Abstract
Macrophages play an important role in the ankylosing spondylitis (AS) auto-inflammatory responses and fibrocartilage destruction. Adenosine is a key modulator of inflammatory conditions. The various effects of adenosine are mediated by its interaction with adenosine receptors (AR). In this study, we investigated the mRNA expression of A1, A2A, A2B, and A3 adenosine receptors, ectonucleoside triphosphate diphosphohydrolase-1 (CD39), and ecto-5′-nucleotidase (CD73) in the monocyte-derived macrophages from AS patients in comparison to healthy controls. We also explored the correlation between analyzed gene expression and patients’ clinical manifestations. Whole blood-separated monocytes from 23 healthy controls and 23 active AS patients were stimulated by macrophage colony-stimulating factor (M-CSF) for 7 days and differentiated to macrophages. Monocyte and macrophage markers were analyzed by flow cytometry. Analysis of adenosine receptors (ADORA1، ADORA2A، ADORA2B، ADORA3), CD39 and CD73 gene expression was performed by SYBR green real-time PCR. Our results demonstrated monocyte-derived macrophages from AS patients expressed increased level of A2AAR and reduced level of A1, A2BAR, and CD39 mRNA compared to healthy controls. We found an inverse correlation between A2AAR mRNA expression and Bath Ankylosing Spondylitis Functional Index (BASFI) score in AS patients. According to our results, altered expression level of adenosine-relying system would be involved in AS macrophage dysfunction and inflammation and correlated with functional status in AS patients.
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This work has been supported by a research grant from Tehran University of Medical Sciences (TUMS); grant no. 95-04-41-33869.
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Supplementary Fig. 1
Flow cytometry analysis of CD14 expression on the cell surface of isolated monocyte. Histograms of PBMC-isolated monocyte staining with PE-conjugated anti-CD14 and isotype control antibodies. The results showed that monocytes were isolated by positive selection with 92–95% purity (GIF 21 kb)
Supplementary Fig. 2
Flow cytometry analysis of the cell surface macrophage markers CD163 and CD206. Histograms of monocyte-derived macrophage staining with a) FITC-conjugated anti-CD163 b) PE-conjugated anti-CD206, and appropriate isotype control antibodies. Results showed that Monocyte-derived macrophages with 7 days’ stimulation by M-CSF were a) 97% CD163 positive and b) 95% CD206 positive (GIF 17 kb)
Supplementary Fig. 3
There was not significant differences in a) A3AR and b) CD73 enzyme mRNA expression in monocyte-derived macrophages between AS patients and healthy controls. Isolated monocytes from 23 AS patients and 23 healthy donors were differentiated to macrophages with M-CSF. The expression was analyzed in monocyte-generated macrophages by real-time PCR and normalized to GAPDH. Data are expressed as the means ± SD (GIF 22 kb)
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Akhtari, M., Zargar, S.J., Mahmoudi, M. et al. Ankylosing spondylitis monocyte-derived macrophages express increased level of A2A adenosine receptor and decreased level of ectonucleoside triphosphate diphosphohydrolase-1 (CD39), A1 and A2B adenosine receptors. Clin Rheumatol 37, 1589–1595 (2018). https://doi.org/10.1007/s10067-018-4055-9
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DOI: https://doi.org/10.1007/s10067-018-4055-9