁻ and on CD14⁺CD16⁺ subpopulations, and the graph shows the frequency of CD14⁺CD16⁺ in analyzed donors.