Received: 14 August 2019 | Revised: 2 December 2019 | Accepted: 6 December 2019 DOI: 10.1111/1756-185X.13783

ORIGINAL ARTICLE

P2 receptors mRNA expression profiles in macrophages from ankylosing spondylitis patients and healthy individuals

Maryam Akhtari1,2 | Seyed Jalal Zargar1 | Mahdi Vojdanian2 | Amir Ashraf-Ganjouei2 | Ali Javinani2 | Elham Hamzeh2 | Alireza Rezaiemanesh3 | Ahmadreza Jamshidi2,4 | Mahdi Mahmoudi2,4

1Department of Cell & Molecular Biology, School of Biology, College of Science, Abstract University of Tehran, Tehran, Iran Background: Ankylosing spondylitis (AS) is a multifactorial rheumatic disease which 2 Rheumatology Research Center, Tehran mainly involves the axial skeleton. Macrophages and extracellular have University of Medical Sciences, Tehran, Iran 3Department of Immunology, School of been shown to contribute to the inflammation process in autoimmune diseases. Medicine, Kermanshah University of Medical Membrane-bound purinergic P2 receptors might be involved in the modulation of Sciences, Kermanshah, Iran immune cells in AS. Therefore, we aimed to analyze the messenger RNA (mRNA) 4Inflammation Research Center, Tehran University of Medical Sciences, Tehran, Iran expression of P2 receptors in the macrophages of AS patients and healthy controls. Methods: Twenty-three AS patients and 23 age- and sex-matched healthy individuals Correspondence Mahdi Mahmoudi, Rheumatology Research were included in our study. Whole blood-separated monocytes of study participants Center, Tehran University of Medical were stimulated by macrophage colony-stimulating factor for 7 days and differenti- Sciences, Shariati Hospital, Kargar Ave., Tehran, Iran. ated to macrophages. Monocyte and macrophage markers were analyzed by flow Email: [email protected]. cytometry. SYBR green real-time polymerase chain reaction was used to measure Seyed Jalal Zargar, School of Biology, the relative expression levels of P2RX1, P2RX2, P2RX3, P2RX4, P2RX5, P2RX6, P2RX7, College of Science, University of Tehran, P.O. Box: 141556455, Tehran, Iran. P2RY1, P2RY2, P2RY4, P2RY6, P2RY11, P2RY12, P2RY13, P2RY14, and PANX1 . Email: [email protected]. Results: P2RY13 and P2RY6 genes had the highest expression levels in mac-

Funding information rophages among P2RY genes. P2RY1 mRNA expression was significantly down- Deputy of Research, Tehran University of regulated (−1.75 fold) and P2RY14 was up-regulated (2.6 fold) in macrophages of AS Medical Sciences, Grant/Award Number: 94-02-41-28991 patients compared to healthy individuals. P2RX4 had the highest expression in

monocyte-derived macrophages, followed by P2RX7 and P2RX1 genes. There was no significant difference in P2X mRNA expression level between macrophages of AS patients and healthy individuals. Conclusions: Our results indicate that AS patients show altered expression levels of genes. Moreover, these changes might be associated with disease activ- ity and patients’ status.

KEYWORDS ankylosing spondylitis, extracellular nucleotides, macrophages, P2 receptors

1 | INTRODUCTION joint, alongside involvement of the peripheral joints.1 Although, the environment has a role in the pathogenesis of AS, studies show that Ankylosing spondylitis (AS) is a complex rheumatic disease mainly char- genetic factors provide up to 90% of the susceptibility to the disease; acterized by the involvement of the axial skeleton including sacroiliac half of them are attributable to major histocompatibility complex

Int J Rheum Dis. 2019;00:1–8. wileyonlinelibrary.com/journal/apl © 2019 Asia Pacific League of Associations for | 1 Rheumatology and John Wiley & Sons Australia, Ltd 2 | AKHTARI et al. genes such as HLA-B27.2 Several studies have focused on the adaptive sex-matched healthy individuals without any familial history of rheu- immune response in AS,3 but due to the polygenic auto-inflammatory matic diseases were included in our study. The included patients had nature of the disease, innate immunity has been proposed to play a Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) ≥4. Mean significant role as well.4 ages for patient and control groups were 32 ± 10 and 35 ± 11 years, re- Macrophages are one of the main cells taking part in the innate spectively. Clinical characteristics of AS patients were collected at the immune response and have several tasks such as antigen presenta- time of sample collection. The ethics committee of Tehran University tion, phagocytosis, cytokine production, and so on. Based on their of Medical Sciences approved the study and we obtained written in- microenvironment, macrophages can be transformed into either M1 formed consents from patients and healthy individuals. or M2 subtypes.5 In the presence of pro-inflammatory cytokines such as interferon (IFN)-γ or tumor necrosis factor (TNF)-α, M1 phenotype has emerged. Macrophage colony-stimulating factor (M- 2.2 | Monocyte separation and CSF), interleukin (IL)-4 and IL-13 promote the development of M2 macrophage derivation macrophages.5 M2 macrophages and M2-type polarized monocytes have been shown to be predominant in synovial membrane and pe- Using ethylenediaminetetraacetic acid (EDTA) tubes, 20 mL of pe- ripheral blood of AS patients, respectively. Moreover, the M2/M1 ripheral blood was collected from each individual. All samples were ratio is correlated with inflammatory biomarkers and disease activity processed within the first 5 hours. Adding phosphate-buffered saline scores.6,7 Studies have demonstrated that macrophages are one of (PBS; GIBCO Invitrogen); samples were diluted with a ratio of 1:2 at the main cells contributing to the inflammation process in AS, es- pH 7.2. Subsequently, peripheral blood mononuclear cells were ex- pecially in joints.8 Therefore, investigating the macrophage function tracted using Ficoll (Lymphodex, Inno-Train) density gradient centrifu- and in patients with AS would be valuable. gation. PBS was used to wash mononuclear cells, and then they were While triphosphate (ATP) is mostly known for its central incubated with magnetic beads. Monocytes were isolated based on role in energy metabolism, extracellular ATP can affect several biolog- being positive for CD14, using magnetic-activated cell sorter (MACS) ical processes such as activation and migration of immune cells.9 ATP columns and MACS CD14 microbeads (Miltenyi Biotec). Isolated cells is released to the extracellular space through Pannexin 1 (Panx1) pores were stained by phycoerythrin (PE)-conjugated anti-CD14 antibody 10 that is induced by P2X7 activation. Extracellular (BD Bioscience). Flow cytometry analysis revealed that isolated cells ATP and related nucleotides act through membrane-bound purinergic have 92%-95% purity.18 Using 24-well plates, monocytes were cul- P2 receptors.11 P2 receptors include G- coupled P2Y recep- tured at 500 000 cells per well for 7 days in complete Roswell Park tors and -gated P2X receptors. Macrophages have Memorial Institute media containing 2 mmol/L L-glutamine (Biosera), been shown to be affected by P2 receptors and rapidly change their 100 U/mL penicillin, 10% fetal bovine serum (Gibco BRL), 50 ng/mL 12 function. The P2X7 was the first member of this family discovered. recombinant human macrophage colony stimulating factor (M-CSF; It affects intracellular concentration in both human and mu- eBioscience) and 100 μg/mL streptomycin (Sigma). rine macrophages.13,14 It is demonstrated that alveolar macrophages have all subtypes of P2 receptors except , P2X2, P2X3 and P2X6.

However, only P2X7, P2Y1, P2Y2 and P2Y11 could increase the intracel- 2.3 | Flow cytometry analysis of macrophage lular calcium level.15 ATP can also affect the production of inflamma- surface markers 16 tory cytokines such as TNF-α by macrophages, which is proven to have a prominent role in AS. Therefore, P2 receptors could contribute After 7 days of stimulation with M-CSF, monocyte-derived mac- to the altered balance of the immune system in AS patients. rophages (2 × 105) were stained with fluorochrome-labeled anti- As was mentioned, P2 receptors are involved in the inflamma- bodies. Subsequently, cells were incubated for 30 minutes with tory processes. However, their role in AS disease pathogenesis is not PE-conjugated anti-human CD206, fluorescein isothiocyanate clear yet. Therefore, we aimed to investigate the mRNA expression (FITC)-conjugated anti-human CD163 (both from BD Bioscience) of P2 receptors and Panx1 hemi-channel in the macrophages of AS and suitable isotype-matched antibodies, avoiding light exposure. patients and healthy controls. Moreover, we have explored the cor- Macrophages were analyzed using a CyFlow ML flow cytometer relation between P2R family mRNA expression and clinical features (Partec, GmbH) and the data were analyzed by FlowJo software of the disease. (Tree Star). Cells were 97% and 95% positive for CD163 and CD206 macrophage markers, respectively.18

2 | MATERIALS AND METHODS 2.4 | Analysis of gene expression using real-time 2.1 | Patients and controls quantitative polymerase chain reaction (PCR)

Twenty-three AS patients (4 female and 19 male) who fulfilled The High Pure RNA Isolation Kit (Roche) was used to extract total the modified New York classification criteria17 and 23 age- and RNA from macrophages after 7 days of stimulation with M-CSF. AKHTARI et al. | 3

TABLE 1 Primer sequences and Gene name Sequence Size (bp) product size of the selected genes GAPDH F: 5′-GAGTCAACGGATTTGGTCGT-3′ 185 R: 5′-GACAAGCTTCCCGTTCTCAG-3′

P2RY1 F: 5′-AATGCGATCTGTATCAGCGTG-3′ 118 R: 5′-TGGTGTCGTAACAGGTGATGG-3′

P2RY2 F: 5′-CCGCTTCAACGAGGACTTCAA-3′ 211 R: 5′-GCGGGCGTAGTAATAGACCA-3′

P2RY4 F: 5′-GGAGCTGGACTGTTGGTTTGA-3′ 105 R: 5′-CATAGGGTTGGGGCGTTAAGG-3′

P2RY6 F: 5′-GTGTCTACCGCGAGAACTTCA-3′ 159 R: 5′-CCAGAGCAAGGTTTAGGGTGTA-3′

P2RY11 F: 5′-AGCTCCTATGTGCCCTACCA-3′ 197 R: 5′-GCGGCCATGTAGAGTAGAGG-3′

P2RY12 F: 5′-GTCATCTGGGCATTCATGTTCT-3′ 243 R: 5′-ACCTACACCCCTCGTTCTTAC-3′

P2RY13 F: 5′-ATCGTGCTGTTAGGGCTCATA-3′ 153 R: 5′-CAAGATCGTATTTGGCAGGGAG-3′

P2RY14 F: 5′-TCTCACCAACCAGAGTGTTAGG-3′ 226 R: 5′-GCGGCTAGATTTCTTTTTGACCG-3′

P2RX1 F: 5′-ATGGTGCTGGTGCGTAATAAG-3′ 217 R: 5′-GGAAGACGTAGTCAGCCACA 3′

P2RX2 F: 5′-AGCTGGGCTTTATCGTGGAGA-3′ 127 R: 5′-TTGGGGTTGCACTCCGATG-3′

P2RX3 F: 5′-TTCTTGCACGAGAAGGCTTAC-3′ 100 R: 5′-CCATGACTCTGTTGGCGTAGA-3′

P2RX4 F: 5′-TGGCGGATTATGTGATACCAGC-3′ 112 R: 5′-GTCGCATCTGGAATCTCGGG-3′

P2RX5 F: 5′-CTGTCGCTGTTCGACTACAAG-3′ 112 R: 5′-CCCATACGACCAGGTACGC-3′

P2RX6 F: 5′-TCAACTTCTCTAAGTCCAATGCC-3′ 88 R: 5′-CAGTAGGGGCTGAATTGTGGT-3′

P2RX7 F: 5′-TATGAGACGAACAAAGTCACTCG-3′ 95 R: 5′-GCAAAGCAAACGTAGGAAAAGAT-3′ PANX1 F: 5′-CCACGGAGTACGTGTTCTCG-3′ 240 R: 5′-CCGCCCAGCAATATGAATCC-3′

Complementary DNA (cDNA) synthesis from the equivalent GraphPad Prism 6 for Windows (GraphPad Software). Compliance quantity of the total RNA was done using the Transcriptor First of variables with normal distribution was tested with Shapiro-Wilk Strand synthesis kit (Roche). StepOnePlus™ Real-Time PCR sys- test and probability graphics. Pearson's Chi-square or Fisher's tem (Applied Biosystems) and SYBR green master mix (Ampliqon) exact test was used to assess nominal variables across groups. were used to analyze the relative expression level of P2RX1, P2RX2, Independent-sample t test (for variables with normal distribution)

P2RX3, P2RX4, P2RX5, P2RX6, P2RX7, P2RY1, P2RY2, P2RY4, P2RY6, and Mann-Whitney U test (for variables without normal distribution)

P2RY11, P2RY12, P2RY13, P2RY14 and PANX1 genes. Glyceraldehyde- was used to assess differences between groups. A simple regression 3-phosphate dehydrogenase (GAPDH) gene was used as the en- model was used to assess the correlation between gene expression dogenous control. The primer sequences are depicted in Table 1. and clinical indices. P values less than .05 were considered statisti- Comparative CT method (2−ΔCT) was used to compare the relative cally significant. expression of the mentioned genes between patients and healthy individuals. 3 | RESULTS

2.5 | Statistical analysis 3.1 | Demographic and clinical features

All statistical analyses were done using IBM SPSS Statistics for Demographic data of AS patients and healthy individuals are de- Windows, version 22 (IBM Corp.). Graphs were created using picted in Table 2. 4 | AKHTARI et al.

TABLE 2 Clinical features of patients and controls Moreover, the association of the clinical characteristic features of AS patients including erythrocyte sedimentation rate (ESR), Healthy controls Characteristic AS patients (n = 23) (n = 23) BASDAI, bath ankylosing spondylitis metrology index (BASMI), bath ankylosing spondylitis functional index (BASFI), bath ankylosing Male/female 19 (82%)/4 (18%) 19 (82%)/4 (18%) spondylitis global score (BAS-G) and patient's disease global assess- ment (PDGA) indices with purinergic receptor mRNA expressions Age, y 32 ± 10 35 ± 11 was also investigated. Among all mentioned indices only BASMI ESR, mm/h 35 ± 27 NA index was positively correlated with P2RY relative mRNA expres- HLA-B27 positivity, 17 (74%) NA 1 sion in AS macrophages (Figure 3). no. (%) Disease duration, y 9 ± 8 NA BASMI score 3.5 ± 1.7 NA 3.3 | mRNA expression of P2X and PANX1 genes in BASFI score 5.2 ± 1.5 NA monocyte-derived macrophages BASDAI score 5.7 ± 2.0 NA

BAS-G score 7.3 ± 1.6 NA To investigate the role of P2X receptor family in AS macrophages, PDGA score 6.8 ± 2.6 NA we have assessed the relative mRNA expression of P2RX1, P2RX2, Biological agents 0 (0%) NA P2RX3, P2RX4, P2RX5, P2RX6 and P2RX7 genes in monocyte-de- Abbreviations: AS, ankylosing spondylitis; BASDAI, bath ankylosing rived macrophages of AS patients and healthy individuals using spondylitis disease activity index; BASFI, bath ankylosing spondylitis quantitative PCR. Our results indicated that P2RX3 and P2RX6 functional index; BAS-G, bath ankylosing spondylitis global score; were not expressed in human macrophages. The expression ratio BASMI, bath ankylosing spondylitis metrology index; ESR, erythrocyte of P2RX :P2RX :P2RX :P2RX :P2RX in healthy macrophages, was sedimentation rate; HLA-B27, human leukocyte antigen (subtypes 1 2 4 5 7 B*2701-2759); NA, not applicable; PDGA, patient's disease global approximately 1:0.0025:20.9:0.035:11.7, where P2RX2 had the assessment. lowest and P2RX4 had the highest expression levels (Figure 4). Moreover, there was no significant difference in P2X receptor 3.2 | mRNA expression of P2Y genes in monocyte- mRNA expression levels between macrophages of AS patients and derived macrophages healthy individuals. Finally, considering the role of Panx1 semi- canal on the withdrawal of ATP from immune cells and its effect

Our results indicated that the expression ratio of P2RY1:P2RY2:P on P2X receptor pathway activity, the expression of PANX1 gene

2RY4:P2RY6:P2RY11:P2RY12:P2RY13:P2RY14 genes in healthy mac- was also studied in monocyte-generated macrophages of patients rophages, was approximately 1.8:1:0.05:3.5:0.9:0.03:5.6:0.0 and healthy subjects. The results showed that the expression of

3, where P2RY12 and P2RY14 had the lowest and P2RY13 had the this gene did not have any significant difference between the highest expression levels (Figure 1). When we analyzed the ex- monocyte-derived macrophages of the patient group and healthy pression of P2Y genes between macrophages from AS patients individuals (Figure 5). and healthy controls, P2RY1 mRNA expression was significantly down-regulated (−1.75 fold with a P value of <.01) and P2RY14 was up-regulated (2.6 fold with a P value of <.05) in macrophages of 4 | DISCUSSION AS patients compared to healthy individuals (Figure 2). There was no significant difference in gene expression of other P2Y genes In this study, to investigate the role of P2 receptors in AS patho- between patients and controls. genesis, we have explored the gene expression of this family in

FIGURE 1 P2RY family relative messenger RNA (mRNA) expression in monocyte-derived macrophages. The receptor mRNA was determined by real-time polymerase chain reaction and normalized to glyceraldehyde-3- phosphate dehydrogenase (GAPDH). The data are presented as the mean ± SD AKHTARI et al. | 5

FIGURE 2 P2RY1 and P2RY14 messenger RNA (mRNA) expression in healthy individuals and ankylosing spondylitis (AS) patients. A, P2RY1 mRNA expression in monocyte-derived macrophages from AS patients was significantly decreased by −1.75-fold as compared to healthy controls. B, P2RY14 mRNA expression in monocyte-derived macrophages from AS patients was significantly increased by 2.6-fold as compared to healthy controls. The data are presented as the mean ± SD

expression of these receptors in dendritic cells (DC), it was found

that P2Y6 and P2Y12 had the highest expression levels, respec- tively.19 In another study by Myrtek et al the expression level of P2 receptors has been analyzed by reverse-transcription PCR on pulmonary macrophages. It was indicated that these macrophages

express P2RY1, P2RY2, P2RY4, P2RY6, P2RY11, P2RY13, and P2RY14 15 genes, but not P2RY12. Eventually, the variances could be the re- sult of using different methods and the different role and function of the mentioned cells in various contexts. We have also compared the expression of P2RY genes in mac- rophages of AS patients with control subjects. It was observed

that a patient's macrophages expressed a lower level of P2RY1 and

a higher level of P2RY14 genes comparing to healthy individuals.

The P2Y1 receptor acts on calcium channels and increases intra- cellular calcium in macrophages.20,21 Previous studies on mouse

leukocytes have shown that P2Y1 receptor plays an important role in triggering the inflammation process and also secretion of FIGURE 3 Correlation between bath ankylosing spondylitis 22,23 TNF-α by these cells. It is also involved in osteoblastogenesis metrology index (BASMI) score and P2RY1 relative messenger and the differentiation of osteoblasts.24 Regarding the effect of RNA (mRNA) expression. Simple linear regression analysis between BASMI score and the P2RY1 relative mRNA expression P2Y1 receptor on the function of osteoblasts, as well as the role in monocyte-derived macrophages from 21 ankylosing spondylitis of this receptor in the process of inflammation, it is proposed that (AS) patients the expression of P2RY1 gene is probably down-regulated in AS patients’ macrophages to act as a negative feedback mechanism in order to suppress the harmful inflammation. As it was shown monocyte-derived macrophages of AS patients and healthy indi- in our subsequent analyses, increased expression of this receptor viduals. To the best of our knowledge, this is the first study in- gene is associated with an increase in the BASMI index in patients, vestigating the role of P2 receptors in the pathogenesis of AS. indicating that the higher levels of the P2Y1 receptor is correlated

According to our results, P2RY13 and P2RY6 genes had the high- with the limitation in joint movements. est expression levels in monocyte-derived macrophages from the The P2Y14 receptor is expressed primarily in neutrophils and control group, and the P2RY12 and P2RY14 genes had the lowest can be activated by diphosphate (UDP) and its metabo- expression levels in these cells. Our results are approximately lites.25,26 Although its function is not fully understood yet, studies in agreement with previous studies. In a study investigating the have shown that P2Y14 receptor activity in B cells is responsible 6 | AKHTARI et al.

FIGURE 4 P2RX family relative messenger RNA (mRNA) expression in monocyte-derived macrophages. The receptor mRNA was determined by real-time polymerase chain reaction and normalized to glyceraldehyde-3- phosphate dehydrogenase (GAPDH). The data are presented as the mean ± SD

for the secretion of the IL-8 pro-inflammatory cytokine.27 It also has a role in mature dendritic cells28 and appears to be effective in regulating immune responses. In the meta-analysis study on spon- dyloarthritis (SpA)/AS patients, P2RY13 and P2RY14 genes were found to have a higher expression level in patients’ whole blood compared to control subjects.29 Regarding the results of our study, the P2Y14 receptor might be involved in macrophage inflammatory responses in AS. In this study, the expression of P2RX genes in monocyte-derived macrophages was investigated as well. Our results indicated that the

P2RX4 gene has the highest expression in monocyte-generated mac- rophages from healthy individuals, followed by P2RX7 and P2RX1 genes. Moreover, P2RX2 and P2RX5 genes had the least expression, and P2RX3 and P2RX6 genes did not express in macrophages. These results are in line with the results of previous studies. In a study by

Gicquel and colleagues on human macrophages, the P2RX4 gene had the highest expression, followed by P2RX7, P2RX1 and P2RX5 genes.

Furthermore, their results showed that the expression of P2RX2, 30 P2RX3 and P2RX6 genes were undetectable. In a study by Myrtek et FIGURE 5 PANX1 messenger RNA (mRNA) expression in al, pulmonary macrophages expressed P2RX , P2RX , P2RX and P2RX 7 1 4 5 healthy individuals and ankylosing spondylitis (AS) patients. The 15 genes and did not express P2RX2 and P2RX3 genes. Another study on mRNA expression of PANX1 did not have any significant difference human monocytes showed that P2RX4 gene expression was the high- between the monocyte-derived macrophages of the patient group 31 and healthy individuals. The data are presented as the mean ± SD est among the P2RX family, followed by P2RX7 and P2RX1 genes.

Due to the role and function of P2X receptors (especially P2X7) 11 in macrophages, it was expected to observe different mRNA levels that there was no significant difference in the expression of the P2X7 of these genes in macrophages of AS patients and healthy individuals. receptor in lymphocytes and monocytes of SLE patients. However,

However, our result indicated there was no significant difference in the monocytes of patients with RA expressed a lower level of P2X7 re- expression of these genes between AS patients and control groups. A ceptor and lymphocytes of these patients showed a higher expres- meta-analysis investigating the results of 4 microarray studies on SpA/ sion level of this receptor.32 Nevertheless, another study by Chen et

AS showed that the expression of P2RX1 and P2RX7 genes were in- al revealed that lymphocytes of SLE patients express a greater level 33 creased in the whole blood sample of patients compared to healthy in- of P2X7 receptor. dividuals.29 There is not enough evidence considering the role of these In addition to the investigation of purine receptors gene expres- genes in AS patients and further studies would be of great value. sions, the mRNA expression of the Panx1 that is associated with ATP Moreover, there are controversies regarding the role of P2X removal from the cell was explored as well. Studies have shown that the 34 receptors in other rheumatic disorders as well. Cervantes and col- function of this channel is necessary for the P2X7 receptor activity. leagues studied the expression and function of the P2RX7 gene in Most studies on this channel have been performed in neural cells and monocytes and B lymphocytes of rheumatoid arthritis (RA) and so far, the association of this channel with diseases such as Alzheimer's, systemic lupus erythematosus (SLE) patients.32 They have indicated diabetes and Crohn's has been investigated.35 Studies have shown that AKHTARI et al. | 7 inhibition of this channel in cells in mouse models can reduce 13. Falzoni S, Munerati M, Ferrari D, Spisani S, Moretti S, Di Virgilio F. The purinergic P2Z receptor of human macrophage cells. neuronal pain in arteritis.36 In another study by Diezmos et al, the ex- Characterization and possible physiological role. J Clin Investig. pression rate of this channel was reduced in the tissues of patients with 1995;95(3):1207-1216. 37 inflammatory bowel disease. However, the results obtained in this 14. Hickman SE, el Khoury J, Greenberg S, Schieren I, Silverstein SC. study demonstrate that the expression of PANX1 gene was not signifi- P2Z receptor activity in cultured human cantly different between patients and healthy individuals. monocyte-derived macrophages. Blood. 1994;84(8):2452-2456. 15. Myrtek D, Muller T, Geyer V, et al. Activation of human alveolar To the best of our knowledge, this is the first study regarding macrophages via P2 receptors: coupling to intracellular Ca2+ in- the role of P2X and P2Y receptors in AS. Alongside with the mRNA creases and cytokine secretion. J Immunol. 2008;181(3):2181-2188. expression level of P2 receptor genes, the current research also has 16. Tonetti M, Sturla L, Giovine M, Benatti U, De Flora A. Extracellular ATP enhances mRNA levels of nitric oxide synthase and TNF-alpha shown that altered expression of the P2RY1 gene in macrophages in lipopolysaccharide-treated RAW 264.7 murine macrophages. of AS patients is associated with clinical features of the disease. Biochem Biophys Res Commun. 1995;214(1):125-130. Therefore, investigating the role of purinergic signaling molecules 17. van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic would be valuable and shed light on AS pathogenesis. criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum. 1984;27(4):361-368. 18. Akhtari M, Zargar SJ, Mahmoudi M, Vojdanian M, Rezaeimanesh ACKNOWLEDGEMENT A, Jamshidi A. Ankylosing spondylitis monocyte-derived macro- This study was supported by a grant from Deputy of Research, phages express increased level of A2A and Tehran University of Medical Sciences (Grant No. 94-02-41-28991). decreased level of ectonucleoside triphosphate diphosphohydro- lase-1 (CD39), A1 and A2B adenosine receptors. Clin Rheumatol. 2018;37(6):1589-1595. CONFLICT OF INTEREST 19. Shin A, Toy T, Rothenfusser S, et al. signaling regulates The authors declare that they have no conflict of interest. phenotype and IFN-alpha secretion of human plasmacytoid den- dritic cells. Blood. 2008;111(6):3062-3069. ORCID 20. Abbracchio MP, Burnstock G, Boeynaems J-M, et al. International Union of Pharmacology LVIII: update on the P2Y G protein-coupled Mahdi Mahmoudi https://orcid.org/0000-0002-8164-8831 receptors: from molecular mechanisms and pathophysi- ology to therapy. Pharmacol Rev. 2006;58(3):281-341. REFERENCES 21. Bowler JW, Bailey RJ, North RA, Surprenant A. P2X4, P2Y1 and 1. Braun J, Sieper J. Ankylosing spondylitis. Lancet. P2Y2 receptors on rat alveolar macrophages. Br J Pharmacol. 2007;369(9570):1379-1390. 2003;140(3):567-575. 2. Reveille JD. The genetic basis of ankylosing spondylitis. Curr Opin 22. Cardoso TC, Pompeu TE, Silva CLM. The P2Y1 receptor-mediated Rheumatol. 2006;18(4):332-341. leukocyte adhesion to endothelial cells is inhibited by melatonin. 3. Kenna TJ, Brown MA. Immunopathogenesis of ankylosing spondyli- Purinergic Signal. 2017;13(3):331-338. tis. Int J Clin Rheumatol. 2013;8(2):265. 23. Amison RT, Momi S, Morris A, et al. RhoA signaling through 4. Vanaki N, Aslani S, Jamshidi A, Mahmoudi M. Role of innate im- P2Y(1) receptor controls leukocyte recruitment in allergic mice. J mune system in the pathogenesis of ankylosing spondylitis. Biomed Allergy Clin Immunol. 2015;135(2):528-538. Pharmacother. 2018;105:130-143. 24. Alvarenga EC, Rodrigues R, Caricati-Neto A, Silva-Filho FC, 5. Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage Paredes-Gamero EJ, Ferreira AT. Low-intensity pulsed ultra- activation: time for reassessment. F1000Prime Rep. 2014;6:13. sound-dependent osteoblast proliferation occurs by via acti- 6. Zhao J, Yuan W, Tao C, Sun P, Yang Z, Xu W. M2 polarization of vation of the P2Y receptor: role of the P2Y1 receptor. Bone. monocytes in ankylosing spondylitis and relationship with inflam- 2010;46(2):355-362. mation and structural damage. APMIS. 2017;125(12):1070-1075. 25. Scrivens M, Dickenson JM. Functional expression of the 7. Baeten D, Demetter P, Cuvelier CA, et al. Macrophages expressing P2Y14 receptor in human neutrophils. Eur J Pharmacol. the scavenger receptor CD163: a link between immune alterations 2006;543(1–3):166-173. of the gut and synovial inflammation in spondyloarthropathy. J 26. Jacobson KA, Balasubramanian R, Deflorian F, Gao ZG. G pro- Pathol. 2002;196(3):343-350. tein-coupled adenosine (P1) and P2Y receptors: ligand design and 8. Bollow M, Fischer T, Reisshauer H, et al. Quantitative analyses receptor interactions. Purinergic Signal. 2012;8(3):419-436. of sacroiliac biopsies in spondyloarthropathies: T cells and mac- 27. Müller T, Bayer H, Myrtek D, et al. The P2Y14 receptor of airway rophages predominate in early and active sacroiliitis- cellularity epithelial cells: coupling to intracellular Ca2+ and IL-8 secretion. Am correlates with the degree of enhancement detected by magnetic J Respir Cell Mol Biol. 2005;33(6):601-609. resonance imaging. Ann Rheum Dis. 2000;59(2):135-140. 28. Skelton L, Cooper M, Murphy M, Platt A. Human immature mono- 9. Di Virgilio F, Chiozzi P, Ferrari D, et al. Nucleotide receptors: an cyte-derived dendritic cells express the G protein-coupled recep- emerging family of regulatory molecules in blood cells. Blood. tor GPR105 (KIAA0001, P2Y14) and increase intracellular calcium 2001;97(3):587-600. in response to its agonist, uridine diphosphoglucose. J Immunol. 10. Iglesias R, Locovei S, Roque A, et al. P2X7 receptor-Pannexin1 2003;171(4):1941-1949. complex: pharmacology and signaling. Am J Physiol Cell Physiol. 29. Park R, Kim T-H, Ji JD. Gene expression profile in patients with axial 2008;295(3):C752-C760. spondyloarthritis: meta-analysis of publicly accessible microarray 11. Ferrari D, Pizzirani C, Adinolfi E, et al. The P2X7 receptor: a key player datasets. J Rheum Dis. 2016;23(6):363-372. in IL-1 processing and release. J Immunol. 2006;176(7):3877-3883. 30. Gicquel T, Robert S, Loyer P, et al. IL-1beta production is dependent 12. Murray PJ, Wynn TA. Protective and pathogenic functions of mac- on the activation of purinergic receptors and NLRP3 pathway in rophage subsets. Nat Rev Immunol. 2011;11(11):723-737. human macrophages. FASEB J. 2015;29(10):4162-4173. 8 | AKHTARI et al.

31. Wang L, Jacobsen SE, Bengtsson A, Erlinge D. P2 receptor mRNA 37. Diezmos EF, Sandow SL, Markus I, et al. Expression and localization expression profiles in human lymphocytes, monocytes and CD34+ of pannexin-1 hemichannels in human colon in health and disease. stem and progenitor cells. BMC Immunol. 2004;5:16. Neurogastroenterol Motil. 2013;25(6):e395-e405. 32. Portales-Cervantes L, Niño-Moreno P, Doníz-Padilla L, et al. Expression and function of the P2X(7) purinergic receptor in pa- tients with systemic lupus erythematosus and rheumatoid arthritis. How to cite this article: Akhtari M, Zargar SJ, Vojdanian M, Hum Immunol. 2010;71(8):818-825. et al. P2 receptors mRNA expression profiles in macrophages 33. Chen G-M, Feng C-C, Ye Q-L, et al. Association of P2X7R gene polymorphisms with systemic lupus erythematosus in a Chinese from ankylosing spondylitis patients and healthy individuals. population. Mutagenesis. 2013;28(3):351-355. Int J Rheum Dis. 2019;00:1–8. https​://doi.org/10.1111/1756- 34. Adamson SE, Leitinger N. The role of pannexin1 in the induction 185X.13783​ and resolution of inflammation. FEBS Lett. 2014;588(8):1416-1422. 35. Penuela S, Harland L, Simek J, Laird DW. Pannexin channels and their links to human disease. Biochem J. 2014;461(3):371-381. 36. Mousseau M, Burma NE, Lee KY, et al. Microglial pannexin-1 channel activation is a spinal determinant of joint. Pain. 2018;4(8):eaas9846.