Alteration of Activated Phenotypes of the Macrophages Treated with Lipopolysaccharide and Interferon- by Sodium Bicarbonate in the Culture Medium

Main Article Content

Fumio Amano, Ph.D.


Macrophage is the immune phagocytic cell, playing variety of immunological and inflammatory reactions. The macrophage activation has been extensively studied in vitro using culture media like Dulbecco's modified Eagle’s medium (DMEM) and Ham's F-12 medium (F-12) in response to bacterial infection, tumor development, cytokines and so on. In the study of macrophage activation during co-culture with EL-4 tumor cells, we found different phenotypes of J774.1 macrophage-like cell line in F-12 and DMEM, when treated with lipopolysaccharide (LPS) and interferon-g (IFN- g). Among these phenotypes, nitric oxide (NO) production with corresponding inducible NO synthase (iNOS) expression was remarkable, showing higher in F-12 than in DMEM. Besides, O2-generating activity and production of interleukin-1b (IL-1b) were also higher in F-12 than DMEM, although production of tumor necrosis factor-a (TNF- a) was higher in DMEM than F-12. RT-PCR analysis revealed significantly higher expression of mRNA of iNOS, IL-1 b, IL-18, IkBa in F-12, but higher that of p65 and p105 in DMEM after treatment with LPS + IFN-g, suggesting these differences being induced at the transcriptional levels. Through investigation of critical factor(s) in these culture media that influence the activation phenotypes of the macrophages, we found that sodium bicarbonate (NaHCO3) concentrations in these culture media, 14 mM in Ham's F-12 and 44 mM in DMEM, were the key. Culture medium-induced differences in macrophage activation were also observed in RAW264.7 macrophage-like cell line and in mouse peritoneal macrophages. The recent studies suggested involvement of carrier (SLC) transporter gene expression and subsequent elevation of JAK/STAT signaling cascades in these NaHCO3 responses. Taken together, these results provide evidence for the importance of NaHCO3 in the culture medium in the macrophage activation in vitro, implying important insights to the NaHCO3 concentration in vivo in the body of patients suffering from inflammation, tumor development or immune disorders where macrophage activation is involved.

Article Details

How to Cite
AMANO, Fumio. Alteration of Activated Phenotypes of the Macrophages Treated with Lipopolysaccharide and Interferon- by Sodium Bicarbonate in the Culture Medium. Medical Research Archives, [S.l.], v. 11, n. 1, jan. 2023. ISSN 2375-1924. Available at: <>. Date accessed: 02 apr. 2023. doi:
Research Articles


1. Nathan CF, Murray HW, Wiebe ME, Rubin BY. Identification of interferon- gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J Exp Med. 1983;158:670–689.
2. Pham TV, MacDonald HR, Mauel J. Macrophage activation in vitro by lymphocytes from Leishmania major infected healer and non-healer mice. Parasite Immunol. 1988;10:353–368.
3. Higashi N, Higuchi M, Hanada N, Oeda J, Kobayashi Y, Osawa T. Identification of human T cell hybridoma-derived macrophage activating factor as interleukin 2. J Biochem. 1993;113:715–720.
4. Ferrante CF, Leibovich SJ. Regulation of macrophage polarization and wound healing. AdvWound Care. 2012;1:10–16.
5. Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature. 2013;496:445–455.
6. Locati M, Curtale G, Mantovani A. Diversity, mechanisms, and significance of macrophage plasticity. Annu Rev Pathol. 2020;15:123-147.
7. Hou LT, Li TI, Liu CM, Liu BY, Liu CL, Mi HW. Modulation of osteogenic potential by recombinant human bone morphogenic protein-2 in human periodontal ligament cells: effect of serum, culture medium, and osteoinductive medium. J Periodontal Res. 2007;42:244–252.
8. Lopez-Cazaux S, Bluteau G, Magne D, Lieubeau B, Guicheux J, Alliot-Licht B. Culture medium modulates the behaviour of human dental pulp-derived cells: technical note. Eur Cells Mater. 2006;11:35–42. Discussion 42.
9. Wu X, Lin M, Li Y, Zhao X, Yan F. Effects of DMEM and RPMI 1640 on the biological behavior of dog periosteum-derived cells. Cytotechnology. 2009;59:103-111.
10. Ginhoux F, Schultze JL, Murray PJ, Ochando J, Biswas SK. New insights into the multidimensional concept of macrophage ontogeny, activation and function. Nat Immunol.2016;17:34–40.
11. Stout RD, Suttles J. Functional plasticity of macrophages: reversible adaptation to changing microenvironments. J Leukoc Biol. 2004;76:509–513.
12. Stout RD, Jiang C, Matta B, Tietzel I, Watkins SK, Suttles J. Macrophages sequentially change their functional phenotype in response to changes in microenvironmental influences. J Immunol. 2005;175:342–349.
13. Chiang CS, Chen FH, Hong JH, Jiang PS, Huang HL, Wang CC, McBride WH. Functional phenotype of macrophages depends on assay procedures. Int Immunol. 2008;20:215–222.
14. Nishijima M, Amano F, Akamatsu Y, Akagawa K, Tokunaga T, Raetz CRH. Macrophage activation by monosaccharide precursors of Escherichia coli lipid A. Proc Natl Acad Sci USA. 1985;82:282-285.
15. Amano F, Nishijima M, Akagawa K, Akamatsu Y. Enhancement of O2-generation and tumoricidal activity of murine macrophages by a monosaccharide precursor of Escherichia coli lipid A. FEBS Lett. 1985;192:263-266.
16. Amano F, Nishijima M, Akamatsu Y. A monosaccharide precursor of Escherichia coli lipid A has the ability to induce tumor-cytotoxic factor production by a murine macrophage-like cell line, J774.1. J Immunol. 1986;136:4122-4127.
17. Amano F, Akamatsu Y. A lipopolysaccharide (LPS)-resistant mutant isolated from a macrophage-like cell line, J774.1, exhibits an altered activated-macrophage phenotype in response to LPS. Infect Immun. 1991;59:2166–2174.
18. Kawakami T, Kawamura K, Fujimori K, Koike A, Amano F. Influence of the culture medium on the production of nitric oxide and expression of inducible nitric oxide synthase by activated macrophages in vitro. Biochem Biophys Rep. 2016;5:328–334.
19. Kawakami T, Koike A, Amano F. Induction of different activated phenotypes of mouse peritoneal macrophages grown in different tissue culture media. Cytotechnology. 2017;69:631-642.
20. Kawakami T, Koike A, Amano F. Sodium bicarbonate regulates nitric oxide production in mouse macrophage cell lines stimulated with lipopolysaccharide and interferon γ. Nitric Oxide. 2018;79:45-50.
21. Kawakami T, Koike A, Maehara T, Hayashi T, Fujimori K. Bicarbonate enhances the inflammatory response by activating JAK/STAT signaling in LPS + IFN--stimulated macrophages. J Biochem. 2020;167:623–631.
22. Cordat E. Casey JR. (2009) Bicarbonate transport in cell physiology and disease. Biochem J. 2009;417:423–439.
23. Sedlyarov V, Eichner R, Girardi E, Essletzbichler P, Goldmann U, Nunes-Hasler P, Srndic I, Moskovskich A, Heinz LX, Kartnig F, Bigenzahn JW, Rebsamen M, Kovarik P, Demaurex N, Superti-Furga G. The bicarbonate transporter SLC4A7 plays a key role in macrophage phagosome acidification. Cell Host Microbe. 2018;23:766–774.
24. Alper SL, Sharma AK. The SLC26 gene family of anion transporters and channels. Mol Aspects Med. 2013;34:494–515.
25. Chan ED, Riches DW. IFN- + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38(MAPK) in a mouse macrophage cell line. Am. J. Physiol. Cell Physiol. 2001;280:C441–C450.
26. Kohama K, Koike A, Amano, F. Triptolide induces cell damage in lipopolysaccharide (LPS)-treated macrophages in an LPS-signaling cascade-dependent manner. J Cytokine Biol. 2018;2:117.
27. Ginhoux F, Guilliams M. Tissue-resident macrophage ontogeny and homeostasis. Immunity 2016;44:439–449.
28. Mosser DM. The many faces of macrophage activation. J Leukoc Biol. 2003;73:209–212.
29. Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep. 2014;6:13.
30. Varol C, Mildner A, Jung S. Macrophages: development and tissue specialization. Annu Rev Immunol. 2015;33:643–675.
31. Buck MD, Sowell RT, Kaech SM, Pearce EL. Metabolic instruction of immunity. Cell. 2017; 169:570–586.
32. Liu YC, Zou XB, Chai YF, Yao YM. Macrophage polarization in inflammatory diseases. Int J Biol Sci. 2014;10:520–529.
33. Romero MF, Chen A-P, Parker MD, Boron WF. The SLC4 family of bicarbonate (HCO3-) transporters. Mol Aspects Med. 2013;34:159–182.
34. Loniewski I, Wesson DE. Bicarbonate therapy for prevention of chronic kidney disease progression. Kidney Int. 2014;85:529–535.
35. De Smet HR, Bersten AD, Barr HA, Doyle IR. Hypercapnic acidosis modulates inflammation, lung mechanics, and edema in the isolated perfused lung. J Crit Care. 2007;22:305–313.
36. Kraut JA, Madias NE. Treatment of acute metabolic acidosis: a pathophysiologic approach. Nat Rev Nephrol.2012;8:589–601.
37. Díaz FE, Dantas E, Geffner J. Unravelling the interplay between extracellular acidosis and immune cells. Mediators Inflamm. 2018;2018:1218297.
38. Halbertsma FJ, Vaneker M, Pickkers P, Snijdelaar DG, van Egmond J, Scheffer GJ, van der Hoeven HG. Hypercapnic acidosis attenuates the pulmonary innate immune response in ventilated healthy mice. Crit Care Med. 2008;36:2403-2406.
39. Jiang W, Le J, Wang P-y, Cheng X, Smelkinson M, Dong W, Yang C, Chu Y, Hwang PM, Munford RS, Lu M. Extracellular acidity reprograms macrophage metabolism and innate responsiveness. J Immunol. 2021;206:3021–3031.
40. Rajamäki K, Nordström T, Nurmi K, Åkerman KEO, Kovanen PT, Öörni K, Eklund KK. Extracellular Acidosis Is a Novel Danger Signal Alerting Innate Immunity via the NLRP3 Inflammasome. J Biol Chem. 2013;288:13410–13419.
41. Torres IM, Patankar YR, Shabaneh TB, Dolben E, Hogan DA, Leib DA, Berwin BL. Acidosis potentiates the host proinflammatory imnterleukin-1β Response to Pseudomonas aeruginosa Infection. Infect Immun. 2014;82:4689–4697.
42. O'Croinin DF, Nichol AD, Hopkins N, Boylan J, O'Brien S, O'Connor C, Laffey JG, McLoughlin P. Sustained hypercapnic acidosis during pulmonary infection increases bacterial load and worsens lung injury. Crit Care Med. 2008;36:2128–2135.
43. El-Kenawi A, Gatenbee C, Robertson-Tessi M, Bravo R, Dhillon J, Balagurunathan Y, Berglund A, Vishvakarma N, Ibrahim-Hashim A, Choi J, Luddy K, Gatenby R, Pilon-Thomas S, Anderson A, Ruffell B, Gillies R. Acidity promotes tumour progression by altering macrophage phenotype in prostate cancer. Br J Cancer. 2019; 121:556–566.
44. Romero-Garcia S, Moreno-AltamiranoMMB, Prado-Garcia H, Sánchez-García FJ. Lactate contribution to the tumor microenvironment: Mechanisms, effects on immune cells and therapeutic relevance. Front Immunol. 2016;7:52.
45. Magor-Elliott SRM, Fullerton CJ, Richmond G, Ritchie GAD, Robbins PA. A dynamic model of the body gas stores for carbon dioxide, oxygen, and inert gases that incorporates circulatory transport delays to and from the lung. J Appl Physiol. 1985;130:1383–1397.
46. Cummins EP, Selfridge AC, Sporn PH, Sznajder JI, Taylor CT. Carbon dioxide-sensing in organisms and its implications for human disease. Cell Mol Life Sci. Cell Mol Life Sci. 2014;71:831–845.
47. West MA, Hackam DJ, Baker J, Rodriguez JL, Bellingham J, Rotstein OD. Mechanism of decreased in vitro murine macrophage cytokine release after exposure to carbon dioxide: relevance to laparoscopic surgery. Ann Surg.1997;226:179–190.
48. Steiner AA, Flatow EA, Brito CF, Fonseca MT, Komegae EN. Respiratory gas exchange as a new aid to monitor acidosis in endotoxemic rats: relationship to metabolic fuel substrates and thermometabolic responses. Physiol Rep. 2017;5:e13100.
49. Helenius IT, Krupinski T, Turnbull DW, Gruenbaum Y, Silverman N, Johnson EA, Sporn PHS, Sznajder JI, Beitel GJ. Elevated CO2 suppresses specific Drosophila innate immune responses and resistance to bacterial infection. Proc Natl Acad Sci USA. 2009;106:18710–18715.