Phorbol myristate acetate induces differentiation of THP-1 cells in a nitric oxide-dependent manner
Introduction
Monocytes and macrophages are both key immune effector cells [1] and regulate immunity through a variety of ways [2,3]. Monocyte-to-macrophage differentiation plays a critical role in many inflammatory diseases, such as atherosclerosis [[4], [5], [6]]. During progress of atherosclerosis, monocytes migrate into subendothelial spaces where they differentiate to macrophages [4,7,8]. Macrophages accumulate in atherosclerotic plaques promoting disease exacerbation [4,7,8]. In this regard, some therapeutic drugs for atherosclerosis target inhibition of monocyte-to-macrophage differentiation or macrophage activities [9,10].
The biology and function of macrophages are widely studied in many in vitro researches. Differentiated THP-1 cells, a human leukemia monocytic cell line, are widely used as surrogate of human macrophages and serve as a reliable cellular model [11,12]. THP-1 cells can be differentiated into macrophages by phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator [13,14]. After differentiated with PMA, THP-1 cells acquire macrophage-like characteristics [14,15]. Increased cell adherence and decreased proliferation rate are found in PMA-differentiated THP-1 cells [[14], [15], [16]]. PMA also enhanced lipopolysaccharide (LPS)-triggered release of pro-inflammatory cytokines [15], and upregulates migration ability of THP-1 cells [17,18].
Nitric oxide (NO) is a crucial intracellular messenger modulating various cellular signaling pathways [19]. The production of NO is generated by nitric oxide synthases (NOS), including inducible nitric oxide synthase (iNOS) [20] primarily found in a variety of immune cells [21]. From previous reports, immune cell activities can be regulated by NO signaling [[22], [23], [24]]. For example, NO regulates differentiation of dendritic cells [23] as well as lymphocyte migration capacity [24]. Though PMA stimulation upregulates NO and iNOS [25,26], whether effects of PMA on THP-1 cells differentiation relate to NO or iNOS remain unstudied.
In this study, we sought to clarify whether the mechanism underlying PMA-induced monocyte-to-macrophage differentiation of THP-1 cells relates to NO or iNOS. Based on previous report, PMA triggers upregulation of calcium levels [27]. Furthermore, there is crosstalk between NO and calcium signaling [28]. For example, NO regulates intracellular calcium levels mediated by acting on NO receptors [29]. On the other hand, calcium-mediated signaling regulates iNOS [30,31]. Therefore, the effect of PMA on mutual regulation between calcium and NO was also investigated in this study.
Section snippets
Cell culture and stimulation
THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were grown in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA). Cells of fewer than 15 passages were incubated in a humidified chamber at 37 °C in a mixture of 95% air and 5% CO2, and employed in this experiment. To facilitate investigation, THP-1 cells were incubated with or without 0-to-200 nM PMA for 24 h
Cell viability was not reduced by PMA up to 200 nM
Fig. 1 illustrated the data of cell viability measured through LDH release assay (n = 5 in each group). The cell viability in groups treated with 50-to-200 nM PMA was not different from that in the PBS group.
PMA increased cell adherence in a dose-dependent manner
The cell adherence was measured and shown in Fig. 2A (n = 5 in each group). Our data revealed that cell adherence in groups treated with PMA all increased significantly compared to the PBS group (all P < 0.001). Furthermore, there was significant difference between groups treated with PMA
Discussion
Our data showed that PMA induces differentiation of THP-1 cells in a NO-dependent manner. The correlation between NO levels and PMA-induced effects was obvious under dose of PMA between 50 nM and 200 nM. Though our results (Fig. 7B) showed l-NAME deceased the NO concentration in the PMA 200 nM group (P = 0.008), level of NO in the PL group was still significantly higher than the PBS group (P < 0.001). Therefore, we may suggest PMA-induced THP-1 cell differentiation partially depends on NO
Author contributions
YYC and TYL contributed to the conception and design, data collection, analysis and interpretation, writing and critical revision of the article. WHJ and CWL contributed to data analysis and interpretation as well as writing of the article. SJS contributed to critical revision of the article.
Acknowledgement
This work was supported by a grant from Far Eastern Memorial Hospital (FEMH-2020-C-022), awarded to YYC.
References (45)
- et al.
LDL accelerates monocyte to macrophage differentiation: effects on adhesion and anoikis
Atherosclerosis
(2016) - et al.
The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus
J. Immunol. Methods
(2016) Nitric oxide synthase in innate and adaptive immunity: an update
Trends Immunol.
(2015)- et al.
Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression
J. Surg. Res.
(2015) Calmodulin-dependent regulation of inducible and neuronal nitric-oxide synthase
J. Biol. Chem.
(1998)- et al.
Increased resting intracellular calcium modulates NF-κB-dependent inducible nitric-oxide synthase gene expression in dystrophic mdx skeletal myotubes
J. Biol. Chem.
(2012) - et al.
Role of endogenous and exogenous nitric oxide, carbon monoxide and hydrogen sulfide in HCT116 colon cancer cell proliferation
Biochem. Pharmacol.
(2018) - et al.
Nitric oxide balances osteoblast and adipocyte lineage differentiation via the JNK/MAPK signaling pathway in periodontal ligament stem cells
Stem Cell Res. Ther.
(2018) - et al.
Development of monocytes, macrophages, and dendritic cells
Science
(2010) - et al.
Human monocytes and macrophages regulate immune tolerance via integrin alphavbeta8-mediated TGFbeta activation
J. Exp. Med.
(2018)
Monocytes and macrophages regulate immunity through dynamic networks of survival and cell death
J Innate Immun
Involvement of monocytes/macrophages as key factors in the development and progression of cardiovascular diseases
Biochem. J.
Inflammatory expression profiles in monocyte-to-macrophage differentiation in patients with systemic lupus erythematosus and relationship with atherosclerosis
Arthritis Res. Ther.
Monocyte and macrophage dysfunction as a cause of HIV-1 induced dysfunction of innate immunity
Curr. Mol. Med.
Macrophages in atherosclerosis: a dynamic balance
Nat. Rev. Immunol.
Molecular interaction of NFkappaB and NICD in monocyte-macrophage differentiation is a target for intervention in atherosclerosis
J. Cell. Physiol.
Macrophage differentiation and function in atherosclerosis: opportunities for therapeutic intervention?
J Innate Immun
Comparative genotypic and phenotypic analysis of human peripheral blood monocytes and surrogate monocyte-like cell lines commonly used in metabolic disease research
PLoS One
Human monocytoid THP-1 cell line versus monocyte-derived human immature dendritic cells as in vitro models for predicting the sensitising potential of chemicals
Int. J. Immunopathol. Pharmacol.
Optimization and evaluation of an inflammatory cell model in LPS-stimulated PMA-differentiated THP-1 cells
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi
The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages
PLoS One
Optimized THP-1 differentiation is required for the detection of responses to weak stimuli
Inflamm. Res.
Cited by (10)
Knockdown of long non-coding RNA MIR155HG suppresses melanoma cell proliferation, and deregulated MIR155HG in melanoma is associated with M1/M2 balance and macrophage infiltration
2022, Cells and DevelopmentCitation Excerpt :These evidences indicate that high expression of MIR155HG in melanoma or other cancers may lead to resistance to apoptosis by modulating apoptosis-associated proteins, and thereby accelerate melanoma cell survival. Human monocytic leukemia THP-1 cells differentiated by PMA are widely used for studying macrophage function and polarization (Chang et al., 2021; Starr et al., 2018). In the present study, THP-1 cells were differentiated into M0 macrophages.
In Vivo Immune Adjuvant Effects of CaCO<inf>3</inf> Nanoparticles through Intracellular Ca<sup>2+</sup> Concentration Regulation
2023, ACS Applied Materials and Interfaces