Effects of UTP on Viability in RAW 264.7 Cell Line

Abstract

Macrophages are important cells of immune system and their deficiency or dysfunction lead to severe diseases. Uridine-5’-triphosphate, a pyrimidine nucleotide, is known to behave as a signaling molecule for macrophages. However, the effect of UTP on macrophage viability or proliferation is yet to be researched. Therefore, we investigated the effects of different concentrations (1, 10 and 100 μM) of UTP on RAW 264.7 mouse macrophage cell viability at different time points using MTT [(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)] assay. We found that while there was no difference among groups at 24 or 48 hours following UTP administration, cell viability was enhanced by 27,7% (p<0,001) compared to controls at 72 hours after 10 μM UTP administration. Interestingly, UTP at 100 μM concentration decreased cell viability significantly (p<0,05) compared to controls. Our findings reveal that UTP enhances macrophage viability in a dose- and time-dependent manner while higher concentrations provide toxic effects. Hence the present data suggest that UTP administered at appropriate concentrations may contribute to immune system functions through enhancing macrophage cell metabolism or proliferation in tissues.

Keywords

• UTP
• RAW 264.7
• macrophage
• viability
• MTT

UTP’nin Raw 264.7 Hücre Hattında Canlılık Üzerine Etkileri

Abstract

Makrofajlar immün sistemin önemli hücreleridir ve eksiklikleri ya da disfonksiyonları vücutta ciddi hastalıklara yol açar. Bir pirimidin nükleotidi olan üridin-5’-trifosfat’ın (UTP) makrofajlar için bir sinyal molekülü gibi davrandığı bilinmektedir. Ancak UTP’nin makrofaj canlılığı ya da proliferasyonu üzerine etkileri henüz araştırılmamıştır. Dolayısıyla bu çalışmada farklı konsantrasyonlarda (1, 10 ve 100 μM) uygulanan UTP’nin RAW 264.7 fare makrofaj hücrelerinin canlılığı üzerine etkileri farklı zaman noktalarında MTT [3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolyum bromür] testi kullanılmak suretiyle incelenmiştir. UTP uygulandıktan 24 ya da 48 saat sonra hücre canlılığında kontrole göre herhangi bir fark gözlenmezken, 10 μM konsantrasyonda uygulanan UTP’nin 72 saat sonra hücre canlılığını kontrole göre %27,7 oranında (p<0,001) artırdığı tespit edilmiştir. İlginç olarak 100 μM konsantrasyonda uygulanan UTP aynı zaman aralığında hücre canlılığını kontrole göre anlamlı (p<0,05) olarak azaltmıştır. Bulgularımız UTP’nin makrofaj canlılığını doza ve zamana bağlı olarak artırdığını ancak yüksek konsantrasyonda toksik etki gösterdiğini ortaya koymaktadır. Dolayısıyla elde ettiğimiz veriler, doğru dozda uygulanan UTP’nin dokularda makrofaj hücre metabolizmasını ya da proliferasyonunu artırarak immün sistem fonksiyonlarına katkıda bulunabileceğini düşündürmektedir.

Keywords

• UTP
• Raw 264.7
• makrofaj
• canlılık
• MTT

References

1. Parkin J, Cohen B. An overview of the immune system. Lancet 2001;357(9270):1777–1789.
2. Netea MG, Schlitzer A, Placek K, Joosten LAB, Schultze JL. Innate and Adaptive Immune Memory: an Evolutionary Continuum in the Host’s Response to Pathogens. Cell Host Microbe 2019;25(1):13–26.
3. Perdiguero EG, Geissmann F. The development and maintenance of resident macrophages. Nat Immunol 2016;17(1):28.
4. Pittet MJ, Nahrendorf M, Swirski FK. The journey from stem cell to macrophage. Ann N Y Acad Sci 2014;1319(1):1–18.
5. Epelman S, Lavine KJ, Randolph GJ. Origin and Functions of Tissue Macrophages. Immunity 2014;41(1).
6. Heyworth PG, Cross AR, Curnutte JT. Chronic granulomatous disease. Curr Opin Immunol 2003;15(5):578–584.
7. Nazir T, Taha N, Islam A, Abraham S, Mahmood A, Mustafa M. Monocytopenia; induction by vinorelbine, cisplatin and doxorubicin in breast, non-small cell lung and cervix cancer patients. Int J Heal Sci 2016;10(4).
8. Tumpey TM, García-Sastre A, Taubenberger JK, Palese P, Swayne DE, Pantin-Jackwood MJ, Schultz-Cherry S, Solórzano A, Van Rooijen N, Katz JM, et al. Pathogenicity of Influenza Viruses with Genes from the 1918 Pandemic Virus: Functional Roles of Alveolar Macrophages and Neutrophils in Limiting Virus Replication and Mortality in Mice. J Virol 2005;79(23).

9. Traut TW. Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 1994;140(1):1–22.
10. Lustig KD, Shiau AK, Brake AJ, Julius D. Expression cloning of an ATP receptor from mouse neuroblastoma cells. In: Proceedings of the National Academy of Sciences of the United States of America. Vol. 90. 1993. p. 5113–5117.
11. Communi D, Pirotton S, Parmentier M, Boeynaems JM. Cloning and functional expression of a human uridine nucleotide receptor. J Biol Chem 1995;270(52):30849–30852.
12. Abbracchio MP, Burnstock G, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Knight GE, Fumagalli M, Gachet C, Jacobson KA, et al. International Union of Pharmacology LVIII: Update on the P2Y G protein-coupled nucleotide receptors: From molecular mechanisms and pathophysiology to therapy. Pharmacol Rev 2006;58(3):281–341.
13. Merz J, Nettesheim A, von Garlen S, Albrecht P, Saller BS, Engelmann J, Hertle L, Schäfer I, Dimanski D, König S, et al. Pro- and anti-inflammatory macrophages express a sub-type specific purinergic receptor profile. Purinergic Signal 2021;17(3):481–492.
14. Albuquerque C, Oliveira SMC, Coutinho-Silva R, Oliveira-Castro GM, Persechini PM. ATP- and UTP-induced currents in macrophages and macrophage polykaryons. Am J Physiol - Cell Physiol 1993;265(6 34-6):C1663-73.
15. Elliott MR, Chekeni FB, Trampont PC, Lazarowski ER, Kadl A, Walk SF, Park D, Woodson RI, Ostankovich M, Sharma P, et al. Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature 2009;461(7261):282–286.
16. Higgins KR, Kovacevic W, Stokes L. Nucleotides regulate secretion of the inflammatory chemokine CCL2 from human macrophages and monocytes. Mediators Inflamm 2014;2014(2014):293925.
17. Wihlborg AK, Malmsjö M, Eyjolfsson A, Gustafsson R, Jacobson K, Erlinge D. Extracellular nucleotides induce vasodilatation in human arteries via prostaglandins, nitric oxide and endothelium-derived hyperpolarising factor. Br J Pharmacol 2003;138(8):1451–1458.
18. Wihlborg AK, Balogh J, Wang L, Borna C, Dou Y, Joshi B V., Lazarowski E, Jacobson KA, Arner A, Erlinge D. Positive inotropic effects by uridine triphosphate (UTP) and uridine diphosphate (UDP) via P2Y2 and P2Y6 receptors on cardiomyocytes and release of UTP in man during myocardial infarction. Circ Res 2006;98(7):970–976.
19. Pooler AM, Guez DH, Benedictus R, Wurtman RJ. Uridine enhances neurite outgrowth in nerve growth factor-differentiated pheochromocytoma cells. Neuroscience 2005;134(1):207–214.
20. Cansev M, Orhan F, Yaylagul EO, Isik E, Turkyilmaz M, Aydin S, Gumus A, Sevinc C, Coskun N, Ulus IH, et al. Evidence for the existence of pyrimidinergic transmission in rat brain. Neuropharmacology 2015;91.
21. Namkung W, Finkbeiner WE, Verkman AS. CFTR-adenylyl cyclase I association responsible for UTP activation of CFTR in well-differentiated primary human bronchial cell cultures. Mol Biol Cell 2010;21(15):2639–2648.
22. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65(1–2):55–63.
23. Stockert JC, Horobin RW, Colombo LL, Blázquez-Castro A. Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochem 2018;120(3):159–167.
24. Ghasemi M, Turnbull T, Sebastian S, Kempson I. The mtt assay: Utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis. Int J Mol Sci 2021;22(23):12827.
25. Loan TT, Do LT, Yoo H. Platinum Nanoparticles Induce Apoptosis on Raw 264.7 Macrophage Cells. J Nanosci Nanotechnol 2017;18(2):861–864.
26. Kwon DH, Cheon JM, Choi E-O, Jeong JW, Lee KW, Kim KY, Kim SG, Kim S, Hong SH, Park C, et al. The Immunomodulatory Activity of Mori folium, the Leaf of Morus alba L., in RAW 264.7 Macrophages In Vitro. J Cancer Prev 2016;21(3):144–151.
27. Hong J, Nakano Y, Yokomakura A, Ishihara K, Kim S, Kang YS, Ohuchi K. Nitric oxide production by the vacuolar-type (H+)-ATPase inhibitors bafilomycin A1 and concanamycin A and its possible role in apoptosis in RAW 264.7 cells. J Pharmacol Exp Ther 2006;319(2):672–681.
28. Soromou LW, Zhang Z, Li R, Chen N, Guo W, Huo M, Guan S, Lu J, Deng X. Regulation of inflammatory cytokines in lipopolysaccharide-stimulated RAW 264.7 murine macrophage by 7-O-methyl-naringenin. Molecules 2012;17(3):3574–3585.
29. Yang EJ, Kim JG, Kim JY, Kim SC, Lee NH, Hyun CG. Anti-inflammatory effect of chitosan oligosaccharides in RAW 264.7 cells. Cent Eur J Biol 2010;5(1):95–102.
30. Yoon S Bin, Lee YJ, Park SK, Kim HC, Bae H, Kim HM, Ko SG, Choi HY, Oh MS, Park W. Anti-inflammatory effects of Scutellaria baicalensis water extract on LPS-activated RAW 264.7 macrophages. J Ethnopharmacol 2009;125(2):286–290.